Gene silencing in Phlebotomine sand flies: xanthine dehydrogenase knock down by dsRNA micro-injections

• Published in: Insect biochemistry and molecular biology Vol. 38; no. 6; pp. 652 - 660
• Main Authors: Sant’ Anna, Mauricio R V, Alexander, Bruce, Bates, Paul A, Dillon, Rod J
• Format: Journal Article
• Language: English
• Published: 04.04.2008
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2008.03.012

Lutzomyia longipalpis are vectors of medically important visceral leishmaniasis in South America. Bloodfed adult females digest large amounts of protein, and xanthine dehydrogenase is thought to be a key enzyme involved in protein catabolism through the production of urate. Large amounts of heme are also released during digestion with potentially damaging consequences, as heme can generate oxygen radicals that damage to lipids, proteins and nucleic acids. However, urate is an anti-oxidant that may prevent such oxidative damage produced by heme. We investigated xanthine dehydrogenase by developing the RNAi technique for sand flies and used this technique to knock down the Lu. longipalpis xanthine dehydrogenase gene to evaluate its role in survival of adult females after blood feeding. The gene sequence of Lu. longipalpis xanthine dehydrogenase is described together with expression in different life cycle stages and RNAi knock down. Semi quantitative RT-PCR of xanthine dehydrogenase expression showed a significant increase in expression after bloodmeal ingestion. Micro-injection of dsRNA via the thorax of 1 day old adult female sand flies resulted in approximately 40% reduction of xanthine dehydrogenase gene expression in comparison to flies injected with a control dsRNA. A significant reduction of urate in the whole body and excretions of Lu. longipalpis was observed after dsRNA xanthine dehydrogenase microinjection and feeding 96h later on rabbit blood. Sand flies injected with XDH dsRNA also exhibit significantly reduced life span in comparison with the mock-injected group when fed on sucrose or when rabbit blood fed, showing that urate could be indeed an important free radical scavenger in Lu. Longipalpis . The demonstration of xanthine dehydrogenase knock down by dsRNA microinjection, low mortality of micro-injected insects and the successful bloodfeeding of injected insects demonstrated the utility of RNAi as a tool for functional analysis of genes in phlebotomine sand flies.