Review
Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review

https://doi.org/10.1016/j.jinsphys.2009.10.004Get rights and content

Abstract

RNA interference already proved its usefulness in functional genomic research on insects, but it also has considerable potential for the control of pest insects. For this purpose, the insect should be able to autonomously take up the dsRNA, for example through feeding and digestion in its midgut. In this review we bring together current knowledge on the uptake mechanisms of dsRNA in insects and the potential of RNAi to affect pest insects. At least two pathways for dsRNA uptake in insects are described: the transmembrane channel-mediated uptake mechanism based on Caenorhabditis elegans’ SID-1 protein and an ‘alternative’ endocytosis-mediated uptake mechanism. In the second part of the review dsRNA feeding experiments on insects are brought together for the first time, highlighting the achievement of implementing RNAi in insect control with the first successful experiments in transgenic plants and the diversity of successfully tested insect orders/species and target genes. We conclude with points of discussion and concerns regarding further research on dsRNA uptake mechanisms and the promising application possibilities for RNAi in insect control.

Introduction

Nearly 10 years ago, Fire et al. (1998) described a process in which the application of exogenous dsRNA silenced the homolog endogenous mRNA in the worm Caenorhabditis elegans and called it RNA interference (RNAi). Although new for animals, the technique was already described as ‘post-transcriptional gene silencing’ in plants and as ‘quelling’ in fungi. Moreover, those three techniques appeared to be remarkably well conserved in several eukaryotes (Fire, 2007). RNAi soon proved to be very promising in several research fields: in genomics for gene function determination and gene knockdown in eukaryotes and in medicine to control cancers and viral disease. In biotechnology it shows great potential because of its high specificity and might therefore serve as a new specific method to control pests in agriculture (Borovsky, 2005, Gordon and Waterhouse, 2007, Price and Gatehouse, 2008). In this branch new techniques are very welcome, due to the continuous threat of resistance development against current insect control products and techniques. The economic importance of pest control is illustrated by the numbers spent on insecticides: 3000 million dollars for the protection of the top five most important agricultural crops in 2007 (Phillips McDougall, 2008).

This relatively new technique of RNAi is already regularly applied in the field of entomology to study the RNAi mechanism and the function, regulation and expression of gene cascades, mostly in Drosophila melanogaster (Roignant et al., 2003, Bischoff et al., 2006, Miller et al., 2008), Tribolium castaneum (Tomoyasu and Denell, 2004, Fujita et al., 2006, Arakane et al., 2008, Konopova and Jindra, 2008, Minakuchi et al., 2009, Parthasarathy and Palli, 2009) and Bombyx mori (Quan et al., 2002, Ohnishi et al., 2006, Hossain et al., 2008). However, most of these experiments have been conducted through experiments in which dsRNA is injected directly in the organism, which is not applicable to control insect pests in the field. For efficient insect control, the organism should be able to autonomously take up the dsRNA, for example through feeding and digestion in the gut. The insect midgut consists of a single layer of columnar cells with microvilli, endocrine cells, and stem cells at the base, grouped in the so-called nidi. The midgut is designed to absorb nutrients from the gut lumen with its large absorption area created by the microvilli, with many channels and endocytosis apparati (Lehane and Billingsley, 1996, Hakim et al., 2010). These characteristics make the tissue very interesting as a potential dsRNA uptake location.

In this review we bring together the current knowledge on the uptake mechanisms of dsRNA in insects and the potential of RNAi to control pest insects. Indeed, research in recent years gave new insights into the dsRNA uptake mechanisms in insects, with emphasis on uptake through the insect midgut and in body tissues; good examples are the transmembrane channel-mediated uptake and the ‘alternative’ endocytosis-mediated uptake. Because of the advanced state of research in C. elegans, much insight on the uptake mechanisms of dsRNA has already been gained, and more progression is possible as more insect genomes are becoming available. The second part of this review presents the dsRNA feeding experiments that have been conducted on insects so far. Here we highlight attempts to implement RNAi in the control of insects and also concerns regarding its application in the field.

Section snippets

Definitions of RNAi in insects

To avoid confusion when talking about different mechanisms involved in RNAi, clear definitions should be determined. We would like to adopt the definitions proposed by Whangbo and Hunter (2008) to discuss RNAi and the uptake mechanisms of dsRNA in insects. RNAi can be divided in cell-autonomous and non-cell-autonomous RNAi (Fig. 1). As the name suggests, in the case of cell-autonomous RNAi, the silencing process is limited to the cell in which the dsRNA is introduced/expressed and encompasses

The transmembrane channel-mediated uptake mechanism

A first approach to search for possible dsRNA uptake mechanisms in insects is to look for systems similar to those already described. In animals, the best studied dsRNA uptake mechanism is that of C. elegans. Research with systemic RNAi defective mutants (sid), resulted in the description of two proteins involved in non-cell-autonomous RNAi. SID-1 is a multispan transmembrane protein essential for systemic RNAi. It functions probably as a multimer, transporting dsRNA passively into the C.

SID-1

Table 1 presents an overview of sid-1 orthologs found in insects. Although orthologs are present in several insects, no conclusion can yet be drawn regarding the involvement of these gene orthologs in the dsRNA uptake due to conflicting results and lack of detailed research.

A sid-1 gene ortholog was found in the cotton aphid (Aphis gossypii). The online analysis of the topological structure showed large similarities with SID-1 of C. elegans, suggesting a possible role in the dsRNA uptake,

Differences in efficiency of RNAi between insects and C. elegans

Comparing the genes known to be involved in cell-autonomous and systemic RNAi between T. castaneum and D. melanogaster, it becomes clear that the inventory of core RNAi components is somewhat larger in T. castaneum than in D. melanogaster. This possibly explains the more sensitive response of the former to dsRNA compared to the latter (Tomoyasu et al., 2008). On the other hand, there is remarkably little similarity between C. elegans and T. castaneum RNAi gene inventories, although they both

Cell line experiments

Cell line experiments can give additional information specific for the target gene in a simplified model. They can also inform us on the possibility and effect of environmental RNAi when the dsRNA is applied through soaking of the cells in a dsRNA-enriched medium. This way the link between Cry1Ac insecticidal protein and an aminopeptidase N in the gut of larvae of the cotton bollworm (Helicoverpa armigera) (HaAPN1) was shown with RNAi cell line experiments. Sf21 cells were modified to express

Conclusions and future perspectives

All the presented data suggest that there are at least two pathways for dsRNA uptake in insects. One is based on the transmembrane SID-1 channel protein, as best known from C. elegans. Many sid orthologs have been found in insects, but their precise role in the uptake mechanism of dsRNA often remains to be determined. The second ‘alternative’ mechanism is possibly based on the endocytosis pathway because it shares several components of its machinery with the dsRNA uptake mechanism. Herein,

Acknowledgements

The authors acknowledge support by the FWO-Vlaanderen (Fund for Scientific Research-Flanders, Brussels) and the Special Research Fund of Ghent University.

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