Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA

Abstract

Reverse genetic studies based on RNA interference (RNAi) have revolutionized analysis of gene function in most insects. However the necessity of injecting double stranded RNA (dsRNA) inevitably compromises many investigations particularly those on immunity. Additionally, injection of tsetse flies often causes significant mortality. We demonstrate, at transcript and protein level, that delivering dsRNA in the bloodmeal to Glossina morsitans morsitans is as effective as injection in knockdown of the immunoresponsive midgut-expressed gene TsetseEP. However, feeding dsRNA fails to knockdown the fat body expressed transferrin gene, 2A192, previously shown to be silenced by dsRNA injection. Mortality rates of the dsRNA fed flies were significantly reduced compared to injected flies 14 days after treatment (Fed: 10.1%+/- 1.8%; injected: 37.9% +/- 3.6% (Mean +/- SEM)). This is the first demonstration in Diptera of gene knockdown by feeding and the first example of knockdown in a blood-sucking insect by including dsRNA in the bloodmeal.

RNAi in Agriculturally-Important Arthropods

“…The first report of oral RNAi in an insect was in a lepidopteran, the light brown apple moth, Epiphyas postvittana, with temporary knockdown of a gut target, carboxylesterase 1, and an adult antennae target, pheromone binding protein 1 (Turner et al, 2006). Since then, additional reports of ingested dsRNA leading to knockdowns in Lepidoptera (Bautista et al, 2009;Mao et al, 2007;Whyard et al, 2009), Coleoptera (Baum et al, 2007;Whyard et al, 2009) , Diptera (Walshe et al, 2009;Whyard et al, 2009), Hemiptera (Price & Gatehouse, 2008), and Isoptera (Zhou et al, 2008) , suggest that knockdown of specific targets may be feasible in most insects. 1 Values are the maximum observed mortality and are approximate (exact values were not given).…”

“…They discovered that reduction of transcripts encoding a type-C lectin, a leucine-rich protein or a serpin (SRPN2) were sufficient to make the mosquito refractory to infection by P. berghei in the midgut. Such studies may inspire the development of new methods to control protozoan infections in humans (Lehane et al, 2008;Solis et al, 2009;Walshe et al, 2009). …”

“…Some are capable of both up-take and systemic spread (Araujo et al, 2006;Dong & Friedrich, 2005;Saleh et al, 2006;Soares et al, 2005;Walshe et al, 2009), while others apparently readily take up dsRNA, but have significant difficulties with systemic distribution (Dietz et al, 2007;Roignant et al, 2003;Van Roessel et al, 2002). However, the inability of an organism to foster systemic effects of dsRNA could be due to shortcomings in methodology which may be mitigated by improved applications, such as extended hairpin RNA-based transgenic RNAi (Artymovich, 2009;Carthew, 2003;Kennerdell et al, 2002), as well as other improvements in RNAi protocols discussed in Section 4.…”

“…The first commercial use of RNAi in agriculture was demonstrated via oral RNAi in transgenic plants (in planta RNAi) toward several economically-important coleopteran pests, including the Western corn rootworm, Diabrotica virgifera virgifera and Colorado potato beetle, Leptinotarsa decemlineata (Baum et al, 2007). Oral RNAi has also been reported in Diptera, Hemiptera, Hymenoptera and Lepidoptera (Araujo et al, 2006;Aronstein et al, 2006;Lehane et al, 2008;Turner et al, 2006;Walshe et al, 2009). Our discussion will focus on the successful use of this strategy, as well as the challenges encountered by researchers seeking to use this sensitive, targeted approach to pest control.…”

Emerging strategies for RNA interference (RNAi) applications in insects

“…For example, for A. pisum, both salivary secretions and the hemolymph were able to degrade the dsRNAs; therefore, dsRNA-induced RNAi effects were not obtained through either ingestion or injection. 81 Oral delivery of RNAi inducers resulted in reduction of targeted mRNAs in several other agriculturally and medically important insects including Epiphyas postvittana, 82 Rhodnius prolixus, 64 Glossina morsitans morsitans, 83 Reticulitermes flavipes,…”