Midgut Allocation

Understanding midgut epithelial regeneration during Plasmodium ookinete invasion in adult female Anopheles mosquito

Background: Malaria is caused by Plasmodium species and transmitted to humans by the bite of an infected female Anopheles mosquito. This infectious disease continues to be a tremendous public health burden, resulting in 627,000 deaths in 2012, causing substantial negative impact on the global socioeconomic development [1,2]. Prior to transmission of Plasmodium to the vertebrate host, the parasite has to undergo a series of obligatory developmental processes inside the mosquito vector. Male and female gametocytes undergo fertilization within the lumen of the midgut. The resulting ookinete breaches through the peritrophic matrix

This is essential as primary midgut culture generation requires large amounts of tissue, is short lived, difficult to propagate, and its utility to study mechanistic aspects is limited due to heterogeneity. Further, in mosquitoes, isolation, culture and establishment of a continuous cell line from differentiated midgut cells did not yield successful results earlier [25], this has been overcome by recent technological advances in virus-transduction systems, which will facilitate the generation of an immortal midgut cell line from primary cultures. Primary cultures will be established from 3-day old mosquito midgut by published methods [26,27]. Lenti-viral transduction system harbouring SV-40 large T antigen is the preferred choice because of its transduction efficiency and host genome integration properties. The commercially available ready-to-use viral stock will be transfected into HEK293 cells to obtain high titres and added to 70-80% confluent primary culture to achieve 100% transduction. Stable cell line generation will be evaluated, by a selection drug or reporter gene, after 48-72 hours incubation. After 10-15 days, clones will be picked for expansion. The cell line at this stage will contain all three cell types, ECs, EEs and stem cells. The mixed cell population culture will be serial diluted such that a single cell is seeded into each well. These single cells will be further propagated to obtain clonal lines, which will serve as parent stocks that will be aliquoted and stored in liquid nitrogen for further use. Parental stocks can be further split and subcultured. Homogenous EC, EE and stem cell lines will be identified by cell-specific markers (Pdm1, Ds or Spectrin for ECs; Prospero or Tachykinin for EEs; and Delta or notch for stem cells) using FACS analysis and further confirmed by RT-PCR analysis