Oral susceptibility of Aedes aegypti mosquitoes to dengue viruses varies between different Aedes species and the genes contributing to susceptibility are unknown. The first goal of this project was to investigate midgut gene expression variation elicited in dengue susceptible (SUS) and refractory (REF) Aedes aegypti mosquitoes during the first 96 hours following dengue infection. The serial analysis of gene expression (SAGE) technique was utilized. Libraries were constructed from midgut RNA obtained from susceptible (SUS) and refractory (REF) strains after DENV-2 (i), naÌÄåøve blood (n) and sucrose meals (SM). The Z-test software scored expression variation between nSUS vs iSUS; nREF vs iREF; SM vs nSUS; SM vs nREF libraries. A total of 65,069 SAGE tags representing 14,474 distinct tag sequences were obtained from the libraries. Variation between DENV-2 infected libraries vs the respective strain-specific naÌÄåøve libraries revealed few transcripts that were common between infected libraries. Among uniquely mapped genes, 31 genes in nSUS vs iSUS and 28 genes in nREF vs iREF showed preferential expression while 12 genes showed shared differential expression between the naÌÄåøve and DENV-infected libraries in both strains, suggesting a common response to infection. Several functional processes were represented but generally varied between strains. Of note, we determined that 29% and 39% of tags mapped to anti-sense transcripts in the SUS and REF strains, respectively, and confirmed anti-sense transcription for a galectin, angitensin converting enzyme and a conserved hypothetical protein gene. In the second goal, expression variation between nSUS and nREF libraries with respect to the SM library was assessed. Of the annotatable genes, 20 and 22 showed preferential expression in the SM vs nSUS and the SM vs nREF comparison, respectively, while 48 genes showed similar expression patterns in blood fed libraries. Various processes, such as proteolysis and transport, showed similar expression patterns in blood fed libraries, although a few others varied. The translation process showed increased expression in the SM library. A proportion of our SAGE tags also mapped on the anti-sense strand of annotated genes. The third goal was to perform functional analysis of two putative dengue candidate genes, a Pellino homologue gene and the Inhibitor of apoptosis 1 (IAP) gene. This is the first in vivo study to investigate the function of any IAP and Pellino homologues during arboviral infection in mosquitoes. While silencing the Pellino gene had little effect on dengue, silencing of IAP had a significant increase in midgut and disseminated infection rates, suggesting IAP is a possible candidate gene for dengue vector competence. Our study highlights new vector-specific molecular factors found in Aedes aegypti midgut transcriptome, that likely contribute to differing capacities for dengue midgut infection in different strains, including those factors that could have potential as effectors against arboviruses.
Comparative Analysis of Midgut Gene Expression Profiles in DENV-2 Susceptible and Refractory Aedes aegypti MosquitoesDoctoral Dissertation
|Author||Irene N Kasumba|
|Advisor||Dr David W Severson|
|Contributor||Dr David W Severson, Committee Chair|
|Degree Level||Doctoral Dissertation|
|Degree Discipline||Biological Sciences|
|Departments and Units|