Dulce Cordeiro dos Santos

Identification, functional characterization and phylogenetic analysis of double stranded RNA degrading enzymes present in the gut of the desert locust, Schistocerca gregaria

RNA interference (RNAi) has become a widely used reverse genetics tool in eukaryotes and holds great potential to contribute to the development of novel strategies for insect pest control. While previous studies clearly demonstrated that injection of dsRNA into the body cavity of the desert locust, Schistocerca gregaria, is highly effective to induce gene silencing effects, we observed that the RNAi response is much less sensitive to orally delivered dsRNA. In line with this, we report on the presence of a potent dsRNA degrading activity in the midgut juice. Four different dsRNase sequences that belong to the DNA/RNA Non-specific Nuclease superfamily were retrieved from a transcriptome database of the desert locust. Surprisingly, we have found that, in the publicly available eukaryote nucleotide sequence databases, the presence of this group of enzymes is restricted to insects and crustaceans. Nonetheless, phylogenetic analyses predict a common origin of these enzymes with the Endonuclease G (EndoG) Non-specific Nucleases that display a widespread taxonomic distribution. Moreover, in contrast to the Sg-endoG transcript, the four Sg-dsRNase transcripts appear to be specifically expressed in the gut. Finally, by means of RNAi, we provide evidence for an important contribution of dsRNase2 to the dsRNA degrading activity that is present in the gut lumen of S. gregaria.

Scavenger receptor‐mediated endocytosis facilitates RNA interference in the desert locust, Schistocerca gregaria

RNA interference (RNAi) has become a widely used loss‐of‐function tool in eukaryotes; however, the delivery of double‐stranded (ds)RNA) to the target cells remains a major challenge when exploiting the RNAi‐technology. In insects, the efficiency of RNAi is highly species‐dependent. Yet, the mechanism of cell entry in insects has only been characterized in a cell line of the fruit fly, Drosophila melanogaster, a species that is well known to be poorly amenable to environmental RNAi. In the present paper, we demonstrate that silencing vacuolar H‐ATPase 16 (vha16) and clathrin heavy chain (clath), two components of the Clathrin‐dependent endocytosis pathway, together with pharmacological inhibition of scavenger receptors with polyinosine and dextran sulphate, can significantly attenuate the highly robust RNAi response in the desert locust, Schistocerca gregaria.

Biological mechanisms determining the success of RNA interference in insects.

Insects constitute the largest group of animals on this planet, having a huge impact on our environment, as well as on our quality of life. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism triggered by double-stranded (ds)RNA fragments. This process not only forms the basis of a widely used reverse genetics research method in many different eukaryotes but also holds great promise to contribute to the species-specific control of agricultural pests and to combat viral infections in beneficial and disease vectoring insects. However, in many economically important insect species, such as flies, mosquitoes, and caterpillars, systemic delivery of naked dsRNA does not trigger effective gene silencing. Although many components of the RNAi pathway have initially been deciphered in the fruit fly, Drosophila melanogaster, it will be of major importance to investigate this process in a wider variety of species, including dsRNA-sensitive insects such as locusts and beetles, to elucidate the factors responsible for the remarkable variability in RNAi efficiency, as observed in different insects. In this chapter, we review the current knowledge on the RNAi pathway, as well as the most recent insights into the mechanisms that might determine successful RNAi in insects.

Lipophorins can adhere to dsRNA, bacteria and fungi present in the hemolymph of the desert locust: a role as general scavenger for pathogens in the open body cavity.

Desert locusts are characterized by a highly sensitive and effective RNA interference (RNAi) response. Moreover, delivery of dsRNA into the open body cavity will elicit potent silencing effects throughout the body. On the other hand, many other insect species, such as Bombyx mori and Drosophila melanogaster, lack the ability to efficiently spread the RNAi-signal. In this study, we demonstrated that, in the serum of the desert locust, lipophorins adhere to dsRNA-fragments. Lipophorins can be subdivided into high density and low density lipophorins (HDLp and LDLp), according to their buoyant density, and we showed that both types of lipophorins can interact with dsRNA fragments. Furthermore, in the presence of (gram-positive) bacteria or fungi, LDLp induce the formation of pathogen aggregates, while no clear aggregation effects were detected in the presence of HDLp.

Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata, but not in the desert locust, Schistocerca gregaria

The responsiveness towards orally delivered dsRNA and the potency of a subsequent environmental RNA interference (RNAi) response strongly differs between different insect species. While some species are very sensitive to dsRNA delivery through the diet, others are not. The underlying reasons for this may vary, but degradation of dsRNA by nucleases in the gut lumen is believed to play a crucial role. The Colorado potato beetle, Leptinotarsa decemlineata, is a voracious defoliator of potato crops worldwide, and is currently under investigation for novel control methods based on dsRNA treatments. Here we describe the identification and characterization of two nuclease genes exclusively expressed in the gut of this pest species. Removal of nuclease activity in adults increased the sensitivity towards dsRNA and resulted in improved protection of potato plants. A similar strategy in the desert locust, Schistocerca gregaria, for which we show a far more potent nuclease activity in the gut juice, did however not lead to an improvement of the RNAi response. Possible reasons for this are discussed. Taken together, the present data confirm a negative effect of nucleases in the gut on the environmental RNAi response, and further suggest that interfering with this activity is a strategy worth pursuing for improving RNAi efficacy in insect pest control applications.

Persistent RNA virus infection of lepidopteran cell lines : interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.

The evolution of animal Argonautes: evidence for the absence of antiviral AGO Argonautes in vertebrates

In addition to mediating regulation of endogenous gene expression, RNA interference (RNAi) in plants and invertebrates plays a crucial role in defense against viruses via virus-specific siRNAs. Different studies have demonstrated that the functional diversity of RNAi in animals is linked to the diversification of the Argonaute superfamily, central components of RISCs (RNA induced silencing complexes). The animal Argonaute superfamily is traditionally grouped into AGO and PIWI Argonautes. Yet, by performing phylogenetic analyses and determining the selective evolutionary pressure in the metazoan Argonaute superfamily, we provide evidence for the existence of three conserved Argonaute lineages between basal metazoans and protostomes, namely siRNA-class AGO, miRNA-class AGO and PIWI Argonautes. In addition, it shown that the siRNA-class AGO lineage is characterized by high rates of molecular evolution, suggesting a role in the arms race with viruses, while the miRNA-class AGOs display strong sequence conservation. Interestingly, we also demonstrate that vertebrates lack siRNA-class AGO proteins and that vertebrate AGOs display low rates of molecular evolution. In this way, we provide supportive evidence for the loss of the antiviral siRNA-class AGO group in vertebrates and discuss the consequence hereof on antiviral immunity and the use of RNAi as a loss of function tool in these animals.

Drosha, Dicer-1 and Argonaute-1 in the desert locust: Phylogenetic analyses, transcript profiling and regulation during phase transition and feeding

In this article, we identify and characterise the miRNA machinery components Drosha, Dicer-1 and Argonaute-1 of the desert locust. By means of phylogenetic analyses, we reveal important insights in the evolutionary context of these components. Our data illustrate that insect Argonaute-1 proteins form a monophyletic group with ALG-1 and ALG-2 of Caenorhabditis elegans and with the four (non-Piwi) Argonaute proteins present in humans. On the other hand, humans apparently lack clear homologues of the insect Argonaute-2 proteins. In addition, we demonstrate that drosha, dicer-1 and argonaute-1 display wide transcript tissue-distribution in adult desert locusts, and that during locust phase transition and feeding of starved locusts the expression levels of the miRNA pathway are regulated at the transcript level.

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.