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Suberin deposition involves both phenolic and aliphatic polymer biosynthesis and deposition in the same tissue. Therefore, any consideration of exploiting suberin for crop enhancement (e.g., enhanced storage, soil borne disease resistance) requires knowledge of both phenolic and aliphatic component biosynthesis and their coordinated, temporal deposition. In the present study, we use a wound-healing potato tuber system to explore global transcriptome changes during the early stages of wound-healing. Wounding leads to initial and substantial transcriptional changes that follow distinctive temporal patterns - primary metabolic pathways were already functional, or up-regulated immediately, and maintained at levels that would allow for precursor carbon skeletons and energy to feed into downstream metabolic processes. Genes involved in pathways for phenolic production (i.e., the shikimate pathway and phenylpropanoid metabolism) were up-regulated early while those involved in aliphatic suberin production (i.e., fatty acid biosynthesis and modification) were transcribed later into the time course. The pattern of accumulation of genes associated with ABA biosynthesis and degradation steps support a role for ABA in regulating aliphatic suberin production. Evaluation of putative Casparian strip membrane-like genes pinpointed wound-responsive candidates that may mediate the suberin deposition process.

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 β-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and β-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 β-cyanoalanine synthase activity. Consistent with the β-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 β-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.

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.

Arabidopsis defenses against herbivores are regulated by the jasmonate hormonal signaling pathway, which leads to the production of a plethora of defense compounds, including tryptophan-derived metabolites produced through CYP79B2/CYP79B3. Jasmonate signaling and CYP79B2/CYP79B3 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, using Arabidopsis mutants that disrupt metabolic pathways downstream of CYP79B2/CYP79B3, and synthetic indole glucosinolates, we identified phytochemicals involved in the defense against T. urticae. We show that Trp-derived metabolites depending on CYP71A12 and CYP71A13 are not affecting mite herbivory. Instead, the supplementation of cyp79b2 cyp79b3 mutant leaves with the 3-indolylmethyl glucosinolate and its derived metabolites demonstrated that the indole glucosinolate pathway is sufficient to assure CYP79B2/CYP79B3-mediated defenses against T. urticae. We demonstrate that three indole glucosinolates can limit T. urticae herbivory, but that they have to be processed by the myrosinases to hinder T. urticae oviposition. Finally, the supplementation of the mutant myc2 myc3 myc4 with indole glucosinolates indicated that the transcription factors MYC2/MYC3/MYC4 induce additional indole glucosinolate-independent defenses that control T. urticae herbivory. Together, these results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple indole glucosinolates, specific myrosinases, and additional MYC2/MYC3/MYC4-dependent defenses.One sentence summaryThree indole glucosinolates and the myrosinases TGG1/TGG2 help protect Arabidopsis thaliana against the herbivory of the two-spotted spider mite Tetranychus urticae.

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., Mites can evolve highly efficient detoxification-based adaptation in approximately 25 generations on an initially unfavorable plant host, revealing that specialization can occur within the ecological timescale.

Spider mites constitute an assemblage of well-known pests in agriculture, but are less known for their ability to spin silk of nanoscale diameters and high Young’s moduli. Here, we characterize silk of the gorse spider mite Tetranychus lintearius, which produces copious amounts of silk with nano-dimensions. We determined biophysical characteristics of the silk fibres and manufactured nanoparticles and biofilm derived from native silk. We determined silk structure using attenuated total reflectance Fourier transform infrared spectroscopy, and characterized silk nanoparticles using field emission scanning electron microscopy. Comparative studies using T. lintearius and silkworm silk nanoparticles and biofilm demonstrated that spider mite silk supports mammalian cell growth in vitro and that fluorescently labelled nanoparticles can enter cell cytoplasm. The potential for cytocompatibility demonstrated by this study, together with the prospect of recombinant silk production, opens a new avenue for biomedical application of this little-known silk.

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., Tis work was supported by the Government of Canada through the Ontario Research Fund (RE08-067), the Natural Sciences and Engineering Research Council of Canada (NSERC) and by the European Union’s Horizon 2020 research and innovation program (773902-SuperPests) awarded to MG and VG; and by the Japan Society for the Promotion of Science KAKENHI (Grant Nos. 18H02203 to TS, 19K22304 to TF and 19K23674 to MT) and the Institute of Global Innovation Research in TUAT to MT, TS, TF and VG. MA was funded through the Global Tesis program, the University of Bari Aldo Moro, Italy.

The two-spotted spider mite Tetranychus urticae is an extremely polyphagous crop pest. Alongside an unparalleled detoxification potential for plant secondary metabolites, it has recently been shown that spider mites can attenuate or even suppress plant defenses. Salivary constituents, notably effectors, have been proposed to play an important role in manipulating plant defenses and might determine the outcome of plant-mite interactions. Here, the proteomic composition of saliva from T. urticae lines adapted to various host plants—bean, maize, soy, and tomato—was analyzed using a custom-developed feeding assay coupled with nano-LC tandem mass spectrometry. About 90 putative T. urticae salivary proteins were identified. Many are of unknown function, and in numerous cases belonging to multimembered gene families. RNAseq expression analysis revealed that many genes coding for these salivary proteins were highly expressed in the proterosoma, the mite body region that includes the salivary glands. A subset of genes encoding putative salivary proteins was selected for whole-mount in situ hybridization, and were found to be expressed in the anterior and dorsal podocephalic glands. Strikingly, host plant dependent expression was evident for putative salivary proteins, and was further studied in detail by micro-array based genome-wide expression profiling. This meta-analysis revealed for the first time the salivary protein repertoire of a phytophagous chelicerate. The availability of this salivary proteome will assist in unraveling the molecular interface between phytophagous mites and their host plants, and may ultimately facilitate the development of mite-resistant crops. Furthermore, the technique used in this study is a time- and resource-efficient method to examine the salivary protein composition of other small arthropods for which saliva or salivary glands cannot be isolated easily.

Two-spotted spider mite (TSSM) Tetranychus urticae (Koch) is an important agricultural pest that causes considerable yield losses to over 150 field and greenhouse crops. Mitochondrial electron transport inhibitors (METI) acaricides are commonly used to control mite species in commercial Canadian greenhouses. Development of resistance to METIs in TSSM populations have been reported worldwide, but not until recently in Canada. The objectives of this study were to: 1) monitor the acaricide-susceptibility in greenhouse TSSM populations, and 2) investigate the resistance to pyridaben, a METI acaricide, in greenhouse resistant and pyridaben-selected (SRS) mite strains. The increased mortality to the pyridaben sub-lethal concentration (LC30) when SRS mites were exposed to piperonyl butoxide (PBO), a general cytochrome P450 monooxygenase inhibitor, and higher P450 activity compared to the greenhouse strain (RS) mites, indicated that P450s may be at least partially responsible for the resistance. The molecular mechanisms of target site insensitivity-mediated resistance in the pyridaben resistant strain of TSSM were investigated by comparing the DNA sequence of NADH dehydrogenase subunits TYKY and PSST, NADH-ubiquinone oxidoreductase chain 1 and 5 (ND1, ND5) and the NADH-ubiquinone oxidoreductase subunit 49 kDa from SRS to the reference strain (SS) and RS. Despite a number of nucleotide substitutions, none correlated with the pyridaben resistance. Understanding the underlying mechanisms of TSSM adaptation to acaricides is an essential part of resistance management strategy in any IPM program. The findings of this study will encourage growers to apply acaricides with different modes of action to reduce the rate at which acaricide resistance will occur in greenhouse TSSM populations.

The two-spotted spider mite (TSSM), Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores, feeding on more than 1,100 plant species. Its wide host range suggests that TSSM has an extraordinary ability to modulate its digestive and xenobiotic physiology. The analysis of the TSSM genome revealed the expansion of gene families that encode proteins involved in digestion and detoxification, many of which were associated with mite responses to host shifts. The majority of plant defense compounds that directly impact mite fitness are ingested. They interface mite compounds aimed at counteracting their effect in the gut. Despite several detailed ultrastructural studies, our knowledge of the TSSM digestive tract that is needed to support the functional analysis of digestive and detoxification physiology is lacking. Here, using a variety of histological and microscopy techniques, and a diversity of tracer dyes, we describe the organization and properties of the TSSM alimentary system. We define the cellular nature of floating vesicles in the midgut lumen that are proposed to be the site of intracellular digestion of plant macromolecules. In addition, by following the TSSM's ability to intake compounds of defined sizes, we determine a cut off size for the ingestible particles. Moreover, we demonstrate the existence of a distinct filtering function between midgut compartments which enables separation of molecules by size. Furthermore, we broadly define the spatial distribution of the expression domains of genes involved in digestion and detoxification. Finally, we discuss the relative simplicity of the spider mite digestive system in the context of mite's digestive and xenobiotic physiology, and consequences it has on the effectiveness of plant defenses.

While mechanisms to detoxify plant produced, anti-herbivore compounds have been associated with plant host use by herbivores, less is known about the role of chemosensory perception in their life histories. This is especially true for generalists, including chelicerate herbivores that evolved herbivory independently from the more studied insect lineages. To shed light on chemosensory perception in a generalist herbivore, we characterized the chemosensory receptors (CRs) of the chelicerate two-spotted spider mite, Tetranychus urticae, an extreme generalist. Strikingly, T. urticae has more CRs than reported in any other arthropod to date. Including pseudogenes, 689 gustatory receptors were identified, as were 136 degenerin/Epithelial Na+ Channels (ENaCs) that have also been implicated as CRs in insects. The genomic distribution of T. urticae gustatory receptors indicates recurring bursts of lineage-specific proliferations, with the extent of receptor clusters reminiscent of those observed in the CR-rich genomes of vertebrates or C. elegans. Although pseudogenization of many gustatory receptors within clusters suggests relaxed selection, a subset of receptors is expressed. Consistent with functions as CRs, the genomic distribution and expression of ENaCs in lineage-specific T. urticae expansions mirrors that observed for gustatory receptors. The expansion of ENaCs in T. urticae to > 3-fold that reported in other animals was unexpected, raising the possibility that ENaCs in T. urticae have been co-opted to fulfill a major role performed by unrelated CRs in other animals. More broadly, our findings suggest an elaborate role for chemosensory perception in generalist herbivores that are of key ecological and agricultural importance.

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.

In Arabidopsis thaliana, the HUA2 gene is required for proper expression of FLOWERING LOCUS C (FLC) and AGAMOUS, key regulators of flowering time and reproductive development, respectively. Although HUA2 is broadly expressed, plants lacking HUA2 function have only moderately reduced plant stature, leaf initiation rate and flowering time. To better understand HUA2 activity, and to test whether redundancy with similar genes underlies the absence of strong phenotypes in HUA2 mutant plants, we identified and subsequently characterized three additional HUA2-LIKE (HULK) genes in Arabidopsis. These genes form two clades (HUA2/HULK1 and HULK2/HULK3), with members broadly conserved in both vascular and non-vascular plants, but not present outside the plant kingdom. Plants with progressively reduced HULK activity had increasingly severe developmental defects, and plants homozygous for loss-of-function mutations in all four HULK genes were not recovered. Multiple mutants displayed reproductive, embryonic and post-embryonic abnormalities, and provide detailed insights into the overlapping and unique functions of individual HULK genes. With regard to flowering time, opposing influences were apparent: hua2 hulk1 plants were early-flowering, while hulk2 hulk3 mutants were late-flowering, and hua2 acted epistatically to cause early flowering in all combinations. Genome-wide expression profiling of mutant combinations using RNA-Seq revealed complex transcriptional changes in seedlings, with FLC, a known target of HUA2, among the most affected. Our studies, which include characterization of HULK expression patterns and subcellular localization, suggest that the HULK genes encode conserved nuclear factors with partially redundant but essential functions associated with diverse genetic pathways in plants.

Germband size in insects has played a central role in our understanding of insect patterning mechanisms and their evolution. The polarity of evolutionary change in insect patterning has been viewed so far as the unidirectional shift from the ancestral short germband patterning of basal hemimetabolous insects to the long germband patterning observed in most modern Holometabola. However, some orders of holometabolic insects display both short and long germband development, though the absence of a clear phylogenetic context does not permit definite conclusions on the polarity of change. Derived hymenoptera, that is, bees and wasps, represent a classical textbook example of long germband development. Yet, in some wasps putative short germband development has been described correlating with lifestyle changes, namely with evolution of endoparasitism and polyembryony. To address the potential reversion from long to short germband, we focused on the family Braconidae, which displays ancestral long germband development, and examined the derived polyembryonic braconid Macrocentrus cingulum. Using SEM analysis of M. cingulum embryogenesis coupled with analyses of embryonic patterning markers, we show that this wasp evolved short germband embryogenesis secondarily, in a way that is reminiscent of embryogenesis in the beetle Tribolium castaneum. This work shows that the evolution of germband size in insects is a reversible process that may correlate with other life-history traits and suggests broader implications on the mechanisms and evolvability of insect development.

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.

RNA interference (RNAi) can be used for the protection against agricultural pests through the silencing of genes required for pest fitness. To assess the potential of RNAi approaches in the two-spotted spider mite, Tetranychus urticae, we compared 5 methods for the delivery of double-stranded RNA (dsRNA). These methods include mite feeding on either (i) leaves floating on a dsRNA solution, (ii) dsRNA-expressing plants, (iii) artificial diet supplemented with dsRNA, or (iv) dsRNA-coated leaves, and (v) mite soaking in a dsRNA solution. In all cases, the gene targeted for method validation was the Vacuolar-type H+-ATPase (TuVATPase), encoding a constitutively expressed ATP-driven proton pump located in the membrane. Down-regulation of TuVATPase increased mortality and/or reduced fecundity in all methods, but with variable efficiency. The most efficient methods for dsRNA delivery were direct soaking of mites in the dsRNA solution and mite feeding on dsRNA-coated leaves that mimics dsRNA application as a sprayable pesticide. Both resulted in a dark-body phenotype not observed in mites treated with a control dsRNA. Although with lower efficiency, dsRNA designed for TuVATPase silencing and expressed in transgenic Arabidopsis plants impacted the fitness of mites feeding on these plants. RNAi may thus be a valuable strategy to control spider mite populations, either as a sprayable pesticide or through transgenic crops. This comparative methodological study focusing on the induction of RNAi-based gene silencing in T. urticae paves the way for reverse genetics approaches in this model chelicerate system and prepares large-scale systematic RNAi screens as a first step towards the development of specific RNA-based pesticides. Such alternative molecules may help control spider mites that cause significant damages to crops and ornamental plant species, as well as other chelicerates detrimental to agriculture and health. © 2017 Suzuki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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 highquality genome sequence and developing genetic tools, coupled with an ease of mite experimental selection to new hosts, make TSSM an outstanding systemto 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. © 2017 The American Phytopathological Society.

The two-spotted spider mite, Tetranychus urticae, is a chelicerate herbivore with an extremely wide host range and an extraordinary ability to develop pesticide resistance. Due to its responsiveness to natural and synthetic xenobiotics, the spider mite is becoming a prime pest herbivore model for studies of the evolution of host range, plant-herbivore interactions and mechanisms of xenobiotic resistance. The spider mite genome has been sequenced and its transcriptional responses to developmental and various biotic and abiotic cues have been documented. However, to identify biological and evolutionary roles of T. urticae genes and proteins, it is necessary to develop methods for the efficient manipulation of mite gene function or protein activity. Here, we describe protocols developed for the delivery of small molecules into spider mites. Starting with mite maintenance and the preparation of the experimental mite populations of developmentally synchronized larvae and adults, we describe 3 methods for delivery of small molecules including artificial diet, leaf coating, and soaking. The presented results define critical steps in these methods and demonstrate that they can successfully deliver tracer dyes into mites. Described protocols provide guidelines for high-throughput setups for delivery of experimental compounds that could be used in reverse genetics platforms to modulate gene expression or protein activity, or for screens focused on discovery of new molecules for mite control. In addition, described protocols could be adapted for other Tetranychidae and related species of economic importance such as Varroa, dust and poultry mites., Correction -- 15 Dec 2017: Suzuki T, España MU, Nunes MA, Zhurov V, Dermauw W, et al. (2017) Correction: Protocols for the delivery of small molecules to the two-spotted spider mite, Tetranychus urticae. PLOS ONE 12(12): e0190025.

GABA(A) functioning has been implicated in anxiety and depressive disorders. In this regard, we suggested that in addition to analyzing GABA(A) and the subunits that comprise the GABA(A) receptor, it might be profitable to assess the coordinated expression of subunits that comprise the GABA(A) receptor cassette. We demonstrate that certain subunits within stress-sensitive brain regions were higher in stressor reactive BALB/cByJ than in hardy C57BL/6ByJ mice, and that a chronic, intermittent, variable stressor (6 days/week over 7 weeks) differentially influenced subunit expression in these strains. Further, mRNA expression of GABA(A) subunits were highly coordinated (inter-correlated), and markedly altered by stressors, once again varying with brain region. At the central amygdala of BALB/cByJ mice the ordinarily high subunit inter-relations were reduced in acutely stressed mice, and this outcome was exacerbated with a chronic stressor. In C57BL/6ByJ mice subunit inter-relations were lower than in BALB/cByJ mice; the acute stressor increased subunit organization, which returned to control levels with following a chronic stressor. The profile of amygdala subunit inter-relations was recapitulated in a step-down behavioral test; anxiety was increased by acute and chronic stressors in BALB/cByJ mice, but in the C57BL/6ByJ strain the elevated anxiety associated with an acute stressor was not apparent after chronic stressor treatment. The anxiety could be dissociated from apparent anhedonia (reflected by free sucrose consumption) where the preference for sucrose was reduced by an acute stressor, but this outcome was more pronounced following a chronic stressor, especially in BALB/cByJ mice. These findings support the view that analyses involving subunit organization, rather than just differences in absolute levels, may be expedient in assessing GABA(A) functioning in stressor-related psychological disturbances.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores feeding on cell contents of over 1100 plant species including more than 150 crops. It is being established as a model for chelicerate herbivores with tools that enable tracking of reciprocal responses in plant-spider mite interactions. However, despite their important pest status and a growing understanding of the molecular basis of interactions with plant hosts, knowledge of the way mites interface with the plant while feeding and the plant damage directly inflicted by mites is lacking. Here, utilizing histology and microscopy methods, we uncovered several key features of T. urticae feeding. By following the stylet path within the plant tissue, we determined that the stylet penetrates the leaf either in between epidermal pavement cells or through a stomatal opening, without damaging the epidermal cellular layer. Our recordings of mite feeding established that duration of the feeding event ranges from several minutes to more than half an hour, during which time mites consume a single mesophyll cell in a pattern that is common to both bean and Arabidopsis plant hosts. In addition, this study determined that leaf chlorotic spots, a common symptom of mite herbivory, do not form as an immediate consequence of mite feeding. Our results establish a cellular context for the plant-spider mite interaction that will support our understanding of the molecular mechanisms and cell signaling associated with spider mite feeding. © 2016 Bensoussan, Santamaria, Zhurov, Diaz, Grbi and Grbi.

Polyembryonic development, where multiple embryos are formed from a single zygote, evolved at least 15 times in six different phyla in animals. The mechanisms leading to polyembryony and the forces that shaped the evolution of the polyembryonic developmental program have remained poorly understood. Recent studies of the polyembryonic development in the endoparasitic wasp Copidosoma floridanum have revealed that the evolution of polyembryony is associated with the evolution of developmental novelties such as total cleavage, early specification of embryonic and extra-embryonic fates, and a specific cell proliferation phase. These changes cumulatively result in the formation of thousands of embryos from a single egg. Laser ablation studies and analysis of early cell fate specification have revealed that a single blastomere representing the progenitor of the primordial germ cell regulates the proliferation of the embryos. We propose that evolutionary changes in cell cleavage, cell interactions, and the cell-differentiation program, reminiscent of interactions between the germinal stem cell and stem cell niche in fly ovaries, underlies the evolution of polyembryony.

International audience; The two-spotted spider mite Tetranychus urticae is one of the most significant mite pests in agriculture, feeding on more than 1,100 plant hosts, including model plants Arabidopsis thaliana and tomato, Solanum lycopersicum. Here, we describe timecourse tomato transcriptional responses to spider mite feeding and compare them with Arabidopsis in order to determine conserved and divergent defense responses to this pest. To refine the involvement of jasmonic acid (JA) in mite-induced responses and to improve tomato Gene Ontology annotations, we analyzed transcriptional changes in the tomato JA-signaling mutant defenseless1 (def-1) upon JA treatment and spider mite herbivory. Overlay of differentially expressed genes (DEG) identified in def-1 onto those from the timecourse experiment established that JA controls expression of the majority of genes differentially regulated by herbivory. Comparison of defense responses between tomato and Arabidopsis highlighted 96 orthologous genes (of 2,133 DEG) that were recruited for defense against spider mites in both species. These genes, involved in biosynthesis of JA, phenylpropanoids, flavonoids, and terpenoids, represent the conserved core of induced defenses. The remaining tomato DEG support the establishment of tomato-specific defenses, indicating profound divergence of spider mite–induced responses between tomato and Arabidopsis.

Polyembryonic development is a unique mode of metazoan development in which a single zygote generates multiple embryos by clonal proliferation1. The polyembryonic parasitic insect Copidosoma floridanum shows one of the most extreme cases of polyembryony, producing up to 2,000 embryos from a single egg. In addition, this wasp exhibits an unusual polyphenism, producing two morphologically distinct larval castes, termed precocious and reproductive, that develop clonally from the same zygote2. This form of development seems incompatible with a model of insect development in which maternal pre-patterning of the egg specifies embryonic axial polarity3. Here we show that maternal pre-patterning in the form of germ plasm creates cellular asymmetry at the four-cell stage embryo of Copidosoma that is perpetuated throughout development. Laser ablations of cells show that the cell inheriting the germ plasm regulates both the fate and proliferation of the reproductive caste. Thus, we have uncovered a new mechanism of caste specification, mediated by the regulatory capacity of a single cell. This study shows that the evolution of mammalian-like regulative development of an insect embryo relies on a novel cellular context that might ultimately enhance developmental plasticity.

The two-spotted spider mite, Tetranychus urticae, is a chelicerate herbivore with an extremely wide host range and an extraordinary ability to develop pesticide resistance. Due to its responsiveness to natural and synthetic xenobiotics, the spider mite is becoming a prime pest herbivore model for studies of the evolution of host range, plant-herbivore interactions and mechanisms of xenobiotic resistance. The spider mite genome has been sequenced and its transcriptional responses to developmental and various biotic and abiotic cues have been documented. However, to identify biological and evolutionary roles of T. urticae genes and proteins, it is necessary to develop methods for the efficient manipulation of mite gene function or protein activity. Here, we describe protocols developed for the delivery of small molecules into spider mites. Starting with mite maintenance and the preparation of the experimental mite populations of developmentally synchronized larvae and adults, we describe 3 methods for delivery of small molecules including artificial diet, leaf coating, and soaking. The presented results define critical steps in these methods and demonstrate that they can successfully deliver tracer dyes into mites. Described protocols provide guidelines for high-throughput setups for delivery of experimental compounds that could be used in reverse genetics platforms to modulate gene expression or protein activity, or for screens focused on discovery of new molecules for mite control. In addition, described protocols could be adapted for other Tetranychidae and related species of economic importance such as Varroa, dust and poultry mites.

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.

Germband size in insects has played a central role in our understanding of insect patterning mechanisms and their evolution. The polarity of evolutionary change in insect patterning has been viewed so far as the unidirectional shift from the ancestral short germband patterning of basal hemimetabolous insects to the long germband patterning observed in most modern Holometabola. However, some orders of holometabolic insects display both short and long germband development, though the absence of a clear phylogenetic context does not permit definite conclusions on the polarity of change. Derived hymenoptera, that is, bees and wasps, represent a classical textbook example of long germband development. Yet, in some wasps putative short germband development has been described correlating with lifestyle changes, namely with evolution of endoparasitism and polyembryony. To address the potential reversion from long to short germband, we focused on the family Braconidae, which displays ancestral long germband development, and examined the derived polyembryonic braconid Macrocentrus cingulum. Using SEM analysis of M. cingulum embryogenesis coupled with analyses of embryonic patterning markers, we show that this wasp evolved short germband embryogenesis secondarily, in a way that is reminiscent of embryogenesis in the beetle Tribolium castaneum. This work shows that the evolution of germband size in insects is a reversible process that may correlate with other life-history traits and suggests broader implications on the mechanisms and evolvability of insect development.

A new family of avian centromeric satellites is described. The highly repeated sequence, designated FCP (Fringilla coelebs PstI element), was cloned from the 500-bp PstI digest fraction of the chaffinch (Fringilla coelebs L.) genomic DNA, sequenced, and characterized. The FCP repeat was found to have 505-506 bp length of monomer, 57% content of GC, to compose about 0.9% of the chaffinch genome, and to be highly methylated. Results of Southern-blot hybridization of cloned FCP element onto genomic DNA digested with different restriction enzymes, and sequencing directly from total genomic DNA using FCP-specific primers and ThermoFidelase enzyme (Fidelity Systems Inc.) were in agreement with a tandem arrangement of this repeat in the chaffinch genome. Five positions of single-nucleotide polymorphism (SNP) were found in the FCP monomers using direct genomic sequencing. Fluorescence in situ hybrid- ization (FISH) with FCP probe and primed in situ labelling (PRINS) with FCP specific primers showed that the FCP elements occupy pericentric regions of all chaffinch chromosomes. On chromosome spreads, the fluorescent signals were also observed in the intercentromeric connectives between nonhomologous chromosomes. The results suggest that the centromeric FCP repeat is responsible for chromosome ordering during mitosis in chaffinch.

Molecular mechanisms behind the etiology and pathophysiology of major depressive disorder and suicide remain largely unknown. Recent molecular studies of expression of serotonin, GABA and CRH receptors in various brain regions have demonstrated that molecular factors may contribute to the development of depressive disorder and suicide behaviour. Here, we used microarray analysis to examine the expression of genes in brain tissue (frontopolar cortex) of individuals who had been diagnosed with major depressive disorder and died by suicide, and those who had died suddenly without a history of depression. We analyzed the list of differentially expressed genes using pathway analysis, which is an assumption-free approach to analyze microarray data. Our analysis revealed that the differentially expressed genes formed functional networks that were implicated in cell to cell signaling related to synapse maturation, neuronal growth and neuronal complexity. We further validated these data by randomly choosing (100 times) similarly sized gene lists and subjecting these lists to the same analyses. Random gene lists did not provide highly connected gene networks like those generated by the differentially expressed list derived from our samples. We also found through correlational analysis that the gene expression of control participants was more highly coordinated than in the MDD/suicide group. These data suggest that among depressed individuals who died by suicide, wide ranging perturbations of gene expression exist that are critical for normal synaptic connectively, morphology and cell to cell communication.

6 páginas, 5 figuras -- PAGS nros. 487-492, Grbic, Miodrag et.al., The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant–herbivore interactions, and provides unique opportunities for developing novel plant protection strategies, M.G. and V.G. acknowledge support from NSERC Strategic Grant STPGP 322206-05, Marie Curie Incoming International Fellowship, OECD Co-operative Research Programme: Biological resource management for Sustainable Agricultural Systems JA00053351, and Ontario Research Fund–Global Leadership in Genomics and Life Sciences GL2-01-035. The genome and transcriptome sequencing projects were funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-046), JGI Community Sequencing Program grant 777506 to M.G., a University of Utah SEED grant (to R.M.C.), and National Science Foundation (NSF) grant 0820985 (to R.M.C., Principal Investigator L. Sieburth); work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under contract No. DE-AC02-05CH11231

Inter-relationships ordinarily exist between mRNA expression of GABA-A subunits in the frontopolar cortex (FPC) of individuals that had died suddenly from causes other than suicide. However, these correlations were largely absent in persons that had died by suicide. In the present investigation, these findings were extended by examining GABA-A receptor expression patterns (of controls and depressed individuals that died by suicide) in the orbital frontal cortex (OFC), hippocampus, amygdala. locus coeruleus (LC),and paraventricular nucleus (PVN), all of which have been implicated in either depression, anxiety or stress responsivity. Results Using QPCR analysis, we found that in controls the inter-relations between GABA-A subunits varied across brain regions, being high in the hippocampus and amygdala, intermediate in the LC, and low in the OFC and PVN. The GABA-A subunit inter-relations were markedly different in persons that died by suicide, being reduced in hippocampus and amygdala, stable in the LC, but more coordinated in the OFC and to some extent in the PVN. Conclusions It seems that altered brain region-specific inhibitory signaling, stemming from altered GABA-A subunit coordination, are associated with depression/suicide. Although, it is unknown whether GABA-A subunit re-organization was specifically tied to depression, suicide, or the accompanying distress, these data show that the co-ordinate expression of this transcriptome does vary depending on brain region and is plastic.

Concealed development of many animal embryos prevents examination of development and limits the application of embryo manipulation techniques aimed at understanding developmental processes. In embryos developing in utero, such as in mammals, it is necessary to dissect embryos from the mother and, upon manipulative intervention, to implant them back into the recipient. Parasitic wasps present a promising system for understanding the evolution of early developmental processes. In basal ectoparasitic species that lay eggs on the surface of the host, it is possible to adapt embryo manipulation techniques developed in Drosophila. However, their derived endoparasitic relatives, which exhibit various modifications of developmental programs, undergo concealed development within the host body. For example, the parasitic polyembryonic wasp Copidosoma floridanum oviposits an egg into the egg of the host moth Trichoplusia ni. The host larva emerges and the parasite undergoes development within the host body, preventing embryo manipulation as a means of examining developmental regulation. Here we present a protocol for embryo transfer that allows the transplantation of C. floridanum egg into the host egg. This approach opens a new avenue in the application of various embryo manipulation techniques aimed at understanding the evolution of embryogenesis in endoparasitic Hymenoptera. In addition, this approach has potential for the development of other tools in C. floridanum, such as transgenesis and reverse genetics, which can also be extended to other endoparasitic species. © Springer-Verlag 2005.

Bio-nanomaterials are one of the fastest developing sectors of industry and technology. Spider silk, a highly attractive light-weight biomaterial, has high tensile strength and elasticity and is compatible with human tissues, allowing for many areas of application. In comparison to spider silk fibers with diameters of several micrometers, spider mite silk fibers have much smaller diameters of tens of nanometers, making conventional tensile testing methods impractical. To determine the mechanical properties of adult and larval Tetranychus urticae silk fibers, we have performed three-point bending tests with an atomic force microscope. We found that because of the small diameters of these fibers, axial tension—due to both the applied force and a pre-existing strain—has a significant effect on the fiber response, even in the small-deformation limit. As a result, the typical Euler-Bernoulli-Timoshenko theory cannot be applied. We therefore follow the approach of Heidelberg et al. to develop a mechanical model...

Díaz-Riquelme, José et al., [Background] The two-spotted spider mite, Tetranychus urticae, is an extreme generalist plant pest. Even though mites can feed on many plant species, local mite populations form host races that do not perform equally well on all potential hosts. An acquisition of the ability to evade plant defenses is fundamental for mite's ability to use a particular plant as a host. Thus, understanding the interactions between the plant and mites with different host adaptation status allows the identification of functional plant defenses and ways mites can evolve to avoid them., [Results] The grapevine genome-wide transcriptional responses to spider mite strains that are non-adapted and adapted to grapevine as a host were examined. Comparative transcriptome analysis of grapevine responses to these mite strains identified the existence of weak responses induced by the feeding of the non-adapted strain. In contrast, strong but ineffective induced defenses were triggered upon feeding of the adapted strain. A comparative meta-analysis of Arabidopsis, tomato and grapevine responses to mite feeding identified a core of 36 highly conserved genes involved in the perception, regulation and metabolism that were commonly induced in all three species by mite herbivory., [Conclusions] This study describes the genome-wide grapevine transcriptional responses to herbivory of mite strains that differ in their ability to use grapevine as a host. It raises hypotheses whose testing will lead to our understanding of grapevine defenses and mite adaptations to them., This Project was funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-046, to MG and VG) and Ontario Research Fund–Global Leadership in Genomics and Life Sciences (GL2-01-035, to MG and VG), the Spanish Ministry of Science and Innovation (AGL2011-24584, to IPM), the Spanish MINECO grant BIO2011-026229 to JMZ. JG was a recipient of a Contract Ramon y Cajal from Spanish MINECO (RYC-2011-07791). SB was partially supported by the Fund for Scientific Research Flanders, grant G.0093.12 N to TVL.