ABSTRACT
Quantitative reverse transcription PCR (RT-qPCR) is a high-throughput method to analyze the transcriptional expression of genes. Currently, no reference genes have been described for evaluating gene expression in Brevipalpus yothersi, the false spider mite, a polyphagous that act as vector of the citrus leprosis virus C (CiLV-C), an important citrus disease. This study aimed to identify the most stable reference genes in B. yothersi. The RT-qPCR expression data for selected genes were evaluated from three conditions: different developmental stages, plant hosts and acquisition of CiLV-C. To analyze the stability of the candidate reference genes we used ΔCq method, GeNorm, NormFinder, BestKeeper and RefFinder. Ubiq and GAPDH are best suited for normalizing gene expression data in viruliferous and non-viruliferous mites. Ubiq, EF1α and GAPDH are the most stable for different developmental stages. RPL13 and RPL32 are the best reference genes for approaches to B. yothersi in different host plants. Considering all the experimental conditions, Ubiq, EF1α, and GAPDH were the most stable genes. Here we developed an accurate and comprehensive RT-qPCR strategy for use in B. yothersi gene expression analysis. These results will improve the understanding of the biology of the false spider mites and their role as virus vectors.
Subject(s)
Citrus/virology , Disease Vectors , Gene Expression Regulation, Viral , Mites/virology , Plant Diseases/virology , Plant Viruses/genetics , Real-Time Polymerase Chain Reaction/methods , Animals , Citrus/growth & development , Gene Expression Profiling , Reference Standards , Reproducibility of ResultsABSTRACT
The false spider mite Brevipalpus yothersi infests a broad host plant range and has become one of the most economically important species within the genus Brevipalpus. This phytophagous mite inflicts damage by both feeding on plants and transmitting plant viruses. Here, we report the first draft genome sequence of the false spider mite, which is also the first plant virus mite vector to be sequenced. The â¼72 Mb genome (sequenced at 42× coverage) encodes â¼16,000 predicted protein-coding genes.
ABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0180658.].
ABSTRACT
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.
Subject(s)
Drug Delivery Systems , Pesticides/pharmacology , Phylogeny , Tetranychidae/drug effects , Animals , Drug Resistance/genetics , Herbivory/drug effects , Host Specificity , Host-Parasite Interactions/drug effects , Pesticides/chemistry , Plants/parasitology , Tetranychidae/pathogenicityABSTRACT
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.
Subject(s)
Acari/genetics , Gene Silencing , Gene Targeting/methods , Animals , Insect Proteins/genetics , Insect Proteins/metabolism , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
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.
Subject(s)
Citrus/virology , Phylogeny , Plant Diseases/virology , Plant Viruses/classification , Plant Viruses/genetics , RNA Viruses/classification , RNA Viruses/genetics , Animals , Commelina/virology , Disease Transmission, Infectious , Genes, Viral , Insect Vectors/virology , Mites/virology , Sequence HomologyABSTRACT
Although it is generally assumed that agriculture negatively influences amphibian populations, few studies on the effects of agricultural cultivations on neotropical anuran have been conducted. As a contribution to the knowledge about anuran in agriculture, the present study sought to identify the anuran species present in three different agrossystems. We used data from anurans captured in pitfall traps initially proposed for a survey of harvestmen fauna in three agrossystems (corn, soybean, and rubber tree). Four anuran species found in the pitfall traps belong to two Families: Leptodactylidae: Leptodactulus fuscus and L. mystacinus; and Leiuperidae: Eupemphix nattereri and Physalaemus cuvieri. In corn plantation, four species and 30 individuals were captured; in rubber trees, four species and 11 individuals; and in soybeans plantation, tree species and eight individuals. Our results show that anurans are present in agrossystems, mainly the generalist anuran species.
Embora seja geralmente assumido que a agricultura influencia negativamente populações de anfíbios, existem poucos estudos sobre os efeitos dos cultivos agrícolas em anuros neotropicais. Visando contribuir para diminuir essa lacuna de conhecimento, no presente estudo buscamos verificar quais espécies de anuros estão presentes nos agrossistemas. Para isso, usamos dados de anuros capturados em armadilhas de queda, inicialmente proposto para o levantamento da fauna de opiliões em três agrossistemas (milho, soja e seringal). Nós registramos quatro espécies de anuros nas armadilhas de queda: Leptodactulus fuscus, L. mystacinus (Leptodactylidae), Eupemphix nattereri e Physalaemus cuvieri (Leiuperidae). Na plantação de milho foram registradas quatro espécies e 30 indivíduos, no seringal quatro espécies e 11 indivíduos e na plantação de soja três espécies e oito indivíduos. Nossos resultados mostram que os anuros estão presentes nos agrossistemas, principalmente espécies de anuros generalistas.
ABSTRACT
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.
