The silk of gorse spider mite Tetranychus lintearius represents a novel natural source of nanoparticles and biomaterials.

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.

Mode of entry of secondary metabolites of the bacteria Xenorhabdus szentirmaii and X. nematophila into Tetranychus urticae, and their toxicity to the predatory mites Phytoseiulus persimilis and Neoseiulus californicus.

The bacterial metabolites in supernatants of Xenorhabdus species have acaricidal activity, but this mode of entry into mites has not yet been elucidated. Herein, we report on the possible mode of entry of Xenorhabdus szentirmaii and Xenorhabdus nematophila supernatants into Tetranychus urticae (Acari: Tetranychidae) adult females. We also assessed the toxicity of the supernatants against the developmental stages of the predatory mites, Phytoseiulus persimilis and Neoseiulus californicus (Acari: Phytoseiidae). Experiments were conducted at 25 ± 1 °C, 70 ± 5% relative humidity, and 16:8h light:dark conditions. Our data showed that the bioactive acaricidal compound is most effective (86.5 to 89% mortality) when the entire integument of T. urticae comes in contact with it compared to contact of the ventral side only (26.5-34%). Against P. persimilis and N. californicus at 6 days post-application (dpa), the eggs were not affected by the X. szentirmaii or X. nematophila supernatant, whereas mortality of the mobile stages (larva, protonymph, deutonymph, adult) was 18.5% to 39.2%. Overall, the predatory mites were less affected by the bacterial metabolites than T. urticae. We hypothesize that the differences in morphology such as longer legs and thicker cuticle, as well as the diet of the predatory mites, reduce the contact of the body parts to the supernatant-treated surfaces. We need to isolate, identify, and characterize the X. szentirmaii and X. nematophila metabolite(s) and demonstrate efficacy to pestiferous mites and safety to plants, non-target organisms and the environment before it can be used as an acaricide.

Substrate specificity and promiscuity of horizontally transferred UDP-glycosyltransferases in the generalist herbivore Tetranychus urticae.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze the addition of UDP-sugars to small hydrophobic molecules, turning them into more water-soluble metabolites. While their role in detoxification is well documented for vertebrates, arthropod UGTs have only recently been linked to the detoxification and sequestration of plant toxins and insecticides. The two-spotted spider mite Tetranychus urticae is a generalist herbivore notorious for rapidly developing resistance to insecticides and acaricides. We identified a set of eight UGT genes that were overexpressed in mites upon long-term acclimation or adaptation to a new host plant and/or in mite strains highly resistant to acaricides. Functional expression revealed that they were all catalytically active and that the majority preferred UDP-glucose as activated donor for glycosylation of model substrates. A high-throughput substrate screening of both plant secondary metabolites and pesticides revealed patterns of both substrate specificity and promiscuity. We further selected nine enzyme-substrate combinations for more comprehensive analysis and determined steady-state kinetic parameters. Among others, plant metabolites such as capsaicin and several flavonoids were shown to be glycosylated. The acaricide abamectin was also glycosylated by two UGTs and one of these was also overexpressed in an abamectin resistant strain. Our study corroborates the potential role of T. urticae UGTs in detoxification of both synthetic and natural xenobiotic compounds and paves the way for rapid substrate screening of arthropod UGTs.

Identification of Saliva Proteins of the Spider Mite Tetranychus evansi by Transcriptome and LC-MS/MS Analyses.

The spider mite Tetranychus evansi has a remarkable ability to suppress and manipulate plant defenses, which makes it an ideal model to investigate plant-herbivores interactions. In this study, a de novo assembly of the transcriptome of T. evansi is performed and the proteins in its secreted saliva by LC-MS/MS are characterized. A total of 29 365 unigenes are assembled and 136 saliva proteins are identified. Comparative analysis of the saliva proteins in T. evansi, T. truncatus, and T. urticae shows that 64 protein groups are shared by at least two Tetranychus species, and 52 protein groups are specifically identified in T. evansi. In addition, some saliva proteins are common in arthropod species, while others are species-specific. These results will help to elucidate the molecular mechanisms by which T. evansi modulates plant defenses.

Transfer of Cry1Ac and Cry2Ab proteins from genetically engineered Bt cotton to herbivores and predators.

With the cultivation of Bt cotton, the produced insecticidal Cry proteins are ingested by herbivores and potentially transferred along the food chain to natural enemies, such as predators. In laboratory experiments with Bollgard II cotton, concentrations of Cry1Ac and Cry2Ab were measured in Lepidoptera larvae (Spodoptera littoralis, Heliothis virescens), plant bugs (Euschistus heros), aphids (Aphis gossypii), whiteflies (Bemisia tabaci), thrips (Thrips tabaci, Frankliniella occidentalis), and spider mites (Tetranychus urticae). Tritrophic experiments were conducted with caterpillars of S. littoralis as prey and larvae of ladybird beetles (Harmonia axyridis, Adalia bipunctata) and lacewings (Chrysoperla carnea) as predators. Immunological measurements (ELISA) indicated that herbivores feeding on Bt cotton contained 5%-50% of the Bt protein concentrations in leaves except whiteflies and aphids, which contained no or only traces of Bt protein, and spider mites, which contained 7 times more Cry1Ac than leaves. Similarly, predators contained 1%-30% of the Cry protein concentration in prey. For the nontarget risk assessment, this indicates that Bt protein concentrations decrease considerably from one trophic level to the next in the food web, except for spider mites that contain Bt protein concentrations higher than those measured in the leaves. Exposure of phloem sucking hemipterans is negligible.

Antioxidant Protection by Astaxanthin in the Citrus Red Mite (Acari: Tetranychidae).

Solar ultraviolet-B (UVB) radiation and radiant heat have lethal effects on plant-dwelling mites, including spider mites, and their natural enemies, such as phytoseiid mites, leading them to reside on lower leaf surfaces. Panonychus spider mites are outcompeted by Tetranychus spider mites and thus exploit upper leaf surfaces, where they are exposed to both UVB radiation and radiant heat. Panonychus spider mites are thought to produce astaxanthin constitutionally. In this study, we compared carotenoid components, antioxidant capacity, lipid peroxidation, survival, and egg production in wild-type (WTS) and albino-type strains (ATS) of Panonychus citri (McGregor). Four carotenoids (neoxanthin, violaxanthin, lutein, and carotene) and their isomers and esters were identified in both strains, but astaxanthin and its esters were present only in WTS. The singlet oxygen scavenging capacity of lipid-soluble ingredients was greater in WTS than in ATS, whereas the oxygen radical absorbance capacities of hydrophilic ingredients were equivalent between them. Lipid peroxide accumulation was clearly higher in ATS than in WTS under both UVB irradiation (25 °C) and high temperature (35 °C) conditions. The findings are consistent with an antioxidant protective function of astaxanthin in this mite. Survival periods at 38 °C were longer in WTS than in ATS, although no difference was shown at 35 °C or under UVB irradiation. Therefore, astaxanthin accumulation was shown to be a major mechanism for survival under radiant heat, although other mechanisms, such as photoreactivation, might play a major role in survival under UVB radiation.

Structural Characterization of a Eukaryotic Cyanase from Tetranychus urticae.

The two-spotted spider mite Tetranychus urticae is a polyphagous agricultural pest and poses a high risk to global crop production as it is rapidly developing pesticide resistance. Genomic and transcriptomic analysis has revealed the presence of a remarkable cyanase gene in T. urticae and related mite species within the Acariformes lineage. Cyanase catalyzes the detoxification of cyanate and is potentially an attractive protein target for the development of new acaricides. Phylogenetic analysis indicates that within the Acariformes, the cyanase gene originates from a single horizontal gene transfer event, which precedes subsequent speciation. Our structural studies presented here compare and contrast prokaryotic cyanases to T. urticae cyanase, which all form homodecamers and have conserved active site residues, but display different surface areas between homodimers in the overall decameric structure.

Variogram analysis of hyperspectral data to characterize the impact of biotic and abiotic stress of maize plants and to estimate biofuel potential.

A considerable challenge in applied agricultural use of reflection-based spectroscopy is that most analytical approaches are quite sensitive to radiometric noise and/or low radiometric repeatability. In this study, hyperspectral imaging data were acquired from individual maize leaves and the main objective was to evaluate a classification system for detection of drought stress levels and spider mite infestation levels across maize hybrids and vertical position of maize leaves. A second objective was to estimate biomass and biofuel potential (heating value) of growing maize plants. Stepwise discriminant analysis was used to identify the five spectral bands (440, 462, 652, 706, and 784 nm) that contributed most to the classification of three levels of drought stress (moderate, subtle, and none) across hybrids, leaf position, and spider mite infestation. Regarding the five selected spectral bands, average reflectance values and standard variogram parameters ("nugget", "sill", and "range" derived from variogram analysis) were examined as indicators of spider mite and/or drought stress. There was consistent significant effect of drought stress on average reflectance values, while only one spectral band responded significantly to spider mite infestations. Different variogram parameters provided reliable indications of spider mite infestation and drought stress. Based on independent validation, variogram parameters could be used to accurately predict spider mite density but were less effective as indicators of drought stress. In addition, variogram parameters were used as explanatory variables to predict biomass and biofuel potential of individual maize plants. The potential of using variogram analysis as part of hyperspectral imaging analysis is discussed.

The predatory mite Neoseiulus womersleyi (Acari: Phytoseiidae) follows extracts of trails left by the two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae).

As it walks, the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) spins a trail of silk threads, that is followed by the predatory mite, Neoseiulus womersleyi Schicha (Acari: Phytoseiidae). Starved adult female N. womersleyi followed T. urticae trails laid down by five T. urticae females but did not follow a trail of one T. urticae female, suggesting that the amount of spun threads and their chemical components should correlate positively with the number of T. urticae individuals. To examine whether chemical components of T. urticae trails are responsible for the predatory mite's trail following, we collected separate T. urticae threads from the exuviae and eggs, and then washed the threads with methanol to separate chemical components from physical attributes of the threads. Female N. womersleyi did not follow T. urticae trails that had been washed with methanol but contained physical residues, but they did follow the direction to which the methanol extracts of the T. urticae trails was applied. These results suggest that the predatory mite follows chemical, not physical, attributes of T. urticae trails.

Characterization of vitellin protein in the twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae).

In mites, vitellogenin synthesis, regulation and uptake by the oocytes as vitellin remain practically unknown. Although a partial sequence of the gene is now available, no previous studies have been conducted that describe the native vitellin protein in mites. The objective of this study was to characterize vitellin in the twospotted spider mite, Tetranychus urticae. The native twospotted spider mite vitellin migrated as a single major band with a molecular weight of 476+/-14.5 kDa as compared to 590+/-25.5 kDa for vitellin from the American dog tick, Dermacentor variabilis. However, isoelectric focusing analysis of native spider mite vitellin showed five bands with pI values slightly acidic to neutral (pH 5.8, 6.2, 6.7, 7.0 and 7.2), as is the case for insect and tick vitellins. Reducing conditions (SDS-PAGE) also revealed multiple subunits ranging from 290.9 to 3.6 kDa and was similar to that found in D. variabilis. Spider mite vitellin weakly bound lipids and carbohydrates compared to the tick. Unlike D. variabilis, the spider mite egg yolk protein does not bind heme. The significance of non-heme binding in mites is discussed.

How to visualize the spider mite silk?

Tetranychus urticae (Acari: Tetranychidae) is a phytophagous mite that forms colonies of several thousand individuals. Like spiders, every individual produces abundant silk strands and is able to construct a common web for the entire colony. Despite the importance of this silk for the biology of this worldwide species, only one previous study suggested how to visualize it. To analyze the web structuration, we developed a simple technique to dye T. urticae'silk on both inert and living substrates. Fluorescent brightener 28 (FB) (Sigma F3543) diluted in different solvents at different concentrations regarding the substrate was used to observe single strands of silk. On glass lenses, a 0.5% dimethyl sulfoxide solution was used and on bean leaves, a 0.1% aqueous solution. A difference of silk deposit was observed depending the substrate: rectilinear threads on glass lenses and more sinuous ones on bean leaves. This visualizing technique will help to carry out future studies about the web architecture and silk used by T. urticae. It might also be useful for the study of other silk-spinning arthropods.

Qualitative and quantitative variation among volatile profiles induced by Tetranychus urticae feeding on plants from various families.

Many plant species are known to emit herbivore-induced volatiles in response to herbivory. The spider mite Tetranychus urticae Koch is a generalist that can feed on several hundreds of host plant species. Volatiles emitted by T. urticae-infested plants of 11 species were compared: soybean (Glycine max), golden chain (Laburnum anagyroides), black locust (Robinia pseudo-acacia), cowpea (Vigna unguiculata), tobacco (Nicotiana tabacum), eggplant (Solanum melalonga), thorn apple (Datura stramonium), sweet pepper (Capsicum annuum), hop (Humulus lupulus), grapevine (Vitis vinifera), and ginkgo (Ginkgo biloba). The degree to which the plant species produced novel compounds was analyzed when compared to the odors of mechanically damaged leaves. Almost all of the investigated plant species produced novel compounds that dominated the volatile blend, such as methyl salicylate, terpenes, oximes, and nitriles. Only spider mite-infested eggplant and tobacco emitted a blend that was merely quantitatively different from the blend emitted by mechanically damaged or clean leaves. We hypothesized that plant species with a low degree of direct defense would produce more novel compounds. However, although plant species with a low direct defense level do use indirect defense to defend themselves, they do not always emit novel compounds. Plant species with a high level of direct defense seem to invest in the production of novel compounds. When plant species of the Fabaceae were compared to plant species of the Solanaceae, qualitative differences in spider mite-induced volatile blends seemed to be more prominent in the Fabaceae than in the Solanaceae.