Evaluation of the function of a type I peritrophic matrix as a physical barrier for midgut epithelium invasion by mosquito-borne pathogens in Aedes aegypti

Abstract

In addition to modulating blood meal digestion and protecting the midgut epithelial cells from mechanical and chemical damage, a biological function attributed to the mosquito type I peritrophic matrix (PM) is preventing or reducing pathogen invasion, especially from Plasmodium spp. Previously, we demonstrated that chitin is an essential component of the PM and is synthesized de novo in response to blood feeding in Aedes aegypti. Therefore, knocking down chitin synthase expression by RNA interference severely disrupts formation of the PM. Utilizing this artificial manipulation, we determined that the absence of the PM has no effect on the development of Brugia pahangi or on the dissemination of dengue virus. However, infectivity of Plasmodium gallinaceum is lower, as measured by oocyst intensity, when the PM is absent. Our findings also suggest that the PM seems to localize proteolytic enzymes along the periphery of the blood bolus during the first 24 hours after blood feeding. Finally, the absence of the PM does not affect reproductive fitness, as measured by the number and viability of eggs oviposited.

FIG. 1.

Effect of Ae. aegypti chitin synthase (AeCs) dsRNA injection on relative transcript levels in Ae. aegypti midguts. Mosquitoes receiving no treatment (control) and mosquitoes injected with green florescent protein double-stranded RNA (dsRNA) (injection control) or AeCs dsRNA (chitin synthase knockdown) were blood fed twice (4 and 12 days after the injection). Total RNA was isolated from midguts at 4 to 16 days after the injection. Transcript abundance was quantified using quantitative polymerase chain reaction and calculated in comparison to the message level of the control (Aeact-1; U20287).

FIG. 2.

Effect of Ae. aegypti chitin synthase (AeCs) double-stranded RNA (dsRNA) injection on peritrophic matrix formation in Ae. aegypti. Mosquitoes received either no treatment, green florescent protein (GFP) dsRNA, or AeCs dsRNA 4 days prior to blood feeding, and the midguts were dissected 24 hours after blood feeding. Samples were stained with hematoxylin and eosin. (A) Midgut from control mosquito (no treatment). (B) Midgut from GFP dsRNA-injected mosquito. (C) Midgut from AeCs dsRNA-injected mosquito.

FIG. 3.

Effect of Ae. aegypti chitin synthase (AeCs) double-stranded RNA (dsRNA) injection on proteolytic activity in the midgut of Ae. aegypti. Mosquitoes were injected with AeCs dsRNA and then fed 4 days later with an artificial blood meal containing a fluorescent-based protease detector. The midguts were dissected 12 and 24 hours after blood feeding, and localization of proteolytic activity in the midgut was visualized histologically. (A) Midgut from control mosquito 12 hours after blood feeding. (B) Midgut from control mosquito 24 hours after blood feeding. (C) Midgut from AeCs dsRNA-injected mosquito 12 hours after blood feeding. (D) Midgut from AeCs dsRNA-injected mosquito 24 hours after blood feeding. Dark areas in each image represent exterior of the midgut.

FIG. 4.

Effect of Ae. aegypti chitin synthase (AeCs) double-stranded RNA (dsRNA) injection on the longevity of Ae. aegypti. Mosquitoes received no treatment (control), green florescent protein dsRNA (injection control), or AeCs dsRNA (chitin synthase knockdown) and were blood fed twice (4 and 12 days after the injection). Mosquito survival was monitored daily.

FIG. 5.

Effect of Ae. aegypti chitin synthase (AeCs) double-stranded RNA (dsRNA) injection on the fecundity of Ae. aegypti. Mosquitoes received no treatment (control), green florescent protein dsRNA (injection control), or AeCs dsRNA (chitin synthase knockdown) and then were blood fed twice (4 and 12 days after the injection) and the number of eggs laid were counted daily.