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

Sant'Anna, Mauricio R.V., Alexander, Bruce, Bates, Paul and Dillon, Rod (2008) 'Gene silencing in phlebotomine sand flies: Xanthine dehydrogenase knock down by dsRNA microinjections'. Insect Biochemistry and Molecular Biology, Vol 38, Issue 6, pp. 652-660.

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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 96 h 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. (c) 2008 Elsevier Ltd. All rights reserved.

Item Type: Article
Additional Information: This work was funded by the Wellcome Trust.
Uncontrolled Keywords: lutzomyia longipalpis rnai urate heme xanthine dehydrogenase oxidative stress functional genomic analysis rna interference lutzomyia-longipalpis anopheles-gambiae drosophila-melanogaster bloodsucking insect oxidative stress blood meal leishmaniasis disruption
Subjects: QX Parasitology > Insects. Other Parasites > QX 505 Diptera
QU Biochemistry > Genetics > QU 475 Genetic processes
WC Communicable Diseases > Tropical and Parasitic Diseases > WC 715 Visceral leishmaniasis
QU Biochemistry > Cells and Genetics > QU 375 Cell physiology
Faculty: Department: Groups (2002 - 2012) > Molecular & Biochemical Parasitology Group
Groups (2002 - 2012) > Vector Group
Digital Object Identifer (DOI): https://doi.org/10.1016/j.ibmb.2008.03.012
Depositing User: Mary Creegan
Date Deposited: 19 Jul 2010 11:16
Last Modified: 06 Feb 2018 13:00
URI: http://archive.lstmed.ac.uk/id/eprint/911

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