Abstract
RNA interference (RNAi) technology uses dsRNAs to silence specific targeted genes by downregulating their expression. It has become a potent tool for functional and regulatory studies of insect genes and has potential to be applied for insect control. Though it has been challenging to generate effective RNAi in lepidopteran insects, in the current study this technology was applied to develop specific RNAi-based molecular tools that could be used to negatively impact the invasive lepidopteran forest pest, gypsy moth (GM). GM midgut-specific genes were selected for dsRNA design from larval transcriptome profiles. Two methods were used to produce specific dsRNAs, bacterial expression and in vitro synthesis, which were then fed per os to GM larvae. Depletion of uncharacterized gene targets known as locus 365 and locus 28365, or their stacked combination, depleted target transcripts in a sequence specific manner and resulted in 60% reduction in body mass. Treated GM females that were able to moult to the adult stage displayed an approximately two-fold reduction in egg masses. These have potential to be developed as molecular biopesticides for GM.
Highlights
Lymantria dispar, the gypsy moth (GM), is a serious insect pest of the North American forests where larvae feed on over 300 tree and shrub species, especially hardwood and shade trees
Systemic RNA interference (RNAi) transmitted through bacterial expression of dsRNA is commonly used in C. elegans (Timmons & Fire, 1998; Kamath et al, 2001)
RNA interference (RNAi) technologies used to silence specific targeted genes by degrading RNAs have great potential to be applied for insect control that is safe and effective
Summary
The gypsy moth (GM), is a serious insect pest of the North American forests where larvae feed on over 300 tree and shrub species, especially hardwood and shade trees. Measures to control the pest and reduce its spread have been implemented in the United States, including augmentative release of natural enemies, chemical pesticides, mating disruption using a chemical pheromone, and use of microbial biopesticides [nucleopolyhedrovirus or Bacillus thuringiensis (Bt)] (Höfte & Whiteley, 1989). These measures have been employed with variable success and resistance to biopesticides has evolved rapidly in larval populations. Fire and colleagues first observed the phenomenon of RNAi by injecting and feeding dsRNA in the nematode, Caenorhabditis elegans to deplete the unc-22 gene generating twitching phenotypes (Fire et al, 1998; Timmons & Fire, 1998). Clemens and colleagues, used RNAi in Drosophila S2 tissue cultures cells to inhibit expression of the DSOR1 (mitogenactivated protein kinase kinase, MAPKK) which prevented the activation of the downstream ERK-A (MAPK) in the insulin signal transduction pathway and vise versa (Clemens et al, 2000)
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