Parental RNA interference as a tool to study genes involved in rostrum development in the Neotropical brown stink bug, Euschistus heros.

In insects, the identity of body segments is controlled by homeotic genes and the knockdown of these genes during embryogenesis can lead to an abnormal development and/or atypical phenotypes. The main goal of this study was to investigate the involvement of labial (lab), deformed (dfd), sex comb reduced (scr), extradenticle (exd) and proboscipedia (pb) in rostrum development in the Neotropical brown stink bug Euschistus heros, using parental RNAi (pRNAi). To achieve this objective, 10-days-old adult females were first microinjected with double-stranded RNAs (dsRNA) targeting these five genes. Then, the number of eggs laid per female, the percentage of hatched nymphs with normal or abnormal phenotype and target gene silencing were evaluated. Except for the dsDfd-treatment, the number of eggs laid per female per day was not affected by the different dsRNA-treatments compared to the control (dsGFP). However, treatment with either dsLab, dsDfd, dsScr or dsExd caused a strong reduction in egg hatching. The dsExd-treatment caused no apparent change in phenotype in the nymphs while hatched nymphs from the dsDfd, dsScr and dsPb-treatment showed abnormalities in the rostrum. Particularly for the dsPb-treatment, 91% of the offspring displayed a bifurcated rostrum with a leg-like structure. Overall, these results indicate that these five genes are involved in E. heros embryonic development and that the knockdown of dfd, scr and pb leads to an abnormal development of the rostrum. Additionally, this study demonstrates the efficiency of pRNAi in studying genes involved in embryogenesis in E. heros, with clear phenotypes and a strong target gene silencing in the next generation, after treatment of the parent female adult with gene-specific dsRNA.

RNAi and CRISPR/Cas9 as Functional Genomics Tools in the Neotropical Stink Bug, Euschistus heros.

The Neotropical brown stink bug, Euschistus heros, is one of the most important stink bug pests in leguminous plants in South America. RNAi and CRISPR/Cas9 are important and useful tools in functional genomics, as well as in the future development of new integrated pest management strategies. Here, we explore the use of these technologies as complementing functional genomic tools in E. heros. Three genes, abnormal wing disc (awd), tyrosine hydroxylase (th) and yellow (yel), known to be involved in wing development (awd) and the melanin pathway (th and yel) in other insects, were chosen to be evaluated using RNAi and CRISPR/Cas9 as tools. First, the genes were functionally characterized using RNAi knockdown technology. The expected phenotype of either deformed wing or lighter cuticle pigmentation/defects in cuticle sclerotization was observed for awd and th, respectively. However, for yel, no obvious phenotype was observed. Based on this, yel was selected as a target for the development of a CRISPR/Cas9 workflow to study gene knockout in E. heros. A total of 719 eggs were injected with the Cas9 nuclease (300 ng/µL) together with the sgRNA (300 ng/µL) targeting yel. A total of six insects successfully hatched from the injected eggs and one of the insects showed mutation in the target region, however, the phenotype was still not obvious. Overall, this study for the first time provides a useful CRISPR/Cas9 gene editing methodology to complement RNAi for functional genomic studies in one of the most important and economically relevant stink bug species.

First transcriptome of the Neotropical pest Euschistus heros (Hemiptera: Pentatomidae) with dissection of its siRNA machinery.

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.

Management of Pest Insects and Plant Diseases by Non-Transformative RNAi.

Since the discovery of RNA interference (RNAi), scientists have made significant progress towards the development of this unique technology for crop protection. The RNAi mechanism works at the mRNA level by exploiting a sequence-dependent mode of action with high target specificity due to the design of complementary dsRNA molecules, allowing growers to target pests more precisely compared to conventional agrochemicals. The delivery of RNAi through transgenic plants is now a reality with some products currently in the market. Conversely, it is also expected that more RNA-based products reach the market as non-transformative alternatives. For instance, topically applied dsRNA/siRNA (SIGS - Spray Induced Gene Silencing) has attracted attention due to its feasibility and low cost compared to transgenic plants. Once on the leaf surface, dsRNAs can move directly to target pest cells (e.g., insects or pathogens) or can be taken up indirectly by plant cells to then be transferred into the pest cells. Water-soluble formulations containing pesticidal dsRNA provide alternatives, especially in some cases where plant transformation is not possible or takes years and cost millions to be developed (e.g., perennial crops). The ever-growing understanding of the RNAi mechanism and its limitations has allowed scientists to develop non-transgenic approaches such as trunk injection, soaking, and irrigation. While the technology has been considered promising for pest management, some issues such as RNAi efficiency, dsRNA degradation, environmental risk assessments, and resistance evolution still need to be addressed. Here, our main goal is to review some possible strategies for non-transgenic delivery systems, addressing important issues related to the use of this technology.

The South American Fruit Fly: An Important Pest Insect With RNAi-Sensitive Larval Stages.

RNA interference (RNAi) technology has been used in the development of approaches for pest control. The presence of some essential genes, the so-called "core genes," in the RNAi machinery is crucial for its efficiency and robust response in gene silencing. Thus, our study was designed to examine whether the RNAi machinery is functional in the South American (SA) fruit fly Anastrepha fraterculus (Diptera: Tephritidae) and whether the sensitivity to the uptake of double-stranded RNA (dsRNA) could generate an RNAi response in this fruit fly species. To prepare a transcriptome database of the SA fruit fly, total RNA was extracted from all the life stages for later cDNA synthesis and Illumina sequencing. After the de novo transcriptome assembly and gene annotation, the transcriptome was screened for RNAi pathway genes, as well as the duplication or loss of genes and novel target genes to dsRNA delivery bioassays. The dsRNA delivery assay by soaking was performed in larvae to evaluate the gene-silencing of V-ATPase, and the upregulation of Dicer-2 and Argonaute-2 after dsRNA delivery was analyzed to verify the activation of siRNAi machinery. We tested the stability of dsRNA using dsGFP with an in vitro incubation of larvae body fluid (hemolymph). We identified 55 genes related to the RNAi machinery with duplication and loss for some genes and selected 143 different target genes related to biological processes involved in post-embryonic growth/development and reproduction of A. fraterculus. Larvae soaked in dsRNA (dsV-ATPase) solution showed a strong knockdown of V-ATPase after 48 h, and the expression of Dicer-2 and Argonaute-2 responded with an increase upon the exposure to dsRNA. Our data demonstrated the existence of a functional RNAi machinery in the SA fruit fly, and we present an easy and robust physiological bioassay with the larval stages that can further be used for screening of target genes at in vivo organisms' level for RNAi-based control of fruit fly pests. This is the first study that provides evidence of a functional siRNA machinery in the SA fruit fly.

Differential impacts of pesticides on Euschistus heros (Hem.: Pentatomidae) and its parasitoid Telenomus podisi (Hym.: Platygastridae).

Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) primarily attack the pods and seeds of soybean plants, causing severe economic losses in Neotropical Region, and chemical control is essential to avoid these losses. Thus, insecticides more effective against this pest and less toxic to Telenomus podisi Ashmead (Hymenoptera: Platygastridae) - the main biological control agent of E. heros - should be used. In this report, we studied the differential acute impacts of pesticides used in Brazilian soybean against E. heros and T. podisi and evaluated their sublethal effects on the parasitoid to identify effective pesticides towards the pest with less harmful effect to the natural enemy. The LC50 of the insecticides to E. heros ranged from 1.20 to 533.74 ng a.i./cm2; the order of toxicity was thiamethoxam + lambda-cyhalothrin > acetamiprid + fenpropathrin > zeta-cypermethrin > acephate > imidacloprid. All pesticides were classified as slightly to moderately toxic to T. podisi based on the risk quotient. The exposure of T. podisi females to imidacloprid and the insecticide pre-formulated mixtures reduced the emergence of the offspring parasitoids by up to 40% whereas zeta-cypermethrin and the insecticides pre-formulated mixtures reduced offspring survival. The preferred order of choice of insecticides for the management of E. heros according to agronomic, toxicological, and environmental feasibility was the following: thiamethoxam + lambda-cyhalothrin > zeta-cypermethrin > acetamiprid + fenpropathrin > acephate > imidacloprid. Our study provides important and pioneer information to select insecticides for effective control of E. heros with lower impacts on T. podisi.

RNA interference technology in crop protection against arthropod pests, pathogens and nematodes.

Scientists have made significant progress in understanding and unraveling several aspects of double-stranded RNA (dsRNA)-mediated gene silencing during the last two decades. Now that the RNA interference (RNAi) mechanism is well understood, it is time to consider how to apply the acquired knowledge to agriculture and crop protection. Some RNAi-based products are already available for farmers and more are expected to reach the market soon. Tailor-made dsRNA as an active ingredient for biopesticide formulations is considered a raw material that can be used for diverse purposes, from pest control and bee protection against viruses to pesticide resistance management. The RNAi mechanism works at the messenger RNA (mRNA) level, exploiting a sequence-dependent mode of action, which makes it unique in potency and selectivity compared with conventional agrochemicals. Furthermore, the use of RNAi in crop protection can be achieved by employing plant-incorporated protectants through plant transformation, but also by non-transformative strategies such as the use of formulations of sprayable RNAs as direct control agents, resistance factor repressors or developmental disruptors. In this review, RNAi is presented in an agricultural context (discussing products that have been launched on the market or will soon be available), and we go beyond the classical presentation of successful examples of RNAi in pest-insect control and comprehensively explore its potential for the control of plant pathogens, nematodes and mites, and to fight against diseases and parasites in beneficial insects. Moreover, we also discuss its use as a repressor for the management of pesticide-resistant weeds and insects. Finally, this review reports on the advances in non-transformative dsRNA delivery and the production costs of dsRNA, and discusses environmental considerations. © 2017 Society of Chemical Industry.

Precipitação artificial após aplicação do inseticida clorantraniliprole associado com adjuvante em plantas de soja / Artificial precipitation after spray of clorantraniliprole insecticide associated with adjuvant application in soybean plants

O controle de insetos-praga com inseticidas é um recurso importante para manutenção das altas produtividades na cultura da soja. Porém, pouco se conhece sobre o desempenho de novos inseticidas, tais como, clorantraniliprole em condições adversas, como é o caso das precipitações pluviométricas após a pulverização agrícola. A ação dos adjuvantes associados aos inseticidas é outro fator relacionado à melhora da sua eficiência. O objetivo deste estudo foi avaliar a influência de quatro períodos de precipitação artificial após a pulverização do inseticida clorantraniliprole (2g de ingrediente ativo ha-1), misturado com diferentes adjuvantes, visando ao o controle de lagartas na cultura da soja. O experimento foi executado com o uso de duas cultivares (blocos) e seis vasos (2 plantas/vaso) por tratamento fatorial (5x5). O fator adjuvante foi constituído por uma testemunha (sem adjuvante) e quatro adjuvantes (Assist(r) - 0,5% do volume de calda, Naturo'il(r) - 0,5%, Nitro LL(r) - 6,6% (10L ha-1) e Silwet L-77(r) - 0,1%). O fator precipitação artificial (20mm) foi composto por uma testemunha (sem precipitação) e quatro tempos (1, 120, 240 e 360 minutos) após a pulverização de clorantraniliprole com adjuvantes. Os resultados sugerem que a precipitação de 20 mm, um minuto após a aplicação de clorantraniliprole, reduz a mortalidade de Anticarsia gemmatalis, e que as precipitações após 240 minutos da aplicação de clorantraniliprole não interferem na mortalidade de A. gemmatalis em soja. Os adjuvantes Assist(r) e Naturo'il(r) associados ao inseticida clorantraniliprole aumentam a mortalidade de A. gemmatalis na cultura da soja.

The insecticide use on insect pest control is an important tool to keep high yields on soybeans. However, little is known about the behavior of the insecticide in adverse conditions, such as spraying followed by precipitation. The use of adjuvants associated with insecticides is another factor that can be related to improvement of its efficiency. This study aimed to evaluate the effects of the influence of four precipitation periods after the pulverization of the insecticide clorantraniliprole (2g of the active ingredient ha-1). A mixed of different adjuvants on the control of caterpillars on soybeans were evaluated. The assay was performed using two cultivars in six pots (2 plants/pots) for a factor scheme of 5x5. The factor adjuvants consisted of one untreated (without adjuvants) and four adjuvants (Assist(r) - 0,5%, Naturo'il(r) - 0,5%, Nitro LL(r) - L ha-1 and Silwet L-77(r) - 0,1% ). The factor artificial precipitation (20mm) consisted of one untreated (without precipitation) and four precipitation intervals (1, 120, 240 and 360 minutes) after the application of clorantraniliprole with adjuvants. The results showed that the precipitation of 20mm, 1 minute after the clorantraniliprole pulverization, reduces the Anticarsia gemmatalis mortality, and that the precipitation 240 min after the pulverization does not interfere in the mortality of A. gemmatalis on soybeans. The adjuvants Assist(r) and Naturo'il(r) associated to the insecticide clorantraniliprole increases the A. gemmatalis mortality on soybeans.