Protocols for the delivery of small molecules to the two-spotted spider mite, Tetranychus urticae

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.


Introduction
The two-spotted spider mite (TSSM) Tetranychus urticae Koch (Acari: Tetranychidae) is an important agricultural pest worldwide. It is a chelicerate herbivore that feeds on an extremely wide host range, including over 100 agricultural crops [1]. Furthermore, T. urticae populations have the highest occurrence of pesticide resistance among arthropods in agricultural habitats [2]. T. urticae genome was recently sequenced [3] and several genomic databases of transcriptome profiles characterizing T. urticae developmental stages and its responsiveness to a variety of xenobiotic and abiotic stresses are available at ORCAE (http://bioinformatics.psb.ugent.be/ orcae/) [4]. In addition, several reports describe both forward and reverse genetics approaches to identify genes of interest [5][6][7].
The rapid responsiveness to natural and synthetic xenobiotics makes T. urticae a prime model for studies of the evolution of host range, plant-herbivore interactions and mechanisms of xenobiotic resistance. However, to fully exploit the potential of spider mite as a model experimental system, it is necessary to develop methods for the efficient manipulation of mite gene function. This includes the delivery of different types of molecules that modulate gene expression, such as double stranded RNAs (dsRNA), morpholinos, or transgenes, or molecules that alter protein activity, including natural or synthetic chemicals that act as agonists or antagonists. Such methods must be robust, reproducible, cost-effective, and designed to record the effects of small molecules on otherwise physiologically normal mites. In addition, these bioassays should be applicable to a large population of mites, so that the effects of small molecules on mite physiology can be quantified phenotypically or using biochemical or molecular analyses. Several approaches have been described for the delivery of small molecules to arthropods. RNA interference (RNAi) triggered by dsRNA has become an important reverse genetics and biotechnological tool for arthropod research and pest control [8,9]. Delivery of dsRNA through artificial diet and microinjections into the hemolymph have been the most widely used methods in insects [10][11][12][13][14]. Alternatively, soaking has been routinely applied to deliver dsRNA into nematodes [15][16][17].
Even though an artificial diet has been developed for T. urticae decades ago [18][19][20], it has not been used for the delivery of dsRNA. dsRNA microinjection and feeding on leaf discs floating on dsRNA solution have been used instead [6,7,21]. However, these methods are not suited for high-throughput applications: microinjection introduces a non-specific stress caused by mechanical damage and is cumbersome because adult female mites are 0.5 mm long; and the leaf-floating method requires large (>20 μg per individual sample) amounts of dsRNA. Other methods for the delivery of small molecules include spraying and leaf dip bioassays and have been most frequently used for the application of synthetic chemicals, e.g. pesticides [22], to a wide range of arthropods including T. urticae. But, these methods require considerable volume of the experimental solution and are not suitable for high-throughput setups.
We report 3 different methods for the delivery of small molecules to T. urticae including artificial diet, leaf coating, and soaking protocols. They are compatible with mites at all developmental stages and can be adapted for high-throughput screens. Small molecule delivery was validated with the demonstration of tracer dyes accumulation in the mite body upon application with the 3 protocols. The protocols described in detail here are a prerequisite for better understanding of mite biology and for the development of novel compounds aimed at spider mite pest control. method, is the preparation of uniform mite populations. The procedure thus includes the rearing of spider mites on bean plants (Step 1) from which adult female mites are collected (Step 2). Adult female mites are used to initiate experimental populations of larvae and adult mites that are tightly synchronized in their development (Step 3). Larvae and adults are subsequently used in separate delivery protocols named: artificial diet, leaf coating and soaking (Step 4).  Stepwise procedure 1.1. Wet soil with tap water and lightly pack the water-soaked soil into pots.  [23]). Mites will rapidly colonize fresh plants.

Maintenance of spider mite populations on bean nursery plants
1.4. Remove old infested bean plants every 7-10 days.
Note Mite population should be carefully monitored to never become too dense otherwise bean plants will be consumed too rapidly and mites will disperse at the risk of contaminating the rearing area.  [23]. Briefly, attach a 1 mL pipette tip to the plastic tubing with a 1.5 mL microcentrifuge tube cut at the bottom as an adapter between the pump/vacuum tube and the tip. Place the piece of Kimwipe between the pipette tip and the tube to keep collected mites inside the tip.

Collection of adult female mites
Stepwise procedure 2.1 Intermix fresh bean plants with infested bean plants (ratio of~2:1) in the afternoon and leave overnight (O/N). Adult mites will rapidly colonize fresh plants.

2.2.
In the morning of the following day, collect the required number of mites directly from newly added bean leaves with the vacuum system using a dissecting microscope to visualize the mites (for illustration see [23]).
2.3. Remove the pipette tip from the tube, making sure that the piece of Kimwipe at the back of the pipette tip is undisturbed and transfer the collected mites into a new 1.5 mL microcentrifuge tube by tapping the tip.
Note 1. T. urticae displays sexual dimorphism: females are bigger than males and have rounded posterior end. Caution is also required to distinguish between the female deutonymphs (last nymph stage) that are smaller but otherwise morphologically similar to adult female mites. For best practice, identify the successive mite developmental stages and train yourself for the recognition of the adult female mites prior to mite collection.
2. Collection of adult mites is hampered by the presence of silk that mites spin on the leaf surface and mites entangled in silk may eventually die. To avoid silk contamination, recover adults from fresh bean plants that were intermixed with infested plants the day before collection. As adult mites are the most mobile developmental stage, the fresh bean plants will mainly carry adults and their leaves will not yet be densely covered with silk.

Preparation of mites synchronized in development
Submergence in water and high humidity were reported to inhibit spider mite egg hatching [24] and molting [25], respectively. We simplified these previously described methods while improving the synchronization of mite embryo and nymph development, with the aim to produce large cohorts of larvae and adults that are coordinated in their development. The procedures are detailed in the following 2 subsections, for larvae and adults, respectively.

Materials and equipment
1. Polystyrene cup (94 mm in diameter, 57 mm in depth) 2. Polyethylene lid with and without venting holes (referred to as vented and non-vented lids, respectively) (V-9, As-one, Osaka, Japan) (S1B and S1C Fig  Stepwise procedure. This protocol is outlined in Fig 2A. 3.1.1. Create an area (approx. 50 mm in diameter) fenced with water-soaked cotton at the bottom of the cup.
3.1.2. Transfer~500 adult females (collected as described in Section 2) into the area and cover the cup with a non-vented lid.
3.1.3. Incubate the adult females at 26˚C for 1 day. During that time females will lay eggs (mites can lay eggs in the vapor saturated environment).
3.1.4. Remove the water-soaked cotton with forceps and all the adult females with vacuum suction (as described in Section 2).
3.1.5. Add water (15 mL) immediately to completely cover the 1-day-old eggs laid at the bottom of the cup.
3.1.6. Cover the cup with the non-vented lid and incubate at 26˚C for 3 to 4 days.
3.1.7. After this period, remove water from the cup with a pipette.
3.1.8. Incubate the eggs in the same cup, but with no lid, at 26˚C and 50% RH.

Production of developmentally synchronized adults
(Timing: 9-10 days) Stepwise procedure. This protocol is outlined in Fig 2B. 3  Delivery of small molecules to the two-spotted spider mite 4. Methods for delivery of small molecules 4.1. Artificial diet. Two holidic diets (i.e. with chemically defined compositions) that were originally formulated for aphids [26,27] were tested for their ability to support mite development. The diet described by Prosser and Douglas [26] was tested in its original formulation. The diet formulated by Febvay et al. [27] was diluted 30-fold in water as described in Jonckheere et al. [28]. While these diets were able to support spider mite larvae and adults for a week, they did not support molting and larval transition to the nymph stage (data not shown).
We thus tested 2 additional meridic diets (i.e. including non-chemically defined constituents such as casein or wheat germs). The first, has been formulated by Gotoh et al. [29] for Panonychus citri, a mite species belonging to the same Tetranychidae family as T. urticae. However, this diet failed to support developmental transition of T. urticae larvae to protonymph, stressing that this developmental transition is a crucial step toward the establishment of an appropriate artificial diet. The second meridic diet we tested was formulated by Van der Geest et al. [18]. These authors reported that diet sustains the entire T. urticae life cycle, from larvae to adult. Indeed, we confirmed mite progression through all developmental stages while feeding on this diet and it is presented below. Composition of chemicals required for the preparation of 100 mL of the artificial diet described in [18] is shown in S1 Table. Preparation of a diet (100 mL) 4.1.1.1. Dissolve wheat germ powder and casein sodium salt in 50 mL of distilled water. Autoclave twice for 20 min at 120˚C and reserve for later. Note: It has been proposed that autoclaving changes certain physical or chemical properties of component(s) in wheat germ powder and/or casein sodium salt that are required for successful mite progression through the life cycle [18]. Thus, this step should not be modified.

Synchronization of egg hatching/larval emergence
The efficiency of synchronization of egg hatching protocol was evaluated by comparing hatchability of eggs that were: a) deposited on bean leaf, b) submerged in water for 4 days, and c) initially submerged in water for 4 days and were subsequently moved to 50% RH for 3 and 24 h, Fig 3A. In the control treatment, adult female mites deposited eggs on a bean leaf. After 1 day, female mites were removed and eggs were incubated at 50% RH. After 3 days, 93.9 ± 2.0% (mean ± SE) of eggs in the control treatment hatched (n = 120-271 with 3 replicates). In contrast, only 3.0 ± 2.0% of eggs of the same age that were submerged in water hatched (n = 86-142 with 3 replicates). However, upon water removal, 73.2 ± 4.6% and 89.3 ± 2.2% of these eggs hatched in first 3 and 24 h, respectively (n = 85-137 with 3 replicates). The majority of larvae emerged within the first 3 h after water removal, yielding a larval population that is tightly synchronized. The hatchability within the first 24 h after water removal was comparable to  that in the control (p>0.05). S1 Video shows the initial 90 minutes of egg hatching upon draining the water from eggs that were submerged for 4 days.

Synchronization of adult emergence
Small differences in RH dramatically impact mite molting. In the Control treatment (cups with vented lid), the RH was 96%, while in No ventilation treatment (in cups with non-vented lids) the RH was 100%. RH was measured with a sensor (HHA-3151, T&D, Nagano, Japan) placed inside the cup. In the Control treatment, 33% and 83% adults emerged 1 and 2 days after deutonymphs became quiescent, respectively. These ratios dropped to 3% and 5% in the No ventilation samples (Fig 3B). When the cups containing the same 2-day teleiochrysalis were moved for 30 minutes, without lids, in a chamber with 50% RH, 90% and 80% of the adults have emerged in the Control and No ventilation treatments, respectively (Fig 3B). Almost all teleiochrysalis females in No ventilation samples molted within 3 h, yielding a uniform population of freshly emerged adults. Thus, ambient relative humidity is a prominent factor controlling the molting of deutonymph and is useful in generating synchronized adult cohorts. S2 Video shows adult molting in No ventilation samples within the first 30 minutes after incubating deutonymphs into 50% RH environment.

Methods for delivery of small molecules
Artificial diet. Several parameters of mite development and physiology, while feeding on the artificial diet, were determined: 1. Ability of the artificial diet to support spider mite development throughout its life cycle was tested by placing 50 females on a cluster of 4 hemispheres. After 24 h, females were removed and around 100 eggs were kept on diet-filled Parafilm 1 M hemispheres at 16:8 L:D, 26˚C and 50% RH. Hemispheres filled with the fresh diet were replaced every 4 days. Mites progressed through all developmental stages (protonymph, deutonymph and adult) and reached adulthood. Mite development occurred at a slower rate relative to bean-leafreared larvae, as adults emerged at day 12 instead of day 9, respectively. Emerged females were not able to lay eggs. However, when placed on bean leaves they regained fecundity. Males reared on the artificial diet had normal mating behavior (see S3 Video) and fertilized newly-emerged females reared on bean leaves that yielded progeny with a normal sex ratio (f:m) of 7:3 (n = 96).
2. Ingestion of the artificial diet and diet distribution within the spider mite body was confirmed in mites reared on beans that were subsequently transferred to feeding on an artificial diet for 24 h (Fig 4). For this purpose, the diet was supplemented with 2 different tracers: a 3% blue food dye or 100 ng/μL of the Alexa Fluor 488 fluorescent dye. Dyes were added to the diet solution immediately before making of diet hemispheres. Mites fed with the fluorescent dye were washed with 0.1% Triton X-100 post-feeding and mounted in a solution of 50% glycerol in PBS buffer (v/v) for observation under an epifluorescence microscope (Zeiss, Oberkochen, Germany). As shown in Fig 4, both dyes localize within the mite digestive tract.
3. Larval and adult female survivorship while feeding on an artificial diet for 5 days was assessed by following the mortality of larvae and adults. Both developmental stages had similar survivorship of~80% in a 5-day period (Fig 5).
Note: Mite containment was not perfect and the rate of mite escapes from the diet-filled hemispheres partially reduced the mite population size. For example, 22% escape rate was observed for larvae (n = 65). Adults were better retained with only 6% of individuals (n = 65) escaping after 5 days of feeding on hemispheres surrounded by a water barrier.
Leaf coating method. This method is designed to evenly apply a defined amount of experimental solution on leaf discs of known surface area. It mimics the delivery of compounds by spraying but does not require specialized equipment such as a Potter spray tower. As two-spotted spider mite feeds from the cell content of the individual mesophyll cells that are internal to the leaf tissue [33], the presence of a surfactant facilitates the spread of the experimental solution on the leaf and its penetration into the leaf tissue. We tested Silwet L-77, a surfactant shown to be a critical component of Agrobacterium suspension solutions for plant transformation that enhances solution infiltration into the plant tissues [34]. According to the recommended concentration range, we tested the potential toxic or deterring effects of Silwet L-77 on mites. Bean leaf discs (10 mm in diameter) were coated with 6 μL of water or 0.0125%, 0.025% and 0.05% of Silwet L-77. Individual newly-molted female adult mites were placed on the bean leaf discs to measure mite mortality and escape percentages at 24 and 48 h of exposure. Non-treated leaf discs were used as the control. We also noted the spreading of the aqueous solution on the leaf surface and the timing of solution absorbance by the leaf tissue as a function of Silwet L-77 concentration. As shown in Table 1, <3% (n = 22-25 with 3 replicates) of adults escaped from leaf discs coated with water or 0.0125-0.025% of Silwet L-77 and the percentages were comparable to the control (0%; n = 25 with 3 replicates) at 48 h of exposure (p>0.05). A slight higher percentage (9.3±5.8%; n = 23-25 with 3 replicates) of adults escaped from leaf discs coated with 0.05% of Silwet L-77 but it was also comparable to the control at 48 h of exposure (p>0.05). After 48 h of exposure, no mite mortality was recorded on leaf discs coated with Silwet L-77 at 0.025% (data not shown) that also showed the best solution spreading and mite retention rate (Table 1). Thus, we used 0.025% Silwet L-77 in the protocol.
Developed leaf coating protocol was tested for its ability to deliver small molecules into the mites and to support mite larval and adult development.
1. Ingestion of the solution applied on the leaf surface was confirmed with solutions supplemented either with 3% of the blue food dye or 100 ng/μL Alexa Fluor 488 fluorescent dye.  Silwet L-77, 0.05% <1 6-7 9.3 ± 5.8 9.3 ± 5.8 Data for the escape percentage were collected from 3 replicates (n = 22-25 females per replicate) and were arcsine square-root transformed before the statistical analysis. All data are shown as mean ± SE (except data for which SE was zero). No significant differences among treatments were detected in each column with p > 0.05 (Tukey's HSD test after one-way ANOVA).
The localization of tracer dyes in mite body was determined 48 h after feeding started. For each treatment, 30 freshly-molted females (prepared as described in Protocol 3) were transferred to 6 leaf discs coated with tracer dyes or water. Mites that fed on bean leaf discs coated with the florescent dye were washed in 0.1% Triton X-100 and mounted in a solution of 50% glycerol in PBS buffer (v/v) for observation under an epifluorescence microscope (Zeiss, Oberkochen, Germany). Both tracer dyes were detected in the mite gut (Fig 6) indicating that the application of the solution on leaf surface does deliver experimental compounds into the mite digestive system. While all mites displayed fluorescent staining in their gut (that is a sensitive detection of a tracer intake), only 60% of mites were blue. This indicates the variability in the tracer dye intake that should be taken into account when delivering small molecules.

2.
Mite performance while feeding on coated leaf discs-including larval and adult female survivorships and fecundity (5 larvae/disc and 1 adult/disc)-were determined over 6 days to investigate whether Silwet L-77 may have any long-term effects. As shown in Fig 7, inclusion of 0.025% Silwet L-77 in the experimental solution did not affect mite viability nor female fecundity.
Soaking. Soaking is an established method for delivery of small molecules (e.g. dsRNA) to Caenorhabditis elegans Maupas (Rhabditidae) or insect cell lines [35][36][37][38], but has been exploited in insects and other arthropods in only few cases [17,39,40]. Thus, the main objective in the development of soaking as a delivery method for spider mites was to secure mite survivorship upon submergence and to ensure penetration of the solution into the mite body. As aqueous solutions have high surface tension preventing mite submergence, we tested the survival of newly-hatched larvae and newly-molted adults in aqueous solutions that contained Delivery of small molecules to the two-spotted spider mite either 0.1% Tween 20 or 0.1% Triton X-100. We focused on a narrow interval of mite development when the chitin exoskeleton is resynthesized with the aim to increase the penetrability of the solution into the body of the mite.
As shown in Table 2, 77.2±7.2% and 70.4±8.0% (n = 19-25 with 5 replicates) or 47.9±3.5% and 45.4±3.4% (n = 20-25 with 5 replicates) of newly-molted adult female mites survived at 24 and 72 h after recovery from soaking at 20˚C for 24 h in 0.1% Tween 20 or 0.1% Triton X-100, respectively. As shown in Table 3, 71.9±2.9% and 65.0±5.2% (n = 23-30 with 5 replicates) or To test the efficiency of the soaking method to deliver small molecules, we used blue food dye and the Alexa Fluor 488 fluorescent dye as tracers to monitor the uptake of compounds by larvae and adults. We also tested the effect of soaking duration (24 h and 10 minutes) and temperature (soaking at 20˚C and 4˚C) on the uptake of the fluorescent dye by adult mites. Mites soaked in the solution containing the fluorescence dye were washed after soaking with 0.1% Triton X-100, were mounted in 50% glycerol in PBS on a slide and were observed by an epifluorescence microscope. Tracer dyes could be detected in both larvae (not shown) and newlymolted adults. In adults, the staining can be observed irrespective of the timing and the temperature of soaking (Fig 8). However, brighter fluorescence was observed in mites soaked for 24 h. Tracer dyes could be detected in the mite gut, but the staining appears to extend beyond the alimentary track. Like with the coating method, variability in number of mites stained with tracer dyes could be observed. While majority of mites were stained with the fluorescent dye, 88% of soaked adults (n = 32) displayed the blue color, indicating variability in dye intake within the treated mite population.

Discussion
This work establishes a detailed and standardized set of protocols for delivery of small molecules (e.g. chemicals, dsRNA, tracer dyes) into spider mites, as supplements to an artificial diet, or constituents of a solution that is either applied on a leaf surface (leaf coating) or used for soaking the mites. In addition, we provide a detailed protocol for the preparation of spider Delivery of small molecules to the two-spotted spider mite mite populations that are tightly synchronized in their development, which is necessary to obtain reproducible results in bioassays.

Artificial diets for spider mites
Artificial diets adequate to sustain TSSM have been published over 30 years ago [18][19][20]41] but have not been widely implemented. Most liquid diets formulated for mites and aphids [26,27,29] could keep mites alive, but did not facilitate progression through a full life cycle. Of The differential interference contrast (DIC) and fluorescent images were merged at opacity of 50% with ImageJ [32]. https://doi.org/10.1371/journal.pone.0180658.g008 Delivery of small molecules to the two-spotted spider mite the diets tested by us, only the diet described by Van Der Geest et al. [18] could support a complete developmental cycle of TSSM. The development of mites feeding on this diet was identical in progression but delayed in time relative to mites maintained on bean leaves. Thus, the diet described herein successfully replaces leaf tissues for the oral delivery of small molecules (Fig 6). Although the mortality and dispersal of mites fed on the artificial diet was relatively high when considering the whole life cycle, the diet supported both larvae and adult mites with moderate mortality (~20%) over a 5 day-period (Fig 7). Female mites reared on this artificial diet were not fecund. However, their ability to lay eggs was restored when transferred to bean leaves, suggesting that the diet supports normal female development but lacks component(s) or signal(s) necessary for oogenesis. Tracing the bean leaf compound(s) that induces/enables mite fecundity would provide important insights into mite oogenesis and would improve the artificial diet. In addition, the identification of compound(s) and processes that are specifically required for mite female oogenesis could be exploited as targets for chemical inhibition, leading to mite contraception as an alternative way to suppress mite population. Despite the failure to support female oogenesis, the same diet supported the normal development of males that were able to mate, produced viable sperm, and gave rise to properly developing diploid female progeny.
Artificial diets have been widely used to deliver dsRNA to insects [8,9]. The diet presented here, based on diet described in [18], was previously applied through the membrane feeding system to deliver dsRNA into T. urticae [7]. However, the high mortality (>50% over the 3-day experimental period) and contamination of diet by fungi and bacteria prevented its use. We tested the membrane feeding system as well, but found that uneven stretching of Parafilm 1 M is introducing variability into the experimental system. Thus, the artificial diet protocol includes the pretreatment of Parafilm 1 M with UV and uses the custom built vacuum device (S1D Fig) to form Parafilm 1 M hemispheres that are compatible with mite feeding. In addition, protocol incorporates diet sterilization and its replacement every 4 days that is sufficient to prevent microbial contamination.
Besides RNAi studies, artificial diets supplemented with purified plant-derived compounds have been used in the identification of plant defense metabolites, for example to show that indole glucosinolate breakdown products act as feeding inhibitors in the aphid Myzus persicae [42,43]. Although other artificial diets tested in this study do not support certain mite developmental transitions, they are still useful for experimental purposes. For example, a chemically defined diet was recently used to collect mite salivary secretions to profile peptides that are secreted into plant tissues during feeding [28].
Leaf coating as a proxy to natural feeding Leaf coating is an alternative method for oral delivery in which mites feed in their natural settings. It is thus most suitable for studies involving the fragile larvae. Since mites feed on mesophyll cells located in inner leaf tissues [33], it was important to confirm their ability to ingest tracers applied on the leaf surface. This was indeed demonstrated because the distribution patterns of dyes within the mite body were similar whether ingested through the artificial diet or after application on the leaf surface (compare Figs 6 and 8).
Furthermore, surfactants such as Silwet L-77 practically improve leaf coating methods. They enhance the penetration of solutions into the leaf internal tissues and thus increase the fraction of compounds delivered into mites. Silwet L-77 also promotes the passive and uniform spread of solutions across the leaf disc surface, which is key to streamline protocols for compound screening.

Soaking, a simple method for spider mite bioassays
Finally, a soaking method has been validated as an alternative protocol for high throughput delivery of small molecules in mites. First, we discovered that mites are resilient to water exposure as eggs and deutonymphs arrest their hatching and molting, respectively, upon submergence. These behaviors were used to prepare tightly synchronized experimental populations of larvae and adults (Protocols 3.1 and 3.2) [24,25]. After optimization, the soaking of larvae and adults in aqueous solution resulted in high survivorship (>70%). In summary, adults sustain soaking for at least 24 h with low mortality. However, larvae only sustain shorter submergence (up to 4 h). Furthermore, larvae and adults were dramatically sensitive to Triton X-100, but only marginally affected by Tween 20.
While soaking is not an oral delivery method per se, tracer dyes were detected in the gut of soaked mites, in a pattern similar of those observed when dyes were administered through artificial diet or leaf coating. However, the tracer distribution appears broader suggesting that compounds may be delivered to a wider domain. This hypothesis can be tested experimentally with compounds that affect mite processes in well-defined organs or tissues. Finally, the soaking procedure is inexpensive, rapid, and can be easily scaled-up.

Perspectives
The 3 methods for which we provide detailed protocols are useful to deliver small molecules into the mite gut, for example chemicals or nucleic acids. They can be adapted to a wide range of applications, including the identification of plant defense compounds specifically targeting TSSM and functional screens based on the perturbation of gene expression initiated in the gut epithelial cells. All methods described are quick and inexpensive, and designed for mid-to high-throughput compound screens that will strengthen and broaden experimental approaches towards a better understanding of TSSM biology.  Table. Composition of chemicals required for the preparation of 100 mL of the artificial diet.

Supporting information
(DOCX) S1 Video. Hatching of T. urticae eggs. Hatching of T. urticae eggs at 26˚C and 50% RH after being submerged in water for 4 days at 26˚C. The recording was conducted over 1.5 h (accelerated 120X) after water removal. (MP4) S2 Video. Molting of teleiochrysalis females. Adult emergence of T. urticae induced by decreasing RH from 100% to 50% and increasing air temperature from 18˚C to 26˚C. The recording was conducted over 30 min (accelerated 120X) after the environmental change. The teleiochrysalis females are molting on a bean leaf in this video, whereas they also molt in an empty Petri dish as in the presented protocol in the text, to avoid unwanted feeding in newlymolted adult females. (MP4) S3 Video. Mites raised using artificial diet. Newly molted male adult grown on artificial diet mating with a newly emerged bean-reared female. (MP4)