Abstract
Malaria parasites have a complex life cycle comprising development in two hosts, the vertebrate and the vector mosquito. In the gut of the mosquito, the parasite develops into the oocyst, which is settled beneath the epithelium and attached to the basal lamina of the gut until the maturation of the cyst and its rupture concomitant with the release of the sporozoites, the infectious form of the parasite. The oocyst represents the longest stage of the parasite life cycle but it is poorly understood, mainly because of the difficulties to separate the oocysts from the mosquito midgut tissue but also the lack of a robust method to reproduce this stage in vitro. Here we describe a simple and reproducible protocol for purification of oocysts from mosquitoes. Midguts were dissected from infected mosquitoes and treated with trypsin which resulted in the degradation of the basal lamina and the release of the oocysts from the midgut tissue. The results obtained showed that the isolated oocysts were free of the mosquito protein E-cadherin. Purified oocysts were alive as judged by a strong GFP signal at least up to 2 h after treatment and furthermore sporozoites that had developed in the cyst were able to glide. Our new method will allow the study of the oocyst composition, formation and development in more details leading to advances in knowledge of this Plasmodium stage.
Highlights
Malaria, spread by Anopheles mosquitoes, is still the most deadly parasitic disease worldwide
Oocysts remain attached to the mosquito gut basal lamina during their development, a process taking about two weeks, and which leads to the formation of sporozoites, the infectious forms of the malaria parasite able to be transmitted to humans
Oocyst development is the longest stage of the Plasmodium life cycle and for this reason it is becoming considered an attractive target for new anti-malarial strategies
Summary
Spread by Anopheles mosquitoes, is still the most deadly parasitic disease worldwide. In cases where oocyst production is reduced or when there is an interest to study the early oocyst, a method for enrichment would allow structural analysis which is limited for practical reasons to parasite strains producing big and many oocysts. In this manuscript we describe a method for oocyst separation from the mosquito midgut tissue. Purified oocysts can be used as “clean” sample for western blot analysis This new method will allow the characterization of the oocyst composition, formation and development in more details leading to advances in knowledge of this Plasmodium stage
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call