Mosquitoes are notorious vectors for multiple diseases like malaria, yellow fever and dengue fever. To manipulate gene expression in mosquito and spread desired genes among natural population for vector control, a thorough understanding of mosquito development and gene regulation is critical. Early embryogenesis is a rapid, complex yet crucial process in the very beginning of development. Previous research in other species indicated genes transcribed that early evolved fast and played essential roles. The study of mosquito early zygotic genes (EZGs) would offer unique insights into mosquito gene evolution as well as potential targets for mosquito control. In this study, I identified 61 pure EZGs (pEZGs) in mosquito Aedes aegypti. These pEZGs were enriched in architectures adapting to the rapid embryonic cell cycles and were over represented by domains or functions related to maternal zygotic transition. Phylogenetic analysis showed that pEZGs originated mainly from duplication, retrotransposition and de novo emergence. The comparison of pEZGs in Ae. aegypti with those in Drosophila revealed an interesting evolutionary paradox where the early zygotic genes turned over fast but the regulatory motif was conserved in two species. Curiously, the motif binding protein in Drosophila (zelda) seemed unable to initiate the earliest zygotic transcription in Ae. aegypti due to late temporal expression. The regulatory motif (VBRGGTA) found in Ae. aegypti pEZGs was shown necessary and sufficient for driving early zygotic gene expression by transient reporter assays and one motif-bearing promoter was tested with success in driving gene expression as early as 2-4h after egg laying in transgenic Ae. aegypti. This was the first characterized promoter with early zygotic but no maternal expression in Ae. aegypti that can be used for future genetic studies and mosquito control strategies. As important gene regulators, miRNAs also play essential roles in early embryogenesis. The genome-wide predictions and systematic analysis of miRNAs in Ae. aegypti and Anopheles stephensi were conducted in this study. The first miRNA profiling in mosquito across all developmental stages was also performed to provide basis for future functional study. Several lineage-specific miRNAs were found highly expressed in embryos, indicating their special roles in the embryogenesis of mosquitoes. / Ph. D.

Aside from few model organisms, little is known about early embryonic development or sex determination in insects, in particular mosquitoes which are major vectors of worldwide disease. The goals of this work were to investigate a mosquito-specific transcription factor and its intronic miRNA cluster and characterize a novel Y chromosome gene in An. stephensi. The aims of these experiments were to expand on the knowledge of genes involved in embryonic development and sex determination with potential application in vector control strategies. In Ae. aegypti a mosquito-specific bZIP1 transcription factor was demonstrated to be conserved among divergent mosquito species. It was maternally and zygotically-expressed and knock-down of bZIP1 mRNA via siRNA microinjection in the embryo resulted in embryonic death. The expression profile of this gene was determined through the use of RT-PCR and qRT-PCR. Additionally, this gene contains a miRNA cluster that is also relatively conserved amongst members of the Culicidae family suggesting its evolutionary importance. The miRNAs are also maternally and zygotically expressed and are the most abundant embryonic miRNAs as determined by small RNA sequencing and Northern analysis. Promoter activity for bZIP1 was characterized and the promoter was used to direct maternal and zygotic transgene expression in An. stephensi. Y chromosome genes were successfully identified in An. stephensi from Illumina sequencing data. This work focused on a gene unique to the Y 1 (GUY1). It was shown that GUY1 was male specific and linked to the Y chromosome. RT-PCR and single embryo analysis suggested that GUY1 was expressed during the maternal to zygotic transition and was only expressed in male embryos. It was shown in multiple transient and transgenic assays that the ectopic expression of GUY1 can influence the sex of subjected individuals and skew sex distribution to a male bias. There is still much to be investigated before a GUY1-based transgenic line can be tested and implemented for use in vector population control. However, the work in this dissertation represents a major step towards novel mosquito control strategies based on the manipulation of Y chromosome genes. / Ph. D.

Insect Odorant-Binding Proteins (OBPs) are small, water-soluble molecules that solubilize hydrophobic odorant molecules in the sensillum lymph and transport them to their cognate receptors in the olfactory receptor neurons. With the availability of the genome sequence of the African malaria mosquito, Anopheles gambiae, there has been a profound interest in the characterization and functional analyses of Obp genes in order to understand the molecular basis of mosquito host-seeking behavior. However, no direct evidence has been found for specific functions of any mosquito OBPs. In this study, I describe the comparative genomics and expression analyses on two mosquito Obp genes (Obp1 and Obp7) as well as efforts to determine their functions. Both of these Obp genes were identified in Anopheles stephensi and only Obp7 gene was identified in Anopheles quadriannulatus by screening bacterial artificial chromosome (BAC) libraries of these species. Comparative analyses revealed several interesting features including segments of conserved non-coding sequences (CNSs) that contain potential regulatory elements relevant to olfactory tissue development and blood-feeding. The expression profiles of these genes were examined in detail in the Asian malaria mosquito An. stephensi. Obp1 and Obp7 transcripts were significantly higher in females than male mosquitoes and they were predominantly found in the antenna, which is the primary olfactory organ of mosquitoes. Twenty-four hours after a blood meal, mRNA levels of these two genes were significantly reduced in the maxillary palp and proboscis, referred to as secondary olfactory organs of mosquitoes. These findings collectively indicate that Obp1 and Obp7 genes in An. stephensi likely function in female olfactory response and may be involved in behaviors related to blood-feeding. To investigate the function of these Obp genes more directly, a Sindbis virus based expression system is established to knockdown the two Obp gene orthologs in Aedes aegypti. The effective knockdown of Obp1 and Obp7 genes (8 and 100-fold, respectively) is accomplished in female mosquito olfactory tissues. The potential for a systematic analysis of the molecular players involved in mosquito olfaction using this newly developed technique is discussed. Such analysis will provide the foundation for interfering with mosquito host-seeking behavior for the prevention of disease transmission. / Ph. D.

Transposable elements (TEs) are mobile genetic elements capable of replicating and spreading within, and in some cases, between genomes. I describe a whole-genome analysis of DD34E TEs, which belong to the IS630-Tc1-mariner superfamily of DNA transposable elements, in the African malaria mosquito, Anopheles gambiae. Twenty-six new transposons as well as a new family, gambol, were identified. The gambol family shares the DD34E catalytic motif with Tc1-DD34E transposons, but is distinct from these elements in their phylogenetic relationships. Although gambol appears to be related to a few DD34E transposons from cyanobacteria and fungi, no gambol elements have been reported in any other insects or animals thus far. This discovery expands the already expansive diversity of the IS630-Tc1-mariner TEs, and raises interesting questions as to the origin of gambol elements and their apparent diversity in An. gambiae. Several DD34E transposons discovered in An. gambiae possess characteristics that are associated with recent transposition, such as high sequence identity between copies, and intact terminal-inverted repeats and open reading frames. One such element, AgTango, was also found in a distantly related mosquito species, Aedes aegypti, at high amino acid sequence identity (79.9%). It was discovered that Tango transposons have patchy distribution among twelve mosquito species surveyed using PCR as well as genomic searches, suggesting a possible case for horizontal transfer. Additionally, it was discovered that in some mosquito genomes, there are several Tango transposons. These observations suggest differential evolutionary scenarios and/or TE-host interaction of Tango elements between mosquito species. This strengthened the case that AgTango may be a functional transposase, and I sought to test its potential activity in a cell culture-based inter-plasmid transposition assay using the Herves plasmids as a positive control (Arensburger et al., 2005). AgTango constructs were successfully constructed; however, no transposition events were detected for Tango or Herves. Because the positive control failed to work, no assessment can be made concerning Tango's transposase. Possible causes and solutions for these results, alternative means to detect transposition, as well as future directions with Tango are discussed. / Ph. D.

Die Haut ist eine oft übersehene Komponente des angeborenen Immunsystems der Mücken. Die Haut der Mücke bildet eine physische Barriere, die die mikrobielle Homöostase aufrechterhält, das Eindringen von Toxinen wie Insektiziden verhindert und das Austrocknen verhindert. Die am meisten untersuchten Akteure des Immunsystems von Stechmücken sind das Fettgewebe und die Blutzellen, aber die Hauttalg-Fabriken, die Oenozyten, werden in Studien nur selten berücksichtigt. Mückenpuppen haben aktiv funktionierende immunitätsbezogene Organe, einschließlich derjenigen, die Hautbarrieren produzieren. Ihre biologische Rolle in diesem Entwicklungsstadium ist kaum bekannt, aber der Übergang von der Puppen- zur Erwachsenenhaut und die Auffälligkeit der talgproduzierenden Zellen machen dieses Stadium zu einem vielversprechenden Entwicklungsstadium für die Untersuchung der Hautbildung. Mit Hilfe der Transkriptomanalyse beschreiben wir die Rolle der Blutzellen bei der Entwicklung des chitinösen Teils der Insektenhaut, die Beteiligung des Fettkörpers an der Immunität und bestätigen die Rolle der talgproduzierenden Zellen im Lipidstoffwechsel. Darüber hinaus beschreiben wir talgsezernierende Zellen als einen bedeutenden Wirkungsort des NF-kappaB-ähnlichen IMD-Rel2-Pathway, in dem der Transkriptionsfaktor Rel2 die Retinoid-Homöostase reguliert. Schließlich bestätigen wir eine 100 Jahre alte Beobachtung, wonach sebumsezernierende Zellen der Stechmücke ihren Zellinhalt in einem Netzwerk von Vesikeln absondern. Wir beschreiben extrazelluläres Chromatin als Fracht in diesem Vesikelnetzwerk und sein antimikrobielles Potenzial. / The skin is an often overlooked component of the mosquito's innate immune system. The mosquito skin provides a physical barrier that maintains microbial homeostasis, prevents the entry of toxins like insecticides, and avoids desiccation. The most studied players in the immune system of mosquitoes are the adipose tissue and blood cells, but studies rarely consider the skin sebum factories, oenocytes. Mosquito pupae have actively functional immunity-related organs, including those producing skin barriers. Their biological roles at this developmental stage are poorly understood, but the pupae-to-adult metamorphic skin transition and the conspicuity of sebum-secreting cells make it a promising developmental stage to study skin formation. We use transcriptomics to describe the role of blood cells in the development of the chitinous section of the insect skin, the involvement of the fat body in immunity, and confirm the lipid metabolism role of sebum-secreting cells. Furthermore, we describe sebum-secreting cells as a significant action site of the NF-kappaB-like IMD-Rel2 pathway where the transcription factor Rel2 regulates retinoid homeostasis. Finally, we confirm a 100-year-old observation of how mosquito sebum-secreting cells secrete their cellular contents in a network of vesicles. We describe extracellular chromatin as cargo inside this vesicle network and its antimicrobial potential.

The intent of this thesis is to develop ordinary differential equation models to better understand the mosquito population. We first develop a framework model, where we determine the condition under which a natural mosquito population can persist in the environment. Wolbachia is a bacterium which limits the replication of viruses inside the mosquito which it infects. As a result, infecting a mosquito population with Wolbachia can decrease the transmission of viral mosquito-borne diseases, such as dengue. We develop another ODE model to investigate the invasion of Wolbachia in a mosquito population. In a biologically feasible situation, we determine three coexisting equilibria: a stable Wolbachia-free equilibrium, an unstable coexistence equilibrium, and a complete invasion equilibrium. We establish the conditions under which a population of Wolbachia infected mosquitoes may persist in the environment via the next generation number and determine when a natural mosquito population may experience a complete invasion of Wolbachia.

Oyster reefs are important estuarine ecosystems that provide habitat to many species including threatened and endangered wading birds and commercially important fishes and crabs. Infaunal organisms (i.e. small, aquatic animals that burrow in the sediment) are also supported by oyster reef habitats. Infaunal organisms are critical to marine food webs and are consumed by many important species that inhabit coastal estuaries. However, over the past century 85% of shellfish reef habitats have been lost, making restoration of these areas vital. Due to their important role in coastal food webs, infauna is hypothesized to be a strong indicator of habitat productivity to document the transition from a dead to a restored and living intertidal oyster reef. Research was conducted in Mosquito Lagoon of the northern Indian River Lagoon system. Three replicate samples were collected from 12 intertidal oyster reefs (four dead, four live, four restored). Samples were collected one-week pre-restoration and one month and six months post-restoration. Infauna was counted and sorted into six taxonomic categories: polychaetes, amphipods, isopods, gastropods, bivalves, and decapods. Reef infaunal abundance increased following restoration: restored reefs became more similar to live reefs one month following restoration. Six months after restoration restored reefs were also significantly different than dead reefs. Live reefs consistently had high infaunal abundance and dead reefs consistently had low abundance, while restored reefs were intermediate. These data suggest restored reefs are more productive than their dead counterparts, with restoration showing a positive trajectory to impact numerous infaunal species and their associated food webs.

Aedes aegypti mosquitoes, vectors for many human diseases, begin life as larvae developing in water, potentially exposed to runoff with herbicides and pesticides. This study serves as a novel investigation into the transstadial effects of exposure to Roundup on A. aegypti life history, immunity, and stress response and aims to account for these effects in an R0 model for vectorborne disease transmission. Prior work has shown that Roundup negatively affects mosquito life history. I hypothesized that larval exposure to the maximum sublethal dose of Roundup (7189µg/L) would negatively impact A. aegypti life history, immunity (candidate gene approach), and stress response (heat shock protein expression and fluctuating asymmetry). No significant differences were found for survival from the larval to adult stages, body size, size or shape fluctuating asymmetry, or sex ratio. However, the Roundup treatment group developed significantly slower for both time to pupation and to adult eclosion (both p < 0.0001). Adult immune gene expression showed no difference between groups, but the larval immune genes Dome (JAK-STAT pathway) and Spatzle (TOLL pathway) were downregulated in the Roundup treatment (p=0.0383 and p=0.0035, respectively), suggesting the larvae have reduced immunity. This study suggests that Roundup may have off-target effects on A. aegypti mosquitoes that are unaccounted for by current models, and these effects may potentially alter disease transmission to human hosts.

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