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A method for purification of Plasmodium oocysts from mosquito midguts.

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.

SUBMITTER: Siden-Kiamos I

PROVIDER: S-EPMC7190746 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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A method for purification of Plasmodium oocysts from mosquito midguts.

Siden-Kiamos Inga I Spanos Lefteris L Currà Chiara C

Scientific reports 20200429 1

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 understoo ...[more]

PMID: 32350329

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Project description:UNLABELLED: BACKGROUND:Oocysts of the malaria parasite form and develop in close proximity to the mosquito midgut basal lamina and it has been proposed that components of this structure play a crucial role in the development and maturation of oocysts that produce infective sporozoites. It is further suggested that oocysts incorporate basal lamina proteins into their capsule and that this provides them with a means to evade recognition by the mosquito's immune system. The site of production of basal lamina proteins in insects is controversial and it is still unclear whether haemocytes or midgut epithelial cells are the main source of components of the mosquito midgut basal lamina. Of the multiple molecules that compose the basal lamina, laminin is known to interact with a number of Plasmodium proteins. In this study, the localisation of mosquito laminin within the capsule and cytoplasm of Plasmodium berghei oocysts and in the midgut epithelial cells of Anopheles stephensi was investigated. RESULTS:An ultrastructural examination of midgut sections from infected and uninfected An. stephensi was performed. Post-embedded immunogold labelling demonstrated the presence of laminin within the mosquito basal lamina. Laminin was also detected on the outer surface of the oocyst capsule, incorporated within the capsule and associated with sporozoites forming within the oocysts. Laminin was also found within cells of the midgut epithelium, providing support for the hypothesis that these cells contribute towards the formation of the midgut basal lamina. CONCLUSION:We suggest that ookinetes may become coated in laminin as they pass through the midgut epithelium. Thereafter, laminin secreted by midgut epithelial cells and/or haemocytes, binds to the outer surface of the oocyst capsule and that some passes through and is incorporated into the developing oocysts. The localisation of laminin on sporozoites was unexpected and the importance of this observation is less clear.

Dataset Information

A method for purification of Plasmodium oocysts from mosquito midguts.

Publications

A method for purification of Plasmodium oocysts from mosquito midguts.

Similar Datasets

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