I am evaluating a notion that stems from a controversial hypothesis of heat stress. The oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis predicts a positive correlation between the tolerance of hypoxia and the tolerance of heat in animals, where the notion claims that these animals must be metabolically active. To evaluate this notion, I tested heat coma recovery in several genetic lines of Drosophila melanogaster and compared it to data collected in prior studies. I hypothesized that the correlations between hypoxia tolerance and heat coma recovery would be similar to correlations found in Teague et al. (2017) and Fredette-Roman et al. (2020). After testing 65 lines from the Drosophila Genetic Reference Panel (DGRP), the notion was supported and provided evidence for the validity of OCLTT. Additional work is needed to enhance our understanding of the limitations of heat tolerance and doing such will generate more accurate models and predictions on how animals will respond to climate change.

Hypoxia-responses help coordinate the growth of oxygen-transporting tissues with the growth of other tissues during development. In Drosophila, hypoxia strongly affects development with flies being reared in a low oxygen environment showing smaller body sizes and diminished tracheal growth. The primary regulator of cellular hypoxic-responses is the hypoxia-inducible factor (HIF), and under normoxic conditions, HIF-alpha is hydroxylated by prolyl hydroxylase domain (PHD) on a proline residue inside the alpha leading to the proteins proteasome degradation downstream. However, in response to reduced oxygen, cells accumulate HIF- alpha, which then joins with the constituent HIF-beta in the cytosol, forming a HIF- alpha/beta heterodimer. Which, in turn, enters the nucleus and binds to hypoxic response elements, activating the hypoxic response genes. Hyperoxia has recently been shown to stimulates metabolic rates only at the last stage Drosophila's larval development (L3), indicating oxygen limitation occurs towards the end of development. Green fluorescent protein (GFP) was added to the oxygen-dependent domain of Drosophila HIF- Alpha (Sima) and a monomeric red fluorescent protein with a nuclear localization signal (mRFP-nls) was added to a protein under the same ubiquitin-69E promoter but is not affected by changing O2 levels. Using a Leica SP5 AOBS Spectral Confocal, third instar larvae were analyzed at the cellular level with attention focused on HIF- signaling in the central nervous system (CNS). L3 Drosophila were divided into groups of 0-12h, 12-24h, 24-48h, and 48-60h corresponding to their development. In each group, flies were either treated for 10-12 hours in 5% O2 or were left normoxic before fixation. What was overwhelmingly found is that HIF-signaling was most prominent during their early development (0-12h), with a significant decline as age increased (P=<0.001). There was also an observed hypoxic effect as animals treated in lower oxygen concentrations had significantly higher HIF signaling (P=<0.001). However, this effect still declines as larvae continued developing. This data supports the idea that internal hypoxia does not become severe during late third instar growth but may occur during the actual molt of the flies.

I am evaluating the genomic basis of a model of heat tolerance in which organisms succumb to warming when their demand for oxygen exceeds their supply. This model predicts that tolerance of hypoxia should correlate genetically with tolerance of heat. To evaluate this prediction, I tested heat and hypoxia tolerance in several genetic lines of Drosophila melanogaster. I hypothesized that genotypes that can fly better at high temperatures are also able to fly well at hypoxia. Genotypes from the Drosophila Genetic Reference Panel (DGRP) were assessed for flight at hypoxia and normal temperature (12% O2 and 25°C) as well as normoxia and high temperature (21% O2 and 39°C). After testing 66 lines from the DGRP, the oxygen- and capacity-limited thermal tolerance theory is supported; hypoxia-resistant lines are more likely to be heat-resistant. This supports previous research, which suggested an interaction between the tolerance of the two environmental variables. I used this data to perform a genome-wide association study to find specific single-nucleotide polymorphisms associated with heat tolerance and hypoxia tolerance but found no specific genomic markers. Understanding factors that limit an organism’s stress tolerance as well as the regions of the genome that dictate this phenotype should enable us to predict how organisms may respond to the growing threat of climate change.

Species survive by adapting to what is demanded by their environment. In constant and fluctuating environments, specialist and generalists should be favored, respectively. However, the costs and benefits of adaptation can depend on a variety of factors that alter the intensity of the specialist-generalist trade-off. We examined flight performance to determine how well flies that evolved in constant and fluctuating temperatures acclimated to hot and cold temperatures. We predicted that flies would perform best at temperatures most similar to the ones the flies evolved at. Best performance was found when rearing and testing temperatures aligned with the temperature at which a genotype had evolved, with the generalist sharing the best and worst performance combination with the constant thermally evolved flies. Interestingly, evolved and reared temperatures had equal impact on flight performance. It was also observed that rearing at 25°C resulted in flies with the best fitness. These results contribute to the specialist-generalist theory and the idea that long term cold development is restricting in terms of range for thermal performance.

Animals are thought to die at high temperatures because proteins and cell membranes lose their structural integrity. Alternatively, a newer hypothesis (the oxygen and capacity limitation of thermal tolerance, or OCLTT) states that death occurs because oxygen supply becomes limited at high temperatures. Consequently, animals exposed to hypoxia are more sensitive to heating than those exposed to normoxia or hyperoxia. We hypothesized that animals raised in hypoxia would acclimate to the low oxygen supply, thereby making them less sensitive to heating. Such acclimation would be expressed as greater heat tolerance and better flight performance in individuals raised at lower oxygen concentrations. We raised flies (Drosophila melanogaster) from eggs to adults under oxygen concentrations ranging from 10% to 31% and measured two aspects of thermal tolerance: 1) the time required for flies to lose motor function at 39.5°C at normoxia (21%), referred to as knock-down time, and 2) flight performance at 37°, 39°, or 41°C and 12%, 21%, or 31% oxygen. Contrary to our prediction, flies from all treatments had the same knock-down time. However, flight performance at hypoxia was greatest for flies raised in hypoxia, but flight performance at normoxia and hyperoxia was greatest for flies raised at hyperoxia. Thus, flight performance acclimated to oxygen supply during development, but heat tolerance did not. Our data does not support the OCLTT hypothesis, but instead supports the beneficial acclimation hypothesis, which proposes that acclimation improves the function of an organism during environmental change.

One hypothesis for why insects are smaller than vertebrates is that the blind-ended tracheal respiratory system challenges oxygen delivery for larger insects. Supporting this hypothesis, several studies have documented that larger insect species have larger gas transport structures than expected by isometric scaling. To further test this hypothesis, we performed the first inter-specific study of the scaling of spiracle size, using ten scarab beetle species, including some of the most massive insects. Using micro-CT, we measured the cross sectional area and depth of all eight spiracles. Areas of large spiracles in the anterior portion of the animal showed hypermetric scaling, varying approximately with mass^0.8. However, because diffusive capacities scaled with lower slopes than metabolic rates, larger beetles had a 10-fold higher required PO2 gradient across the spiracles to sustain oxygen consumption by diffusion. Despite this trend, calculations suggest that large beetles can exchange oxygen by diffusion across the spiracles at rest, but likely no beetles can do so during flight. Advective capacities through the spiracles scale with mass^1.8, suggestive of a switch toward greater use of convection and/or reduced required pressures in larger beetles.