Physiological and Molecular Responses to Environmental Stressors in Emerging Eco-molecular Models

Abstract

The perception of stress and the stress responses of animals are complex. My dissertation examines stress response in the context of different environmental stressors. Ecosystems host a multitude of stressors that impact the organisms inhabiting them. It is understood that different environmental stressors can elicit varied effects on these organisms. This raises the question: To what degree is the stress response uniform, or is the stress response specifically adapted to each type of stressor? Research on vertebrates shows that in response to many different stressful situations, levels of corticosterone/cortisol produced by the endocrine system will rise in a similar manner. However, stress responses are multifaceted, involving numerous biological levels, with the endocrine system being just one component. My dissertation aims to characterize sources of variation in stress response using diverse approaches in both vertebrate and invertebrate organisms. I utilize the Eastern fence lizard, Sceloporus undulatus, in my second and third chapters. The fence lizard is an emerging ecological model organism used to understand the molecular and physiological mechanisms of stress response and how ectotherms respond to their environments through climate change or invasive species. In the second chapter, I aim to investigate the relative effects of acute heat exposure and inadvertent stress from experimental procedures on female fence lizards using two experiments. In the first experiment, lizards were exposed to a temperature of 42.5°C for up to 1.5 hours, followed by a 1-hour recovery period without heat exposure. We predicted that increased exposure duration at high temperatures would elevate corticosterone (CORT) levels, which would then decrease during the recovery period.Contrary to our predictions, CORT levels remained elevated and showed significant variation between individuals. To further understand these results, I conducted a secondary experiment testing the hypothesis that stress from general experimental procedures, such as handling, in part explains the CORT response. In this experiment, lizards were exposed to a control temperature of 30°C or a heat stress temperature of 43°C, with exposure times set to either 60 minutes or 90 minutes. The results indicated that control temperatures alone raised CORT levels above baseline, suggesting that the experimental procedure itself induced a measurable stress response. I identified heat shock protein gene expression as a specific biomarker for heat stress, noting an elevation in heat shock protein 70 (HSP70) levels, suggesting the presence of heat stress in the animals. These data support the concept that while CORT may serve as a general indicator of stress, future research should focus on more precise biomarkers tailored to the specific stressors being investigated in experiments to understand if their treatments have an effect. The findings and experimental approach from chapter two lay the groundwork for my third chapter, whose aim was to identify shared and unique responses to two ecologically relevant stressors—acute heat and fire ant envenomation—in male fence lizards. At the endocrine level, both stressors showed a shared response of elevated CORT. At the liver transcriptome level, I found a very robust response to heat stress, having many differentially expressed genes, such as heat shock proteins. However, the response was relatively mild in the fire ant group compared to the control, supporting the idea of unique responses. Several commonly expressed genes associated with the upregulation of CORT were identified in both treatment groups, including NR4A1, FOS, and DUSP1. These genes were further validated through their significant correlation with the CORT-linked module identified via weighted gene correlation network analysis (WGCNA). At the pathway level, stress-induced cellular pathways like the P53 pathway and Tumor Necrosis Factor Alpha (TNFA) signaling were shared across both treatment groups. Interestingly, the fire ant treatment downregulated pathways related to immune responses, such as the complement system. Meanwhile, glycolysis—a process for the metabolic breakdown of glucose to produce energy—was upregulated in the heat treatment group. Glucocorticoid receptor motifs were identified in promoter regions of differentially expressed genes and regions of the genome that became accessible due to heat stress. To investigate the heat stress response further, ATAC-seq was used for differential peak analysis to identify genome regions accessible for transcription. We overlapped the glucocorticoid response element (GRE) sites, differentially expressed genes, and differentially accessible regions. This analysis suggests that in response to heat stress, epigenetic modifications facilitate genome accessibility, enabling the glucocorticoid receptor (GR) to bind to the GRE and modulate the transcription of adjacent genes by either upregulating or downregulating their expression. These methodologies examine the transcriptomic and epigenetic responses to these two independent natural stressors and support the idea that stress responds at multiple biological levels and at the genomic level these responses can be largely unique. In the fourth chapter, we shift our focus from examining how animals respond to stressors to exploring the interactions between genotype and environments and the divergence in stress responses among animals with different genetic backgrounds. Daphnia is a well-known model organism in ecology and eco-toxicology, predominantly found in freshwater habitats such as lakes and ponds, and they can survive extreme environments like acidic swamps. Due to its phenotypic plasticity, this organism is extensively utilized to study both natural and anthropogenic environmental stressors. The diverse phenotypes exhibited by Daphnia are strongly influenced by their environment, making it an ideal model for addressing evolutionary genomic questions. Strains of Daphnia pulicaria show varying levels of tolerance to the toxic cyanobacterium Microcystis aeruginosa, which is known for producing hepatotoxins called microcystins. D. pulicaria strains from nutrient-rich lakes have greater tolerance to M. aeruginosa compared to their counterparts from nutrient-poor lakes, which are more susceptible to microcystins. This chapter aimed to assess the effect of microcystins on lifespan and reproduction across different strains of D. pulicaria. I hypothesized that the toxin-produced by M. aeruginosa will reduce the lifespan and reproductive rates of D. pulicaria strains. I predicted tolerant strains would exhibit better survival and reproductive outcomes than susceptible strains. I conducted two replicate experiments where I tested four strains of D. pulicaria, two characterized as tolerant and two as susceptible, exposing them to either a strain of M. aeruginosa that was previously reported to produce the microcystin or a strain for which the microcystin gene was knocked out and could not produce the microcystin, measuring survival, time to reproduction, and lifetime fitness. While the M. aeruginosa microcystin treatment demonstrated the predicted results, interestingly, we observed a significant decrease in life-history traits for all D. pulicaria strains when exposed to the M. aeruginosa that could NOT produce the microcystin. I conclude there may be a molecular component other than general microcystins (toxins) produced by the M. aeruginosa that led to significant mortality in D. pulicaria strains. Future directions of this project will involve investigating the genetic components of both M. aeruginosa genomes to identify potential drivers responsible for the observed decline in fitness within “nontoxic” Microcystis strains. My dissertation investigated stress in a vertebrate model by comparing different types of stressors and in an invertebrate model by examining evolved differences in stress responses across genotypes. Exploring stress responses at multiple biological levels provides a deeper understanding of stress and how organisms interact with their complex environments.