3D imaging of undissected optically clearedAnopheles stephensimosquitoes infected withPlasmodiumparasites

Abstract

Malaria is a life-threatening disease, caused by Apicomplexan parasites of the Plasmodium genus. The Anopheles mosquito is necessary for the sexual replication of these parasites and for their transmission to vertebrate hosts, including humans. Imaging of the parasite within its insect vector has been attempted using multiple microscopy methods, yet most have been hampered by the presence of the mosquito's opaque cuticle, which scatters light. So far, imaging of the Plasmodium mosquito stages has depended on either sectioning or surgical dissection of important anatomical sites, such as the midgut and the salivary glands. Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) enable imaging fields of view in the centimeter scale whilst providing micrometer resolution. In this paper, we present reconstructions of the whole body of Plasmodium-infected, optically cleared Anopheles stephensi mosquitoes and midguts. The 3D-reconstructions from OPT imaging show detailed features of the mosquito anatomy relevant for transmission, and enable overall localization of the parasite within the vector throughout the entire mosquito life cycle. Additionally, LSFM imaging of mosquito midguts shows detailed distribution of oocysts in the midgut of intact mosquitoes.