Publications

@article{Cagliari2020,

title = {First transcriptome of the Neotropical pest Euschistus heros (Hemiptera: Pentatomidae) with dissection of its siRNA machinery},

author = {Deise Cagliari and Naymã Pinto Dias and Ericmar Ávila Santos and Leticia Neutzling Rickes and Frederico Schmitt Kremer and Juliano Ricardo Farias and Giuvan Lenz and Diogo Manzano Galdeano and Flávio Roberto Mello Garcia and Guy Smagghe and Moisés João Zotti},

url = {https://www.nature.com/articles/s41598-020-60078-3},

doi = {10.1038/s41598-020-60078-3},

issn = {2045-2322},

year  = {2020},

date = {2020-01-01},

journal = {Scientific Reports 2020 10:1},

volume = {10},

issue = {1},

pages = {1-16},

publisher = {Nature Publishing Group},

abstract = {Over the past few years, the use of RNA interference (RNAi) for insect pest management has attracted considerable interest in academia and industry as a pest-specific and environment-friendly strategy for pest control. For the success of this technique, the presence of core RNAi genes and a functional silencing machinery is essential. Therefore, the aim of this study was to test whether the Neotropical brown stinkbug Euschistus heros has the main RNAi core genes and whether the supply of dsRNA could generate an efficient gene silencing response. To do this, total mRNA of all developmental stages was sequenced on an Illumina platform, followed by a de novo assembly, gene annotation and RNAi-related gene identification. Once RNAi-related genes were identified, nuclease activities in hemolymph were investigated through an ex vivo assay. To test the functionality of the siRNA machinery, E. heros adults were microinjected with ~28 ng per mg of insect of a dsRNA targeting the V-ATPase-A gene. Mortality, relative transcript levels of V-ATPase-A, and the expression of the genes involved in the siRNA machinery, Dicer-2 (DCR-2) and Argonaute 2 (AGO-2), were analyzed. Transcriptome sequencing generated more than 126 million sequenced reads, and these were annotated in approximately 80,000 contigs. The search of RNAi-related genes resulted in 47 genes involved in the three major RNAi pathways, with the absence of sid-like homologous. Although ex vivo incubation of dsRNA in E. heros hemolymph showed rapid degradation, there was 35% mortality at 4 days after treatment and a significant reduction in V-ATPase-A gene expression. These results indicated that although sid-like genes are lacking, the dsRNA uptake mechanism was very efficient. Also, 2-fold and 4-fold overexpression of DCR-2 and AGO-2, respectively, after dsRNA supply indicated the activation of the siRNA machinery. Consequently, E. heros has proven to be sensitive to RNAi upon injection of dsRNA into its hemocoel. We believe that this finding together with a publically available transcriptome and the validation of a responsive RNAi machinery provide a starting point for future field applications against one of the most important soybean pests in South America.},

keywords = {RNAi, Sequencing},

pubstate = {published},

tppubtype = {article}

}

@article{Pacheco2020,

title = {Gene silencing of Diaphorina citri candidate effectors promotes changes in feeding behaviors},

author = {Inaiara Souza Pacheco and Diogo Manzano Galdeano and Nathalie Kristine Prado Maluta and Joao Roberto Spotti Lopes and Marcos Antonio Machado},

url = {https://www.nature.com/articles/s41598-020-62856-5},

doi = {10.1038/s41598-020-62856-5},

issn = {2045-2322},

year  = {2020},

date = {2020-01-01},

journal = {Scientific Reports 2020 10:1},

volume = {10},

issue = {1},

pages = {1-14},

publisher = {Nature Publishing Group},

abstract = {Insect effectors are mainly secreted by salivary glands, modulate plant physiology and favor the establishment and transmission of pathogens. Feeding is the principal vehicle of transmission of Candidatus Liberibacter asiaticus (Ca. Las) by the Asian citrus psyllid (ACP), Diaphorina citri. This study aimed to predict putative ACP effectors that may act on the Huanglongbing (HLB) pathosystem. Bioinformatics analysis led to the identification of 131 candidate effectors. Gene expression investigations were performed to select genes that were overexpressed in the ACP head and modulated by Ca. Las. To evaluate the actions of candidate effectors on D. citri feeding, six effectors were selected for gene silencing bioassays. Double-stranded RNAs (dsRNAs) of the target genes were delivered to D. citri adults via artificial diets for five days. RNAi silencing caused a reduction in the ACP lifespan and decreased the salivary sheath size and honeydew production. Moreover, after dsRNA delivery of the target genes using artificial diet, the feeding behaviors of the insects were evaluated on young leaves from citrus seedlings. These analyses proved that knockdown of D. citri effectors also interfered with ACP feeding abilities in planta, causing a decrease in honeydew production and reducing ACP survival. Electrical penetration graph (EPG) analysis confirmed the actions of the effectors on D. citri feeding behaviors. These results indicate that gene silencing of D. citri effectors may cause changes in D. citri feeding behaviors and could potentially be used for ACP control.},

keywords = {Entomology, RNAi},

pubstate = {published},

tppubtype = {article}

}

@article{Rogerio2019,

title = {Reference genes for gene expression studies by RT-qPCR in Brevipalpus yothersi (Acari: Tenuipalpidae), the mite vector of citrus leprosis virus},

author = {Luana Aparecida Rogerio and Diogo Manzano Galdeano and Gabriella Dias Arena and Maria Andreia Nunes and Marcos Antonio Machado and Valdenice Moreira Novelli},

url = {https://www.nature.com/articles/s41598-019-42993-2},

doi = {10.1038/s41598-019-42993-2},

issn = {2045-2322},

year  = {2019},

date = {2019-01-01},

journal = {Scientific Reports 2019 9:1},

volume = {9},

issue = {1},

pages = {1-9},

publisher = {Nature Publishing Group},

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.},

keywords = {Gene expression, Gene expression profiling, RNAi},

pubstate = {published},

tppubtype = {article}

}