RNA-interference methods for gene-knockdown in the sea louse, Lepeophtheirus salmonis: studies on a putative prostaglandin E synthase.
Harnessing the full utility of extensive gene sequences recently available for the economically important sea louse, Lepeophtheirus salmonis, requires the adaptation of modern molecular biology approaches to this non-model organism. Using a putative microsomal prostaglandin E synthase type-2 (PGES2) as a candidate gene, we investigated gene-knockdown by double-stranded RNA interference (dsRNAi) in the small free-living and the larger parasitic stages of L. salmonis. dsRNA was administered to nauplius and copepodid stages by immersion for 7 h. Pre-adult and adults received dsRNA by intra-haemocoelic injection. The extent, speed and persistence of the knockdown effects were determined by RT-PCR. LsPGES2 was abundantly expressed in all life stages, including the non-parasitic stages. Administration of dsRNA to nauplius and copepodids by immersion had no effect on mortality rates and moulting through to copepodids was observed. Dramatic knockdown of LsPGES2 was observed within 7 h and persisted for at least 48 h. Injection of dsRNA had no effect on mortality in pre-adults and adults, but knockdown of LsPGES2 was apparent within 24 h, reaching 95% over the 72 h and was persistent for at least 120 h. The methods developed resulted in rapid and persistent knockdown in L. salmonis suitable for studies in the different stadia.
Campbell
E M
EM
School of Biological Sciences (Zoology), University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK.
Pert
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CC
Bowman
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AS
D000818:Animals; D033342:Copepoda / Q000235:genetics*; D005260:Female; D055785:Gene Knockdown Techniques / Q000379:methods*; D050259:Genes, Mitochondrial / Q000235:genetics; D007267:Injections; D019746:Intramolecular Oxidoreductases / Q000235:genetics*; D000071876:Prostaglandin-E Synthases; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D020317:Salmo salar / Q000469:parasitology
D012330:RNA, Double-Stranded; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases