Visualisation of double-stranded RNA in the insect whole body

To guarantee the success of RNA-based biocontrols as bio-insecticides, the efficient delivery of the double-stranded RNA (dsRNA) to the intracellular RNAi machinery is critical. The gut epithelium constitutes the first cell layer encountered by the orally ingested dsRNA, and whether the spread from there is necessary for an efficient RNAi effect, is not yet known. Moreover, the delivery to the gut epithelium remains poorly understood in most insect species. To investigate these mechanisms, a diverse range of ADME-related techniques have been developed and utilized. Of these, fluorescent imaging techniques are particularly attractive to track dsRNA.

In this work, we used two fluorescence microscopy techniques to track dsRNA in situ throughout the juvenile body of key agronomical pests from the coleopteran and hemipteran orders. Confocal microscopy, on the one hand, allowed localization of the dsRNA in respect to different tissues and structures surrounding the epithelium cell layer. Light sheet microscopy, on the other hand, resulted in a more global 3D spatial distribution pattern of the dsRNA throughout the body, due to its ability to scan thicker tissues and even whole organisms. The detailed visualization of the active ingredient in the whole insect provides learnings about the early stages of RNAi-induced phenotypes in insect pests and aids the development of RNA-based pest management products.