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

Abstract

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene 98 function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) 99 RNAi has many times proven to be difficult to achieve. Most of the negative results have been 100 anecdotal and the positive experiments have not been collected in such a way that they are 101 possible to analyze. In this review, we have collected detailed data from more than 150 102 experiments including all to date published and many unpublished experiments. Despite a 103 large variation in the data, trends that are found are that RNAi is particularly successful in the 104 family Saturniidae and in genes involved in immunity. On the contrary, gene expression in 105 epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding 106 dsRNA requires high concentrations for success. Possible causes for the variability of success 107 in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further 108 investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the 109 innate immune response. Our general understanding of RNAi in Lepidoptera will be further 110 aided in the future as our public database at http://insectacentral.org/RNAi will continue to 111 gather information on RNAi experiments.

Use of chromatin remodeling ATPases as RNAi targets for parental control of western corn rootworm (Diabrotica virgifera virgifera) and Neotropical brown stink bug (Euschistus heros)

“…In other orders such as Diptera, generally higher doses are needed to achieve an effective RNAi response. For example, in the African malaria mosquito, Anopheles gambiae, doses on the order of 1 μg/g to 10 μg/g are necessary to achieve an effective RNAi response (Boisson et al, 2006); and Lepidoptera needs a vast range of concentrations that span from <0.01 μg/g to > 1 μg/g depending on the species (Terenius et al, 2011). Drosophila lack systemic RNAi (Roignant et al, 2003), and therefore are not as sensitive to injection or oral administration of dsRNAs (e.g., most RNAi assays in D. melanogaster are performed with transgenic flies designed to express hairpin RNAs (Dietzl et al, 2007)).…”

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

“…Lepidoptera (including H. armigera) (Bellés, 2010). This disparity in terms of RNAi sensitivity has partly been attributed to the presence or lack of certain cellular import and export mechanisms for dsRNA, as well as RNAi signal amplification among insect orders (Bellés, 2010;Gatehouse and Price, 2011;Gu and Knipple, 2013;Terenius et al, 2011).…”

Parental RNA interference of genes involved in embryonic development of the western corn rootworm, Diabrotica virgifera virgifera LeConte

“…This pathway has been implicated as a mechanism of defense against invasive nucleic acids from viruses or from mobile genetic elements, and has been conclusively shown to regulate gene expression in virtually all eukaryotic organisms (Fire, 2007;Hussain et al, 2010;Huvenne and Smagghe, 2010;Scott et al, 2013;Terenius et al, 2011).…”

“…In insects, the effectiveness of RNAi has been confirmed in a number of species but varies across different taxa and among different tissues (Burand and Hunter, 2013;Terenius et al, 2011). Most of the studies with insects have involved injection of long dsRNA directly in the insect hemocoel to achieve silencing which has become a routine method for assessing gene function.…”

“…Most of the studies with insects have involved injection of long dsRNA directly in the insect hemocoel to achieve silencing which has become a routine method for assessing gene function. While injection of dsRNA for functional genomics studies has been successful in a variety of insects, uptake of dsRNA from the gut environment through oral exposure to dsRNA and subsequent downregulation of essential genes is required in order for RNAi to be Terenius et al, 2011). Systemic RNAi through oral administration effective as a pest management tool (Auer and Frederick, 2009 has been documented in a number of different species representing seven different orders (Huvenne and Smagghe, 2010).…”