Functional genomics tool: gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

Abstract

Background: Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation.

Results: The genes encoding the putative Phospholipase A2 (PLA2), cytoplasmic Cystatin, Syntaxin-5, beta-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of beta-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA2, cytoplasmic Cystatin, Syntaxin-5, beta-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs.

Conclusions: Our study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks.

Figure 1

A) Schematic depicting in vivo assay system for gene silencing in tick nymphs and eggs. All RNAi experiments using candidate tick gene dsRNA followed the same procedure. B: A schematic transdermal delivery of dsRNA by electroporation in tick nymphs. A) Unfed nymphs trapped in dsRNA and, B) electric pulse discharged through electrodes. a) Arrow indicates the size of unfed nymph, b) schematics theoretical influx of labeled long dsRNA in the unfed nymphs and, c) partially feed nymphs after feeding on mouse.

Figure 2

Visualization of Cy3-labeled dsRNA electroporated into unfed I. scapularis nymphs: A) ventral view, unfed nymph electroporated with Cy3 dye (10×); B) dorsal view of nymphal tick electroporated with Cy3 dye after 24 hrs feeding on mouse (5×); C) dorsal view of nymphal tick electroporated with Cy3 dye after 36 hrs feeding on mouse (10×); D) Ventral view of nymphal tick electroporated with Cy3 labeled GAPDH control siRNA after 48 hrs feeding on mouse (10×); E) Ventral view of 48 hrs fed nymph electroporated with Cy3 labeled Actin dsRNA (10×); F) dorsal view of 48 hrs fed nymph electroporated with Cy3 labeled β-Actin dsRNA; and G) Cy3 labeled Actin dsRNA in the dissected tick salivary gland acini (20×). Arrows indicate the detection of electroporated dsRNA all over the nymphal ticks.

Figure 3

Visualization of Cy3-labeled dsRNA electroporated into I. scapularis eggs: A) eggs electroporated with Cy3 dye; B) eggs electroporated with Cy3 Actin dsRNA; C) eggs electroporated with Cy3-labeled Actin dsRNA after 2 weeks; D) eggs electroporated with labeled Cy3 GAPDH control siRNAs. Arrows indicate the presence of labeled siRNA or dsRNA in the tick eggs.

Figure 4

A) Gene silencing in tick eggs. Representative Ixodes scapularis eggs electroporated, 1) No treatement (3×) 2) water (3×), 3) dsRNA-gfp (3×), 4. dsRNA-LacZ (2×), 5-6) dsRNA-β-Actin (3× & 1×). Photographs were taken after 15 days of post-electroporation, B) Representative images taken as larvae started hatching from the electroprated eggs. 1 dsRNA-LacZ (1×), 2. dsRNA-gfp (1×), 3. dsRNA-Actin (1×).