MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in post-transcriptional gene expression regulation in eukaryotic organisms. miRNA function is driven by base-pair binding to a complementary mRNA strand which in turn results in mRNA silencing. Here, we employed a novel co-immunoprecipitation technique (CLIP-seq) to identify miRNA-mRNA interactions during the post-eclosion (PE) and blood meal stages of Aedes aegypti, and validated miRNA binding using a dual luciferase assay. Next, we investigated the transcriptional factors that govern miRNA expression regulation. We used a double-stranded RNA (dsRNA) knockdown approach to silence drosha to enrich for pri-miRNAs in the total RNA pool. Three pri-miRNAs (miR-276, miR-2940 and miR-252) were identified and their respective promoter regions were selected for transcription factor binding analysis using a luciferase based assay. Only one transcription factor, E75, induced luciferase activity and we further explored E75 as part of the juvenile hormone (JH) hierarchical network. E75 transcript levels could be induced by topical application of JH, and reduced through the blockage of JH pathway by RNAi targeting of the key receptor Met. Using small-RNA sequencing, we found that iMet and iE75 treatments both had a global negative impact on miRNA expression. These findings established the role of E75 within the JH hierarchical network in miRNA activation during the PE phase. We next examined the functional roles of E75 regulated miRNAs, miRNA-276, miRNA-2940 and miR-252, during the gonotrophic cycle. Antagomir treatment with Ant-2940-3p resulted in reduced follicle size and number of eggs laid. We performed computational mRNA-miRNA target predictions to elucidate the mRNA gene(s) affected by miR-2940 knockdown and confirmed by RT-qPCR that the putative target Clumsy (AAEL002518) had elevated gene levels from Ant-2940-3p treated fatbody tissues. This mRNA-miRNA interaction was confirmed using an in vitro dual luciferase assay in Drosophila S2 and in native Ae. aegypti Aag2 cell lines. Finally, we performed a phenotypic rescue experiment to demonstrate that the elevated expression of Clumsy was responsible for the disruption we noted in egg development upon Ant-2940-3p treatment. Collectively, these results confirm the importance of miR-2940-3p in the fatbody tissue during the PE developmental phase of the gonotrophic cycle.

Despite recent progress in genetic manipulations of mosquitoes, there is a much needed improvement and development of specific research tools to further facilitate research of these disease vectors. The focus of this dissertation is to extend and refine upon the Gal4/UAS system for the yellow fever mosquito, Aedes aegypti. The first part of the dissertation focuses on the development the blood-meal-activated, gut-specific Gal4/UAS system by utilizing a 1.1-kb, 5' upstream region of the carboxypeptidase A (CP) gene to genetically engineer the CP-Gal4 driver mosquito line. The generated driver successfully drive the expression of the effect gene followed a similar pattern as endogenous CP gene. Using the CP-Gal4/UAS-EGFP mosquitoes, we demonstrated that the CP gene is regulated by insulin and amino acids. In the second part of the dissertation, the gut-specificCP-Gal4/UAS and the fat-body specific Vg-Gal4/UAS systems were utilized in a combination with microRNA decoy to investigate the function of the, aae-miR-275 (miR-275). This approach allowed demonstrating a gut-specific function of miR-275.Specific miR-275 silencing in the midgut resulted in dramatic defects in blood digestion and egg development in female mosquitoes. The predicted miR-275 target gene- sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)-was confirmed to directly bind miR-275 by means of luciferase assay. RNA interference silencing of SERCA or application of SERCA-specific inhibitor Thapsigargin (TG) resulted in an apparent lack of blood digestion, inhibition of egg development, as well as diminished egg number in comparison to controls. Application of the miR-275 mimic in the CP-Gal4/UAS-miR275decoy mosquitoes restored SERCA transcript level and rescued the phenotype. In the last part of this thesis, I described two additional midgut-specific Gal4 driver lines that are currently under development. Together, my work provide considerable advance in developing the binary Gal4/UAS system for research of essential gene and microRNA functions in the mosquito Ae. aegypti.

Protein trafficking through the endomembrane system of plants is a highly dynamic and transient process. Protein trafficking plays a role in several signal transduction and developmental pathways such as gravitropic response, plant pathogen resistance, cell pattern formation and autophagy. One of the most dynamic compartments of the endomembrane system is the endosome. The endosome is comprised on proteins that transit to and from the plasma membrane, the trans-Golgi network, and the prevacuolar compartment. Given the highly dynamic nature of this compartment, chemical genomics is a useful approach to slow down trafficking of proteins or inhibit trafficking through specific compartments. Since high-throughput cellular phenotyping is not available in Arabidopsis thaliana, tobacco pollen was used as a model system for membrane cycling to identify putative trafficking inhibitors for Arabidopsis. Tobacco pollen displays polarized growth that is dependent on the delivery and recycling of protein to the apical tip, and tobacco pollen is highly amenable to high-throughput screening. Utilizing tobacco pollen, over 46,000 natural and synthetic compounds were assayed and 378 were found to inhibit pollen germination and/or morphology. Of these 378, 365 were novel compounds. These 378 were then screened on three highly characterized Arabidopsis plasma membrane protein known to cycle through endosomal compartments, the brassinosteroid receptor BRI1:BRI1:GFP, and two PINFORMED auxin efflux proteins PIN2:PIN2:GFP and PIN2:PIN1:GFP. With just these three markers, 129 compounds from the pollen screen disrupted the normal localization patterns of at least one of these proteins. To examine a specific physiological response, these 129 were screened for effects on root gravitropic response. Sixteen compounds were identified as strongly disrupting gravity response. From these, the compound Endosidin2 (ES2) was characterized. ES2 disrupted several specific plasma membrane markers without affecting any internal protein markers, with the exception of the auxin homeostasis protein PINFORMED5, which localizes to the endoplasmic reticulum. ES2 specifically inhibits endosomal recycling to the plasma membrane without affecting endocytosis. ES2 allows for specific interrogation into the effects of endosomal recycling without affecting any other internal compartments as Brefeldin A does, the only other compound known to inhibit endosomal recycling.

Mosquitoes are vectors of many parasitic and viral diseases that continue to burden the global public health system. The innate immune system of insects is the first line of defense against invading pathogens. This system is governed by two major pathways, Toll, and IMD pathways. This dissertation involves the investigation of these pathways. Aim 1 involves the exploration of the Toll and PPO activation pathway in response to pathogen invasion. Aim 2 seeks to investigate the components of the IMD pathway and lastly, Aim 3 provides a comparative genomic study of the mosquito immune system. The results of the first aim identified an interleukin receptor associated kinase (IRAK), IRAK4, in the mosquito, Aedes aegyti. Furthermore, it was found that IRAK4 serves an important immune function in the Toll immune pathway. Addtionally, several cytoplasmic components of the IMD pathway were identified including PGRPLC, IMD, IKKβ, and FADD (Aim 2).

Female Aedes aegypti mosquitoes spread pathogens for various vector-borne diseases while feeding on vertebrate blood. Being anautogenous, these mosquitoes require a blood meal for vitellogenesis, the process of yolk formation during the maturation of eggs. A clear understanding of the molecular mechanism of reproduction, such as detailed transcription mechanisms, should contribute significantly to the vector control strategies. The aim of this project is to decipher how complex transcription programs govern genes that are differentially expressed during female mosquito reproduction. We have identified 89 putative transcription factor biding sites (TFBSs) using bioinformatics tools, on the promoter regions of more then 1K differentially regulated genes. The putative TFBSs were screened for positional bias, orientation bias and evolutionary conservation, properties that are often associated with real TFBSs. Promoters from orthologous genes in closely related species Anopheles gambiae and Culex quinquefascitus, were used to check for evolutionary conservation. The above-mentioned screens were used to increase the accuracy of the in-silico predictions. JASPAR database was searched to identity the known TFBSs and their corresponding TFs. GeneMANIA webtool was used for the construction of putative regulatory networks. Molecular biology techniques such as RNA-interference mediated depletion of selected TFs and quantitative reverse-transcription polymerase chain reaction have been used for verifying and evaluating the functionality of some of these putative TFBSs.

In addition to identifying cis-regulatory elements this dissertation also describes an approach developed for prediction of miRNA targets involved in mosquito reproduction. miRNAs are 21 to 24 nucleotide long non-coding RNAs that degrade or inhibit by coupling with target sites on mRNAs. Since the discovery huge numbers of miRNAs have been reported. However, the functions of only a few of these miRNAs are well understood. The study of miRNA targets is critical step for determining the functionality of miRNAs. There are many miRNA target prediction tools that are readily available, but almost all of these have a problem of producing huge number of false-positive results. Here, we have developed a new approach for computational target prediction that involves five different target prediction programs, the results have been experimentally verified in separate studies and found to be highly reliable.

Overall we have been able to identify binding sites for TFs and targets for miRNAs that are involved in female mosquito reproduction. We realize that future work remains, for a complete understanding of the complex molecular mechanisms, during the reproductive period. However this study can be regarded as a significant step towards the accomplishment of that goal.

No need for Master's Thesis