Towards the elements of successful insect RNAi.

TL;DR: An enhanced conceptual understanding ofRNAi function in insects will facilitate the application of RNAi for dissection of gene function, and to fast-track the application to both control pests and develop effective methods to protect beneficial insects and non-insect arthropods from viral and parasitic diseases.

About: This article is published in Journal of Insect Physiology.The article was published on 2013-12-01 and is currently open access. It has received 396 citations till now. The article focuses on the topics: RNA interference.

Summary

1. Introduction

• RNA interference (RNAi) has transformed insect science research because it enables the researcher to suppress a gene of interest and thereby link a phenotype to gene function.
• All too often, the application of RNAi technology is an empirical exercise: ‘‘try it, for it might work’’.
• The article is divided into three sections.
• Importantly, there is no single protocol for the perfect RNAi experiment, partly because the efficacy of RNAi strategies varies among insect groups.
• In the third section, the authors turn to the application of RNAi for the management of pest and beneficial insects, and discuss the unique opportunities and challenges associated with each of these applications.

2. Mechanisms of RNAi

• RNAi refers to the suppression of gene expression by small noncoding RNA molecules, predominantly by the cleavage of a target mRNA in a sequence-specific manner (Fire et al., 1998), and the general steps involved in this process are shown in Fig.
• The experimental use of RNAi exploits the siRNA pathway, specifically the capacity of cells to degrade a single-stranded RNA (including mRNAs) with sequence identity to the administered dsRNA molecules.
• The difficulty is that the authors have little or no understanding of whether or how dsRNAs are amplified within insect cells or disseminated among insect cells.
• Analysis of gene orthology between C. elegans and insects has been a productive approach to identify the core RNAi machinery in insects, but far less informative for understanding the molecular basis of intracellular amplification and systemic spread of RNAi.
• An insect infected with an asymptomatic, persistent virus that codes for an RNAi suppressor would display limited responsiveness to experimental RNAi (Berry et al., 2009).

3. Designing a RNAi experiment

• As described above, RNAi application and efficacy remains variable between genes, organisms and life stages, despite the tremendous utility that RNAi presents for improving their understanding of fundamental biological questions and for pest control.
• In mosquitoes, most tissues can be reached by the injection of dsRNA, however the success of knockdown in the central nervous system varies highly between genes and may be dose-dependent (Lycett et al., 2006; Biessmann et al., 2010).
• Tissue differences in RNAi efficacy may be overcome by the design of new delivery methods, including transgenesis or viral transduction, which eliminate the requirement for cellular uptake of the RNAi trigger.
• Development of such technologies is lacking for the majority of species (Fig. 2 and Section 3.2 below).
• Often, these challenges can be mitigated by experimental factors including the design of the RNAi molecule, the mode of delivery and the dose of the dsRNA molecule.

3.1. The RNAi molecule

• Experiments should include an RNAi molecule against a heterologous sequence absent from the target insect’s genome (typically green fluorescent protein (GFP) or LacZ), to control for both the administration of the experimental dsRNA and the physiological impact of triggering the RNAi cascade.
• In some cases, a positive control can be incorporated into the experimental design.
• A crucial consideration is the choice of sequence for dsRNA preparation, especially its length and sequence identity to the target transcript of the insect.
• Comparisons among gene regions (e.g., 5’ end) to which RNAi molecules are designed have yielded variable results.

3.2. RNAi delivery

• The most widely used routes for administering RNAi to insects are injection into the hemolymph and feeding.
• Microinjection was used in the first successful application of RNAi in an insect, to obtain knockdown of frizzled in Drosophila melanogaster (Kennerdell and Carthew, 1998).
• The diluent may require adjustment to the particular osmotic pressure of the hemolymph.
• In addition, oral delivery of RNAi molecules in species where systemic RNAi cannot be achieved limits its application to genes expressed in gut cells (Fig. 2).

3.3. RNAi dosage

• The requisite dose of RNAi molecules varies with insect species, life stage, the target gene transcript abundance and its spatial and temporal expression profiles, and according to the delivery method of choice.
• The viscosity of high dsRNA concentrations limits the injectable concentrations to 6 lg ll 1 (K. Michel, unpub data), and the cost of synthesizing large amounts of dsRNA presents a challenge for high concentrations in artificial diets.
• The mode of uptake, ability to spread RNAi molecules and ability to process the RNAi molecules are other important considerations that no doubt strongly influences the requisite dose required to induce a RNAi response.
• In D. melanogaster larvae, cell autonomous RNAi can be induced readily by the expression of short hairpin RNAs from a transgene; however, injected dsRNAs fail to trigger RNAi in most tissues with the exception of hemocytes (Miller et al., 2008).
• Further research is required to establish the incidence and significance of inducibility in RNAi function.

3.4. Choice of gene: transcript abundance and protein stability

• In principle, the ideal gene target for RNAi produces an mRNA pool with high turnover that codes for a protein with a short half-life.
• The use of RNAi for phenotypic analysis of gene function in any life stage could be more difficult if the protein product of the target gene has a long half-life.
• Nicotinic acetylcholine receptors can be stable for >2 weeks (Lomazzo et al., 2011) and this protein stability may explain the weak phenotypic response associated with RNAi-mediated knockdown of Da6 (nicotinic acetylcholine receptor subunit) expression in both D. melanogaster and T. castaneum (Rinkevich and Scott, 2013).
• For the great majority of genes, mRNA turnover and protein half-life are not known.
• This gap in their knowledge presents a major challenge for RNAi experiments.

3.5. Evaluation of RNAi experiments

• The desired result of an RNAi experiment varies with the purpose of the study.
• For many analyses of gene function, physiological indices of predicted function should be central to the analysis.
• For some experiments, it may be necessary to reduce the RNAi dose to obtain a reliable physiological signal of gene function obtained by an intermediate expression knockdown, because strong knockdown could result in secondary, deleterious effects on insect fitness that obscure the primary lesion.
• The choice of reference or housekeeping genes for calculating relative transcript levels is challenging.
• The effect of RNAi on the protein may not be well-correlated to the level of transcript suppression.

4. Application of RNAi for the management of insect populations

• The potential of RNAi for the management of pest insects and protection of domesticated beneficial insects, especially the honey bee, is widely recognized (Xue et al., 2012).
• In other words, RNAi offers exquisite specificity and flexibility that cannot be matched by traditional chemical insecticides, biological control by natural enemies, or plants bearing protein-coding transgenes.

4.1. RNAi and the control of insect pests

• Proof of principle for the application of RNAi in insect crop pest control comes from early studies conducted on the western corn rootworm, Diabrotica virgifera virgifera (WCRW) (Baum et al., 2007), and cotton bollworm Helicoverpa armigera (CBW) (Mao et al., 2007).
• The studies of Baum et al. (2007) and Mao et al. (2007) illustrate two key issues for successful RNAi of insect crop pests: choice of the target sequence(s) for RNAi; and mode of delivery.
• These analyses can be conducted in silico, by comparing a 21–25 bp moving window along the candidate dsRNA sequence to both the target gene in all target insect taxa, and to all predicted protein-coding genes in all other publiclyavailable genomes.
• Because siRNA molecules can inhibit translation of transcripts with less than perfect sequence identity, the threshold for concern about non-target effects could be less than 100% sequence identity.
• Recent breakthroughs in dsRNA production methods, which can produce kilogram quantities, continues to reduce the cost associated with dsRNA production and makes it feasible to start discussing strategies which will apply dsRNA products as baits, sprays, or through irrigation systems (Hunter et al., 2010, 2012).

4.2. RNAi and the protection of insects against parasites and pathogens

• The susceptibility of many eukaryotic parasites to RNAi offers a novel strategy to enhance the health of beneficial insects.
• Nosema causes high morbidity and mortality of honey bees (Martin-Hernandez et al., 2011).
• Proof-of-principle successes have been achieved in the laboratory (Dong et al., 2011).
• Furthermore, viral suppression is promoted by enhancing the RNAi pathway, achieved by engineering the insects to express an inverted-repeat RNA that triggers production of dsRNA specific to the virus sequence (Franz et al., 2011; Mathur et al., 2010).
• Evidence that exogenous dsRNA can supplement the endogenous RNAi machinery comes from the demonstration that IAPV infection of honey bees can be eliminated by orally-delivered dsRNA corresponding to two different sequences of the IAPV genome (Maori et al., 2009).

4.3. The evolutionary stability of RNAi-based management of insect populations

• The relationship between viruses and RNAi-based insect immunity is evolutionarily dynamic.
• This is indicated by both the presence of viral suppressors of RNAi (see above) and the positive selection on the genes contributing to RNAi-machinery interacting with siRNAs, but not the endogenous miRNAs (Obbard et al., 2006).
• This implies that resistance to a dsRNA specific to one gene cannot be prevented by pyramiding multiple genes with different function, nor overcome by switching to a different gene or gene set.
• Furthermore, single nucleotide polymorphisms (SNPs) that result in lower effectiveness of the RNAi, could potentially be selected for and lead to the evolution of resistance.
• The long-term benefits of RNAi-based applications in insect pest management will require new and independent thought on effective resistance management strategies designed to minimize selective pressures and delay the evolution of resistance.

4.4. RNAi risks and regulation

• The above examples offer clear evidence for potential applications for RNAi for the control of insect pests, manipulation of insect disease vectors, and management of beneficial insects, together with concerns about the stability of RNAi strategies in the face of selection for resistance.
• Overlying these considerations is a very real uncertainty regarding the environmental and ecological risks posed by these technologies.
• The Federal regulatory framework for estimating the ecological risks associated with RNAi technologies is still in development, and a number of critical gaps remain including potential toxicity to non-target organisms (see Section 4.1), environmental fate, and importantly, the risk of resistance evolution in target pests (Section 4.3).
• Insecticide resistance presents a major challenge for the sustainable control of pests.
• Predictions that resistance could not develop to a new control strategy (e.g., Williams, 1967) have proven to be wrong time and time again.

5. Concluding comments

• A decade of research on RNAi in insects has demonstrated the great power of the technology for discovery-led science and potential for improved management of insect populations.
• As the science has matured, it has equally become evident that RNAi is no panacea, but introduces a range of new conceptual and technological challenges for insect scientists.
• Insects vary widely in their amenability to RNAi, and no single protocol is suitable for all species.
• The authors still have only a weak understanding of whether and how the RNAi signal is amplified in individual cells and disseminated between cells in insects.

Figures

Fig. 2. Current methodologies for RNAi delivery in insects: a guide on the performance of different delivery methods. All approaches yield transient RNAi other than the transgenic method. ? = lack of data prevents evaluation. RNAi efficacy in Drosophila is limited to hemocytes if delivered by injection (Miller et al., 2008). Delivery by feeding can be highly effective (Whyard et al., 2009), but transgenesis is the preferred methodology. In Aedes aegypti, RNAi has been most successful by injection (e.g. Campbell et al., 2008), transgenesis (e.g. Bian et al., 2005), and viral transduction (e.g. Adelman et al., 2001), with a few reported instances of success by feeding (Coy et al., 2012; Mysore et al., 2013) or topical application (Pridgeon et al., 2008). Most cases of successful RNAi in Tribolium castaneum have been by injection (e.g. Arakane et al., 2005). RNAi efficacy in Manduca sexta is most effective by injection for immune-related genes, and there is evidence to support successful gene suppression by delivery by feeding (Terenius et al., 2011). RNAi in A. pisum has been successful by both injection (Mutti et al., 2006) and feeding (Shakesby et al., 2009). RNAi in Apis mellifera is effective for a variety of genes and life stages both by injection and feeding (Amdam et al., 2003; Hunter et al., 2010). Even where a specific method of application has shown success for RNAi of certain genes, the effective suppression of all genes by that technique is not guaranteed.

Fig. 1. The RNAi process in insects. See text for additional details

Frequently asked questions

Q1. What is the threshold for concern about non-target effects?

Because siRNA molecules can inhibit translation of transcripts with less than perfect sequence identity, the threshold for concern about non-target effects could be less than 100% sequence identity. 

Q2. What have the authors contributed in "Towards the elements of successful insect rnai" ?

In this paper, the authors provide a roadmap for the application of RNAi for experimental analysis of gene function, management of pests and protection of beneficial arthropods. 

Q3. What have the authors stated for future works in "Towards the elements of successful insect rnai" ?

A priority for the future is for the insect research community to apply their persistence and ingenuity to solve the fundamentals of how insect RNAi works, in the context of the physiology of the insect body, and apply that to the pressing problems posed by pests and beneficial insects. 

Q4. What is the target gene for the study of Mao et al. (2007)?

In the study of Mao et al. (2007) on CBW, the target gene was a cytochrome P450, CYP6AE14, which is expressed in the larval midgut and detoxifies gossypol, a secondary metabolite common to cotton plants. 

Q5. What is the role of RdRP in RNAi in C. elegans?

Efficient amplification of RNAi by RdRP can drive the abundance of the target ssRNA molecule to undetectable levels, and RdRP is essential for RNAi in C. elegans (Sijen et al., 2001). 

Q6. What are the main factors that influence the RNAi response?

The mode of uptake, ability to spread RNAi molecules and ability to process the RNAi molecules are other important considerations that no doubt strongly influences the requisite dose required to induce a RNAi response. 

Q7. Why do some insects have low responsiveness to dsRNA?

Some insects or cell types may have low responsiveness to exogenously-applied dsRNA because they utilize alternative anti-viral defenses (e.g. apoptosis of infected cells, symbiont-mediated protection) (Merkling and van Rij, 2013). 

Q8. What are the main reasons for the reduction of RNAi in midgut?

The efficacy of RNAi of midgut transcripts may be reduced due to low or inconsistent doses taken up by individual insects, frequency and size of feeding, plus GI tract morphology and physiology will affect the actual dose of RNAi that reaches the midgut epithelium. 

Q9. What is the way to prevent RNAi from causing damage to the insect?

Eukaryotic parasites that exploit insect organs other than the gut would be susceptible to RNAi only where the insect host displays systemic spread of the RNAi signal. 

Q10. What is the commercial potential of dsRNA delivery?

The commercial potential of these methods depends critically on the ability to deliver dsRNA to the target insect, which is in part determined by stability of the dsRNA in the environment, its concentration in the baits and take-up rates by the insects, as offset against the production costs for dsRNA. 

Q11. What is the way to control for RNAi in insect species?

Experiments should include an RNAi molecule against a heterologous sequence absent from the target insect’s genome (typically green fluorescent protein (GFP) or LacZ), to control for both the administration of the experimental dsRNA and the physiological impact of triggering the RNAi cascade. 

Q12. What is the main reason for the use of microinjection in insects?

An important barrier to the use of microinjection in some insects is non-specific damage caused by mechanical damage, which is most often pronounced when targeting embryos. 

Q13. How can dsRNAs be induced in hemocytes?

In D. melanogaster larvae, cell autonomous RNAi can be induced readily by the expression of short hairpin RNAs from a transgene; however, injected dsRNAs fail to trigger RNAi in most tissues with the exception of hemocytes (Miller et al., 2008). 

Q14. What is the way to deliver RNAi triggers to plants?

in Lepidoptera, feeding as a mode of delivery necessitates the provision of high doses of RNAi trigger (Terenius et al., 2011). 

Q15. What is the importance of RNAi in honey bees?

Further research is required to establish the frequency and dose of RNAi applications required to sustain protection of colonies, and whether this approach offers a cost-effective strategy for the control of Varroa mite, which is of first-order importance in compromising the health of honey bee colonies. 

Q16. What is the minimum length required to obtain maximum biological activity?

Generally speaking, greater success with insect RNAi has been obtained with dsRNA molecules >50–200 bp in length (Huvenne and Smagghe, 2010), although the minimal length required to obtain maximal biological activity varies among insect species (Bolognesi et al., 2012). 

Q17. What is the way to study dsRNA?

It may be appropriate to obtain genomic or transcriptomic data for other non-target taxa that currently lack genomic resources, so that the in silico analysis of the proposed dsRNA sequences includes ecologically-relevant organisms.