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

ABSTRACT

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.

KEYWORDS:

• Plasmodium berghei
• sporozoite
• mosquito
• midgut sporozoites
• salivary gland sporozoites
• Anopheles stephensi
• ex vivo invasion

Acknowledgments

We gratefully acknowledge Dr Chris J. Janse at the Leiden University Medical Center from providing Plasmodium berghei PbANKA-Cherry-2204cl. This work was funded by [805618-1] Pb. infected mice for mosquito infection studies (R-15-78) project, Institute for Bioscience and Biotechnology Research, University of Maryland-College Park, Rockville, MD, United States. MIH was supported by The Culture Affairs and Mission Sector, Ministry of Higher Education and Scientific Research, Egypt.

Authors’ contributions

Mai I. Hussein performed the whole practical protocols, data analysis and wrote the manuscript; Belal A. Soliman revised the manuscript; Maha K. Tewfick provided direction, contributed in data analysis and reviewed the manuscript; David A. O’Brochta suggested the protocol idea, designed the experiments, supervised the practical work step by step and co-wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Ministry of Scientific Research, Egypt [Js-3623]; University of Maryland Foundation [805618-1].