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 the insect vector has been attempted using multiple microscopy methods, most of which are hampered by the presence of the light scattering opaque cuticle of the mosquito. So far, imaging of the Plasmodium mosquito stages 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 their midguts. The 3D-reconstructions from OPT imaging show detailed features of the mosquito anatomy and enable overall localization of parasites in midguts. Additionally, LSFM imaging of mosquito midguts shows detailed distribution of oocysts in extracted midguts.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Various diseases are transmitted by mosquitoes but their imaging is hindered by heavy light scattering. We present here 3D reconstructions of Plasmodium-infected, optically cleared mosquitoes, imaged using optical projection tomography and light sheet fluorescence microscopy. This manuscript has been revised to a) justify the decision to use BABB as a clearance method; b) include quantitative information in a new figure (Figure 2) showing quantifications of fluorescence loss, morphological modifications and time for clearance; c) include the results of cleared midguts, showing Plasmodium oocysts. Moreover, the manuscript was extensively reformatted to deliver the message more concisely, and clearly separate the result and discussion sections, which were together in the previous manuscript version.