RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection

https://doi.org/10.1016/j.ibmb.2006.05.012Get rights and content

Abstract

Mass sequencing of cDNA libraries from salivary glands of triatomines has resulted in the identification of many novel genes of unknown function. The aim of the present work was to develop a functional RNA interference (RNAi) technique for Rhodnius prolixus, which could be widely used for functional genomics studies in triatomine bugs. To this end, we investigated whether double-stranded RNA (dsRNA) can inhibit gene expression of R. prolixus salivary nitrophorin 2 (NP2) and what impact this might have on anticoagulant and apyrase activity in the saliva. dsRNA was introduced by two injections or by ingestion. RT-PCR of the salivary glands showed that injections of 15 μg of NP2 dsRNA in fourth-instar nymphs reduced gene expression by 75±14% and that feeding 1 μg/μL of NP2 dsRNA into second-instar nymphs (approx. 13 μg in total) reduced gene expression by 42±10%. Phenotype analysis showed that saliva of normal bugs prolonged plasma coagulation by about four-fold when compared to saliva of knockdown bugs. These results and the light color of the salivary gland content from some insects are consistent with the knockdown findings. The findings suggest that RNAi will prove a highly valuable functional genomics technique in triatomine bugs. The finding that feeding dsRNA can induce knockdown is novel for insects.

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.

References (45)

  • M.F. Moreira et al.

    Changes in salivary nitrophorin profile during the life cycle of the blood-sucking bug Rhodnius prolixus

    Insect Biochem. Mol. Biol.

    (2003)
  • J.M. Ribeiro et al.

    Exploring the sialome of the blood-sucking bug Rhodnius prolixus

    Insect Biochem. Mol. Biol.

    (2004)
  • M.R. Sant’Anna et al.

    Feeding behaviour of morphologically similar Rhodnius species: influence of mechanical characteristics and salivary function

    J. Insect Physiol.

    (2001)
  • J. Sun et al.

    Characterization and cDNA cloning of a hemoprotein in the salivary glands of the blood-sucking insect, Rhodnius prolixus

    Insect Biochem. Mol. Biol.

    (1998)
  • P.D. Tracey-Patte et al.

    Boophilus microplus: passage of bovine immunoglobulins and albumin across the gut of cattle ticks feeding on normal or vaccinated cattle

    Res. Vet. Sci.

    (1987)
  • A. Vermehren et al.

    The nicotinic alpha subunit MARA1 is necessary for cholinergic evoked calcium transients in Manduca neurons

    Neurosci. Lett.

    (2001)
  • S. Ackerman et al.

    Passage of host serum components, including antibody, across the digestive tract of Dermacentor variabilis (Say)

    J. Parasitol.

    (1981)
  • E.S. Baginski et al.

    Microdetermination of inorganic phosphate, phospholipids, and total phosphate in biologic materials

    Clin. Chem.

    (1967)
  • D. Ben-Yakir

    Quantitative studies of host immunoglobulin G in the hemolymph of ticks (Acari)

    J. Med. Entomol.

    (1989)
  • S. Blandin et al.

    Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene

    EMBO Rep.

    (2002)
  • A. Fire et al.

    Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

    Nature

    (1998)
  • A.G. Fraser et al.

    Functional genomic analysis of C. elegans chromosome I by systematic RNA interference

    Nature

    (2000)
  • Cited by (219)

    • Transcript level is a key factor affecting RNAi efficiency

      2021, Pesticide Biochemistry and Physiology
    View all citing articles on Scopus
    View full text