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Gene silencing in phlebotomine sand flies:Xanthine dehydrogenase knock down by dsRNA microinjections

Sant'Anna, Mauricio R and Alexander, Bruce and Bates, Paul A and Dillon, Rod J (2008) Gene silencing in phlebotomine sand flies:Xanthine dehydrogenase knock down by dsRNA microinjections. Insect biochemistry and molecular biology, 38 (6). pp. 652-660. ISSN 0965-1748

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Abstract

Lutzomyia longipalpis are vectors of medically important visceral leishmaniasis in South America. Blood-fed 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 lipids, proteins and nucleic acids. However, urate is an antioxidant 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. Microinjection 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 microinjected 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.

Item Type: Article
Journal or Publication Title: Insect biochemistry and molecular biology
Uncontrolled Keywords: Lutzomyia longipalpis ; RNAi ; Urate ; Heme ; Xanthine dehydrogenase ; Oxidative stress
Subjects: Q Science > QR Microbiology > QR355 Virology
Departments: Faculty of Health and Medicine > Biomedical & Life Sciences
ID Code: 56023
Deposited By: ep_importer_pure
Deposited On: 17 Jul 2012 11:48
Refereed?: Yes
Published?: Published
Last Modified: 06 Aug 2012 09:19
Identification Number:
URI: http://eprints.lancs.ac.uk/id/eprint/56023

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