One of the largest problems facing modern medicine is drug resistance. Many classes of drugs can be rendered ineffective if their target is able to acquire beneficial mutations. While this is an excellent showcase of the power of evolution, it necessitates the development of increasingly stronger drugs to combat resistant pathogens. Not only is this strategy costly and time consuming, it is also unsustainable. To contend with this problem, many multi-drug treatment strategies are being explored. Previous studies have shown that resistance to some drug combinations is not possible, for example, resistance to a common antifungal drug, fluconazole, seems impossible in the presence of radicicol. We believe that in order to understand the viability of multi-drug strategies in combating drug resistance, we must understand the full spectrum of resistance mutations that an organism can develop, not just the most common ones. It is possible that rare mutations exist that are resistant to both drugs. Knowing the frequency of such mutations is important for making predictions about how problematic they will be when multi-drug strategies are used to treat human disease. This experiment aims to expand on previous research on the evolution of drug resistance in S. cerevisiae by using molecular barcodes to track ~100,000 evolving lineages simultaneously. The barcoded cells were evolved with serial transfers for seven weeks (200 generations) in three concentrations of the antifungal Fluconazole, three concentrations of the Hsp90 inhibitor Radicicol, and in four combinations of Fluconazole and Radicicol. Sequencing data was used to track barcode frequencies over the course of the evolution, allowing us to observe resistant lineages as they rise and quantify differences in resistance evolution across the different conditions. We were able to successfully observe over 100,000 replicates simultaneously, revealing many adaptive lineages in all conditions. Our results also show clear differences across drug concentrations and combinations, with the highest drug concentrations exhibiting distinct behaviors.

Hundreds of thousands of people die annually from malaria; a protozoan of the genus Plasmodium is responsible for this mortality. The Plasmodium parasite undergoes several life stages within the mosquito vector, the transition between which require passage across the lumen of the mosquito midgut. It has been observed that in about 15% of parasites that develop ookinetes in the mosquito abdomen, sporozoites never develop in the salivary glands, indicating that passage across the midgut lumen is a significant barrier in parasite development (Gamage-Mendis et al., 1993). We aim to investigate a possible correlation between passage through the midgut lumen and drug-resistance trends in Plasmodium falciparum parasites. This study contains a total of 1024 Anopheles mosquitoes: 187 Anopheles gambiae and 837 Anopheles funestus samples collected in high malaria transmission areas of Mozambique between March and June of 2016. Sanger sequencing will be used to determine the prevalence of known resistance alleles for anti-malarial drugs: chloroquine resistance transporter (pfcrt), multidrug resistance (pfmdr1) gene, dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr). We compare prevalence of resistance between abdomen and head/thorax in order to determine whether drug resistant parasites are disproportionately hindered during their passage through the midgut lumen. A statistically significant difference between resistance alleles in the two studied body sections supports the efficacy of new anti-malarial gene surveillance strategies in areas of high malaria transmission.

Mosquitoes are estimated to kill roughly 700,000 people each year through the transmission of vector-borne diseases. Vector control via insecticides is a widely used method in order to combat the spread of mosquito populations; however, this comes at a cost. Resistance to insecticides has the potential to increase vector-borne disease rates. Aedes aegypti is an invasive mosquito species in Arizona and is a known potential vector for a variety of infectious diseases including dengue, chikungunya, Zika, and yellow fever. In contrast to many other mosquito species Ae. aegypti mosquito eggs can undergo quiescence, an active state of dormancy, over long periods of time. Variation in quiescent periods correlates to climatic rainfall alterations and can ultimately influence hatching and mating between multiple generations. I have studied the effect of quiescence on larvicide (i.e., temephos) susceptibility using mosquito eggs collected from a susceptible lab strain and stored under optimal temperature and humidity conditions. After undergoing various quiescent periods (3, 7, 14, 28, 84, and 182 days), the experimental eggs as well as 7-day quiescent control eggs were hatched and reared to 3rd instar larvae. Temephos susceptibility was tested using the WHO bioassay procedure at lethal concentration (LC) 20, LC50, LC80, diagnostic dose (twice LC99), plus an untreated control. Each concentration dose was replicated four times with 20 larvae each. The 3-day experimental group was excluded from analysis because the mortality was significantly lower than the 7-day for both the experimental and control groups. The 3 day experimental eggs displayed decreased mortality which did not align with the hypothesis, as the quiescence period elongates under optimal conditions, susceptibility to insecticides decreases, and this could have likely resulted from unintentional selection for increased fitness and faster developing eggs because the larvae that developed to 3rd instar first were those used for larvicide testing. ANOVA testing demonstrates variability in the LC80 experimental group which suggests the need for further investigation into high dose temephos concentrations. For the experimental LC20 linear regression, there were significant differences in mortality. The results indicate mortality gradually decreases when the quiescence period elongates, therefore there are significant differences in insecticide susceptibility when quiescence is 182 days (or longer), compared to when quiescence is 7 days. Further investigation into field mosquito’s genetic diversity, insecticide resistance profile, and environmental conditions should be considered.

Vector-borne diseases, such as Zika, chikungunya, dengue, and yellow fever, cause a significant portion of the global infectious disease problem, thereby representing an enormous public health threat worldwide. The threat has become more concerning as Aedes aegypti, who serve as primary vectors for these infectious diseases, continue to thrive in highly populated, urban environments. To solve this problem, insecticides have commonly been used, but this has brought forward additional issues. The overreliance on insecticides has resulted in insecticide resistant individuals emerging within once susceptible populations. Insecticide resistance in Ae. aegypti is a worldwide problem because it compromises the ability to control Ae. aegypti populations, thus increasing the spread of vector-borne diseases. With pyrethroids being commonly used worldwide, the mechanisms behind the knock-down resistance (kdr) are essential to investigate. Investigating the fitness of kdr resistant Ae. aegypti is essential in order to better understand their ability to reproduce and survive in a natural environment. Kdr resistant mutations are known to come with fitness costs: a highly energetic cost or a significant disadvantage that diminishes an aspect of the individual’s fitness. Although it is known that resistance comes with a cost, many research gaps remain. Still, it is unknown whether resistant genotypes differ in larval development times, immature survival, and adult qualities (body weight and wing length). As such, this study observed the impact of the larval development of Ae. aegypti genotypes with varying resistance at loci 1016 and 1534 of the voltage gated sodium channels. The 1016 kdr mutation results in a valine to isoleucine amino acid substitution at position 1016 (V1016I), and the 1534 kdr mutation results in a phenylalanine to cysteine amino acid substitution at position 1534 (F1534C). All strains included in this study were homozygous resistant for the 1534 mutation, while genotype varied at the 1016 locus. Mosquito strains were named after their genotype and are VVCC, VICC, and IICC. Mosquito larvae of each genotype were placed at three temperatures (22℃, 27℃, 32℃) and time to pupation, emergence, immature mortality, sex ratio, dry weight, and wing length was measured. In congruence with previous data, larval pupation and emergence occurred at a faster rate in hotter temperatures (32℃) than in colder temperatures (22℃) for all genotypes. Furthermore, the observed data shows that male mosquitos generally emerged before female mosquitos, regardless of temperature or strain. Interestingly, there were no significant differences between different genotypes in any of the fitness parameters, although the times to pupation suggest a potential trend of increased developmental time with increased resistivity. Ultimately, this data brings important implications to come up with better solutions in vector control programs in order to decrease the likelihood of adult mosquitoes becoming infected and delivering more infective bites. The study also brings light into on where future studies should take place, such as immature competition experiments, and reproductive fitness parameters in order to provide a more complete picture of the life history traits of Ae. aegypti with kdr mutations.

Aedes aegypti are vectors for common arthropod-borne-diseases (arboviruses) such as Zika, yellow fever, dengue, and chikungunya, which are of significant public health concern. The management of vectors is critical to mitigating the incidence, reemergence, and expansion of these diseases. Vector control has been complicated by the emergence of insecticide resistance within vectors, which threatens the effectiveness of control efforts. Furthermore, vector management is also complicated by the interaction between insecticide susceptibility and abiotic factors, such as temperature. While it is well-documented that environmental factors affect insecticide susceptibility, it is poorly understood how insecticide resistant vectors with different genetic backgrounds respond to insecticides at different temperatures. This study aims to establish the relationship between deltamethrin susceptibility at varying temperatures across Ae. aegypti lines that differ in their susceptibility due to knockdown resistance (kdr) mechanism. This was done through exposures using the “WHO tube test method” using simulated climate environments (22°C, 27 °C, and 32 °C) on mosquitoes of varying resistance at 1016 and homozygous resistance at 1534. This experiment is still ongoing. This study found that IICC was the most resistant genotype, VVCC the least resistant, and VICC and intermediate. There was found to be no statistically significant relationship between temperature and insecticide susceptibility across kdr genotypes.

A mutation rate refers to the frequency at which DNA mutations occur in an organism over time. In organisms, mutations are the ultimate source of genetic variation on which selection may act. However, a large number of mutations over time can be detrimental to the cell. Mutation rates are the frequency at which these new mutations arise over time. This can give great insight into DNA repair mechanisms abilities as well as the mutagenic abilities of selected factors. CRISPR-Cas9 is a powerful tool for genome editing, but its off-target effects are not yet fully understood and studied. With its increasing implementation in science and medicine, it is crucial to understand the mutagenic potential of the tool. S. cerevisiae is a model organism for studying genetics due to its fast growth rate and eukaryotic nature. By integrating CRISPR-Cas9 systems into S. cerevisiae, the mutational burden of the technology can be measured and quantified using fluctuation assays. In this experiment, a fluctuation assay using canavanine selective plates was conducted to determine the mutational burden of CRISPR-Cas9 in S. cerevisiae. Multiple trials revealed that various strains of CRISPR-Cas9 had a mutation rate up to 3-fold higher than that of wild-type S. cerevisiae. This information is essential in improving the precision and safety of CRISPR-Cas9 editing in various applications, including gene therapy and biotechnology.

The majority of the public is not aware that common objects in their backyard can be mosquito breeding sites, thus leading to an increase in mosquitoes and mosquito-borne diseases affecting humans and animals during the peak seasons. An engaging app that instructs people of all ages how to identify, prevent, and eliminate breeding sites may be of use in increasing positive behavioral changes in people, and therefore reducing available breeding sites for mosquitoes. The Embodied Games Lab in Psychology at Arizona State University created an educational game phone app using machine learning to teach students how to identify and eliminate mosquito breeding sites. Skeeter Breeder is an interactive, educational game that teaches participants about potential mosquito breeding sites and how to eliminate them from the immediate environment as documented by smartphone imagery. Currently, there is no educational game phone app that uses machine learning to teach this topic. This Thesis describes a pilot study focused on educating about common mosquito breeding sites and increasing the knowledge of 5th graders on the topic through an agentic (by taking their own pictures), engaging (game-like platform with rewards), and interactive (receiving immediate feedback on pictures) game developed from scratch at ASU.