Genome-wide studies are prone to false positives due to inherently low priors and statistical power. One approach to ameliorate this problem is to seek validation of reported candidate genes across independent studies: genes with repeatedly discovered effects are less likely to be false positives. Inversely, genes reported only as many times as expected by chance alone, while possibly representing novel discoveries, are also more likely to be false positives. We show that, across over 30 genome-wide studies that reported Drosophila and Daphnia genes with possible roles in thermal adaptation, the combined lists of candidate genes and orthologous groups are rapidly approaching the total number of genes and orthologous groups in the respective genomes. This is consistent with the expectation of high frequency of false positives. The majority of these spurious candidates have been identified by one or a few studies, as expected by chance alone. In contrast, a noticeable minority of genes have been identified by numerous studies with the probabilities of such discoveries occurring by chance alone being exceedingly small. For this subset of genes, different studies are in agreement with each other despite differences in the ecological settings, genomic tools and methodology, and reporting thresholds. We provide a reference set of presumed true positives among Drosophila candidate genes and orthologous groups involved in response to changes in temperature, suitable for cross-validation purposes. Despite this approach being prone to false negatives, this list of presumed true positives includes several hundred genes, consistent with the “omnigenic” concept of genetic architecture of complex traits.

Geographic variation in thermal tolerance in Daphnia seems to represent genetic load at the loci specifically responsible for heat tolerance resulting from conditional neutrality. We see no evidence of trade-offs between fitness-related traits at 25 °C vs. 10 °C or between two algal diets across Daphnia magna clones from a variety of locations representing the opposite ends of the distribution of long-term heat tolerance. Likewise, we found no evidence of within-environment trade-offs between heat tolerance and fitness-related traits in any of the environments. Neither short-term and long-term heat tolerance shows any consistent relationship with lipid fluorescence polarization and lipid peroxidation across clones or environments. Pervasive positive correlations between fitness-related traits indicate differences in genetic load rather than trade-off based local adaptation or thermal specialization. For heat tolerance such differences may be caused by either relaxation of stabilizing selection due to lower exposure to high temperature extremes, i.e., conditional neutrality, or by small effective population size followed by the recent range expansion.

Examples of phenotypic plasticity—the ability of organisms of identical genotypes to produce different phenotypes in response to the environment—are abundant, but often lack data on the causative physiology and biochemistry. Phenotypes associated with increased protection against or reduced damage from harmful environments may, in fact, be downstream effects of hidden adaptive responses that remain elusive to experimental measurement or be obscured by homeostatic or over-compensatory effects. The freshwater zooplankton crustacean Daphnia drastically increases its heat tolerance as the result of acclimation to high temperatures, an effect often assumed to be based on plastic responses allowing better protection against oxidative stress. Using several geographically distant Daphnia magna genotypes, we demonstrate that the more heat tolerant individuals have a higher total antioxidant capacity (TAC) both in the comparison of heat-acclimated vs. non heat-acclimated females and in the comparison of females to age- and body size-matched males, which show lower heat tolerance than females. However, experimental manipulations of hypothesized antioxidant pathways by either glutathione addition or glutathione synthesis inhibition had no effect on heat tolerance. Lipid peroxidation (LPO), contrary to expectations, did not appear to be a predictive measure of susceptibility to thermal damage: LPO was higher, not lower, in more heat tolerant heat-acclimated individuals after exposure to a lethally high temperature. We hypothesize that LPO may be maintained in Daphnia at a constant level in the absence of acute exposure to elevated temperature and increase as a by-product of a possible protective antioxidant mechanism during such exposure. This conclusion is corroborated by the observed short-term and long-term changes in phospholipid composition that included an increase in fatty acid saturation at 28 °C and up-regulation of certain long-chain polyunsaturated fatty acids. Phospholipid composition was more strongly affected by recently experienced temperature (4-day transfer) than by long-term (2 generations) temperature acclimation. This is consistent with partial loss of thermal tolerance after a short-term switch to a reciprocal temperature. As predicted under the homeoviscous adaptation hypothesis, the more heat tolerant Daphnia showed lower membrane fluidity than their less heat tolerant counterparts, in comparison both between acclimation temperatures and among different genotypes. We conclude that thermal tolerance in Daphnia is influenced by total antioxidant capacity and membrane fluidity at high temperatures, with both effects possibly reflecting changes in phospholipid composition.

The hierarchy of biological organization, from molecules to ecosystems, describes the relationships among various biological systems. Of particular interest is assessing how the factors that primarily determine the nature of one hierarchical level also have transcendent qualities that affect the ecology and evolution of higher hierarchical levels. The goal of this dissertation was to use a bottom-up approach to examine the transcendent effects of two factors that strongly determine the nature of their associated level of biological organization. The first, phenotypic plasticity, is a primary factor that determines the phenotype of an individual. The second factor, genetic diversity, largely determines the phenotypic distributions associated with populations. Controlled laboratory experiments on taxa from a freshwater tri-trophic food web were employed to examine the transcendent effects of phenotypic plasticity and genetic diversity on the biological hierarchy because relationships between individuals and populations from different trophic levels are well documented for numerous freshwater species. The results show that phenotypic plasticity can induce changes in population means and variances that promote population persistence and evolvability, and that plasticity provides a mechanistic explanation of community stability in response to changing environments. Similarly, genetic diversity may act as a signal that induces phenotypic plasticity in individuals, modulates community richness and ecosystem properties, and suggests a potential mechanism for the changes in biodiversity. Thus, results from this dissertation show that plasticity and genetic variation can shape the attributes of other biological groups higher in the biological hierarchy, and, in some cases, may also provide a mechanistic explanation for variability observed in higher levels of the biological hierarchy. These results highlight the importance of integrating traditionally disparate biological disciplines and may help to unify biology as a field.

With increasing global plastics production, the amounts of fragmenting, microscopic plastic debris (microplastics, MPs) are anticipated to rise in aquatic environments. The ecological consequences of this pollution are currently unknown. Studies are being conducted at present to assess these risks but many have been shown to be uninformative from a risk assessment perspective due to flawed and environmentally unrealistic experimental designs; the main problem being confounding effects of food dilution due to the use of particle free controls. Natural particles, such as clay are in the microplastic-size range and ubiquitous in the environment. Hence, to counteract and improve on the poor experimental design to test microplastic effects, the aim of the study was to develop a simple, high throughput screening method which accounts for naturally occurring microparticles like suspended clay in mixtures with MP. Lethal and sub lethal effects of MP alone and in mixtures at different ratios were investigated. Single particle exposures revealed a significant and up to a hundred times higher LC50 for clay compared to the plastics. Among the plastics, weathered plastics were four to five times more toxic than the pristine forms. The mixtures indicated interaction effects of all particles and revealed toxic microplastic-specific effects. A mean decrease of 19 % in the protein content across all particle types was observed after 96 h, however, one plastic type showed a higher reduction in the protein content. Testing MPs effects in mixtures with natural particulates is important due to various interaction effects but more comparative studies with environmental relevant concentrations are required in future. / <p>Master thesis is used for further publication.</p> / WEATHER-MIC, irPLAST, MICROPOLL

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The response of aquatic species to contaminants is often context dependent as illustrated by the influence that predation cues can have on the toxicity of some chemicals. We sought to gain additional insight into this interaction by examining how predation cues (alarm cue and fish kairomone) influence metabolic rate and the acute toxicity of sodium chloride and cadmium to fathead minnow larvae (Pimephales promelas) and sodium chloride to Daphnia pulex neonates. Consistent with a “flight or fight” response, the metabolic rate of fish larvae was elevated in the presence of alarm cue and growth of the minnows was also significantly reduced when exposed to alarm cue. The average 48-h LC50 for fathead minnows exposed to sodium chloride was significantly lower in the presence of alarm cue and kairomone combined as compared to tests with the salt alone. Analysis of the dose and survival response indicated alarm cue increased sensitivity of the fish to mid-range salt concentrations in particular. These results suggest an energetic cost of exposure to predation cues that resulted in enhanced toxicity of NaCl. Exposure to kairomone alone had no significant effect on salt toxicity to the minnows, which could be related to a lack of previous exposure to that cue. The acute toxicity of cadmium to the fish larvae was also not affected by the presence of predation cues which could be due to a metal-induced sensory system dysfunction or reduced bioavailability of the metal due to organic exudates from the predation cues. In contrast to the fathead minnow results, the metabolic rate of D. pulex and toxicity of NaCl to the daphnids were reduced in the presence of certain predator kairomones. This suggests an anti-predator response that enhanced tolerance to the salt. This study illustrates that the effect of predation cues on toxicity of aquatic contaminants can vary significantly based on the prey species, type of cue, and chemical stressor.

Poikilothermic organisms experience trade-offs associated with life at different temperatures caused by incompatible physiological and biochemical demands caused by temperature extremes. As the result many such organisms exhibit acclamatory effects, adjusting their metabolism and physiology to recently experiences temperatures. One such acclamatory effect is the metabolic compensation that allows an organism to withstand increases in temperature by decelerating biological rates below Arrhenius expectations, presumably reducing energetic demand and reducing stress. Daphnia magna is resilient across a wide temperature range, and if acclimated to mildly stressful temperatures first, exhibits longer survival in lethal temperatures. Certain genotypes of Daphnia also exhibit higher acute thermal tolerance than others, indicating the presence of genetic variation and local adaptation in heat tolerance. This study examined the effect of ambient temperature (5°C-37°C) and acclimation history (2 generations at 10°C or 25°C) on the oxygen consumption rates of 8 genotypes of Daphnia (4 with high acute temperature tolerance, and 4 low). There are nonlinear decelerations of Daphnia respiratory rates across a temperature gradient when acclimated to 25°C or following short 8-hour acclimation to measurement temperatures. Furthermore, Daphnia exposed to a near-lethal temperature (35°C) with a subsequent 24-hour recovery period at their native 25°C-acclimation temperature shows no indication of respiratory damage. Genotype showed no difference in metabolic compensation, indicating the process is genetically constrained. Regulation of mitochondrial and membrane function are promising areas to further characterize the mechanism of metabolic compensation found in this study.

Poikilothermic organisms experience trade-offs by differential physiological demands generated by temperature extremes. Many such organisms exhibit acclimatory effects, adjusting their metabolism and physiology to recently experienced temperatures. One such acclimatory effect is metabolic compensation, the deceleration of biological rates below Arrhenius expectations. Daphnia magna is eurythermal, and if acclimated to mildly stressful temperatures first, survives longer in lethal temperatures. This study examined the effect of ambient temperature (5°C-37°C) and acclimation history (lifetime at 10°C or 25°C) on the oxygen consumption rates of 8 genotypes of Daphnia with high or low acute temperature tolerance. There were decelerations of respiratory rates across a temperature gradient when acclimated to 25°C or following short 8- hour acclimation to measurement temperatures. Daphnia exposed to a near-lethal temperature (35°C) with a 24-hour recovery period at 25°C-acclimation temperature showed no difference in respiratory control compared to unexposed 25°C-acclimated Daphnia. Genotypes showed no difference in potential compensatory ability.