RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection
Introduction
Introduction of double-standed DNA (dsRNA) induces gene-specific silencing in living organisms producing a knockdown of the corresponding protein (Hammond et al., 2001). As a consequence, RNA interference (RNAi) mediated by dsRNA has emerged as one of the most promising techniques to study gene function in diverse experimental systems, particularly non-model organisms where other methods of investigation are often very limited (Fraser et al., 2000; Gonczy et al., 2000). Most of this success derives from the simple, convenient and inexpensive methods for producing and introducing dsRNA into organisms. The phenomenon was first clearly demonstrated in animals in the nematode Caenorhabditis elegans where knockdown can be achieved by injection of dsRNA, by feeding bacteria expressing the dsRNA to the worm or simply by feeding dsRNA directly to the worm (Fire et al., 1998). RNAi has subsequently been adapted for use in insects, including Anopheles gambiae, Drosophila melanogaster, Manduca sexta, Periplaneta americana, Oncopeltus fasciatus (Blandin et al., 2002; Hughes and Kaufman, 2000; Kennerdell and Carthew, 1998; Marie et al., 2000; St. Johnston, 2002; Vermehren et al., 2001; Zhou et al., 2002). The technique was also used for reducing salivary gene expression in the ticks Ixodes scapularis (Narasimhan et al., 2004) and Amblyomma americanum (Karim et al., 2004).
Triatomine bugs are the vectors of Chagas disease. They take very large blood meals which can take up to 15 min to ingest and so the bugs are likely to have extensive anti-hemostatic mechanisms. Discovery programs on triatome salivary proteins have been undertaken (Ribeiro and Francischetti, 2003). Among them, mass sequencing of cDNA libraries of Rhodnius prolixus have identified several novel genes with unknown functions (Ribeiro et al., 2004). A functional RNAi tool for triatomine bugs would provide a potentially powerful means of investigating the function of the many uncharacterized molecules discovered. In this paper, we describe the development of such a tool using nitrophorins (NPs) as our subject matter. NPs are among the most remarkable proteins in R. prolixus saliva. They are salivary hemeproteins with multifunctional activities presenting a reddish color because of the presence of the heme group in the molecule (Ribeiro et al., 1993; Champagne et al., 1995). Four of them, named NP1–NP4, store and transport nitric oxide (NO), which when released in tissues induces vasodilatation and reduced platelet aggregation (Champagne et al., 1995). Recently, two other NPs were described (NP5 and NP6), but anticoagulant activity has been associated only with NP2 (Moreira et al., 2003). To demonstrate that RNAi, achieved by injection or ingestion of dsRNA, can be a functional genomic study tool for triatomine bugs and to further characterize salivary bioactive molecules, we have investigated R. prolixus salivary nitrophorin 2 (NP2) and its impact on anticoagulant and apyrase activity in saliva.
Section snippets
Insects
The colony of R. prolixus was reared under controlled conditions of temperature (26±2.0 °C) and humidity (65±5.0%) and the insects fed on chickens or rats weekly. The specimens selected for use in the experiments were standardized as 7 days after the last molt and for weight (1.8±0.4 mg for second and 20±2.5 mg for fourth-instar nymphs).
RNA extractions and RT-PCR
Total RNA was extracted from bug salivary glands using the RNeasy® Micro Kit (Qiagen, USA). Synthesis of cDNA was performed using the M-MLV reverse transcriptase
Expression of NPs
RT-PCR from R. prolixus eggs amplified only NP2 transcripts. Except for NP3 expression that could not be detected in unfed first instars, RT-PCR detected transcripts for NP1-4 in both fed and unfed first, second, third, fourth and fifth instars. The 390 bp fragment of the constitutive gene UGALT was also amplified in eggs and all fed and unfed instars and was used as housekeeping control in the experiments.
Effect of dsRNA resuspended in saline or water for injection
RT-PCR results from three replicates (pools of three pairs of glands each) showed no
Discussion
NPs regulate the levels of NO which, when released in the feeding wound, induces vasodilatation and reduced platelet aggregation (Ribeiro et al., 1993; Champagne et al., 1995). Anticoagulant activity has been associated only with NP2 (Moreira et al., 2003). The above results support these findings, showing that introduction of NP2 dsRNA significantly reduces the anticoagulant activity of R. prolixus saliva and that this reduction can be achieved by injection or ingestion of the dsRNA. Although
Acknowledgments
This work was supported by a grant from the Wellcome Trust (UK) and by the Brazilian research agencies FAPEMIG, CNPq and CAPES.
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