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


CONCLUSIONS
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.
Karim
Shahid
S
Center for Vector-Borne Disease, University of Rhode Island, 9 East Alumni Ave, Kingston, RI 02881, USA. kshahid@uri.edu
Kenny
Bronwyn
B
Troiano
Emily
E
Mather
Thomas N
TN
D000199:Actins / Q000032:analysis / Q000235:genetics; D000818:Animals; D002490:Central Nervous System / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D007267:Injections; D007413:Intestinal Mucosa / Q000378:metabolism; D018884:Ixodes / Q000235:genetics*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000008:administration & dosage / Q000493:pharmacokinetics; D012469:Salivary Glands / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000032:analysis / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000235:genetics; D014158:Transcription, Genetic
D000199:Actins; D012330:RNA, Double-Stranded; D012471:Salivary Proteins and Peptides; D000254:Sodium-Potassium-Exchanging ATPase