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.

Spider mites (Acari: Tetranychidae) are pests of a wide range of agricultural crops, vegetables, and ornamental plants. Their ability to rapidly develop resistance to synthetic pesticides has prompted the development of new strategies for their control. Evaluation of synthetic pesticides and bio-pesticides-and more recently the identification of RNA interference (RNAi) target genes-requires an ability to deliver test compounds efficiently. Here we describe a novel method that uses a sheet-like structure mimicking plant leaves and allows for oral delivery of liquid test compounds to a large number of individuals in a limited area simultaneously (~100 mites cm −2). The main component is a fine nylon mesh sheet that holds the liquid within each pore, much like a plant cell, and consequently allows for greater distribution of specific surface area even in small amounts (10 µl cm −2 for 100-µm mesh opening size). The nylon mesh sheet is placed on a solid plane (e.g., the undersurface of a Petri dish), a solution or suspension of test compounds is pipetted into the mesh sheet, and finally a piece of paraffin wax film is gently stretched above the mesh so that the test mites can feed through it. We demonstrate the use of the method for oral delivery of a tracer dye (Brilliant Blue FCF), pesticides (abamectin and bifenazate), dsRNA targeting the Vacuolar-type H +-VATPase gene, or fluorescent nanoparticles to three species of Tetranychus spider mites (Acari: Tetranychidae) and to the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae). The method is fast, easy, and highly reproducible and can be adapted to facilitate several aspects of bioassays.

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