Juvenile hormone (JH) plays pivotal roles in the development and reproduction of insects. Efforts to characterize the mechanisms of JH regulation are complicated due to JH pathways often being intertwined with those of 20-hydroxyecdysone (20E). Upon adult emergence, female Aedes aegypti enter a period of development during which they gain competence for mating, bloodfeeding, and egg production. JH levels rise dramatically and peak during the first 2-3 days post-emergence and remain relatively high until a bloodmeal is consumed, while 20E titers remain very low throughout the entire stage. Thus, post-emergence development offers a unique opportunity to study the effects of JH in the absence of 20E. In this study, four potential JH response genes were identified in newly emerged females. One such gene, AaKr-h1, is a homologue of Kr-h1, a zinc-finger transcription factor which has been characterized in Manduca sexta, Drosophila melanogaster, Tribolium castaneum, and Apis mellifera, and is involved in a diverse range of JH-regulated pathways. AaKr-h1 demonstrated a dose-dependent transcriptional response to JHIII as well as two JH mimics in abdominal ligation assays. The findings of this study indicate that Kr-h1 may be regulated by JH independently of any 20E regulation and suggests a fundamental, conserved role for Kr-h1 in JH-regulated pathways. / Master of Science in Life Sciences

The transcription factor Krüppel homolog 1 (Kr-h1) is crucial in multiple reproductive processes of Aedes aegypti mosquitoes, including previtellogenesis, vitellogenesis, and oogenesis. This study explores the interaction between Kr-h1 and its potential associated proteins (KAPs), with a specific focus on the dimerization partner (DP-1), and how this interaction regulates gene expression pathways critical for mosquito reproduction. Utilizing RNA interference (RNAi), the research identifies DP-1 as a significant regulator of follicle growth post-eclosion (PE), highlighting its vital role in the mosquito reproductive regulatory pathway. The experimental approach included RNAi-mediated knockdown of DP-1, accompanied by evaluations using quantitative PCR (qPCR), Western blotting (WB), co-immunoprecipitation (Co-IP), follicle length measurement, and egg counting to assess the role of DP-1 in reproductive functions. For the first time, the inhibition of DP-1 expression was found to significantly impede A. aegypti follicular development. The elucidation of the mechanistic roles of Kr-h1 and DP-1 provides valuable insights that could lead to innovative strategies for mosquito population control and effective disease vector management. / Master of Science in Life Sciences / Mosquitoes can spread serious insect-borne diseases such as dengue, Zika, and malaria, etc. These diseases can infect hundreds of millions of individual and cause around a million of death annually. This study focuses on a specific mosquito species, Aedes aegypti, which is a major carrier of these diseases. To manage their populations and reduce the spread of these diseases, scientists are constantly seeking new methods to control their reproduction. Chemical insecticides are one of the most efficient and widely used strategies. However, these insecticides face significant challenges, including the development of resistance in mosquito populations and the potential damage to non-target species to affect the ecosystem. To address this issue, the development of new insecticides is crucial. We can identify new targets to pave the way to research novel effective insecticides. Inside mosquitoes, there are various proteins that help control their ability to reproduce. One of these proteins is called Krüppel homolog 1 (Kr-h1). Kr-h1 plays a crucial role in the development and reproductive processes of mosquitoes. Our research looked at how Kr-h1 interacts with other types of protein to control mosquito reproduction. Through various experiments, including gene expression analysis and protein studies, we found that DP-1 is essential for the proper development of mosquito eggs. This insight helps us understand more about the biological processes and hormonal pathways during mosquito reproduction, therefore provide greater opportunity to develop insecticides to reduce their populations and the spread of the diseases they carry.

Female adults of many mosquito species possess distinct physiological features adapting to blood feeding for successful reproduction. The disease pathogens that are transmitted by mosquitoes have evolved to take advantages of the indispensable blood feedings to complete their transmission cycles and to survive attacks from the mosquito's innate immune system. Normal egg development and mosquito immunity are tightly controlled by tissue- and stage-specific gene expression and coordinated by many signal molecules in the mosquito. Understanding gene regulation affecting mosquito reproduction and malaria parasites infection is of paramount importance for developing novel malaria control strategies. A growing body of evidence indicates that microRNAs (miRNAs) are involved in egg maturation and immune reactions against invading pathogens in mosquitoes. However, the molecular mechanisms by which specific miRNAs selectively modulate reproduction and the survival of pathogens are largely unknown. The miRNA-induced gene-silencing pathway in mosquitoes was mostly extrapolated from the studies of flies. To explore the dynamics of miRNAs in reproduction, I used small RNAs sequencing to monitor miRNAs expression and their association with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) in the malaria mosquito Anopheles gambiae (An. gambiae) during the 72-h period immediately after blood feeding. I found the abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. I confirmed that miR-309 is essential for normal egg development by depletion of endogenous miR-309 with a specific antagomir. In addition, my results showed that the Ago association of some miRNAs was not proportional to their cellular abundance implying additional regulation at miRNA integration. To investigate the functional roles of miRNAs and define context-dependent miRNA-mRNA interactions during the reproductive process, I have applied an innovative experimental approach to study miRNA-mRNA interactome. CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP can generate miRNA-mRNA chimeras from UV-irradiation stabilized Ago-miRNA-mRNA complex. My results have defined tens of thousands of miRNA-mRNA interactions in mosquitoes, including novel targets for mosquito-specific miRNAs. Verification of the predicted interactions using mRNA-seq, ribosome-profiling, and luciferase reporter assay revealed a reliable miRNA-mRNA interaction network. Based on the detected interactions, I refined the paring rules for mosquito miRNAs and illustrated the dynamic pairing between different regions of miRNAs with their targets in vivo. The miRNA-mRNA interactions were compared using this approach at multiple time points before and after blood feeding. Importantly, this study showed that the interactions were dynamic and enriched in genes that are involved in metabolisms, supporting the proposed functions of miRNAs in coordinating the gene regulation in mosquito reproduction. Plasmodium falciparum (P. falciparum) is a major human malaria parasite. To understand the functions of miRNAs in the mosquito resistance to Plasmodium infection, we analyzed the miRNA-mRNA interactions after female mosquitoes taking a P. falciparum-infected blood meal or an uninfected blood meal. Comparison of the interactions revealed enhanced miRNA-mRNA interactions after P. falciparum infection involving a group of immunity-related genes. In summary, this study has provided a systematic view and significantly advanced our understanding of the miRNA functions in mosquito reproduction and P. falciparum infection. / PHD