Chelicerata are the second largest group of arthropods after insects. Chelicerates branch first from the root of the arthropod phylum (Boore et al. 1995; Friedrich and Tautz 1995), they aremc1 important comparative group for understanding the deep evolutionary processes underlying the varieties of arthropods. Apart from understanding their developmental patterning, chelicerates comprise many species that are important for agriculture and medicine such as spider mites and ticks. Both of these groups mc2 belong to the order Acari, the most species-rich chelicerate order. While spider mites represent major pests in agriculture, ticks are vectors of human diseases. mc3 In contrast with their predatory relatives (the spiders and scorpions), the two-spotted spider mite, Tetranychus urticae, belongs to a gathering of web-spinning species that feed on plants. Furthermore, unlike spiders and scorpions, which have long developmental times, T. urticae has a short generation time. T. urticae completes its embryonic development in only 39 hours with full maturation to the reproductive adult in less than 7 days (Rao et al. 1996). The two-spotted spider mite Tetranychus urticae Koch is one of the most polyphagous herbivore and agricultural pest around worldwide (Jeppson et al., 1975; Migeon and Dorkeld, 2012; Van Leeuwen et al., 2010). mc4 It is a chelicerate herbivore that feeds on an extremely wide host range, with over 100 agricultural crops (Migeon at al., 2017).
The two-spotted spider mite (Tetranychus urticae Koch (Acari: Tetranychidae) is a cosmopolitan agricultural pest, feeding on more than 140 different plant families and 1100 plant species. (Dermauw et al. 2012). Spider mites feed by inserting their stylets into the leaf tissue and removing cell contents; the loss of leaf chlorophyll and the subsequent reduction in net photosynthetic rate causes leaf discoloration often called bronzing, a decline in overall plant health, or even death (Sances et al. 1981; Tomczyk and Kropczynska 1985; Park and Lee 2002). Spider mites can feed directly on fruit at mature green and breaker stages, and this damage can increase unmarketable fruit and economic losses (Ghidiu et al. 2006). Warm, dry conditions are the optimum conditions for spider mite population, mites increase their feeding habit, if uncontrolled, causes severe yield losses up to 90 % in greenhouse and open field tomato crops in Southeast and West Africa (Sibanda et al. 2000). In greenhousesmc5 resistance develops even faster because of the relativeisolation of mite populations, the extended growing season and the frequency of spraying (Cranham & Helle, 1985). Biological control of spider mites through natural enemies has only been widely practiced in closed greenhouses (Perdikis et al. 2008). Current spider mite control relies mainly on frequent applications of synthetic acaricides. However, spider mites have the capacity to quickly develop acaricide resistance (Van Leeuwen et al. 2004; Dermauw et al. 2012). T. urticae populations have the highest occurrence of pesticide resistance among arthropods in agricultural habitats (Leeuwen at al., 2010). New alternative management strategies should be defined to decrease the resistance problems and side effects of chemical acaricides. The capacity of fast immune responses to xenobiotics in T. urticae plays a crucial role to use it as a model organism in further studies including host-range evolution, the interactions of plant-herbivore, and xenobiotic resistance mechanism (Kwon at al., 2013).
The Insecticide Resistance Action Committee (IRAC; http://www.irac-online.com) defines resistance as: ‘a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label’s recommendation for that pest species’. Resistance arises through the overuse of a pesticide against a pest species, killing the susceptible individuals which resultsmc6 in the evolution of populations that are resistant to that pesticide (Jeppson et al., 1975).
The first case of insecticide resistance was reported 100 years ago by Melander (1914), who described resistance of orchard pests to sulfur-lime (one of many inorganic chemicals used in those days). Since then, resistance has been spreading an alarming rate. Pesticide users wrongly assumed that survivors had escaped spraying, increasing the dose and rate of application. Currently, more than 500 arthropod species are reported to have developed resistance to commonly used chemicals (Georghiou and Saito, 1983; Hoy, 2011). Up-to-date information about resistant species can be found in the Arthropods Resistant to Pesticides Database (ARPD, by 2013, there were 407 cases of pesticide resistance among two-spotted spider mite populations, spread over 92 compounds with unique active ingredients (Onstad, 2013). mc7 Spider mites were minor pests and resistance was a rare phenomenon but introduction of synthetic organic pesticides, like DDT and organophosphates, after the 2nd World War, resulted in a steady increase of resistance cases. The sudden increase of spider mites infestations and resistance can be explained by a combination of different factors including overuse of pesticides destroying the natural enemy population, overuse of fertilizers resulting in plants suitable for higher rates of spider mite reproduction and low concentrations of pesticides having a stimulatory effect on spider mite reproduction (Hoy, 2011)mc8 .
Resistance is not only presents the selecting compound ineffective, but also result in cross resistance between compounds who usually share a common mode of action, so it is a high risk that developing target-site resistance will confer cross-resistance to all compounds of the same chemical group (Chapman and Penman, 1979). The majority of cases involve changes in the sensitivity of the target site due to point mutations, or sequestration/metabolism of the insecticide before it reaches the target site due to quantitative or qualitative changes in major detoxification enzymes (esterases, P450 monooxygenases and glutathione-S-transferases, reviewed in Oakeshott et al. (2005), Feyereisen (2005), Enayati et al. (2005) and Li et al. (2007)). Spider mites rapidly develop resistance to pesticides because of their high fecundity, short generation time, high rate of inbreeding and arrhenotokous reproduction. Haploid males express both dominant and recessive traits, allowing favorable mutations to become more rapidly fixed at male haploid loci when selected with a pesticide (Hoy, 2011; Jeppson et al., 1975; Van Leeuwen et al., 2010; Zhang, 2003). Resistance mechanisms in mites are similar to those found in insects, and include reduced penetration through the cuticle, metabolic resistance and target site insensitivity. Target-site resistance has been also associated with sodium channel alternate transcriptional variation and/or posttranscriptional modifications in some species (Tan et al., 2002; Du et al., 2006), although the possible broader role of such changes in other species remains unclear. resistance refers to increased sequestration/excretion and increased detoxification of the pesticide, decreasing the effective dose of the pesticide at the target site. This type of resistance involves several major protein families including cytochrome P450 monooxygenases, esterases, glutathione-S-transferases and ABC transporters. Target site insensitivity or modification will present the pesticide ineffective, since it will not be able to bind to its target (Knowles, 1997; Pittendrigh et al., 2013; Ranson et al., 2002).
Abamectin has also been developed as an acaricide/insecticide. It has a broad spectrum of activity against arthropods, including major insect pests in the orders Coleoptera, Homoptera, Diptera, Orthoptera, Isoptera, Hymenoptera, and Lepidoptera (Putter et al., 1981) and some mite species including the two-spotted spider mite Tetranychus urticae, the most important mite pest in glasshouses throughout the world. This species is extremely polyphagous species including major economic crops such as cotton, maize, tomatoes, sweet pepper, fruits and a range of ornamentals (Van Leeuwen et al., 2010). The mode of action of abamectin is the activation of glutamate gated chloride channels (GluCls) (Wolstenholme and Rogers, 2005; Lynagh and Lynch, 2012), which are only found in invertebrates (Kehoe et al., 2009). Target-site resistance to abamectin in T. urticae has been attributed to a G323D point mutation in the GluCl which was tightly associated with a moderately resistant phenotype (17-fold resistance) in isogenic lines from Korea (Kwon et al., 2010).
Cytochrome P450s belong to a diverse family of hemecontaining enzymes that catalyze the mono-oxygenation of xenobiotics and endogenous compounds. They have been implicated in resistance in agricultural pests and insects of public health importance (Feyereisen, 2005, 2006). Eighty-six cytochrome P450 (CYP) genes were detected in the T. urticae genome, a total number similar to insects but with an expansion of the CYP2 clan (Grbic et al., 2011). Several cytochrome P450 genes were shown to be associated with the abamectin resistance phenotype of the Mar-ab strain by a full genome microarray analysis (Dermauw et al., 2013). Among them, the cytochrome P450 genes CYP392D8, CYP392D10 and CYP392A16 stood out as being remarkably over-expressed in Mar-ab as well as an additional resistant strain (MR-VP), and their expression was also confirmed by real-time PCR (Dermauw et al., 2013)
Spirodiclofen is one of the most recently developed acaricides and belongs to the new family of spirocyclic tetronic acids (ketoenols). This new acaricidal family is an important chemical tool in resistance management strategies providing sustainable control of spider mites such as Tetranychus urticae. Spirodiclofen targets lipid biosynthesis mediated by direct inhibition of acetyl coenzyme A carboxylase (ACCase) (Dermauw et al., 2013).
The fast ability of host changing makes Tetranychus Urticae an interesting species to study. For polyphagous species that can easy to be exposed to new hosts, colonizing a new host plant species often involves tolerating or resisting defenses specific to the new host plant, and thus quickly responding to diverse and possibly strong selection. The herbivore ability to get around host plant defenses is particularly important for polyphagous species, which can feed and reproduce on several plant families (Schoonhoven et al. 2005 ). For polyphagous species that can frequently be exposed to new hosts, colonizing a new host plant species often involves tolerating or resisting defenses specific to the new host plant, and thus quickly responding to diverse and possibly strong selection. Responses of herbivore populations to environmental changes, such as a host plant shift, can range from local extinction to adaptation. After host shifting from tomato to cucumber, the host plant on which mite populations had evolved did not affect the performance of the mites, but only affected their sex ratio. Females that lived on tomato plants for 24 generations produced a higher percentages of daughters than did females that lived on cucumber plants (Marinosci et al, 2015). After 15 generations, mite populations evolving and tested on detached leaves of a novel host species (tomato or pepper) performed better on their host than did populations evolving on leaves of another host (cucumber), indicating the occurrence of local adaptation (Magalh~aes et al. 2007). Recent studies revealed that adaptation of this herbivore to one host plant could facilitate exploitation of other host plant species and therefore broaden its host range (Magalh~aes et al. 2009; Fellous et al. 2014). Further, adaptation to a new host plant for this species did not always result in its decreased performance on the original host (Gould 1979; Fry 1990; Agrawal 2000; Magalh~aes et al. 2009).
One of the main reasons why T. urticae is such a devastating pest is its ability to feed and develop on so many different host plants, including many agricultural crops. Although the genome of some agriculturally important plant-feeding species has been sequenced (e.g. the pea aphid) and more will become available soon, spider mites are probably best suited to study of the underlying mechanisms of polyphagy. They feed on several experimentally amenable plants for which genetic and genomic resources are available, such as tomato, Arabidopsis and Medicago. Together with the availability of the techniques described above, this clearly opens up opportunities to study interactions between mites and plants on a whole-genome expression scale. Interestingly, some spider mite species, such as Tetranychus evansi, seem actively to manipulate plant defences, but the molecular mechanisms behind such adaptation have not been documented. A better understanding of how mites and plants interact might yield new ideas for control strategies in the future.
The two spotted spider mite, T. urticae, is one of the most economically damaging pests of cultivated tomato, particularly in Southeast Africa, West Africa, South Asia, Southeast Asia, Europe and Mediterranean countries. Sustainable chemical control of spider mites is difficult because of its capacity to rapidly develop resistance to new acaricides (Dermauw et al. 2012).
Spider mite resistant tomato cultivars, would offer growers an inexpensive and easier method of pest control. In general, some wild tomato species are far less attractive to pests than cultivated tomato due to their glandular trichomes and production of effective defense compounds. Most previous studies focused on identification of spider mite resistance from distant wild tomato species including accessions of S. habrochaites f. glabratum (PI 134417, PI 251304, PI 134418 and PI 126449), S. habrochaites (PI 127826, LA1777, LA 1740 and LA 2860) and S. pennellii (LA 716, LA 2963 and LA 2580) (Gentile et al. 1969; Snyder and Carter 1984; Antonious and Snyder 2006; Saeidi and Mallik 2006; Onyambus et al. 2011; Bleeker et al. 2012; Lucini et al. 2015).
Resistance to spider mite and other insects has been found in some accessions of wild tomato species particularly S. habrochaites Knapp et Spooner (syn. Lycopersicon hirsutum Dunal), and S. pennellii Correll (syn. L. pennellii (Correll) d’ Arcy) (de Azevedo et al. 2003; Resende et al. 2006). The resistance in these species was related to the presence of glandular trichomes (Frelichowski and Juvik 2001; Muigai et al. 2002; Oriani et al. 2011; Bleeker et al. 2012), which are reported to be absent in cultivated tomato (Luckwill 1943; Antonious 2001). Trichomes are consideredthe most important factor conferring pest resistance. The genus Solanum possesses seven types of trichomes of which types II, III and V are non-glandular, and types I, IV, VI and VII are glandular (Gurr and McGrath 2001; Simmons and Gurr 2005). Glandular trichomes secrete secondary metabolites including acylsugars, methylketones and sesquiterpenes that can be toxic, repellent, trap insects or act as a physical barrier, and interfere with insect feeding and oviposition (Sharma et al. 2009).
T. Urticae has only three chromosomes (Oliver 1971) and possesses the smallest genome determined thus far within the arthopods— only 75 Mbp, or 0.08 pg per haploid genome. This is roughly equivalent to 60% of the Drosophila genome (Dearden et al. 2002) and makes T. urticae a potential candidate for a model chelicerate. Recently, T. urticae genome was completely analyzed and serves as a good model genome (Girbic at al., 2017) for understanding Arachnid biology. The availability of T. urticae genome sequences will also facilitate the investigation of gene regulation mechanisms in Acari, including post-transcriptional gene silencing by RNAi. A genome-wide screen in T. urticae revealed that all machinery (Dicer, Argonautes and RISC components) for RNAi is present in T. urticae. Strikingly, T. urticae also has five duplicated copies of an RNA-directed RNA polymerase (RdRP), an enzyme not yet described in insects. The presence of RdRP in mites such as T. urticae suggests that spider mites could be efficiently controlled via plant-mediated RNAi. However, it remains to be experimentally validated whether dsRNA is efficiently taken up and distributed after feeding upon plants containing dsRNA, a prerequisite for the success of such strategies (Van Leeuwen, 2012).
Gene suppression via RNA interference (RNAi) provides an alternative strategy for insect pest management. RNAi can become an important tool for crop protection against two-spotted spider mite when it applies on the leaf surface and dsRNA expressed transgenic plants. RNAi triggered by dsRNA has become a valuable genetics and biotechnological tool for pest control and arthropod reseach. (Kim at al., 2015; Kola at al., 2015).
A promising approach to knockout (silence) genes in various plants and animals includes RNA interference (RNAi)-mediated gene silencing (Meister and Tuschl 2004). Although the RNAi mode of action is conserved, delivery protocols of experimental RNAi vary depending upon the model system. They range from direct delivery to a specific developmental stage (transfection of cell lines or injections into individual embryos) to the introduction of double-stranded RNA (dsRNA) into earlier reproductive stages, thereby allowing gene silencing in the progeny of injected females.(Khila,2007). Injection of dsRNA/siRNA into the spider mite female abdomen was distributed throughout the abdominal cavity and was passed to the eggs. Developing embryos inside these eggs displayed the expected RNAi phenotype, suggesting strong systemic RNAi mechanisms.
Some novel and applicable pest management strategies will be essential to control pest in agricultural in the future. Recently, RNA interference (RNAi) gene-silencing approach provides a powerful tool in managing insect pest relations.
Vacuolar-type H+-ATPase (V-ATPase) gene was used as a target gene which encodes a protein that works as a pump and it ensures exchanging protons across cellular membranes. This mechanism uses the energy which is released by ATP hydrolysis ( Finbow at al.1997). TuVATPase is a valuable target for comparative analysis for different reasons. It has been used as a target gene silencing in lots of arthropod systems; e.g. the Western corn rootworm (Diabrotica virgifera virgifera), the pea aphid (Acyrthosiphon pisum), the red flour beetle (Tribolium castaneum), the tobacco hornworm (Manduca sexta), the whitefly (Bemisia tabaci) and the Colorado potato beetle (Leptinotarsa decemlineata) (Baum at al., 2007; Kwon at al., 2013; Upadhyay at al., 2011). Conducting studies witnessed an important mission of V-ATPase, showing the effective V-APTase silencing that gives rise to a significant reduction of fitness in the arthropod species. Moreover, previous studies illustrate that orally-delivered dsRNAs against VATPase induce RNA interference. Finally; TuVATPase has already been shown to increase mite mortality when silenced in T. Urticae through the leaf floating method (Kwon at al., 2013). Additionally, RNAi can become an important tool for crop protection against two-spotted spider mite when it applies on the leaf surface and dsRNA expressed transgenic plants. RNAi triggered by dsRNA has become a valuable genetics and biotechnological tool for pest control and arthropod research. (Kim at al., 2015; Kola at al., 2015).
The most common methods for the delivery of dsRNA through artificial diet and microinjections into the hemolymph (Whyard at al., 2009; Ghanim at al., 2007). Soaking has been routinely applied to deliver dsRNA into nematodes (Tabara at al., 1998; Campbell at al., 2010). Although an artificial diet has been used for T. urticae decades ago (Van at al., 1983; Ekka at al., 1971), it has not been used for the delivery of dsRNA. Alternatively, dsRNA microinjection and feeding on leaf discs floating on dsRNA solution have been used instead ( Kwon at al., 2015; Kwon at al., 2013; Khila at al., 2007). On the other hand, these methods are not suitable for new applications: microinjection gives rise to a non-specific stress which caused by mechanical damage and it is hard because adult female mites are 0.5 mm long; and the leaf-floating method requires large (>20 ?g per individual sample) amounts of dsRNA. Other method for delivering the small molecules are spraying and leaf dip bioassays. They have been used for the application of synthetic chemicals e.g. pesticides (Kranthi at al., 2005) with a large scale of arthropods including T. urticae. On the other hand, these methods need a high amount of experimental solution so it is not suitable for high-throughput.