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 Illumine 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 postembryonic 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

RNAi and CRISPR/Cas9 as functional genomics tools in the neotropical stink bug, Euschistus heros

Understanding the biology of insect pests is an important step towards developing appropriate control strategies. In this study, a CRISPR/Cas9 gene knockout work flow was established for the first time and was together with RNAi used as tools to study gene functions in the Neotropical stink bug, Euschistus heros. RNAi was first employed to study the function of three genes, abnormal wing disc (awd), tyrosine hydroxylase (th) and yellow (yel). Targeting awd and th resulted in distinct malformed phenotypes such as a deformed wing or a lighter cuticle pigmentation/defects in cuticle sclerotization, respectively. However, no distinct phenotype was observed for yel. To further investigate the function of yel, a CRISPR/Cas9 gene editing protocol was developed for E. heros. A total of 719 eggs were microinjected with single-guide (sgRNA) and Cas9 and total of six insects hatched. Out of these six nymphs, one insect showed mutation in yel but no clear phenotype was visible. Although, we were unable to generate insects with a distinct phenotype for yel, a successful gene editing workflow was established to complement RNAi for future functional gene studies in E. heros. Additionally, we provided recommendations to improve the established gene editing workflow. 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/mu L) together with the sgRNA (300 ng/mu L) 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

Nontransformative Strategies for RNAi in Crop Protection

RNAi in crop protection can be achieved not only by plant-incorporated protectants through plant transformation (transgenic) but also by nontransformative strategies such as formulations of sprayable dsRNAs used as direct control agents, resistance factor repressors, or developmental disruptors. Therefore, the RNAi-based biopesticides are expected to reach the market also in the form of nontransgenic strategies such as sprayable products, stem injection, root drenching, seed treatment, or powder/granule. While the delivery of dsRNA by transgenic expression is well established, it requires generations of crop plants and is costly, which may take years and delays for practical application, depending on the regulatory rules, plant transformability, genetic stability, and public acceptance of genetically modified crop species. DsRNA delivery as a nontransgenic approach was already published as a proof-of-concept work, so it is time to point out some directions on how the real potential for agriculture and crop protection is

Flutuação populacional de Nysius simulans associado com soja e buva no Brasil

This paper reports the first record of the occurrence and the populational fluctuation of Nysius simulans (Stål) (Hemiptera: Lygaeidae) associated with hairy fleabane (Conyza bonariensis) and soybean (Glycine max) in Brazil. Fortnightly samplings were carried out in a soybean field weeded with hairy fleabane, in São Vicente do Sul, Rio Grande do Sul, Brazil, between December 2010 and November 2011. N. simulans was found on the plants from the beginning of November to the end of June. The largest population density was observed during low rainfall periods and the reproductive stage of soybean. After soybean maturation and harvest, the largest populations of N. simulans were found on the weed, indicating that hairy fleabane serves as a host for this species in the absence of soybean from the field.Este trabajo presenta el primer registro de ocurrencia y la fluctuación poblacional de Nysius simulans (Stål) (Hemiptera: Lygaeidae) asociado con rama negra (Conyza bonariensis) y soja (Glycine max) en Brasil. Se tomaron muestras quincenales en un campo de soja con rama negra como maleza, en São Vicente do Sul, Rio Grande do Sul, Brasil, entre diciembre 2010 y noviembre 2011. Se encontró N. simulans desde principios de noviembre hasta finales de junio. Las poblaciones más abundantes se observaron en períodos de escasez de precipitaciones y durante la etapa reproductiva de la soja. Después de la maduración y la cosecha de soja, densidades grandes de N. simulans se encuentran en rama negra, lo que indica que esta planta alberga a esta especie cuando la soja está ausente del campo.Este trabalho relata o primeiro registro de ocorrência e a flutuação populacional de Nysius simulans (Stål) (Hemiptera: Lygaeidae) associado a plantas de buva (Conyza bonariensis) e de soja (Glycine max) no Brasil. Foram realizadas amostragens quinzenais em São Vicente do Sul, Rio Grande do Sul, Brasil, entre dezembro 2010 e novembro 2011. A praga foi encontrada desde o início de novembro até o final de junho. As maiores populações foram observadas em períodos de menor precipita- ção e durante a fase reprodutiva da soja. Após a maturação e colheita da soja, grandes densidades populacionais de N. simulans foram encontradas sobre a buva, indicando que essa planta hospeda a praga na ausência da soja.Fil: Dalazen, Giliardi. Universidade Federal do Rio Grande do Sul; BrasilFil: Guedes, Jerson Vanderlei Carús. Universidade Federal de Santa Maria; BrasilFil: Carpintero, Diego Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Stacke, Regis Felipe. Universidade Federal de Santa Maria; BrasilFil: Cagliari, Deise. Universidade Federal de Santa Maria; Brasi

Insecticidal Gene Silencing by RNAi in the Neotropical Region.

Insecticidal gene silencing by RNA interference (RNAi) involves a post-transcriptional mechanism with great potential for insect control. Here, we aim to summarize the progress on RNAi research toward control of insect pests in the Neotropical region and discuss factors determining its efficacy and prospects for pest management. We include an overview of the available RNAi information for Neotropical pests in the Lepidoptera, Coleoptera, Diptera, and Hemiptera orders. Emphasis is put on significant findings in the use of RNAi against relevant Neotropical pests, including diamondback moth (Plutella xylostella L.), Asian citrus psyllid (Diaphorina citri Kuwayama), and the cotton boll weevil (Anthonomus grandis Boheman). We also examine the main factors involved in insecticidal RNAi efficiency and major advances to improve screening of lethal genes, formulation, and delivery. Few studies detail resistance mechanisms to RNAi, demonstrating a need for more research. Advances in formulation, delivery, and resistance management tools for insecticidal RNAi in the Neotropics can provide a basis for efficient field application

Análise do transcriptoma e exploração do RNAi e CRISPR no percevejo neotropical, Euschistus heros

Um dos principais fatores limitantes a produção agrícola, são o ataque de insetos, patógenos e plantas daninhas, os quais podem causar perdas significativas. Euschistus heros (Heteroptera: Pentatomidae) é uma das espécies de maior importância encontradas na cultura da soja, com ocorrência distribuída em toda área produtora brasileira. Na tentativa de reduzir os danos causados por esse e outros insetos, produtores dependem quase que exclusivamente do uso de inseticidas. Entretanto, o uso indiscriminado desses produtos vem causando sérios problemas de manejo, como a seleção de populações resistentes levando a ineficiência dos produtos disponíveis no mercado. Diante disso, é necessário o desenvolvimento de ferramentas alternativas de controle, tais como silenciamento gênico baseado em RNA de interferência (RNAi) e CRISPR (Repetições Palindrômicas Curtas Agrupadas e Regularmente Interespaçadas). O RNAi é um mecanismo altamente conservado em organismos eucariotos e pelo qual a molécula de RNA mensageiro (RNAm) é clivada pela maquinaria de silenciamento, levando a inativação da expressão gênica (Knockdown). Já o CRISPR é uma ferramenta de edição gênica com a qual é possível realizar modificação a nível de DNA, introduzindo ou eliminando sequências específicas do DNA (knockout). Nos últimos anos, a utilização do RNAi e CRISPR tem atraído o interesse de pesquisadores no estudo das funções dos genes no desenvolvimento e reprodução dos insetos, o que permitirá o desenvolvimento de estratégias alternativas de controle. Assim, considerando a importância do RNAi e do sistema CRISPR/Cas9 no estudo da genômica funcional e no desenvolvimento de novas ferramentas para o manejo de insetos-praga, os objetivos deste estudo incluem: i) identificar os principais componentes da maquinaria de RNAi presentes em E. heros, assim como validar a funcionalidade dessa ferramenta em E. heros; ii) uma revisão mostrando o uso potencial da ferramenta RNAi em uma abordagem nãotransformativa; iii) o uso do RNAi parental como ferramenta no estudo das funções gênicas em E. heros; iv) a combinação de RNAi e CRISPR/Cas9 como ferramentas genômicas funcionais no percevejo Neotropical, E. heros. Essas informações fornecem novos e importantes conjuntos de dados sobre a maquinaria de RNAi e sua eficiência, a perspectiva futura da utilização do RNAi via não-transformativa e do uso de RNAi e CRISPR como ferramentas em estudos genéticos funcionais, apoiando estratégias futuras para melhorar RNAi e CRISPR em E. heros e outras espécies de insetos perfuradores e sugadores importantes na agricultura.Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPqOne of the main factors limiting agricultural production is the attack of insects, pathogens and weeds, which can cause significant losses in the culture. Euschistus heros (Heteroptera: Pentatomidae) is one of the most important stink bug species found in soybean, with occurrence distributed throughout the Brazilian production area. In an attempt to reduce the damage caused by this and other insects, producers depend almost exclusively on the use of insecticides. However, the indiscriminate use of these products has been causing serious management problems, such as the selection of resistant populations leading to the inefficiency of products available on the market. Therefore, the development of alternative control tools, such as gene silencing based on RNA interference (RNAi) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is necessary. RNAi is a highly conserved mechanism in eukaryotic organisms and by which the messenger RNA molecule (mRNA) is cleaved by the gene-silencing machinery, leading to inactivation of gene expression (Knockdown). CRISPR, on the other hand, is a gene editing tool with which it is possible to carry out modification at the DNA level, introducing or eliminating specific DNA sequences from the genome (knockout). During the last years, the use of RNAi and CRISPR has attracted the interest of researchers, mainly in the study of the functions of genes during insect’s development and reproduction, which will allow the future development of alternative control strategies. Thus, considering the importance of RNAi and the CRISPR / Cas9 system in the study of functional genomics and in the development of new tools for the management of insect pests, the objectives of this study include: i) to identify the main components of the RNAi machinery present in E. heros, as well as validating the functionality of this tool in E. heros; ii) a review showing the potential use of the RNAi tool in a non-transformative approach; iii) the use of parental RNAi as a tool in the study of gene functions in E. heros; iv) the combination of RNAi and CRISPR/Cas9 as functional genomic tools in the Neotropical bug, E. heros. This information provides novel and important dataset on RNAi machinery and its efficiency, the future perspective of non-transformative RNAi approaches and the use of RNAi and CRISPR as tools in functional genetic studies, underpinning future strategies to improve RNAi and CRISPR in E. heros and other piercing-sucking insects species important in agriculture

Faunistic analysis of pest insects and their natural enemies associated with hairy fleabane in soybean crop1

<div><p>ABSTRACT In addition to competing with crops, weeds can provide shelter for arthropods in cropping fields and adjacent areas. This study aimed to investigate the occurrence and population fluctuation of insect pests and their natural enemies associated with hairy fleabane (Conyza spp.), in soybean farming areas. The predominant species were Schizaphis graminum, Taylorilygus apicalis, Empoasca spp. and Nysius simulans. Species that comprise important pest complexes, including stink bugs and caterpillars, can develop on fleabane plants. Among them, the stink bugs Edessa meditabunda, Dichelops spp., Piezodorus guildinii, Nezara viridula and Euschistus heros, as well as the caterpillars Anticarsia gemmatalis, Spodoptera frugiperda, Spodoptera eridania, Chrysodeixis includens and Helicoverpa spp. have prevailed. The most common natural enemies were Orius spp., Eriopis connexa, Cycloneda sanuiinea and Chrysoperla spp. The population fluctuation analysis indicated that hairy fleabane mainly hosted stink bugs at the beginning of the crop cycle and after soybean maturation. The caterpillars, however, use this weed as an alternative host throughout the crop cycle, continuing after soybean maturation and harvesting. For natural enemies species, the population fluctuation depends on the occurrence of insects that serve as a food source for them. Thus, it is possible to conclude that hairy fleabane is an important alternative host for pest insects and their natural enemies in soybean crops.</p></div