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.

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.