A massive incorporation of microbial genes into the genome of Tetranychus urticae, a polyphagous arthropod herbivore.

A number of horizontal gene transfers (HGTs) have been identified in the spider mite Tetranychus urticae, a chelicerate herbivore. However, the genome of this mite species has at present not been thoroughly mined for the presence of HGT genes. Here, we performed a systematic screen for HGT genes in the T. urticae genome using the h-index metric. Our results not only validated previously identified HGT genes but also uncovered 25 novel HGT genes. In addition to HGT genes with a predicted biochemical function in carbohydrate, lipid and folate metabolism, we also identified the horizontal transfer of a ketopantoate hydroxymethyltransferase and a pantoate ß-alanine ligase gene. In plants and bacteria, both genes are essential for vitamin B5 biosynthesis and their presence in the mite genome strongly suggests that spider mites, similar to Bemisia tabaci and nematodes, can synthesize their own vitamin B5. We further show that HGT genes were physically embedded within the mite genome and were expressed in different life stages. By screening chelicerate genomes and transcriptomes, we were able to estimate the evolutionary histories of these HGTs during chelicerate evolution. Our study suggests that HGT has made a significant and underestimated impact on the metabolic repertoire of plant-feeding spider mites.

Mechanisms and ecological consequences of plant defence induction and suppression in herbivore communities.

BACKGROUND: Plants are hotbeds for parasites such as arthropod herbivores, which acquire nutrients and energy from their hosts in order to grow and reproduce. Hence plants are selected to evolve resistance, which in turn selects for herbivores that can cope with this resistance. To preserve their fitness when attacked by herbivores, plants can employ complex strategies that include reallocation of resources and the production of defensive metabolites and structures. Plant defences can be either prefabricated or be produced only upon attack. Those that are ready-made are referred to as constitutive defences. Some constitutive defences are operational at any time while others require activation. Defences produced only when herbivores are present are referred to as induced defences. These can be established via de novo biosynthesis of defensive substances or via modifications of prefabricated substances and consequently these are active only when needed. Inducibility of defence may serve to save energy and to prevent self-intoxication but also implies that there is a delay in these defences becoming operational. Induced defences can be characterized by alterations in plant morphology and molecular chemistry and are associated with a decrease in herbivore performance. These alterations are set in motion by signals generated by herbivores. Finally, a subset of induced metabolites are released into the air as volatiles and function as a beacon for foraging natural enemies searching for prey, and this is referred to as induced indirect defence. SCOPE: The objective of this review is to evaluate (1) which strategies plants have evolved to cope with herbivores and (2) which traits herbivores have evolved that enable them to counter these defences. The primary focus is on the induction and suppression of plant defences and the review outlines how the palette of traits that determine induction/suppression of, and resistance/susceptibility of herbivores to, plant defences can give rise to exploitative competition and facilitation within ecological communities "inhabiting" a plant. CONCLUSIONS: Herbivores have evolved diverse strategies, which are not mutually exclusive, to decrease the negative effects of plant defences in order to maximize the conversion of plant material into offspring. Numerous adaptations have been found in herbivores, enabling them to dismantle or bypass defensive barriers, to avoid tissues with relatively high levels of defensive chemicals or to metabolize these chemicals once ingested. In addition, some herbivores interfere with the onset or completion of induced plant defences, resulting in the plant's resistance being partly or fully suppressed. The ability to suppress induced plant defences appears to occur across plant parasites from different kingdoms, including herbivorous arthropods, and there is remarkable diversity in suppression mechanisms. Suppression may strongly affect the structure of the food web, because the ability to suppress the activation of defences of a communal host may facilitate competitors, whereas the ability of a herbivore to cope with activated plant defences will not. Further characterization of the mechanisms and traits that give rise to suppression of plant defences will enable us to determine their role in shaping direct and indirect interactions in food webs and the extent to which these determine the coexistence and persistence of species.

Parallel evolution of cytochrome b mediated bifenazate resistance in the citrus red mite Panonychus citri.

Bifenazate is a recently developed acaricide that is mainly used to control spider mites on a variety of crops. Although first thought to be a neurotoxin, genetic evidence obtained from bifenazate resistant Tetranychus urticae strains suggested an alternative mode of action as a Qo pocket inhibitor of the mitochondrial complex III. In this study, we reveal how bifenazate resistance in strains of Panonychus citri is maternally inherited and can confer cross-resistance to the known Qo inhibitor acequinocyl. The mitochondrial genome of P. citri was sequenced and Qo pocket mutations were shown to be linked with the resistant trait. Parallel evolution of cytochrome b mediated bifenazate resistance corroborates the alternative mode of action and yet again illustrates that care should be taken when employing Qo inhibitors as crop protection compounds.

Wolbachia induces strong cytoplasmic incompatibility in the predatory bug Macrolophus pygmaeus.

Macrolophus pygmaeus is a heteropteran predator that is widely used in European glasshouses for the biological control of whiteflies, aphids, thrips and spider mites. We have demonstrated that the insect is infected with the endosymbiotic bacterium Wolbachia pipientis. Several gene fragments of the endosymbiont were sequenced and subsequently used for phylogenetic analysis, revealing that it belongs to the Wolbachia supergroup B. The endosymbiont was visualized within the ovarioles using immunolocalization. Tetracycline treatments were used to cure M. pygmaeus from its infection. Although a completely cured line could not be obtained by this approach, the application of a constant antibiotic pressure over 13 generations resulted in a line with a significantly reduced Wolbachia concentration. Crosses performed with this tetracycline-treated line revealed that the endosymbiont causes severe cytoplasmic incompatibility. This is the first report of a reproductive effect induced by Wolbachia in an economically important heteropteran predator that may have vital implications for its commercial production and use in biological control.

Abamectin is metabolized by CYP392A16, a cytochrome P450 associated with high levels of acaricide resistance in Tetranychus urticae.

Abamectin is one of the most important insecticides worldwide. It is used against major agricultural pests and insects of public health importance, as well as against endoparasites in animal health. Abamectin has been used successfully for the control of the spider mite Tetranychus urticae, a major agricultural pest with global distribution, an extremely diverse host range, and a remarkable ability to develop resistance against insecticides including abamectin. Target site resistance mutations may explain a large part of resistance, although genetic evidence and transcriptomic data indicated that additional mechanisms may also be implicated in the abamectin resistant phenotype. To investigate a functional link between cytochrome P450-mediated metabolism and abamectin resistance, we recombinantly expressed three cytochrome P450s (CYP392A16, CYP392D8 and CYP392D10) that have been associated with high levels of abamectin resistance in a resistant T. urticae strain isolated from Greece. CYP392A16 was expressed predominately in its P450 form however, both CYP392D8 and CYP392D10 were expressed predominately as P420, despite optimization efforts on expression conditions. CYP392A16 catalyses the hydroxylation of abamectin (Kcat=0.54 pmol/min/pmol P450; Km=45.9 µM), resulting in a substantially less toxic compound as confirmed by bioassays with the partially purified metabolite. However, CYP392A16 did not metabolize hexythiazox, clofentezine and bifenthrin, active ingredients that also showed reduced toxicity in the abamectin resistant strain. Among a number of fluorescent and luminescent substrates screened, Luciferin-ME EGE was preferentially metabolized by CYP392A16, and it may be a potential diagnostic probe for metabolic resistance detection and monitoring.

A horizontally transferred cyanase gene in the spider mite Tetranychus urticae is involved in cyanate metabolism and is differentially expressed upon host plant change.

The genome of the phytophagous two-spotted spider mite Tetranychus urticae was recently sequenced, representing the first complete chelicerate genome, but also the first genome of a highly polyphagous agricultural pest. Genome analysis revealed the presence of an unexpected high number of cases of putative horizontal gene transfers, including a gene that encodes a cyanase or cyanate lyase. In this study we show by recombinant expression that the T. urticae cyanase remained functionally active after horizontal gene transfer and has a high affinity for cyanate. Cyanases were also detected in other plant parasitic spider mites species such as Tetranychus evansi and Panonychus citri, suggesting that an ancient gene transfer occurred before the diversification within the Tetranychidae family. To investigate the potential role of cyanase in the evolution of plant parasitic spider mites, we studied cyanase expression patterns in T. urticae in relation to host plant range and cyanogenesis, a common plant defense mechanism. Spider mites can alter cyanase expression levels after transfer to several new host plants, including the cyanogenic Phaseolus lunatus. However, the role of cyanase is probably not restricted to cyanide response, but likely to the plant nutritional quality as a whole. We finally discuss potential interactions between cyanase activity and pyrimidine and amino acid synthesis.

The cys-loop ligand-gated ion channel gene family of Tetranychus urticae: implications for acaricide toxicology and a novel mutation associated with abamectin resistance.

The cys-loop ligand-gated ion channel (cysLGIC) super family of Tetranychus urticae, the two-spotted spider mite, represents the largest arthropod cysLGIC super family described to date and the first characterised one within the group of chelicerates. Genome annotation, phylogenetic analysis and comparison of the cysLGIC subunits with their counterparts in insects reveals that the T. urticae genome encodes for a high number of glutamate- and histamine-gated chloride channel genes (GluCl and HisCl) compared to insects. Three orthologues of the insect γ-aminobutyric acid (GABA)-gated chloride channel gene Rdl were detected. Other cysLGIC groups, such as the nAChR subunits, are more conserved and have clear insect orthologues. Members of cysLGIC family mediate endogenous chemical neurotransmission and they are prime targets of insecticides. Implications for toxicology associated with the identity and specific features of T. urticae family members are discussed. We further reveal the accumulation of known and novel mutations in different GluCl channel subunits (Tu_GluCl1 and Tu_GluCl3) associated with abamectin resistance in T. urticae, and provide genetic evidence for their causality. Our study provides useful toxicological insights for the exploration of the T. urticae cysLGIC subunits as putative molecular targets for current and future chemical control strategies.

Systemic use of spinosad to control the two-spotted spider mite (Acari: Tetranychidae) on tomatoes grown in rockwool.

Spinosad is a reduced-risk insecticide derived as a fermentation product from the soil actinomycete Saccharopolyspora spinosa. It is toxic by ingestion and contact and has a unique mode of action on the insect nervous system. Spinosad exhibits a high degree of selective toxicity towards the insect orders Lepidoptera, Diptera and Thysanoptera, but is less toxic to many beneficial arthropods. To determine if spinosad could be valuable as an alternative acaricide for the control of Tetranychus urticae, laboratory toxicity experiments with leaf-disk bio-assays were performed on a laboratory susceptible and several resistant strains. LC50 values were rather high in comparison with newly developed commercial acaricides. Surprisingly, when spinosad was applied to the roots of tomato plants in rock wool, excellent control of spider mites was obtained. Apparently, spinosad has systemic properties and quantities as low as 1 mg/plant could protect tomato plants from mite infestation. Different substrates with varying percentage of clay and organic matter were tested in comparison with rockwool and showed that sufficient control was restricted to the rockwool substrate. Consequently, a dose-response experiment with tomato plants grown in rockwool was set up. The persistence of spinosad toxicity when applied via the roots was determined, and pointed to a long lasting control (up to 30 DAT). Spinosad amounts in leaves after systemic application were determined with an immunological technique to quantify spinosad uptake. Correlations between mite control, spinosad uptake and leaf concentrations can be helpful to determine the necessary dose in field situations.