3D imaging of undissected optically cleared Anopheles stephensi mosquitoes and midguts infected with Plasmodium parasites

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, most 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 compare different optical clearing protocols and 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. This work was submitted as a pre-print to bioRxiv , available at https://www.biorxiv.org/content/10.1101/682054v2 .

Shared genetic etiology between cortical brain morphology and tobacco, alcohol, and cannabis use

“…Individuals who have greater genetic liability for using cannabis may use cannabis more frequently, which has been associated with cortical alterations. 40,41 For example, work by Chye et al 12,41 indicated that adolescents who used cannabis showed reduced thickness in the bilateral insula, a brain region that has been closely linked to addictive behaviors, including craving and drug-seeking, interoceptive processing, response to reward, and impulsive decision making. 42,43 Other work has shown that individuals who initiate cannabis use had a smaller insula surface area in the right hemisphere.…”

Autofluorescent Biomolecules in Diptera: From Structure to Metabolism and Behavior

“…This approach applied to chemically-cleared Drosophila adult male individuals allowed to just use the AF of the sample to image both surface (e.g., hexagons of the lens facets, bristles on inter-ommatidial, maxillary palp and distal labellum) and internal (e.g., elements of the compound eye, the optic lobe, cardia) structures, to be made then observable with a highly sharp resolution in a 3D virtual reconstruction [165]. The 3D imaging analysis has been thus used as a technique additional to conventional optical or scanning electron microscopy, and its ability to provide variable views of the target organism has stimulated both new investigations to improve sample preparations, or applications to improve the detection of parasites (e.g., Plasmodium in infected A. stephensi) [36,166,167]. Sample clearing with KOH has been thus recommended as the preferred preparation procedure to obtain specimens suitable to be preserved for long time and thus for reexaminations by confocal microscopy, favoring sample exchange among scientists and comparative and taxonomic studies.…”

“…In this context, efforts have been devoted to circumvent the problem. Different optical clearing methods and microscope imaging procedures have been compared, in order to improve the localization of Plasmodium parasites in the midgut of Anopheles vectors [ 36 ]. On the other hand, Koga and colleagues have developed a protocol based on the use of hydrogen peroxide able to strongly reduce AF in insect tissues and allow a reliable FISH detection of endosymbionts of aphids, lice and bat flies [ 37 ].…”

“…On the other hand, Koga and colleagues have developed a protocol based on the use of hydrogen peroxide able to strongly reduce AF in insect tissues and allow a reliable FISH detection of endosymbionts of aphids, lice and bat flies [ 37 ]. Anyway, several studies performed in insects on the development of transgenic individuals through germline transformation and consequent expression of fluorescent protein markers (e.g., green and red fluorescent proteins) have reported that AF signals did not impair the detection of the experimentally-induced fluorescence [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ]. It is also worth to recall the particular case of the AF rising from the yolk granules and the vitelline membrane of dechorionated D. melanogaster embryos, which, together with the embryo thickness, size, and opacity, seemed to affect the application of optical fluorescence microscopy in the study of this developmental stage.…”

“…In this context, efforts have been devoted to circumvent the problem. Different optical clearing methods and microscope imaging procedures have been compared, in order to improve the localization of Plasmodium parasites in the midgut of Anopheles vectors [36].…”