effective-oral-rna-interference-rnai-administration-to-adult

Effective Oral RNA Interference (RNAi) Administration to Adult <em>Anopheles gambiae</em> Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

Johns Hopkins Bloomberg School of Public Health

Johns Hopkins School of Medicine

Idaho College of Osteopathic Medicine

Aedes aegypti is the vector of some of the most important vector-borne diseases like dengue, chikungunya, zika and yellow fever, affecting millions of people worldwide. The cellular processes that follow a blood meal in the mosquito midgut are directly associated with pathogen transmission. We studied the homeostatic response of the midgut against oxidative stress, as well as bacterial and dengue virus (DENV) infections, focusing on the proliferative ability of the intestinal stem cells (ISC). Inhibition of the peritrophic matrix (PM) formation led to an increase in reactive oxygen species (ROS) production by the epithelial cells in response to contact with the resident microbiota, suggesting that maintenance of low levels of ROS in the intestinal lumen is key to keep ISCs division in balance. We show that dengue virus infection induces midgut cell division in both DENV susceptible (Rockefeller) and refractory (Orlando) mosquito strains. However, the susceptible strain delays the activation of the regeneration process compared with the refractory strain. Impairment of the Delta/Notch signaling, by silencing the Notch ligand Delta using RNAi, significantly increased the susceptibility of the refractory strains to DENV infection of the midgut. We propose that this cell replenishment is essential to control viral infection in the mosquito. Our study demonstrates that the intestinal epithelium of the blood fed mosquito is able to respond and defend against different challenges, including virus infection. In addition, we provide unprecedented evidence that the activation of a cellular regenerative program in the midgut is important for the determination of the mosquito vectorial competence.

Regulation of midgut cell proliferation impacts Aedes aegypti susceptibility to dengue virus.

 sporozoites (SPZs) must traverse the mosquito salivary glands (SGs) to reach a new vertebrate host and continue the malaria disease cycle. Although SGs can harbor thousands of sporozoites, only 10 to 100 are deposited into a host during probing. To determine how the SGs might function as a bottleneck in SPZ transmission, we have characterized  SGs infected with the rodent malaria parasite  using immunofluorescence confocal microscopy. Our analyses corroborate findings from previous electron microscopy studies and provide new insights into the invasion process. We identified sites of SPZ accumulation within SGs across a range of infection intensities. Although SPZs were most often seen in the distal lateral SG lobes, they were also observed in the medial and proximal lateral lobes. Most parasites were associated with either the basement membrane or secretory cavities. SPZs accumulated at physical barriers, including fused salivary ducts and extensions of the chitinous salivary duct wall into the distal lumen. SPZs were observed only rarely within salivary ducts. SPZs appeared to contact each other in many different quantities, not just in the previously described large bundles. Within parasite bundles, all of the SPZs were oriented in the same direction. We found that moderate levels of infection did not necessarily correlate with major SG disruptions or abundant SG cell death. Altogether, our findings suggest that SG architecture largely acts as a barrier to SPZ transmission. Malaria continues to have a devastating impact on human health. With growing resistance to insecticides and antimalarial drugs, as well as climate change predictions indicating expansion of vector territories, the impact of malaria is likely to increase. Additional insights regarding pathogen migration through vector mosquitoes are needed to develop novel methods to prevent transmission to new hosts. Pathogens, including the microbes that cause malaria, must invade the salivary glands (SGs) for transmission. Since SG traversal is required for parasite transmission, SGs are ideal targets for transmission-blocking strategies. The work presented here highlights the role that mosquito SG architecture plays in limiting parasite traversal, revealing how the SG transmission bottleneck is imposed. Further, our data provide unprecedented detail about SG-sporozoite interactions and gland-to-gland variation not provided in previous studies.

Article	Year
Regulation of midgut cell proliferation impacts Aedes aegypti susceptibility to dengue virus. PLoS neglected tropical diseases\| PubMed ID: 29782512	2018
Salivary Gland Architecture Shapes Sporozoite Availability for Transmission. mBio\| PubMed ID: 31387905	2019

Michael Wells

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