The role of stem-cell mediated midgut repair in the dynamics of mosquito infections

Project Summary
Vector-borne diseases account for more than 17% of all infectious disease and cause more than 700,000 deaths
annually 1. Mosquitoes alone cause 400,000 malaria deaths and transmit viruses to hundreds of millions 2. The
vectorial capacity of mosquitoes depends on their ability to survive infection. The damaging effects of pathogenic
invasion of the mosquito midgut are well-documented 15-22, but little is known about how mosquitoes tolerate this
stress. Intestinal stem cell (ISC) mediated midgut epithelial repair is essential for Drosophila survival following
oral ingestion of pathogens 33. The mosquito midgut epithelium contains ISC-like cells 27, 34, 37-39, but their functional
significance for infection outcomes and mosquito survival is unknown. We propose to address this knowledge
gap in vector biology by investigating the mosquito gut regenerative response to pathogenic invasion.
The “black box” regarding the functional significance of ISCs in the mosquito midgut is part of a fundamental
knowledge gap: physiological studies treat the mosquito midgut as a homogeneous whole, rather than a
complex, regionally compartmentalized tissue comprised of multiple cell populations (e.g. enterocytes,
enteroendocrine cells, and ISCs). The specific contributions of these cell types to gut-pathogen interactions have
not been investigated. The proposed work will not only illuminate mosquito epithelial responses to infection at
the cellular level but will lead to the creation of new and innovative tools for the broader vector biology community.
The first two aims of this project are (A) to characterize gut epithelial cell dynamics in mosquitoes under
conditions of homeostasis and oral infection and (B) to evaluate the role of midgut epithelial repair in
mosquito infection outcomes. Aedes aegypti will be used as a model to determine what stimuli (including
human pathogens) affect gut epithelial turnover rates, whether post-infection repair rebuilds the gut
homeostatically or alters epithelial composition, what genetic pathways control midgut epithelial repair, and what
role epithelial repair plays in vector survival and competence. Our third aim is (C) to determine the specific
contributions of functionally differentiated cell populations to epithelial dynamics and infection
response. We will use single-cell RNAseq/ATACseq to establish how many cell types compose the midgut
epithelium, create new transgenic lines expressing fluorescent markers for important cell types (enteroendocrine
cells and ISCs), and, using these lines, couple fluorescence-activated cell sorting with RNAseq to examine the
transcriptional response of the three major cell types (enterocytes, enteroendocrine cells, and ISCs) to infection.
Our study will fill a critical gap in our understanding of mosquito midgut regenerative responses to pathogenic
invasion. Our long-term goal is to identify new targets for vector control strategies that disrupt gut regeneration
and reduce survival of infected mosquitoes below the critical incubation threshold required for pathogen
transmission. In addition to laying the groundwork for innovative control targets, we will create tools for the
broader vector biology community, paving the way for novel discoveries in mosquito midgut physiology.

Grant Number: 5R01AI148529-05
NIH Institute/Center: NIH
Principal Investigator: Nicolas Buchon