Mosquito-borne helminth infections are responsible for a significant worldwide disease burden in both humans and animals. Accordingly, development of novel strategies to reduce disease transmission by targeting these pathogens in the vector are of paramount importance. We found that a strain of
Aedes aegypti that is refractory to infection by Dirofilaria immitis, the agent of canine heartworm disease, mounts a stronger immune response during infection than does a susceptible strain. Moreover, activation of the Toll immune signaling pathway in the susceptible strain arrests larval development of the parasite, thereby decreasing the number of transmission-stage larvae. Notably, this strategy also blocks transmission-stage Brugia malayi, an agent of human lymphatic filariasis. Our data show that mosquito immunity can play a pivotal role in restricting filarial nematode development and suggest that genetically engineering mosquitoes with enhanced immunity will help reduce pathogen transmission.
Brugia; Dirofilaria; Malpighian tubule; immunity; mosquito.
Copyright © 2020 the Author(s). Published by PNAS.
Conflict of interest statement
The authors declare no competing interest.
Immune genes are more robustly up-regulated in
D. immitis-infected Malpighian tubules of refractory Ae. aegypti compared to an infection susceptible strain. ( A) Overlay of light and fluorescent photomicrographs of representative portions of Ae. aegypti (Susceptible) and S Ae. aegypti (Refractory) Malpighian tubules containing R D. immitis (arrows) 3 d postinfection. The nuclei of tubule principal cells, stained with Hoechst, are indicated with white arrowheads. (Scale bar, 25 µm.) ( B) Volcano plot showing differentially regulated genes in Malpighian tubules of Ae. aegypti compared to R Ae. aegypti 3 d post S D. immitis infection. Different color dots indicate members of Immune (open), Rel1 up-regulated (blue), Rel2 up-regulated (green), or up-regulated by B. malayi (cyan) or by Wolbachia infection (yellow) gene sets that contribute to significant GSEA scores. Dots appearing with more than one color are present in multiple lists. Panel B appears in high resolution format in . ( SI Appendix, Fig. S3 B C) Clustered heat map of differentially expressed immune genes in either Ae. aegypti (Susceptible) or S Ae. aegypti (Refractory) Malpighian tubules 3 d post R D. immitis infection relative to its uninfected control. The key indicates the row z score, with red corresponding to up-regulated, white corresponding to neutral, and blue corresponding to down-regulated relative to its control. Horizontal lines separate three distinct clusters of expression profiles. Dots indicate genes that are known targets of Rel1 (blue) or Rel2 (green).
Toll pathway activation reduces emerging transmission-stage
D. immitis. ( A) Schematic diagram of the experimental workflow where mosquitoes are infected with D. immitis, then immediately injected with dsRNA. ( B) Graph of the average prevalence of emerging transmission-stage D. immitis larvae. The error bar indicates the SD, and the P value is from a χ 2 test. ( C) Dots indicate the number of transmission-stage D. immitis larvae emerging from individual mosquitoes. The P value is from a Kruskal−Wallis test with Dunn’s correction. Data in B and C are pooled from three independent experiments with ds Cactus and ds Caspar and a fourth where only ds Cactus was injected. ( D and F) Graph of the average prevalence of individuals with at least one emerging parasite assayed 17 d postinfection. The error bar indicates the SD. The P value is from a χ 2 test. ( E and G) Dots indicate the number of transmission-stage larvae emerging per mosquito assayed 17 d postinfection. The P value is from a Kruskal−Wallis test with Dunn’s correction for multiple comparisons. Data in D and E and F and G are pooled from two and three independent experiments, respectively.
Toll pathway activation blocks
D. immitis development in Malpighian tubules. ( A) Schematic diagram of the experimental workflow. After performing an emergence assay with whole mosquitoes, larvae were assayed in dissected Malpighian tubules. The head and carcass were placed separately into wells of a multiwell plate to capture larvae migrating in the hemocoel. ( B) Graph of the average prevalence of emerging transmission-stage D. immitis larvae assayed 17 d postinfection. The error bar indicates the SD, and the P value is from a χ 2 test. ( C) Dots are the number of transmission-stage D. immitis larvae emerging from individual mosquitoes assayed 17 d postinfection. Data are pooled from three independent experiments. ( D) Dots indicate the number of larvae present in dissected Malpighian tubules. ( E) Dots indicate the number of larvae emerging from the combined head and carcass following Malpighian tubule dissection. The P value in C– E is from a Mann−Whitney test. ( F) Dots indicate pooled parasite numbers present across all tissues, which are normally distributed. Red lines indicate the mean. The populations are not significantly different (ns) using an unpaired t test with Welch’s correction. ( G) Representative images of dissected Malpighian tubules from ds Cactus- and ds GFP-treated (control) mosquitoes after performing an emergence assay. Larvae are indicated with white arrowheads. Note that the larvae in the ds Cactus-treated mosquitoes are typically stunted compared to the more elongated larva in the ds GFP-treated controls. Also indicated are deposits of melanin that appear specifically in the hindguts of some of the ds Cactus-treated mosquitoes. The scale for both images is the same. (Scale bar, 100 µm.)
Toll pathway activation reduces emerging transmission-stage
B. malayi. ( A) Graph of the average prevalence of emerging transmission-stage B. malayi larvae assayed 12 d postinfection. The error bar indicates the SD, and the P value is from a χ 2 test. ( B) Dots are the number of transmission-stage B. malayi larvae emerging from individual mosquitoes assayed 12 d postinfection. The P value is from a Mann−Whitney test. Data are pooled from three independent experiments.
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