• Behavioural Thermoregulation and Energetics in Two Intermediate Hosts of Trematode Parasites

      Wang, Susan Yao Shan; Department of Biological Sciences
      Infection by macroparasites, such as trematodes (flatworms), can negatively impact survival of hosts such as larval amphibians, potentially altering host energy use in response to infection, and also through alterations of host behaviour that may increase infection tolerance or instead benefit the parasite. However, physiological consequences of macroparasite infections are not well studied, despite heavy parasite burdens in the field. The purpose of this work was to examine altered thermoregulatory behaviours in two taxa (snails and larval amphibians) used as intermediate hosts by trematodes, as well as to study the metabolism of naturally-infected tadpoles. Both infected and uninfected tadpoles (Lithobates sylvaticus and L. pipiens) and snails (Helisoma trivolvis) were placed in thermal gradients to observe thermal preferences in hosts. Oxygen consumption in naturally-infected bullfrog tadpoles (L. catesbeiana) was measured to determine whether macroparasites could impact host metabolism. The trematode-infected, L. sylvaticus tadpoles exhibited “behavioural fever” by choosing warmer temperatures by the end of the experiment compared to uninfected tadpoles, but this did not occur in L. pipiens. Active, infected snails also selected warmer temperatures relative to inactive snails and active uninfected snails. Trematode infection intensity did not affect respiration in L. catesbeiana tadpoles, but those with higher metabolic rates and larger fat bodies had lower parasite counts. These results suggest that behavioural fever may occur in ectotherms infected with macroparasites, but may be more important for species which are relatively intolerant of infection given that fever was not seen in L. pipiens. As infected snails selected warmer temperatures, this may be a case of parasite manipulation to increase production and emergence of infectious stages in warm microhabitats to facilitate transmission. Metabolic rate increased with fat body content, and larger fat bodies were observed in tadpoles with lower parasite intensity, suggesting more heavily parasitized animals had lower energy stores. Globally, infectious diseases are known to contribute to amphibian declines, thus more research is needed to understand the possible consequences of parasitism and mechanisms by which hosts to may defend themselves.
    • Flood Survival Strategies of Overwintering Snakes

      Yagi, Anne .R.; Department of Biological Sciences
      This thesis investigates snake flood survival during hibernation and how anthropogenic habitat alteration and climate variability may affect habitat quality and overwintering survival. Chapter one reviews the current understanding of ecophysiology of hibernation in snakes. In chapter two, I introduce a winter habitat model of a subterranean space that remains flood and frost-free, referred to as the “life zone,” where snakes survive winter. I analyzed 11- winters of hibernation habitat data and 18-yrs of population mark-recapture data to assess the effects of the first flood event on an endangered Massasauga population. Following the flood event, snake observations declined despite hundreds of hours of search-effort. At the population level this was evidence of poor winter survival and recruitment post flood. The direct cause of mortality was not determined but poor winter survival in areas with a depleted life zone was statistically supported. In the third chapter, I measured the metabolic rate (M ̇_(O_2 )) at 5°C for three snake species that inhabit my study area. I varied water level conditions and measured activity and dive behaviours continuously during experiments. I found differences between species in their resting metabolic rate, which I attributed to body size differences. I confirmed, cutaneous respiration occurs at a low rate and was significantly upregulated during a forced dive (flood event). Therefore, there is an intrinsic physiological response to a flood event in neonatal snakes. However, post-flood recovery indicated a greater oxygen demand after the short-forced dive. An oxygen debt was incurred during a short-forced dive under normoxic conditions. My conclusions are, 1) hibernation habitat (i.e., life zone) must include a non-freezing, non-flooding aerobic space throughout winter to maintain snake survival. 2) cutaneous respiration is a short-term flood survival strategy. I found no support for a complete aquatic hibernation strategy 3) the energy costs of a full-dive is additive to the recovery energetic costs of a flood event. A neonatal snake wintering energy budget is proposed, and winter mortality conservation issues are discussed in chapter 4.
    • Interactions with bioenergetics by the mitochondria-targeted anti-apoptotic imidazole fatty acid derivative ‘TPP-IOA’.

      Maddalena, Lucas; Department of Biological Sciences
      The mitochondrial pathway of apoptosis contributes to cell death and tissue degeneration in a variety of human diseases. Targeting the molecular events of this pathway represents a promising therapeutic strategy under pathological scenarios, particularly in tissues with limited regenerative capability (e.g. brain, heart). Recently, the small molecule ‘TPP-IOA’ was designed to target mitochondria and therein inhibit the peroxidase activity of cytochrome c that promotes activation of the apoptotic death pathway (Atkinson et al., Nat. Commun. 2011; 2:497). Therefore, TPP-IOA holds promise as a potential therapeutic agent in many pathological scenarios of cell loss. However, TPP-IOA’s target protein and organelle perform critical functions in aerobic ATP production via oxidative phosphorylation, and yet little is known about TPP-IOA’s interaction with mitochondrial energetics. This is an important consideration for TPP-IOA’s potential future therapeutic utility, as avoiding interference to mitochondrial energetics will be essential for many target cell types that feature a high rate of oxidative phosphorylation (e.g. neurons, cardiomyocytes). Using the purified protein target, isolated organelle target, and live cultured cells, this thesis investigated TPP-IOA’s interaction with fundamental components of energetics-related mitochondrial biology. Assessments with pure cytochrome c revealed that TPP-IOA can inhibit the respiration-associated reduction activity of cytochrome c, and that this occurred at concentrations marginally higher than those required to inhibit the protein’s pro-apoptotic peroxidase activity. In isolated rat liver mitochondria, TPP-IOA impaired oxidative phosphorylation via inhibition of both ADP-phosphorylating-respiration and FCCP-uncoupled respiration, and stimulation of non-phosphorylating respiration. Critically, TPP-IOA was unable to inhibit pro-apoptotic peroxidase activity at concentrations that avoided interference to oxidative phosphorylation, suggesting it may be unable to perform its desired pharmacological activity without causing toxicity to mitochondrial energetics. In live cultured cells, TPP-IOA caused effects associated with impaired oxidative phosphorylation, including a collapsed mitochondrial membrane potential, fragmented mitochondrial network morphology, and apparent loss of mitochondria. Furthermore, TPP-IOA effectively inhibited apoptotic death under lethal conditions in cells that preferentially utilized anaerobic glucose metabolism, but not in cells manipulated to be heavily reliant on oxidative phosphorylation and thus more sensitive to the perturbations to mitochondrial energetics. Altogether, these findings indicate that relative cellular dependence on oxidative phosphorylation is a critical factor influencing TPP-IOA’s pharmacological efficacy. Furthermore, potential therapeutic applications may be limited to pathologies involving predominantly less aerobic tissues/cell types or depressed aerobic metabolism. Additionally, these findings have broader implications for mitochondrial medicine based on similar mitochondrial drug targeting strategies.