**The title, authors, and abstract for this completion report are provided below.  For a copy of the completion report, please contact the author at Steve.Whyard@ad.umanitoba.ca or via telephone at 204-474-9418. Questions? Contact the GLFC via e-mail or via telephone at 734-662-3209**

Gene silencing technologies to control sea lampreys – A proof-of-concept

Steve Whyard and Margaret Docker

Department of Biological Sciences, University of Manitoba,

Winnipeg MB, Canada

September 2012

Abstract

Since its arrival into the upper Great Lakes of North America, the parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks. The Great Lakes Fishery Commission (GLFC) sea lamprey management program was initiated in the 1950s, and initially, sea lampreys were controlled by barriers to impede migration, traps to capture spawning phase adults, and the use of a selective lampricide (3-trifluoromethyl-4-nitrophenol, TFM) to kill the filter-feeding larvae. With growing concerns about the long-term use of this chemical and it possible effects on non-target species including native lamprey species, more species-specific methods of controlling sea lampreys are being sought.

RNA interference (RNAi) technologies are being developed to control a variety of invertebrate pests, and in this study, we examined the possibility of using RNAi to kill sea lamprey larvae, as a proof-of-concept to develop a sea lamprey-specific lampricide. RNAi is a method of gene silencing that is induced by the delivery of double-stranded RNA to an organism’s cells, which then results in the destruction of the target gene’s mRNA molecules. In this study, we delivered six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos and found that four of the siRNAs, targeting α-actinin, calmodulin, splicing factor, and γ–tubulin, induced a significant reduction in their respective mRNAs, and three of the siRNAs (α-actinin, calmodulin, and splicing factor) induced a higher level of mortality than the negative control siRNA that is specific for the bacterial gene, β-glucuronidase (gus). These findings represent the first demonstration of RNAi in lamprey embryos, and given the utility of this technology to suppress gene expression in many other model organisms, there is now considerable promise for RNAi to be used as a molecular genetics tool to study gene functions in lampreys.

Two different age classes of sea lamprey larvae (~10 weeks and 1 year olds) were exposed to formulations of the siRNAs incorporating a liposome transfection reagent and a yeast emulsion to encourage larval lamprey feeding. Two days after a 6 h exposure to the feeding formulation, the larvae were dissected and tissues examined for evidence of RNAi, using quantitative RT-PCR. Three of the siRNAs, targeting elongation factor, calmodulin, and α- actinin, reduced their respective genes’ transcript levels 2.5, 3.6, and 5.0–fold, respectively. This is not only the first demonstration of RNAi in lamprey larvae, but it is also the first example of delivery of siRNAs to a vertebrate through feeding formulations. The prospects for delivering siRNAs to other vertebrates are far-reaching, not just for control technologies, but also for the delivery of novel therapeutic siRNAs to other species.

Of the six siRNAs fed to lamprey larvae, the splicing factor-siRNA induced a significant increase in mortality of the 10 week old lamprey larvae, eight days post-treatment. Even though only a modest knockdown of this gene’s expression had been observed at day 2 post-treatment, there were lasting effects that resulted in the subsequent death of a significant percentage (48%) of the larvae. These results are remarkable in that the larvae only endured a relatively brief single exposure to the siRNAs, and it is possible that longer, more prolonged exposures, or more frequent exposures, could have greater impacts on survival.

Given that RNAi is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to different species. This study focused on only a few highly conserved genes to demonstrate that ingested siRNAs could kill sea lamprey larvae, but with thousands of genes to consider, there are undoubtedly many potential genes that could be targeted for a sea lamprey-specific siRNA-based lampricide. While there is still a considerable amount of work to be done on developing the best siRNAs and the most effective delivery method, this technology offers some exciting possibilities to control this serious pest in a species-specific manner.