Project description:Canine heartworm is a widespread and potentially fatal mosquito-borne disease caused by infections with the parasitic nematode, Dirofilaria immitis. We have previously shown that systemic activation of the Toll immune pathway via silencing of the negative regulator Cactus in Aedes aegypti blocks parasite development in the Malpighian tubules, the mosquito renal organ. However, it was not established whether the Malpighian tubules were directly responding to Toll activation or were alternatively responding to upregulated proteins or other changes to the hemolymph driven by other tissues. Distinguishing these possibilities is crucial for developing more precise strategies to block D. immitis while potentially avoiding the fitness cost to the mosquito associated with Cactus silencing. This study defines the transcriptional response of Ae. aegypti Malpighian tubules after systemic Toll activation via intra-thoracic injection of dsCactus and found, like the response of whole mosquitoes, a significant increase in expression of Toll pathway target genes. Additionally, we identified a significant overlap between the transcriptional response of the Malpighian tubules and proteins upregulated in the hemolymph. Our data show that Malpighian tubules are capable of RNAi-mediated gene silencing and directly respond to dsCactus treatment by upregulating canonical Toll pathway targets. Though not definitive, the strong correspondence between the Malpighian tubule transcriptional and the hemolymph proteomic responses provides evidence that the tubules may contribute to mosquito humoral immunity.
Project description:Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts and are central to RNA interference (RNAi). AGO-crosslinking immunoprecipitation (AGO-CLIP) has illuminated RNAi networks, but bioinformatic analysis is laborious and lack of experimental tools hinders its application outside of model organisms. RNAi is critical for regulation of gene expression and defense against viral infection in the Aedes aegypti mosquito, which transmits Zika, chikungunya, dengue, and yellow fever viruses to cause human disease. We developed AGO-CLIP for both mosquito AGO proteins and a universal, streamlined software package for CLIP analysis, identifying 230 novel small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map. We used this unique resource to predict repression of small RNA targets in specific mosquito tissues. Notably, this resource revealed unexpected AGO target preferences and uncovered a new mode of AGO-mediated repression, findings that have broad implications for the study of antiviral RNAi.