Confocal microscopy for analysis of dsRNA uptake in Lepidoptera midgut cells

Abstract: Confocal laser scanning microscopy (CLSM) is a powerful tool which has vast applications in biology. It is rapidly gaining acceptance as an important technology because of its capacity to produce images free of out-of-focus information which provides a better understanding of the cell structure. Using this technology we ambition to understand one of the limiting factors for RNAi efficiency in Lepidopteran insects. These include uptake of dsRNA molecules from the outside environment to the inside of the insects, specifically uptake into the target cells, intracellular transport and also stability of dsRNA inside the gut of the insect. In the present research, we investigated the use of CLSM as (1) a tool to study the uptake of fluorescently labeled dsRNA by insect cells, and (2) to visualize the different cell organelle structures of these cells for a better intracellular localization of dsRNA later on. Specifically, we used living cells of the midgut from an important pest lepidopteran, the spruce budworm Choristoneura fumiferana (CF203). Taken together, our results confirm that CLSM is an appropriate life cell-imaging tool to study uptake in insect midgut CF203 cells and that dsRNA is taken up by these cells.