RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection

Abstract

Mass sequencing of cDNA libraries from salivary glands of triatomines has resulted in the identification of many novel genes of unknown function. The aim of the present work was to develop a functional RNA interference (RNAi) technique for Rhodnius prolixus, which could be widely used for functional genomics studies in triatomine bugs. To this end, we investigated whether double-stranded RNA (dsRNA) can inhibit gene expression of R. prolixus salivary nitrophorin 2 (NP2) and what impact this might have on anticoagulant and apyrase activity in the saliva. dsRNA was introduced by two injections or by ingestion. RT-PCR of the salivary glands showed that injections of 15 microg of NP2 dsRNA in fourth-instar nymphs reduced gene expression by 75+/-14% and that feeding 1 microg/microL of NP2 dsRNA into second-instar nymphs (approx. 13 microg in total) reduced gene expression by 42+/-10%. Phenotype analysis showed that saliva of normal bugs prolonged plasma coagulation by about four-fold when compared to saliva of knockdown bugs. These results and the light color of the salivary gland content from some insects are consistent with the knockdown findings. The findings suggest that RNAi will prove a highly valuable functional genomics technique in triatomine bugs. The finding that feeding dsRNA can induce knockdown is novel for insects.

Primary culture of Rhodnius prolixus (Hemiptera: Reduviidae) salivary gland cells

“…Five minutes after feeding, salivary gland cells were highly active, as characterised by an increased size, resumption of secretion and protoplasmatic regeneration (Barth 1954). These findings are in agreement with our results, in which more cells were adherent to plates in Araujo et al (2006). Bars = mean ± standart error of 5 pools.…”

RNAi Trigger Delivery into <em>Anopheles gambiae</em> Pupae

“…Overall, the goal of this method is to provide researchers the ability to expand the timeframe during which reverse genetic analyses can be performed, further enabling research that will support the development of novel vector control strategies. Interestingly, experiments in other species, such as Rhodnius prolixus and Spodoptera frugiperda, reveal that gene silencing effects tend to be much greater when initiated during pre-adult stages 38,39 . During all stages of development, RNAi-mediated gene knockdown is subject to considerations regarding the rapidity and persistence of gene silencing, and the stability of proteins encoded by targeted genes.…”

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

“…Likewise, microinjection of 15 µg dsRNA achieved 75% silencing of nitrophorin 2 in Rhodnius prolixus, as compared to 42% knockdown when fed 13 µg dsRNA (Araujo et al, 2006).…”