Gene silencing in phlebotomine sand flies: Xanthine dehydrogenase knock down by dsRNA microinjections
Introduction
Phlebotomine sand flies are the vectors of Leishmania species, parasitic protozoans responsible for the leishmaniases, a group of diseases affecting human and various animal populations throughout much of the tropics and subtropics (Bates, 2007). Leishmaniases have several diverse clinical manifestations ranging from ulcerative skin lesions, destructive mucosal inflammation to the potentially fatal disseminated visceral infection (Bañuls et al., 2007). During the last 20 years, zoonotic visceral leishmaniasis due to Leishmania (Leishmania) infantum and transmitted by Lutzomyia longipalpis (Lutz & Neiva 1912) has become a serious public health problem in several Brazilian cities (Alexander et al., 2002; Michalsky et al., 2007).
The female sand fly ingests blood meals to supply protein for egg production. When feeding on vertebrate blood, haematophagous insects are adapted to overcome the pro-oxidant action of the large amounts of heme generated during digestion of haemoglobin inside their midguts (Paiva-Silva et al., 2006; Graça-Souza et al., 2006). Urate is the main end-product of nitrogen metabolism, including protein and purine break down (Glantzounis et al., 2005). It is released into the haemolymph and absorbed by the Malpighian tubules via a pH gradient to constitute a major component of the insect's urine (Souza et al., 1997). Xanthine dehydrogenase (XDH) is a key enzyme as it catalyses the oxidation of xanthine to uric acid.
Mosquito microarray experiments suggest that XDH mRNA levels increase upon blood feeding in Aedes aegypti (Sanders et al., 2003). XDH enzyme activities increase in blood-fed A. aegypti and maximal levels precede digestive protease activities (von Dungern and Briegel, 2001), with XDH and urate synthesis correlating directly with bloodmeal size. In insects, urate has been shown to have important antioxidant properties that prevent lipid peroxidation (Souza et al., 1997). In Drosophila melanogaster, urate-null mutants defective for XDH/XOD are more sensitive to paraquat, ionising radiation and hyperoxia, suggesting this gene plays a major role against oxidative stress in this insect species (Hilliker et al., 1992). Kim et al. (2001), also working with D. melanogaster rosy mutants, suggested that XDH plays an important role in the innate response to infection and that age-associated deterioration of the innate immune response may be associated to some extent with loss of XDH activity.
In organisms as diverse as nematodes, trypanosomes, plants, fungi, insects and humans, introduction of double-stranded RNA triggers the destruction of homologous mRNA, producing knock down of the corresponding protein (Hutvágner and Zamore, 2002; Tomari and Zamore, 2005). The technique of RNA interference has emerged as a powerful tool to study gene function in a wide variety of experimental systems, particularly in non-model organisms for which other methods of investigation are not available (Fraser et al., 2000; Gönczy et al., 2000). Gene silencing using dsRNA has been used to knock down a variety of genes in arthropods including Anopheles gambiae (Vlachou et al., 2005; Boisson et al., 2006) the honeybee Apis mellifera (Maleszka et al., 2007), Bemisia tabaci (Ghanim et al., 2007), D. melanogaster (Kennerdell and Carthew, 1998), Manduca sexta (Eleftherianos et al., 2006) and Ixodes scapularis (Narasimhan et al., 2004). Recently, Araújo et al. (2006) described a nitrophorin 2 knock down in a triatomine species (Rhodnius proxilus) by both dsRNA injection and ingestion, the latter proving to be a non-invasive way to deliver dsRNA into a biological organism. Jaubert-Possamai et al. (2007) described the development of RNAi by microinjection of dsRNA in the pea aphid Acyrthosiphon pisum, showing that specific gene knock down is feasible for small insects, even when minor volumes of dsRNA are injected. For Lu. longipalpis, a viral vector driven RNA silencing method was tested on cultured embryonic cells (Pitaluga et al., 2007). Silencing of a luciferase gene was achieved but unrelated dsRNAs also reduced expression of the gene.
The recent completion of a Lu. longipalpis mass sequencing EST programme (Dillon et al., 2006) and cDNA microarray analysis of blood-fed and Leishmania-infected Lu. longipalpis (unpublished data) provided an extensive list of differentially expressed sand fly genes. The development of a reverse genetics technique for phlebotomine sand flies is of crucial importance as a functional genomic tool to study genes and proteins that might have an effect on Leishmania development inside the gut of the sand fly.
The aim of this study was to develop the double-stranded RNA knock down approach for the diminutive Lu. longipalpis and to characterise the XDH gene that is thought to play a key role in bloodmeal digestion and response to oxidative stress.
Section snippets
Insects
A laboratory colony of Lu. longipalpis established from flies caught in Jacobina (Bahia, Brazil) and kept at the Liverpool School of Tropical Medicine was used in all experiments and maintained using standard methods (Modi, 1997). Insects were reared under controlled conditions of temperature (28±1 °C) and humidity (80–95%).
RNA extractions and RT-PCR
Total RNA was extracted from whole bodies of Lu. longipalpis using the RNaqueous 4-PCR Kit (Ambion®). RT-PCR was performed using the AccessQuick RT-PCR System (Promega®)
Lu. longipalpis XDH cDNA
The full-length LuloXDH cDNA sequence contains 4023 bp (EMBL Accession no. AM887864 and encodes a peptide of 1331 amino acids and a predicted mass of 146.4 kDa when putatively translated. A partial alignment of eight available XDH proteins of insect species and one aldehyde oxidase from A. aegypti shows that this enzyme is highly conserved within the regions thought to bind iron–sulfur (2Fe/2S), the NAD-binding domain conferring substrate specificity to XDH and molybdenum cofactor (MoCo) domain
Discussion
This study showed for the first time that specific gene knock down using RNAi is feasible for phlebotomine sand flies using an injected volume of 140 ng dsRNA per fly. Microinjection of dsRNA via the thorax of 1-day-old adult female sand flies resulted in approximately 40% reduction of XDH gene expression in comparison to flies injected with a control dsRNA. Examination of the expression profile post-injection suggested that expression of this gene was not reduced until 48 h after injection. This
Acknowledgements
The authors would like to thank Dr. Pedro L. Oliveira for helpful suggestions, Dr. Craig Wilding for helping with XDH phylogenetic analysis, Dr. Ray Wilson for his help during the Lu. longipalpis XDH cloning and sequencing and Dr. Matthew Rogers for helping with sand fly survival analysis. We gratefully acknowledge the technical assistance of Davina Moor and Jan Lewis. This work was funded by the Wellcome Trust.
References (43)
- et al.
RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection
Insect Biochem. Mol. Biol.
(2006) - et al.
Brasiliensin: a novel intestinal thrombin inhibitor from Triatoma brasiliensis (Hemiptera: Reduviidae) with an important role in blood intake
Int. J. Parasitol.
(2007) Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies
Int. J. Parasitol.
(2007)- et al.
Gene silencing in mosquito salivary glands by RNAi
FEBS Lett.
(2006) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding
Anal. Biochem.
(1976)- et al.
Analysis of ESTs from Lutzomyia longipalpis sand flies and their contribution toward understanding the insect–parasite relationship
Genomics
(2006) - et al.
Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference
Insect Biochem. Mol. Biol.
(2006) - et al.
Tissue-specific gene silencing by RNA interference in the whitefly Bemisia tabaci (Gennadius)
Insect Biochem. Mol. Biol.
(2007) - et al.
Adaptations against heme toxicity in blood-feeding arthropods
Insect Biochem. Mol. Biol.
(2006) - et al.
RNAi: nature abhors a double-strand
Curr. Opin. Genet. Dev.
(2002)