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

Abstract

Background: 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). 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.

Results: Tick beta-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 beta-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.

Conclusion: Abdominal 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.

Figure 1

Visualization of Cy3 labeled β-Actin dsRNA in unfed adult female Ixodes scapularis ticks: Unfed female adult ticks were injected with Cy3 labeled Actin dsRNA and kept at 37°C overnight. Unfed ticks injected with labeled dsRNA were visualized under confocal microscope. A-B: Control ticks injected with Cy3 dye; C. Cy3 labeled Actin-dsRNA readily detectable in the unfed tick after injection. Arrow indicates the Cy3 labeled dsRNA; D) Labeled dsRNA diffused and degraded after 24 hrs feeding on host. Red-dots on the lower side of the tick may have been excreted labeled dsRNA. Arrow indicates the degradation of labeled dsRNA. E) Uptake of Cy3 labeled dsRNA in the dissected salivary glands.

Figure 2

In vivo visualization of Cy3 labeled β-Actin dsRNA: Labeled dsRNA was injected in the unfed adult female ticks and fed for 72 hrs on rabbit. 72 hrs fed ticks were dissected and synganglia were removed and visualized under confocal microscopy. A) light microscopic image of tick synganglia; B) Low infrared image showed a mesh of neurons; C-D) tick synganglia stained with DAPI showing neuronal nuclei, and superimposed image (arrows indicate nuclei labeling), E) Cy3 labeled dsRNA spread in the whole synganglia (10×) and F) 40× image (arrows indicate the spread of labeled dsRNA in the synganglia).

Figure 3

In vitro RNAi in tick synganglia. Adult female I. scapularis ticks were dissected and synganglia pooled in two groups and incubated with buffer alone or with (A) dsRNA-β-actin or (B) dsRNA Na⁺-K⁺-ATPase for 6 hrs at room temperature. Total RNA was extracted and cDNA produced. RT-PCR was conducted followed by gel electrophoresis. The I. scapularis Cyclophilin A or G was used as a normalizing factor (lanes 6–7). A) β-actin transcript level demonstrating the efficiency of RNAi in tick synganglia. (Lane 1, Low DNA Mass™ Ladder, 2–3: Mock and gene disrupted samples amplified with β-actin gene specific primers, 4–5: mock and gene silenced samples amplified with Na⁺-K⁺-ATPase gene specific primers, 6–7: mock and gene silenced amplified Cyclophilin A gene specific primers). B) Na⁺-K⁺-ATPase transcription demonstrated the efficiency of RNAi. (Lane 1, Low DNA Mass™ Ladder, 2–3: Mock and gene suppressed samples amplified with Na⁺-K⁺-ATPase gene specific primers, 4–5: mock and gene silenced samples amplified with β-actin gene specific primers, 6–7: mock and gene silenced samples amplified Cyclophilin G gene specific primers). Low DNA Mass™ ladder (100–2000 bp; 2000 bp, 1200 bp, 800 bp, 400 bp, 200 bp, 100 bp) was used from Invitrogen.

Figure 4

In vivo RNAi and expression of Dicer 1 like gene in tick synganglia. Unfed, adult female I. scapularis ticks were first micro-injected with either dsRNA-β-Actin or buffer or dsRNA Na⁺-K⁺-ATPase or irrelevant LacZ gene and then allowed to blood feed for 3–4 days on rabbits. Synganglia were dissected from partially fed mock-injected and gene silenced ticks and pooled into two groups. Total RNA was extracted and cDNA produced. RT-PCR was conducted followed by gel electrophoresis. A) β-Actin transcript levels demonstrating the efficiency of RNAi in tick synganglia, (Lane 1, AmpliSize Molecular Ruler, 2: buffer injected, 3: LacZ injected, 4: Actin-dsRNA injected, 5: Na⁺-K⁺-ATPase-dsRNA injected synganglia amplified with β-actin gene specific primers, B) Na⁺-K⁺-ATPase transcript levels demonstrating the effectiveness of RNAi in tick synganglia, (Lane 1, AmpliSize Molecular Ruler, 2: buffer injected, 3: LacZ injected, 4: Actin-dsRNA injected, 5: Na K ATPase-dsRNA injected synganglia amplified with Na+-K+-ATPase gene specific primers.

Figure 5

In vivo RNAi in tick salivary glands. Unfed, adult female I. scapularis ticks were first micro-injected with either A) dsRNA-β-Actin or buffer or B) dsRNA Na⁺-K⁺-ATPase or buffer and then allowed to blood feed for 3–4 days on rabbits. Salivary glands were dissected from partially fed mock-injected and knockout ticks and pooled into two groups. Total RNA was extracted and cDNA produced. RT-PCR was conducted followed by gel electrophoresis. A) β-Actin transcript levels demonstrating the efficiency of RNAi in tick salivary glands, (Lane 1, Low DNA Mass™ Ladder, 2–3: Mock and gene silenced samples amplified with β-actin gene specific primers, 4–5: mock and gene silenced samples amplified with Cyclophilin A gene specific primers), B) Na⁺-K⁺-ATPase transcript levels (Lane 1, Low DNA Mass™ Ladder, 2–3: Mock and gene silenced samples amplified with Na⁺-K⁺-ATPase gene specific primers, 4–5: mock and gene silenced samples amplified with cyclophilin G gene specific primers). Low DNA Mass™ ladder (100–2000 bp; 2000 bp, 1200 bp, 800 bp, 400 bp, 200 bp, 100 bp) was used from Invitrogen.

Figure 6

RNAi-mediated silencing of β-Actin expression in female adult I. scapularis affects the ability of ticks to feed and lay eggs. A) Ticks injected with buffer (mock), B) irrelevant LacZ dsRNA and C) β-Actin-dsRNA were allowed to oviposit. D) Engorged tick female adults (N = 45 and N = 45, respectively) were recovered and weighed.

Figure 7

RNAi-mediated suppression of Na⁺ K⁺ ATPase expression in adult female I. scapularis affects the ability of ticks to feed successfully and oviposit. A) Ticks injected with buffer (mock), B) irrelevant LacZ dsRNA, and C) β-Actin-dsRNA were allowed to oviposit. D) Engorged tick female adults (N = 45 and N = 45, respectively) were recovered and weighed.