https://biblio.ugent.be/publication/1101411/file/1101624.pdf

RNA interference in Lepidoptera: An overview of successful and unsuccessful studies and implications for experimental design

Ingested double-stranded RNAs can act as species-specific insecticides.

RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7) as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si) RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality) comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.

/pdf/characterizing-the-mechanism-of-action-of-double-stranded-1g9nsbkvjn.pdf

Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte).

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1366&context=entomologyfacpub

Towards the elements of successful insect RNAi.

Background: RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs. Methodology/Principal Findings: In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions. Conclusions/Significance: Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plantmediated RNAi approach for aphid control.

/pdf/silencing-of-aphid-genes-by-dsrna-feeding-from-plants-3gautocmjc.pdf

Silencing of Aphid Genes by dsRNA Feeding from Plants

Mosquito-borne diseases threaten over half the world’s human population, making the need for environmentally-safe mosquito population control tools critical. The sterile insect technique (SIT) is a biological control method that can reduce pest insect populations by releasing a large number of sterile males to compete with wild males for female mates to reduce the number of progeny produced. Typically, males are sterilized using radiation, but such methods can reduce their mating competitiveness. The method is also most effective if only males are produced, but this requires the development of effective sex-sorting methods. Recent efforts to use transgenic methods to produce sterile male mosquitoes have increased interest in using SIT to control some of our most serious disease vectors, but the release of genetically modified mosquitoes will undoubtedly encounter considerable delays as regulatory agencies deal with safety issues and public concerns. Testis genes in the dengue vector Aedes aegypti were identified using a suppression subtractive hybridization technique. Mosquito larvae were fed double-stranded RNAs (dsRNAs) that targeted both the testis genes and a female sex determination gene (doublesex) to induce RNA interference (RNAi) -mediated sterility and inhibition of female development. Fertility and mating competiveness of the treated males were assessed in small-scale mating competition experiments. Feeding mosquito larvae dsRNAs targeting testis genes produced adult males with greatly reduced fertility; several dsRNAs produced males that were highly effective in competing for mates. RNAi-mediated knockdown of the female-specific isoform of doublesex was also effective in producing a highly male-biased population of mosquitoes, thereby overcoming the need to sex-sort insects before release. The sequence-specific gene-silencing mechanism of this RNAi technology renders it adaptable for species-specific application across numerous insect species. We envisage its use for traditional large-scale reared releases of mosquitoes and other pest insects, although the technology might also have potential for field-based control of mosquitoes where eggs deposited into a spiked larval site lead to the release of new sterile males.

/pdf/silencing-the-buzz-a-new-approach-to-population-suppression-3req5zrngw.pdf

Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs

RNA interference has already proven itself to be a highly versatile molecular biology tool for understanding gene function in a limited number of insect species, but its widespread use in other species will be dependent on the development of easier methods of double-stranded RNA (dsRNA) delivery. This study demonstrates that RNA interference can be induced in the mosquito Aedes aegypti L. (Diptera: Culicidae) simply by soaking larvae in a solution of dsRNA for two hours. The mRNA transcripts for β-tubulin, chitin synthase-1 and -2, and heat shock protein 83 were reduced between 30 and 50% three days post-dsRNA treatment. The dsRNA was mixed with a visible dye to identify those individuals that fed on the dsRNA, and based on an absence of RNA interference in those individuals that contained no dye within their guts, the primary route of entry of dsRNA is likely through the gut epithelium. RNA interference was systemic in the insects, inducing measurable knock down of gene expression in tissues beyond the gut. Silencing of the β-tubulin and chitin synthase-1 genes resulted in reduced growth and/or mortality of the larvae, demonstrating the utility of dsRNA as a potential mosquito larvicide. Silencing of chitin synthase-2 did not induce mortality in the larvae, and silencing of heat shock protein 83 only induced mortality in the insects if they were subsequently subjected to a heat stress. Drosophila melanogaster Meigen (Diptera: Drosophilidae) larvae were also soaked in dsRNA designed to specifically target either their own β-tubulin gene, or that of A. aegypti, and significant mortality was only seen in larvae treated with dsRNA targeting their own gene, which suggests that dsRNA pesticides could be designed to be species-limited.

/pdf/oral-delivery-of-double-stranded-rna-in-larvae-of-the-yellow-38p4hlyrc6.pdf

Oral Delivery of Double-Stranded RNA in Larvae of the Yellow Fever Mosquito, Aedes aegypti: Implications for Pest Mosquito Control

With twelve Drosophila genomes now sequenced, there is a growing need to develop higher-throughput methods for identifying the functions of the many newly identified genes. Genetic transformation and RNA interference are two technologies that have been used extensively to facilitate gene-function studies in Drosophila melanogaster, to introduce genes or block the expression of endogenous genes, respectively. Both of these technologies typically require the delivery of nucleic acids into developing insect embryos, and virtually all studies to date have relied on microinjection as the DNA delivery method of choice. In this study, we describe the use of biolistics as a higher-throughput method of nucleic acid delivery. By bombarding dechorionated D. melanogaster embryos with 1 microm gold beads coated with P-element or piggyBac transformation vectors, we observed transformation frequencies (3-4%) that are comparable to those achieved using microinjection methods, but in only a fraction of the time required for the DNA delivery. Biolistic delivery of double-stranded RNA (dsRNA) specific to a beta-glucuronidase (gus) transgene resulted in a significant (71%) reduction in gus transcripts in embryos and the RNA interference (RNAi) persisted through two successive larval molts, albeit at reduced levels. DsRNAs specific to four essential genes were delivered to embryos and resulted in arrested development and phenotypes that closely match that of null mutations. These results suggest that biolistic delivery of dsRNA into embryos could be adapted for high throughput RNAi screens of early Drosophila developmental genes.

Aditi D. Singh

Papers

Ingested double-stranded RNAs can act as species-specific insecticides.

Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs

Oral Delivery of Double-Stranded RNA in Larvae of the Yellow Fever Mosquito, Aedes aegypti: Implications for Pest Mosquito Control

Biolistics for high-throughput transformation and RNA interference in Drosophila melanogaster.