Early exposure to hypoxia is related to a variety of physiological and metabolic changes that have lasting effects on organisms’ physiology and life history. We measured the effects of maternal and embryonic mild, intermittent hypoxia on the life history of four clones of microcrustacean Daphnia magna, an emerging model organism for the studies of senescence and longevity. Daphnia individuals were produced parthenogenically, maintained in individual vials, and fed standard algal concentration daily. The cohort consisted of 189 individuals. We measured body size at first reproduction, fecundity (including late-life fecundity peak), offspring sex ratio, and longevity. We found no effect of maternal and embryonic hypoxia on body size and longevity; however, there was a slight but statistically significant increase in age-specific mortality in the early hypoxia treatment cohort. Daphnia from the hypoxia group showed higher early fecundity which disappeared by the age of 100 days. A late-life spike in fecundity was observed at the age of 100 days when hypoxia group individuals showed significantly lower fecundity. There was little evidence of a trade-off between early- and late-life fecundity. Finally, early hypoxia affected mid-life male production in one of the four clones, and we discuss possible physiological changes triggered by maternal and embryonic exposure to hypoxia.

The mitochondria are organelles where energy in the form of ATP (Adenosine Triphosphate) is produced. During low oxygen supply (hypoxia) and mitochondrial uncoupling, ATP synthesis is reduced and AMP (Adenosine Monophosphate) accumulates in the cells. This increase in AMP: ATP ratio stimulates the AMP-Activated Kinase (AMPK) pathway, known to improve healthspan and lifespan by increasing mitochondrial biogenesis (making new mitochondria), decreasing oxidative stress and inflammation, and proteotoxicity (by degrading non-functional organelles and proteins). Here, the life/healthspan extending potential of chronic mild intermittent hypoxia (CMIH) and mitochondrial uncoupling using 2,4 -Dinitrophenol (DNP) was investigated in an emerging model organism, an aquatic crustacean, Daphnia magna. First, the effect of CMIH (4mgO2/L) on longevity in four different genotypes of Daphnia magna was investigated. All individuals were kept in similar conditions with controls in normoxia (8mgO2/L). Hypoxia was created by bubbling compressed nitrogen gas through the water twice daily. Survival was assessing through censuses conducted every 3 days and gene expression changes in response to CMIH were assessed by RNA sequencing using Oxford Nanopore Technology. Briefly, RNA was isolated from genotypes after hypoxic treatments and reverse transcribed to cDNA, libraries were multiplexed and sequenced using Oxford Nanopore MinION for 24-48 hours. Lastly, the effect of prolonged exposure to DNP on longevity was evaluated. Daphnia were chronically exposed to either 0 (control), or 0.1, 1, and 5μM of DNP. Genotypes displayed different tolerance to hypoxia and DNP treatments. Contrary to the expectations, CMIH and DNP reduced longevity, but only in genotypes from permanent ponds, while having no effect on the survival of genotypes from intermittent ponds, arguably better adapted to naturally occurring hypoxic conditions. We uncovered 11 candidate genes that were differentially expressed in these genotypes. In particular, genes involved in mTOR, p53, and sirtuin pathways showed patterns of expression consistent with protection against hypoxia. These pathways are known to regulate autophagy, apoptosis, inflammation, and cell cycle. Because our findings elucidate genotype-specific physiological and transcriptomic responses to respiratory perturbations (CMIH and DNP) we may be able to make a step towards the understanding of a model organism’s response to respiratory stress.

The diploma thesis is primarily focused on the standardization of Daphnia magna breeding in laboratory conditions of the Department of Zoology, Fisheries, Hydrobiology and Beekeeping at the Faculty of Agronomy of the Mendel University in Brno. The main aim of the work is to simplify the preparation of medium for breeding Daphnia magna due to time saving. During testing, we were interested in births and mortality of individuals observed during 14-day tests in two media. We focus on chemical and physical properties of the media, namely the pH, temperature and intensity of the light.

The aim of this thesis was to derermine the values of toxic effect of chemical sub-stance chemical on the tested organism Daphnia magna. For testing, these materials were selected: polyaluminiumchloride (PAX 18), its aqueous solution (PAX 19) and potassium dichromate. The thesis is divided into two parts, in the first half I devote to theory of the basic terminology of the issue and the general characterization of the tested organism. In the practical part I focus on the characteristics of the used substances, the course of the tes-ting and the results of the work together with the discussion. Experimental concentrations for PAX 18 were between 1-100 mg.l-1, for PAX 19 10-300 mg.l-1 and for potassium dichromate 40-150 mg.l-1. Testing was perfomed in 3 repeatings with ten individuals + control. 24hEC50 values were: for PAX 18 45,06 mg.l-1, for PAX 19 97,82 mg.l-1 and for potassium dichromate 1,569 mg.l-1. The values of 48hEC50 for PAX 18 byly 22,62 mg.l-1, for PAX 19 37,20 mg.l-1 and for potassium dichromate 0,864 mg.l-1. In all tests, fluctuations in pH and dissolved oxygen content, as well as the number of inhibited individuals, were observed. The results of the observations were plotted and then commented verbally.

Hypoxia has become a subject of interest among the many environmental stressors as its role in biology is complex and diverse. Hypoxia is a significant low oxygen condition that causes many pathologies and adaptive responses in organisms. It can lead to a moderate or dangerous loss of respiration and can be an indication of tumorigenesis as many tumors lack adequate blood supply. Organisms possess adaptive responses to hypoxia that include hypoxia-inducible factors (HIFs) that activate several downstream pathways that are responsible for altering metabolism and maintaining homeostasis. Within aquatic organisms, hypoxia is an important ecological constraint as oxygen availability within bodies of water can vary greatly over time and space. Therefore, adaptation to hypoxia is likely pervasive, especially in genotypes originating from bodies of water that are prone to hypoxia. Here we report the transcriptional response to acute hypoxia in the clonal freshwater crustacean Daphnia magna. Daphnia were subjected to 1mg/O2 for 12 hours. Then, RNA was extracted, reverse transcribed, and sequenced using Oxford Nanopore MinION. We find that severe hypoxia significantly up-regulates key enzymes in the gluconeogenesis pathway. Additionally, we report genotype-by-environment interactions showing that Daphnia clones from habitats that are hypoxia prone survive better in hypoxia.

Although the microcrustacean Daphnia is becoming an organism of choice for proteomic studies, protein expression across its life cycle have not been fully characterized. Proteomes of adult females, juveniles, asexually produced embryos, and the ephippia-resting stages containing sexually produced diapausing freezing- and desiccation-resistant embryos are analyzed. Overall, proteins with known molecular functions are more likely to be detected than proteins with no detectable orthology. Similarly, proteins with stronger gene model support in two independent genome assemblies can be detected, than those without such support. This suggests that the proteomics pipeline can be applied to verify hypothesized proteins, even given questionable reference gene models. In particular, upregulation of vitellogenins and downregulation of actins and myosins in embryos of both types, relative to juveniles and adults, and overrepresentation of cell-cycle related proteins in the developing embryos, relative to diapausing embryos and adults, are observed. Upregulation of small heat-shock proteins and peroxidases, as well as overrepresentation of stress-response proteins in the ephippium relative to the asexually produced non-diapausing embryos, is found. The ephippium also shows upregulation of three trehalose-synthesis proteins and downregulation of a trehalose hydrolase, consistent with the role of trehalose in protection against freezing and desiccation.

Ecotoxicology aims to understand toxic effects of chemicals in the environment. Effects can be observed at different levels of the biological organization, from molecular to ecosystem level. Biomarkers on molecular and biochemical levels are used in ecotoxicology as early warning signals of chemical exposure, possible toxic effects and underlying mechanisms. As methods and technologies improve, more biomarkers are being implemented in ecotoxicological studies, due to the general interest in early detection and thus efficient prevention of environmental risks. However, to be of value in ecotoxicological assessment, a connection between biomarker response and effects at higher levels of biological organization should be established. Also, baseline variability for the biomarker in question as well as response to natural fluctuations of environmental factors should be evaluated. The aim of this thesis was to increase value and understanding of biomarkers in ecotoxicological assessment by (1) linking responses across different levels of biological organization, and (2) gaining better understanding of the relative importance of ecological and physiological factors affecting oxidative biomarkers. Paper I is focused on evolutionary conserved drug targets and the toxicity of pharmaceuticals for non-target organisms. The main conclusion from this study is that pharmaceuticals with conserved drug targets in non-target organisms have a higher toxicity than pharmaceuticals for which drug-targets have not been identified in the species. The effects were evaluated using end points at molecular, biochemical and individual levels. Consistent with the expected higher sensitivity of molecular and biochemical end points, the effects on the low-level biomarkers were observed at lower concentrations than at the individual level.   Paper II is focused on delineating effects of feeding and toxic exposure on oxidative biomarkers commonly used in ecotoxicology. The results are in agreement with the theory of caloric restriction that links enhanced caloric intake to increased pro-oxidative processes in animals. In our experiments with the cladoceran Daphnia magna, we observed positive effects for both antioxidant capacity and oxidized lipids in response to enhanced feeding rates. This have implications for the use of oxidative stress biomarkers in ecotoxicology as many substances have inhibitory effects on feeding rate and thus, changes in oxidative biomarkers can result from the altered feeding rate rather than other toxic mechanisms. Therefore, possible changes in feeding rate should be assessed when conducting exposure experiments or interpreting field data in studies employing oxidative stress biomarkers. However, it was concluded that the ratio between antioxidative capacity and protein content was independent of feeding rate. Thus, this biomarker is suitable for xenobiotic exposure in D. magna. This thesis have contributed to better understanding of molecular and biochemical biomarkers in ecotoxicological studies in regard to the connections between effects at different biological levels and confounding factors in biomarker response. / <p>At the tiem of defence the following papers wer unpublished and had a status as follows:</p><p>Paper 1: Manuscript; Paper 2: Manuscript</p>

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Climate change has caused an increase in extreme rain events and flooding in certain regions across the globe. During rain events, water flows over impervious surfaces structures such as roads and sidewalks, picking up contaminants such as metals, fertilizers and other nutrients, and various organics that which may impact organisms in such as streams, river, and lakes. Previous work has found significant differences in survival of organisms that were exposed to contaminated stormwater runoff. This study investigated stormwater chemistry parameters at collection sites on the East Tennessee State University campus. Sites were selected based on the extent of human interaction and traffic in the areas. Additionally, acute toxicity of stormwater samples was investigated through 48-h bioassays with the cladoceran, Daphnia magna. In September and November 2022, water chemistry and toxicity analyses were conducted across multiple rain events and over a six-hour time course of an individual rain event. For each of the events and the time course, chlorophyll levels, specific conductivity, pH, temperature, and dissolved oxygen were measured. No statistical difference between the water chemistry parameters between sampling sites or between rain events were observed. Additionally, no significant differences in 48-h survival of D. magna were detected between sampling locations or during the single event time course study. These data suggest that there were no pollutant surges at the collection sites and that D. manga survival was not affected by the contaminants.

No description available.