The Study of Cell-Penetrating Peptides to Deliver dsRNA and siRNA by Feeding in the Desert Locust, Schistocerca gregaria.

RNA(i) interference is a gene silencing mechanism triggered by double-stranded (ds)RNA, which promises to contribute to species-specific insect pest control strategies. The first step toward the application of RNAi as an insecticide is to enable efficient gene silencing upon dsRNA oral delivery. The desert locust, Schistocerca gregaria is a devastating agricultural pest. While this species is responsive to dsRNA delivered by intra-hemocoelic injection, it is refractory to orally delivered dsRNA. In this study, we evaluated the capacity of five cell-penetrating peptides (CPPs) to bind long dsRNA and protect it from the locust midgut environment. We then selected the CPP EB1 for further in vivo studies. EB1:dsRNA complexes failed to induce RNAi by feeding. Interestingly, we observed that intra-hemocoelic injection of small-interfering (si)RNAs does not result in a silencing response, but that this response can be obtained by injecting EB1:siRNA complexes. EB1 also protected siRNAs from midgut degradation activity. However, EB1:siRNA complexes failed as well in triggering RNAi when fed. Our findings highlight the complexity of the dsRNA/siRNA-triggered RNAi in this species and emphasize the multifactorial nature of the RNAi response in insects. Our study also stresses the importance of in vivo studies when it comes to dsRNA/siRNA delivery systems.

Identification and profiling of stable microRNAs in hemolymph of young and old Locusta migratoria fifth instars.

Since the discovery of the first microRNA (miRNA) in the nematode Caenorhabditis elegans, numerous novel miRNAs have been identified which can regulate presumably every biological process in a wide range of metazoan species. In accordance, several insect miRNAs have been identified and functionally characterized. While regulatory RNA pathways are traditionally described at an intracellular level, studies reporting on the presence and potential role of extracellular (small) sRNAs have been emerging in the last decade, mainly in mammalian systems. Interestingly, evidence in several species indicates the functional transfer of extracellular RNAs between donor and recipient cells, illustrating RNA-based intercellular communication. In insects, however, reports on extracellular small RNAs are emerging but the number of detailed studies is still very limited. Here, we demonstrate the presence of stable sRNAs in the hemolymph of the migratory locust, Locusta migratoria. Moreover, the levels of several extracellular miRNAs (ex-miRNAs) present in locust hemolymph differed significantly between young and old fifth nymphal instars. In addition, we performed a 'proof of principle' experiment which suggested that extracellularly delivered miRNA molecules are capable of affecting the locusts' development.

PIWI Proteins Play an Antiviral Role in Lepidopteran Cell Lines.

Insect antiviral immunity primarily relies on RNAi mechanisms. While a key role of small interfering (si)RNAs and AGO proteins has been well established in this regard, the situation for PIWI proteins and PIWI-interacting (pi)RNAs is not as clear. In the present study, we investigate whether PIWI proteins and viral piRNAs are involved in the immunity against single-stranded RNA viruses in lepidopteran cells, where two PIWIs are identified (Siwi and Ago3). Via loss- and gain-of-function studies in Bombyx mori BmN4 cells and in Trichoplusia ni High Five cells, we demonstrated an antiviral role of Siwi and Ago3. However, small RNA analysis suggests that viral piRNAs can be absent in these lepidopteran cells. Together with the current literature, our results support a functional diversification of PIWI proteins in insects.

RNAs on the Go: Extracellular Transfer in Insects with Promising Prospects for Pest Management.

RNA-mediated pathways form an important regulatory layer of myriad biological processes. In the last decade, the potential of RNA molecules to contribute to the control of agricultural pests has not been disregarded, specifically via the RNA interference (RNAi) mechanism. In fact, several proofs-of-concept have been made in this scope. Furthermore, a novel research field regarding extracellular RNAs and RNA-based intercellular/interorganismal communication is booming. In this article, we review key discoveries concerning extracellular RNAs in insects, insect RNA-based cell-to-cell communication, and plant-insect transfer of RNA. In addition, we overview the molecular mechanisms implicated in this form of communication and discuss future biotechnological prospects, namely from the insect pest-control perspective.

The presence of extracellular microRNAs in the media of cultured Drosophila cells.

While regulatory RNA pathways, such as RNAi, have commonly been described at an intracellular level, studies investigating extracellular RNA species in insects are lacking. In the present study, we demonstrate the presence of extracellular microRNAs (miRNAs) in the cell-free conditioned media of two Drosophila cell lines. More specifically, by means of quantitative real-time PCR (qRT-PCR), we analysed the presence of twelve miRNAs in extracellular vesicles (EVs) and in extracellular Argonaute-1 containing immunoprecipitates, obtained from the cell-free conditioned media of S2 and Cl.8 cell cultures. Next-generation RNA-sequencing data confirmed our qRT-PCR results and provided evidence for selective miRNA secretion in EVs. To our knowledge, this is the first time that miRNAs have been identified in the extracellular medium of cultured cells derived from insects, the most speciose group of animals.

Insights into RNAi-based antiviral immunity in Lepidoptera: acute and persistent infections in Bombyx mori and Trichoplusia ni cell lines.

The control of viral infections in insects is a current issue of major concern and RNA interference (RNAi) is considered the main antiviral immune response in this group of animals. Here we demonstrate that overexpression of key RNAi factors can help to protect insect cells against viral infections. In particular, we show that overexpression of Dicer2 and Argonaute2 in lepidopteran cells leads to improved defense against the acute infection of the Cricket Paralysis Virus (CrPV). We also demonstrate an important role of RNAi in the control of persistent viral infections, as the one caused by the Macula-like Latent Virus (MLV). Specifically, a direct interaction between Argonaute2 and virus-specific small RNAs is shown. Yet, while knocking down Dicer2 and Argonaute2 resulted in higher transcript levels of the persistently infecting MLV in the lepidopteran cells under investigation, overexpression of these proteins could not further reduce these levels. Taken together, our data provide deep insight into the RNAi-based interactions between insects and their viruses. In addition, our results suggest the potential use of an RNAi gain-of-function approach as an alternative strategy to obtain reduced viral-induced mortality in Lepidoptera, an insect order that encompasses multiple species of relevant economic value.