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Philosophiae Doctor - PhD / Blood feeding is a habit exhibited by many insects. Considering the devastating impact of these insects on human health, it is important to focus research on understanding the biology behind blood-feeding, disease transmission and host-pathogen interactions. Such knowledge would pave the way for developing efficient preventative measures. Iron an important element for species survival, is at the center of events controlling tsetse’s fitness and reproductive success. Hence, targeting genes involved in iron trafficking and sequestration would present possible means of preventing disease transmission. Considering the dynamic and multi-factorial nature of iron metabolism, a well-coordinated regulatory system is expected to be at work. Despite extensive literature on the mechanism of iron regulation and key factors responsible in maintaining its homeostasis in human, less attention has been given to understand such system in insects, especially the blood-feeding insects. The availability of the genome sequences for several insect disease vectors allows for a more detailed analysis on the identification and characterization of events controlling and preventing iron-induced toxicity following a blood-meal. The International Glossina Genome Initiative (IGGI) has coordinated the sequencing and annotation of the Glossina morsitans genome that has led to the identification of 12220 genes. This knowledge-base along with current understanding of the IRE system in regulating iron metabolism, allowed for investigating the UTRs of Glossina genes for the presence of these elements. Using a combination of motif enrichment and IRE-stem loop structure prediction, an IRE-mediated regulation was inferred for 150 genes, among which, 72 were identified with 5’-IREs and 78 with 3’-IREs. Of the identified IRE-regulated genes, the ferritin heavy chain and MRCK-alpha are the only known genes to have IREs, while the rest are novel genes for which putative roles in regulating iron levels in tsetse fly have been assigned in this study. Moreover, the functional inference of the identified genes further points to the enrichment of transcription and translation. Furthermore, several hypothetical proteins with no defined functions were identified to be IRE-regulated. These include TMP007137, TMP009128, TMP002546, TMP002921, TMP003628, TMP004581, TMP008259, TMP012389, TMP005219, TMP005827, TMP007908, TMP009332, TMP01- 3384, TMP009102, TMP010544, TMP010707, TMP004292, TMP006517, TMP014030, TMP009821 and TMP003060 for which an iron-regulatory mechanism of action may be inferred. We further report 26 IRE-regulated secreted proteins in Glossina, that present good candidates for further investigation pertaining to the development of novel vector control strategies. Using the predicted data on the identified IRE-regulated genes and their functional classification, we derived at 29 genes with putative roles in iron trafficking, where several unknown and hypothetical proteins are included. Thus a novel role is inferred for these genes in cellular binding and transport in the context of iron metabolism. It is therefore possible that these genes may have evolved in Glossina, such that they compensate for the absence of an IRE- regulated mechanism for transferrin. Additionally, we propose 14 IRE-regulated genes involved in immune and stress response, which may indeed play crucial roles at the host pathogen interface through their possible mechanisms of iron sequestration. Using the subcellular localization analysis, we further categorized the putative IRE regulated genes into several subcellular localizations, where the majority of genes were found within the nucleus and the cytosol. The detection of the conserved motifs in a set of genes, is an interesting yet sophisticated area of research, that allows for identifying either co-regulated or orthologous genes, while further providing support for the putative function of a set of genes that would otherwise remain uncharacterized. This is based on the notion that co-regulated genes are often coexpressed to carry out a specific function. As such, 14 regulatory elements were identified in the 5’- and 3’-UTRs of IRE-regulated genes, involved in embryonic development and reproduction, inflammation and immune response, signaling pathways and neurogenesis as well as DNA repair. This study further proposes several IRE-regulated genes as targets for micro-RNA regulation through identifying micro-RNA binding sites in their 3’UTRs. Using a motif clustering approach we clustered IRE-regulated genes based on the number of motifs they share. Significantly co-regulated genes sharing two or more motifs were determined as critical targets for future investigation. The expression map of IRE-regulated genes was analyzed to better understand the events taking place from 3 hours to 15 days following a blood meal. Re-analysis of Anopheles microarray chip showed the significant expression of three cell envelope and transport genes as early response and six as late response to a blood meal, which could indeed be assigned a putative role in iron trafficking. Genes identified in this study with implications in iron metabolism, whose timely expression allows for maintaining iron homeostasis, represent good targets for future work. Considering the important role of evolution in species adaptation to habits such as Hematophagy, it is of importance to identify evolutionary signatures associated with these changes. To distinguish between evolutionary forces that are specific to iron-metabolism in blood-feeding insects and those that are found in other insects, the IRE-regulated genes were clustered into orthologous groups using several blood feeding and non-blood feeding insect species. Assessment of different evolutionary scenarios using the Maximum Likelihood (ML) approach, points to variations in the evolution of IRE-regulated genes between the two insect groups, whereby several genes indicate an increased mutation rate in the BF-insect group relative to their non-blood feeding insect counterparts. These include TMP003602 (phosphoinositide3-kinase), TMP009157 (ubiquitin-conjugating enzyme9), TMP010317 (general transcription factor IIH subunit1), TMP011104 (serine-pyruvate mitochondrial), TMP013137 (pentatricopeptide Transcription and translation), TMP013886 (tRNA(uridine-2-o-)-methyl-transferase-trm7) and TMP014187 (mediator 100kD). Additionally, we have indicated the presence of positively selected sites within seven blood-feeding IRE-regulated genes namely TMP002520 (nucleoporin), TMP008942 (eukaryotic translation initiation factor 3), TMP009871(bruno-3 transcript) , TMP010317 (general transcription factor IIH subunit1), TMP010673 (ferritin heavy-chain protein), TMP011104 (serine-pyruvate mitochondrial) and TMP011448 (brain chitinase and chia). Thus the results of this study provides an in depth understanding of iron metabolism in Glossina morsitans and confers important targets for future validations based on which innovative control strategies may be designed.

The family Bunyaviridae is the largest of vertebrate diseases and includes the mosquito-borne disease La Crosse (LAC) virus. Vectors include the major vector Aedes triseriatus and two accessory vectors Aedes albopictus and Aedes japonicus. In the past several decades there has been an increase in the number of LAC cases, implication of new vectors, and the appearance of new foci of disease in the southeastern U.S. To better understand the vectors and the relationship between vectors and the virus, laboratory and field studies were conducted to determine oviposition preferences, effect of virus infection on blood-feeding behavior, and compare the efficacy of various methods to determine minimum infection rates of vectors. In laboratory studies of oviposition preference, only Ae. japonicus demonstrated a preference when presented with preexisting eggs. They deposited more eggs in cups containing either conspecifics or Ae. albopictus. The presence of 1st instar larvae Ae. albopictus larvae deterred oviposition by Ae. triseriatus and Ae. japonicus. Ae. japonicus and Ae. triseriatus preferred cups containing larval rearing water (LRW) of conspecifics and Ae. albopictus. Aedes albopictus preferred LRW regardless of species compared to control cups. Field experiments with fresh egg papers and preexisting eggs did not show significant differences, although the unequal population densities of species in the study area confounded the analysis. More work is needed to elucidate the interaction among these species and its effect on oviposition in the field. Blood-feeding experiments showed that LAC virus-infected Ae. triseriatus and Ae. albopictus imbibed significantly less blood compared to uninfected mosquitoes. Because blood meal size affects the subsequent inhibition of host seeking, experiments were done to ascertain the effect of virus infection on refeeding. Significantly more infected Ae. triseriatus mosquitoes refed but there was no effect on the refeeding rate of Ae. albopictus. Thus, the detrimental effect of virus infection, i.e., reduction in blood meal size, may lead to increased host exposure by Ae. triseriatus, enhancing horizontal transmission. Collecting adult mosquitoes was more efficient to detect virus in field populations than the collection of eggs. Maximum likelihood estimation-infection rates (MLE-IR) were calculated using bias-corrected maximum likelihood estimation. Adult collections yielded significantly more positive pools compared with egg collections. Virus was isolated from pools from Ae. canadensis, Ae. triseriatus, and Ae. albopictus. These results are comparable to other studies. / Ph. D.

The pitcher plant mosquito Wyeomyia smithii maintains a broad geographic range from the Gulf of Mexico to central Canada, and throughout its range is genetically and phenotypically variable, though fully interfertile. Many of the traits that vary across the broad range of this mosquito owe their diversity to selection on populations, which maximize fitness in the unique environment in which each populations finds itself. While a diversity of traits vary by latitude and merit the interest of evolutionary biologists, including critical photoperiod, voltinism, and thermal tolerance, of interest in the following thesis is the variation in blood-feeding propensity within this single species of mosquito. In no other mosquito species are some populations obligate non-biters while in other populations willingly hematophagous. This thesis explores the evolutionary transition from biting to non-biting in the pitcher plant mosquito at multiple levels of biological integration, starting first by establishing a heritable basis for the transition, then moving to the fitness and life historical consequences of both the natural system and of a line artificially selected in the lab. The latter half of this thesis moves on to probe the genetic architecture underlying the shift in phenotype and ends after examining the transition to non-biting at the level of the gene using an RNA-sequencing experiment. The results stemming from this thesis are thoroughly discussed: in short, we find that fitness differs between biting and non-biting populations, that complex genetic architectures underlie the transition to non-biting in nature, but not under artificial selection, and finally, that many candidate loci are differentially regulated in biting populations relative to non-biting populations and that these loci most often cluster with metabolic biological pathways.

The evolution of the blood feeding adaptation has required precise coordination of multiple physiological processes in the insect, such as reproduction, behavior, digestion and diuresis. These processes are under careful synchronous hormonal control. For rapid excretion, multiple diuretic hormones are known. Although originally described based on their ability to stimulate hindgut contractions, the Aedes kinins have been shown to stimulate fluid secretion in female mosquitoes of Aedes aegypti. Aedes kinins are leucokinin-like neuropeptides released from neurosecretory cells in the brain and abdominal ganglia. They act by binding to the Aedes kinin receptor, a G proteincoupled receptor (GPCR). The Aedes kinin receptor has been cloned, sequenced, functionally characterized, and immunolocalized to stellate cells in the Malpighian tubules of Ae. aegypti. In addition to their myotropic and diuretic roles, leucokinin-like peptides and/or their receptors have been also been discovered in the nervous, digestive, and reproductive systems of other arthropod species. Therefore, the Aedes kinins have the potential to function in several simultaneous physiological processes that are stimulated by blood feeding. This thesis aims to understand better their role in the whole mosquito by investigating the Aedes kinin receptor's global expression as well as its in vivo contribution to post-prandial diuresis. Presence of the Aedes kinin receptor was investigated in the head, posterior midgut (stomach), hindgut, ovaries, and Malpighian tubules of both non blood-fed and blood-fed females by western blot using anti-receptor antibodies. The receptor was then immunolocalized in the posterior midgut and rectum. Finally, RNAi was employed to knock down kinin receptor expression, followed by measurement of in vivo urine excretion post blood feeding in a precision humidity chamber. Transcript and protein knockdown were confirmed by qPCR and immunohistochemistry, respectively. Results indicate widespread expression of the Aedes kinin receptor protein in organs novel for hematophagous insects and demonstrate the receptor's fundamental role in rapid diuresis. These findings strongly point to the Aedes kinins as integrative signaling molecules that could coordinate multiple physiological systems. The Aedes kinins could therefore have contributed to the success of the blood feeding adapation in mosquitoes.

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Aedes aegypti mosquitoes are found globally and also act as the primary vector of Zika, dengue, and Chikungunya viruses, for which there are limited treatment options and no vaccines available. The use of insecticides as the main control strategy against diseases transmitted by this mosquito, is increasingly challenged by emerging resistance. Thus, there is a dire need for the development of novel approaches informed by an improved understanding of mosquito biology, to control mosquito populations and, ultimately, disease transmission. Rhythmic biological processes in mosquitoes help optimize resource exploitation by coordinating behaviors and physiology with fluctuating environmental conditions. Such synchronization enables organisms to adjust their physiology, metabolism, and behavior to predictable external cycles. In mosquitoes, circadian rhythmicity has been demonstrated in their biting and oviposition behavior, as well as their locomotor activity. However, little is known regarding how responses to long-range host cues are modulated by the circadian system. Here we show that both antennal sensitivity and olfactory behavior are time-of-day and odor-specific in Ae. aegypti females. Global transcriptomic analysis in whole heads of Ae. aegypti females reveal chemosensory genes differentially expressed throughout the day, providing insight into the molecular mechanisms behind daily variations in olfactory sensitivity and behaviors. We additionally show an odor-induced activation of mosquito behavior. Mosquito locomotion and behavior are also mediated by physiological state, and activity decreases after blood-feeding. Since the central clock components have been shown in other organisms to be redox-sensitive, we explored the role that diet heme plays in mediating behavioral changes following blood ingestion using artificial blood diets. We found that the transcription of the timekeeping gene period is reduced in the head immediately after feeding on a meal containing hemoglobin, but peripheral period transcription is reduced throughout the course of digestion following ingestion of a protein meal independent of hemoglobin inclusion. Overall, our results show that Ae. aegypti behavioral rhythms mediated by rhythmic gene expression are plastic and susceptible to external host cues and host blood digestion. This work can be leveraged for future studies investigating mosquito host-seeking and blood digestion to identify novel targets for vector control. / Doctor of Philosophy / Female mosquitoes rely on blood-feeding in order to produce eggs, but can unfortunately act as vectors of disease if they transmit pathogens when biting. Insecticides are currently our strongest main tool for controlling mosquito disease vectors such as Aedes aegypti, the yellow fever mosquito. However, increasing cases of insecticide resistance present new challenges in vector control, and new strategies to prevent vector-borne disease are needed. The Ae. aegypti mosquito is found globally and transmits Zika, dengue, and Chikungunya viruses, for which there are limited treatment options and no vaccines available. Mosquitoes exhibit rhythms in their gene expression and behaviors such as biting and activity patterns, in order to optimize energy efficiency and coordinate their biology and behaviors with daily fluctuations in the environment. However, it is unknown how their responses to human host odor cues are modulated by their central timekeeping system in the brain. Mosquitoes primarily find a human host via their sense of smell, or olfaction. Odor molecules in the air, emitted by humans, can be detected by mosquitoes' antennae. Here we show that both antennal sensitivity and behavioral responses to odors are time of day and odor-specific in Ae. aegypti females. We quantified gene transcripts in whole heads of Ae. aegypti females as a measure of gene expression, which revealed that genes involved in odor detection are expressed differently throughout the day, providing insight into the molecular mechanisms behind behavioral observations. We also show that mosquito behavior can be activated by odor exposure, and that their behavioral patterns can be influenced for multiple days following exposure. Mosquito behavior is also influenced by blood-feeding, which reduces mosquito activity and flight. Time-keeping genes in the fly brain have been shown to be sensitive to oxidative stress. Blood contains the protein hemoglobin, which can lead to oxidative stress when digested. Using artificial blood diets that allowed us to include or exclude hemoglobin in the meal, we found that the transcription of the timekeeping gene period is reduced in the head immediately after feeding on a diet containing hemoglobin, but is reduced in the rest of the body throughout the course of digestion following ingestion of a protein meal, whether hemoglobin was included or not. This work can be leveraged for future studies investigating mosquitoes' rhythms in host-seeking and blood digestion to identify new effective targets for vector control.

Argulus foliaceus (Linnaeus, 1758) is a member of the branchiuran family Argulidae and has a worldwide distribution, causing major economic impacts for freshwater aquacultured fish species worldwide. In the UK, it has economic impacts for both aquaculture and sports fishing industries. Previous studies observed haemorrhagic and inflammatory responses after Argulus infection, which has been taken to support the idea that the parasite secretes chemicals during the feeding process to assist with the ingestion of blood and epithelial tissue. The present study suggests that the blood-feeding ectoparasite of fish, A. foliaceus, may use similar mechanisms for evading host immune responses to those used by sea lice and other haematophagous arthropods. No previous studies have directly investigated the nature of the bioactive compounds / proteins, assumed to be released from these ectoparasites, and which are considered to contribute to feeding processes and host-parasite interactions during infection. Thus, the work described in this thesis was undertaken with the objective of identifying, describing and characterising the secretory components that have previously been suggested to be secreted from glandular cells associated with the feeding appendages of Argulus foliaceus. The current study applied transcriptomic and proteomic techniques in conjunction with in situ methods to investigate known immunomodulatory genes that may serve a function in parasite-host interactions. Overall, the findings of this project have generated considerable additional knowledge concerning the biology of Argulus spp. and have provided a list of proteins that may be used by the parasite to facilitate feeding processes by secreting these active molecules into the host and hence modulating their immune defence mechanisms. This information can be used as a baseline for developing freshwater lice control strategies to help prevent the spread of Argulosis in aquaculture by applying vaccination as means of control using the candidate antigens described in this study to specifically target Argulus spp. Knowledge generated by the work described in this thesis can also contribute to the development of drugs for controlling Argulus or functional components of feed that may serve to protect fish against this parasite. Furthermore, data from this thesis enhances the knowledge of the distribution of toxin/venom or venom-like substances in crustaceans and arthropods in general.