ReviewMechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review
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.
References (64)
- et al.
Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum
Mechanisms of Development
(2008) - et al.
RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection
Insect Biochemistry and Molecular Biology
(2006) - et al.
RNA interference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin
Insect Biochemistry and Molecular Biology
(2009) - et al.
Gene silencing in mosquito salivary glands by RNAi
FEBS Letters
(2006) - et al.
Eater: a big bite into phagocytosis
Cell
(2005) - et al.
A chitinase structurally related to the glycoside hydrolase family 48 is indispensable for the hormonally induced diapause termination in a beetle
Biochemical and Biophysical Research Communications
(2006) - et al.
RNA interference with the allatoregulating neuropeptide genes from the fall armyworm Spodoptera frugiperda and its effects on the JH titer in the hemolymph
Journal of Insect Physiology
(2008) RNAi mechanisms in Caenorhabditis elegans
FEBS Letters
(2005)- et al.
Expression of 20-hydroxyecdysone-induced genes in the silkworm brain and their functional analysis in post-embryonic development
Insect Biochemistry and Molecular Biology
(2008) - et al.
Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila
Cell
(2005)
Silencing of acetylcholinesterase gene of Helicoverpa armigera by siRNA affects larval growth and its life cycle
Journal of Insect Physiology
RNA interference suggests sulfakinins as satiety effectors in the cricket Gryllus bimaculatus
Journal of Insect Physiology
Kruppel homolog 1, an early juvenile hormone-response gene downstream of Methoprene-tolerant, mediates its anti-metamorphic action in the red flour beetle Tribolium castaneum
Developmental Biology
A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions
Insect Biochemistry and Molecular Biology
RNAi-mediated crop protection against insects
Trends in Biotechnology
Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor
Journal of Biological Chemistry
Drosophila scavenger receptor Cl is a pattern recognition receptor for bacteria
Immunity
A water-specific aquaporin involved in aphid osmoregulation
Insect Biochemistry and Molecular Biology
On the role of RNA amplification in dsRNA-triggered gene silencing
Cell
Knockdown of aminopeptidase-N from Helicoverpa armigera larvae and in transfected Sf21 cells by RNA interference reveals its functional interaction with Bacillus thuringiensis insecticidal protein Cry1Ac
Journal of Biological Chemistry
Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells
Journal of Biological Chemistry
Environmental RNA interference
Trends in Genetics
RNA interference in the termite Reticulitermes flavipes through ingestion of double-stranded RNA
Insect Biochemistry and Molecular Biology
SID-1 is implicated in systemic gene silencing in the honey bee
Journal of Apicultural Research
Control of coleopteran insect pests through RNA interference
Nature Biotechnology
Down regulation of the Drosophila immune response by peptidoglycan-recognition proteins SC1 and SC2
PLoS Pathogens
Insect peptide hormones and RNA-mediated interference (RNAi): promising technologies for future plant protection
Phytoparasitica
The promises and pitfalls of RNA-interference-based therapeutics
Nature
Nymphal RNAi: systemic RNAi mediated gene knockdown in juvenile grasshopper
BMC Biotechnology
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans
Nature
Gene silencing by double-stranded RNA (Nobel lecture)
Angewandte Chemie-International Edition
RNAi for insect-proof plants
Nature Biotechnology
Cited by (737)
Knockdown of double-stranded RNases (dsRNases) enhances oral RNA interference (RNAi) in the corn leafhopper, Dalbulus maidis
2023, Pesticide Biochemistry and PhysiologyIdentification and functional characterisation of N-acetylglucosamine kinase from Helicoverpa armigera divulge its potential role in growth and development via UDP-GlcNAc salvage pathway
2023, International Journal of Biological MacromoleculesFunctional characterization of tyrosine melanin genes in the white-backed planthopper and utilization of a spray-based nanoparticle-wrapped dsRNA technique for pest control
2023, International Journal of Biological Macromolecules