Steven Whyard

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

Abstract 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 &bgr;-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 &bgr;-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 &bgr;-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.

Delivery of DNA to early embryos of the Kuruma prawn, Penaeus japonicus

We examined the relative efficiency of microinjection, electroporation and particle bombardment for introducing DNA into the embryos of the Kuruma prawn, Penaeus japonicus. The amount of DNA that could be delivered into one- to four-celled embryos and subsequently recovered was examined using plasmids with selectable antibiotic resistance. Microinjection proved to be the most reliable technique. The mean value for plasmid recovery from injected embryos ranged from 225 to 243 plasmids per embryo. Electroporation was effective in delivering small, but measurable amounts of DNA into embryos with values ranging from 0.5 to 0.9 plasmids per embryo. Bombardment failed to introduce any significant amount of DNA into viable embryos.

RNA interference technology to control pest sea lampreys - A proof-of-concept

The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0–fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

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

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 µm 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 β-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.

Preventing Genetic Pollution and the Establishment of Feral Populations: A Molecular Solution

Aquaculture animals that escape from farms have the potential to create major environmental problems. These include establishment of potentially destructive feral populations (e.g., Pacific oysters [Crassostrea gigas] in Australia, Atlantic salmon [Salmo salar] in British Columbia) and genetic contamination of wild stocks. The latter includes introgression of foreign genes into natural populations from both hatchery-reared fish and genetically modified fish and invertebrates. Concern about these environmental and genetic effects has already led to restrictions on aquaculture industry development and is likely to grow as demand for genetically improved stocks escalates to fulfill production objectives. To circumvent these problems, we have developed a genetic construct that, when properly integrated into production-line fish or invertebrates, should render individuals functionally sterile outside of hatchery conditions. In the hatchery, however, provision of a simple repressor compound at a particular life-history stage allows the animals to be bred and reared as normal. We Theresa M. Bert (ed.), Ecological and Genetic Implications of Aquaculture Activities, 103–114. 2007 Springer. 103 are developing this ‘‘Sterile Feral’’ technology for both invertebrate and fish species, and we anticipate practical commercial application within a few years.