RNA Interference in Insects: Protecting Beneficials and Controlling Pests

Insects constitute the largest and most diverse group of animals on Earth with an equally diverse virome. The main antiviral immune system of these animals is the post-transcriptional gene-silencing mechanism known as RNA(i) interference. Furthermore, this process can be artificially triggered via delivery of gene-specific double-stranded RNA molecules, leading to specific endogenous gene silencing. This is called RNAi technology and has important applications in several fields. In this paper, we review RNAi mechanisms in insects as well as the potential of RNAi technology to contribute to species-specific insecticidal strategies. Regarding this aspect, we cover the range of strategies considered and investigated so far, as well as their limitations and the most promising approaches to overcome them. Additionally, we discuss patterns of viral infection, specifically persistent and acute insect viral infections. In the latter case, we focus on infections affecting economically relevant species. Within this scope, we review the use of insect-specific viruses as bio-insecticides. Last, we discuss RNAi-based strategies to protect beneficial insects from harmful viral infections and their potential practical application. As a whole, this manuscript stresses the impact of insect viruses and RNAi technology in human life, highlighting clear lines of investigation within an exciting and promising field of research

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

Generation of virus- and dsRNA-derived siRNAs with species-dependent length in insects

Double-stranded RNA (dsRNA) molecules of viral origin trigger a post-transcriptional gene-silencing mechanism called RNA interference (RNAi). Specifically, virally derived dsRNA is recognized and cleaved by the enzyme Dicer2 into short interfering RNAs (siRNAs), which further direct sequence-specific RNA silencing, ultimately silencing replication of the virus. Notably, RNAi can also be artificially triggered by the delivery of gene-specific dsRNA, thereby leading to endogenous gene silencing. This is a widely used technology that holds great potential to contribute to novel pest control strategies. In this regard, research efforts have been set to find methods to efficiently trigger RNAi in the field. In this article, we demonstrate the generation of dsRNA- and/or virus-derived siRNAs-the main RNAi effectors-in six insect species belonging to five economically important orders (Lepidoptera, Orthoptera, Hymenoptera, Coleoptera, and Diptera). In addition, we describe that the siRNA length distribution is species-dependent. Taken together, our results reveal interspecies variability in the (antiviral) RNAi mechanism in insects and show promise to contribute to future research on (viral-based) RNAi-triggering mechanisms in this class of animals

Role of small RNAs in antiviral immunity and systemic RNA interference of insects

RNA(i) interference is a post-transcriptional gene silencing mechanism that is guided by non-coding small (s)RNA molecules. These sRNAs can be classified, based on their role in distinct biological processes, in three main cell-autonomous pathways: miRNAs regulate endogenous gene expression, piRNAs control retrotransposon activity and siRNAs reduce virus accumulation. In insects, the latter is activated by dsRNA molecules (i.e. products of viral replication), which are then cleaved into small interfering (si)RNAs and used by an Argonaute2 (Ago2) protein to target and degrade complementary RNA. In addition, this pathway can also be induced by artificial long dsRNA and has therefore been widely used as a reverse genetics research tool. Due to its strong gene-silencing capacity and specificity, RNAi holds great potential to contribute to the development of novel ways to control pest insects and combat viral infections in beneficial and disease-vectoring insects. Nonetheless, obtaining a potent RNAi response in the entire organism upon uptake of exogenous dsRNA requires spreading of this response to different cells and tissues, a process designated as systemic (sys)RNAi. However, as with every technique, RNAi also has some limitations, including the refractory response that several insect species present to dsRNA administered at an extracellular level. In this context, understanding the mechanism by which the RNAi signal is transferred intercellularly could be of major importance. In addition, despite the fact that it is widely accepted that the spread of RNAi is crucial for antiviral immunity in insects, the mechanisms involved in this systemic immune response remain to be elucidated. Moreover, it is also not clear if artificial dsRNA molecules and viral replication intermediates are processed and systemically spread in the same manner. Therefore, this thesis focuses on the identification of sRNA species (16-36 nt) upon dsRNA treatment or viral infection and their subsequent sysRNAi signalling mechanism(s), both in vivo and in vitro. This was investigated in species and cell lines that are known to have a robust (systemic) RNAi response: the migratory locust Locusta migratoria, the fruit fly Drosophila melanogaster S2 cells and the red flour beetle Tribolium castaneum TcA cells. First, we report on the production of dsRNA- and virus-derived siRNAs in L. migratoria in vivo, TcA cells and S2 cells. In addition, our data combined with the currently available literature, demonstrate a specific siRNA length preference in species belonging to five economically important insect orders (Lepidoptera, Diptera, Coleoptera, Orthoptera and Hymenoptera). Next, the presence of dsRNA- and virus-derived sRNAs was investigated in the cell-free haemolymph of L. migratoria and in the medium of cultured insect cell lines, as well as in specific extracellular fractions, such as extracellular vesicles (EVs), lipophorins and extracellular (ex)Ago2 containing complexes. In this context, clear interspecies differences were observed for both the dsRNA- and virus-derived sRNAs. In L. migratoria haemolymph, a clear dsRNA-derived siRNA profile was observed at different time points, which could not be detected in chromatographic fractions containing EVs or lipophorins. On the contrary, the culture medium of S2 cells and its derived EVs did not display a typical pattern of dsRNA-derived siRNAs and, subsequently, these EVs did not induce a significant silencing response in recipient cells. However, we have detected antisense siRNA peaks referring to two persistent viral infections in the culture medium and EVs of these cells. Moreover, virus-derived siRNAs were also identified in the cell-free culture medium, EVs and exAgo2 fractions of TcA cells. Lastly, we demonstrate for the latter that, upon dsRNA treatment, these cells process this dsRNA into siRNAs, after which their presence can be identified in EVs. Furthermore, these EVs were able to trigger a significant RNAi silencing response in recipient TcA cells, but not in other insect cell lines (i.e. Drosophila S2 cells, Bombyx mori BmN4 cells, Trichoplusia ni High Five cells and Spodoptera frugiperda Sf9 cells). In addition, other non-coding sRNAs (16-36 nt) were identified in EVs derived from these cells, including miRNAs, sRNAs corresponding to transposable elements, small nucleolar RNAs, small nuclear RNAs and both ribosomal RNA-derived and transfer RNA-derived sRNAs. Taken together, this doctoral thesis provides new fundamental insights in the RNAi-based antiviral immunity and the systemic spreading of the RNAi signal in insects.status: publishe

Insights into the small RNA pathways of locusts

status: publishe

RNA Interference in Insects: Protecting Beneficials and Controlling Pests

Insects constitute the largest and most diverse group of animals on Earth with an equally diverse virome. The main antiviral immune system of these animals is the post-transcriptional gene-silencing mechanism known as RNA(i) interference. Furthermore, this process can be artificially triggered via delivery of gene-specific double-stranded RNA molecules, leading to specific endogenous gene silencing. This is called RNAi technology and has important applications in several fields. In this paper, we review RNAi mechanisms in insects as well as the potential of RNAi technology to contribute to species-specific insecticidal strategies. Regarding this aspect, we cover the range of strategies considered and investigated so far, as well as their limitations and the most promising approaches to overcome them. Additionally, we discuss patterns of viral infection, specifically persistent and acute insect viral infections. In the latter case, we focus on infections affecting economically relevant species. Within this scope, we review the use of insect-specific viruses as bio-insecticides. Last, we discuss RNAi-based strategies to protect beneficial insects from harmful viral infections and their potential practical application. As a whole, this manuscript stresses the impact of insect viruses and RNAi technology in human life, highlighting clear lines of investigation within an exciting and promising field of research.status: publishe

RNAi-based interactions: a latent viral infection in a lepidopteran cell line

RNA interference (RNAi), the main antiviral immune response in insects, is a post-transcriptional gene silencing mechanism triggered by dsRNA. In brief, long dsRNA molecules are processed into short RNA duplexes by a Dicer enzyme. These duplexes are then unwound and the ‘guide strand’ is loaded into the RNA-induced silencing complex (RISC). Subsequently, an Argonaute protein contained within the RISC cleaves or blocks the messenger RNA with sequence homology to the guide strand, which finally results in specific gene silencing. Interestingly, several cytoplasmic RNA viruses have been reported to be persistently present in insects and insect cell lines. However, although RNAi has been demonstrated to play a role in the immunity against acute infections, it is not clear whether it plays a role in the host-pathogen equilibrium that characterizes persistent infections. In this context, making use of the Trichoplusia ni High Five™ cell line, we set out to study whether the overexpression of key components of the RNAi machinery would result in decreased levels of a persistent viral infection. Importantly, this cell line has been demonstrated to be persistently infected with the Macula-like Latent Virus (MLV). However, upon overexpression of Dcr-2, Ago-2 and R2D2 (siRNA pathway); and of Ago-3 and Piwi/Aub (piRNA pathway), it was not possible to observe a decrease in the MLV transcript levels. In addition, we investigated if a Cricket Paralysis Virus (CrPV) infection would affect the MLV levels. Noteworthy, the CrPV is able to induce an acute infection in High Five cells and is known to codify a suppressor of RNAi, namely the A1 protein. Interestingly, when infected with this virus, an increase of the MLV levels in the cells could be observed. To conclude, these results contribute to a better understanding of the RNAi-based interactions between insect hosts and persistent viral infections.status: publishe

Extracellular vesicles spread the RNA interference signal of Tribolium castaneum TcA cells

The potential utility of RNA interference (RNAi) to control insect pests and viral infections depends largely on the target organism's ability to systemically spread the RNAi response. The efficacy of systemic RNAi varies among insects, though it has been shown to be high in the red flour beetle, Tribolium castaneum. We identified an extracellular RNAi signal that is present in the culture medium of T. castaneum (TcA) cells after treatment with long dsRNA specific for a luciferase reporter gene. Luciferase-specific siRNAs were detected in extracellular vesicles (EVs) that were purified from the culture medium of these dsRNA-treated cells. Furthermore, by measuring the silencing of luciferase expression, we showed that these siRNA-containing EVs can act as an RNAi signal for recipient TcA cells. We have therefore shown that a systemic RNAi response upon dsRNA treatment can be effectively spread through EVs

Extracellular vesicles spread the RNA interference signal of Tribolium castaneum TcA cells

The potential utility of RNA interference (RNAi) to control insect pests and viral infections depends largely on the target organism's ability to systemically spread the RNAi response. The efficacy of systemic RNAi varies among insects, though it has been shown to be high in the red flour beetle, Tribolium castaneum. We identified an extracellular RNAi signal that is present in the culture medium of T. castaneum (TcA) cells after treatment with long dsRNA specific for a luciferase reporter gene. Luciferase-specific siRNAs were detected in extracellular vesicles (EVs) that were purified from the culture medium of these dsRNA-treated cells. Furthermore, by measuring the silencing of luciferase expression, we showed that these siRNA-containing EVs can act as an RNAi signal for recipient TcA cells. We have therefore shown that a systemic RNAi response upon dsRNA treatment can be effectively spread through EVs.status: Published onlin