RNAi-mediated gene silencing in tick synganglia: a proof of concept study

Shahid Karim,Emily Troiano,Thomas N Mather,Bronwyn Kenny

doi:10.1186/1472-6750-8-30

Shahid Karim, Emily Troiano + Show 2 more

Open Access

https://doi.org/10.1186/1472-6750-8-30

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Abstract

BackgroundProgress in generating comprehensive EST libraries and genome sequencing is setting the stage for reverse genetic approaches to gene function studies in the blacklegged tick (Ixodes scapularis). However, proving that RNAi can work in nervous tissue has been problematic. Developing an ability to manipulate gene expression in the tick synganglia likely would accelerate understanding of tick neurobiology. Here, we assess gene silencing by RNA interference in the adult female black-legged tick synganglia.ResultsTick β-Actin and Na+-K+-ATPase were chosen as targets because both genes express in all tick tissues including synganglia. This allowed us to deliver dsRNA in the unfed adult female ticks and follow a) uptake of dsRNA and b) gene disruption in synganglia. In vitro assays demonstrated total disruption of both tick β-Actin and Na+-K+-ATPase in the synganglia, salivary glands and midguts. When dsRNA was microinjected in unfed adult female ticks, nearly all exhibited target gene disruption in the synganglia once ticks were partially blood fed.ConclusionAbdominal injection of dsRNA into unfed adult female ticks appears to silence target gene expression even in the tick synganglia. The ability of dsRNA to cross the blood-brain barrier in ticks suggests that RNAi should prove to be a useful method for dissecting function of synganglia genes expressing specific neuropeptides in order to better assess their role in tick biology.

Highlights

Progress in generating comprehensive EST libraries and genome sequencing is setting the stage for reverse genetic approaches to gene function studies in the blacklegged tick (Ixodes scapularis)
The fluorescence was detected in the tick synganglia at various intensity levels (Fig. 2) indicating that molecules of dsRNA injected into adult female ticks can be up-taken by the synganglia where they would be expected to disrupt target gene expression
The result suggests that depletion of gene expression by dsRNA correlated with localization of fluorescently labeled dsRNA in the synganglia

Summary

Introduction

Progress in generating comprehensive EST libraries and genome sequencing is setting the stage for reverse genetic approaches to gene function studies in the blacklegged tick (Ixodes scapularis). We assess gene silencing by RNA interference in the adult female black-legged tick synganglia. Sequencing of the I. scapularis genome, along with development of new investigative tools such as expressed sequence tags, microarrays, and RNA interference offer new alternatives for researching tick and tick-borne disease control [9,10,11,12,13,14]. One very promising method for generating targeted down-regulation of gene expression in a wide range of organisms is RNA interference (RNAi). Previous experiments showed the feasibility of using RNAi to abrogate expression of gene transcripts in salivary glands of the ticks, Amblyomma americanum and Ixodes scapularis [18,1214,18,15-19]. Hatta et al, [21] demonstrated the RNA interference of cystosolic leucine aminopeptidase in the salivary glands, and in the midgut, ovary and epidermis of Haemaphysalis longicornis

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