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Plasmodium  sporozoites associated with the midgut and in the hemolymph of mosquitoes differ from sporozoites in the secretory cavities and ducts of the insects’ salivary glands in their transcriptome, proteome, motility, and infectivity. Using an  ex vivo Anopheles stephensi  salivary gland culture system incorporating simple microfluidics and transgenic  Plasmodium berghei  with the fluorescent protein gene  mCherry  under the transcriptional control of the  Pbuis4  promoter whose expression served as a proxy for parasite maturation, we observed rapid parasite maturation in the absence of salivary gland invasion. While  in vivo Pbuis4::mCherry  expression was only detectable in sporozoites within the salivary glands (mature parasites) as expected, the simple exposure of  P. berghei  sporozoites to dissected salivary glands led to rapid parasite maturation as indicated by mCherry expression. These results suggest that previous efforts to develop  ex vivo  and  in vitro  systems for investigating sporozoite interactions with mosquito salivary glands have likely been unsuccessful in part because the maturation of sporozoites leads to a loss in the ability to invade salivary glands.

An ex vivo system for investigation of Plasmodium berghei invasion of the salivary gland of Anopheles stephensi mosquitoes