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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.

[This corrects the article DOI: 10.1371/journal.pone.0180658.].

RNA interference (RNAi) can be used for the protection against agricultural pests through the silencing of genes required for pest fitness. To assess the potential of RNAi approaches in the two-spotted spider mite, Tetranychus urticae, we compared 5 methods for the delivery of double-stranded RNA (dsRNA). These methods include mite feeding on either (i) leaves floating on a dsRNA solution, (ii) dsRNA-expressing plants, (iii) artificial diet supplemented with dsRNA, or (iv) dsRNA-coated leaves, and (v) mite soaking in a dsRNA solution. In all cases, the gene targeted for method validation was the Vacuolar-type H+-ATPase (TuVATPase), encoding a constitutively expressed ATP-driven proton pump located in the membrane. Down-regulation of TuVATPase increased mortality and/or reduced fecundity in all methods, but with variable efficiency. The most efficient methods for dsRNA delivery were direct soaking of mites in the dsRNA solution and mite feeding on dsRNA-coated leaves that mimics dsRNA application as a sprayable pesticide. Both resulted in a dark-body phenotype not observed in mites treated with a control dsRNA. Although with lower efficiency, dsRNA designed for TuVATPase silencing and expressed in transgenic Arabidopsis plants impacted the fitness of mites feeding on these plants. RNAi may thus be a valuable strategy to control spider mite populations, either as a sprayable pesticide or through transgenic crops. This comparative methodological study focusing on the induction of RNAi-based gene silencing in T. urticae paves the way for reverse genetics approaches in this model chelicerate system and prepares large-scale systematic RNAi screens as a first step towards the development of specific RNA-based pesticides. Such alternative molecules may help control spider mites that cause significant damages to crops and ornamental plant species, as well as other chelicerates detrimental to agriculture and health.

Leprosis is a serious disease of citrus caused by Citrus leprosis virus C (CiLV-C, genus Cilevirus) whose transmission is mediated by false-spider mites of the genus Brevipalpus. CiLV-C infection does not systemically spread in any of its known host plants, thus remaining restricted to local lesions around the feeding sites of viruliferous mites. To get insight into this unusual pathosystem, we evaluated the expression profiles of genes involved in defense mechanisms of Arabidopsis thaliana and Citrus sinensis upon infestation with non-viruliferous and viruliferous mites by using reverse transcriptase-qPCR. These results were analyzed together with the production of reactive oxygen species (ROS) and the appearance of dead cells as assessed by histochemical assays. After interaction with non-viruliferous mites, plants locally accumulated ROS and triggered the salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathways. ERF branch of the JA/ET pathways was highly activated. In contrast, JA pathway genes were markedly suppressed upon the CiLV-C infection mediated by viruliferous mites. Viral infection also intensified the ROS burst and cell death, and enhanced the expression of genes involved in the RNA silencing mechanism and SA pathway. After 13 days of infestation of two sets of Arabidopsis plants with non-viruliferous and viruliferous mites, the number of mites in the CiLV-C infected Arabidopsis plants was significantly higher than in those infested with the non-viruliferous ones. Oviposition of the viruliferous mites occurred preferentially in the CiLV-C infected leaves. Based on these results, we postulated the first model of plant/Brevipalpus mite/cilevirus interaction in which cells surrounding the feeding sites of viruliferous mites typify the outcome of a hypersensitive-like response, whereas viral infection induces changes in the behavior of its vector.

Citrus leprosis virus C (CiLV-C) causes a severe disease affecting citrus orchards in the Western hemisphere. This study reveals the molecular variability of the virus by analyzing four genomic regions (p29, p15, MP and RNA2-intergenic region) distributed over its two RNAs. Nucleotide diversity (π) values were relatively low but statistically different over the analyzed genes and subpopulations, indicating their distinct evolutionary history. Values of πp29 and πMP were higher than those of πp15 and πRNA2–IR, whereas πMP was increased due to novel discovered isolates phylogenetically clustered in a divergent clade that we called SJP. Isolate BR_SP_SJP_01 RNA1 and RNA2 sequences, clade SJP, showed an identity of 85.6% and 88.4%, respectively, with those corresponding to CiLV-C, the type member of the genus Cilevirus, and its RNA2 5′-proximal region was revealed as a minor donor in a putative inter-clade recombination event. In addition to citrus, BR_SP_SJP_01 naturally infects the weed Commelina benghalensis and is efficiently transmitted by Brevipalpus yothersi mites. Our data demonstrated that negative selection was the major force operating in the evaluated viral coding regions and defined amino acids putatively relevant for the biological function of cilevirus proteins. This work provides molecular tools and sets up a framework for further epidemiological studies.

The B-strain of Bemisia tabaci Gennadius is a key pest of several crops and chemical control is the main control method used by growers, although reduction in efficacy due to insecticide resistance has already been reported. The aim of this work was to investigate the insecticidal effect of an array of synthetic sucrose esters with the aliphatic and aromatic groups on whitefly adults. Sucrose butyrate, caprate, octanoate, palmitate, oleate, octaacetate, phthalate, benzoate, and sucrose diacetate hexaisobutyrate were tested. The solutions were prepared and applied on the adults caught on yellow sticky traps using the Potter spray tower. Long-chains sucrose aliphatic esters were more effective against the silverleaf whiteflies and the highest mortality was obtained with sucrose oleate and sucrose octanoate. Since these compounds were tensoactive, sodium dodecylsulphate was also tested for the comparison but no effect was observed. Sucrose butyrate and other aliphatic and aromatic sucrose polyesters showed negligible effect on the silverleaf whiteflies.O biótipo B de B. tabaci Gennadius tem se destacado como uma praga-chave de diversas culturas. O controle químico tem sido a principal tática de controle utilizada, embora já se tenha observado redução na eficiência dos produtos devido ao desenvolvimento de resistência. Assim, o objetivo do presente trabalho foi avaliar o efeito de diversos ésteres de sacarose com grupos alifáticos ou aromáticos sobre adultos de mosca-branca. Butirato de sacarose, caprato, octanoato, palmitato, oleato, actaacetato, ftlato, benzoato e diacetato hexaisobutirato de sacarose foram testados. Soluções de éster de sacarose foram preparadas e aplicadas sobre adultos capturados em armadilhas adesivas utilizando Torre de Potter. Ésteres alifáticos de sacarose com longas cadeias foram mais efetivos contra mosca-branca e as maiores taxas de mortalidade foram obtidas com oleato e octanoato de sacarose. Uma vez que estes compostos são caracterizados como tensoativos, dodecilsulfato de sódio foi testado para comparação e não se observou qualquer efeito. Butirato de sacarose e outros poliésteres de sacarose alifáticos ou aromáticos foram praticamente inócuos para mosca-branca.

O objetivo deste trabalho foi avaliar a forma de ação de duas preparações de extrato pirolenhoso aplicadas diretamente sobre Brevipalpus phoenicis, que é o ácaro vetor da leprose dos citros, um dos principais problemas da citricultura Paulista. Para o experimento, foram utilizados ácaros adultos mantidos numa criação-estoque no laboratório de Acarologia da UNESP - Universidade Estadual Paulista, em Jaboticabal-SP. Os tratamentos foram constituídos por duas diferentes preparações (destilado e decantado) de extrato pirolenhoso de eucalipto nas proporções EP:água de 1:600; 1:300 (normalmente recomendadas); 1:150; 1:75; 1:38; 1:19 e de água (testemunha), com 7 repetições. Cada parcela foi constituída de 10 ácaros mantidos sobre um fruto de laranja, em arena de 2,5 cm de diâmetro, delimitada com cola adesiva tipo Tanglefoot®. As aplicações foram efetuadas em Torre de Potter, pulverizando-se 2 mL por fruto das soluções correspondentes aos diferentes tratamentos. Os frutos foram mantidos em sala climatizada a 27±1ºC, e as avaliações foram realizadas 24 e 48 horas após a aplicação dos tratamentos, determinando-se o número de ácaros mortos (mortalidade) e retidos na barreira adesiva (repelência). Os dois tipos de extrato pirolenhoso testados não apresentaram repelência significativa sobre Brevipalpus phoenicis; ambos induziram mortalidade significativa somente para concentrações acima de 1:150, com efeito mais pronunciado para o destilado; há um aumento na mortalidade de 24 para 48 horas após a aplicação; a ação protetora preconizada pela aplicação de baixas doses (1:300 a 1:600) nas plantas não é devida à mortalidade e repelência pelo contato direto do extrato pirolenhoso sobre os ácaros.The aim of this work was to evaluate the acaricide and repellent effects of two different pyroligneous extract preparations (PE) applied directly on Brevipalpus phoenicis. This mite is the vector or citrus leprosies, which is one of the main problems of São Paulo State citrus culture. This experiment was carried out with mites derived of the colony-supply maintained in the laboratory of Acarology of the Unesp - São Paulo State University, at Jaboticabal. The experiment consisted of application of water (control) and two distinct way of preparations (distilled and decanted) of pyroligneous extract (PE) of eucalyptus in 6 volumetric ratios PE: water of 1:600, 1:300, 1:150, 1:75, 1:38 and 1:19, with 7 replications. Each plot with 10 mites maintained on an orange fruit, in enclosure of 2.5 cm diameter isolated by Tanglefoot® (The Tanglefoot Company: USA) adhesive barrier. After confection of the enclosures and mites transference, 2 mL of the solutions corresponding to the different treatments were applied per fruit, by using a Potter´s Tower. Then, the fruits were maintained in a room at 27±1ºC, and evaluations of the number of dead mites (mortality) and restrained in the adhesive barrier (repellence) were carried out 24 and 48 hours after treatment application. The distilled PE presented more mortality in comparison to decanted PE for concentrations higher than 1:150. None of two types of extract showed significant repellence effect on B. phoenicis. The protective action recognized for low doses (1:300 to 1:600, normally recommended), is not due to mortality or repellence by direct contact of these products with mites.

ABSTRACT: Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV.