Daphnia Hypoxia Closing Worksheet 10 points 3-1-24 (1)

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Apr 3, 2024

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Daphnia Hypoxia Closing Worksheet 10 points Day 2 Welcome back! Let’s begin by recording the same data that we did in day 1 of our experiment. Carefully obtain your daphnia from the instructor and record the data carefully. Daphnia Behavioral Observations Day 2: (1 points) Please observe at least 3 different beakers of Daphnia (roughly 30 individuals) and fill out the following chart. Include which treatment they received as well! Behavior Control (# of individuals) Experimental (# of individuals) Active (Swimming vertically or horizontally, rocking, rolling) 5 vertically 8 swimming vertically Stationary 5 dead, 4 alive 2 dead Daphnia Color Phenotype Day 2: (1 points) Please observe the color phenotype of our Daphnia before the experiment begins. If the Daphnia appear to be completely clear in color you’d mark 0. When any pink/red shows, follow the scale below from 1 to 5 in intensity. Please view at least 30 individuals. Include which treatment they received as well! Color Number Control (# of individuals) Experimental (# of individuals) 0 10 0 1 0 10 2 0 0 3 0 0 4 0 0 5 0 0 RNA Isolation, Reverse Transcription, PCR, and Gel Electrophoresis Now that we have our hypoxic Daphnia and we’ve recorded the visible phenotypes, it’s time to isolate the RNA from our Daphnia to analyze the gene expression. Please follow the RNA isolation protocol as found in the lab manual. After isolating the RNA, we’ll then convert it into a usable product called complementary DNA (cDNA). We can then run a polymerase chain reaction (PCR) to analyze the expression of HIF, Hemoglobin, and Beta tubulin genes. For the Reverse Transcription and PCR protocols please see the lab manual (you’ll also find the specific annealing temperatures that we’ll need to perform the PCR). Once the PCR is finished, we’ll run an agarose gel and examine the gene expression changes that may have happened in exposing our Daphnia to hypoxic conditions. 5 4 3 2 1

Review Questions 1. What specifically causes HIF1 (known as HIF1-alpha in Homo sapiens) to become activated from hypoxic conditions? (Include the biochemistry behind the mechanism of action for this amazing transcription factor). (1 points) The HIF1 is determined by the levels of HIF1-α proteins and any inhibitors. HIF1-α proteins are bound by another protein that uses O2 as a substrate. When under hypoxic conditions, this binding protein (PHD2) is ineffective and the unbound HIF1- α proteins initiate the activation of HIF1. 2. Dense population size is one way to decrease the oxygen content of water, can you think of other stimuli occurring in nature that may result in hypoxic conditions for Daphnia? (1 points) If there are other gases in the water instead of oxygen like nitrogen, then it will create hypoxic conditions. 3. What changes did you observe in the behavior of Daphnia after they were exposed to hypoxic conditions? (1 points) They moved in a vertical direction, and they survived better than the control. 4. Any changes in the phenotype of Daphnia color? How would this change if we were to expose them to a lower oxygen concentration? (1 points) The Daphnia in hypoxic conditions were slightly darker than the non-hypoxic conditions. The Daphnia in non-hypoxic conditions were all clear. If they had an even lower oxygen concentration, then they would probably be even darker. 5. RNA interference is a way to functionally silence the expression of specific genes. What would happen if we were to use RNAi to silence HIF and then subject our Daphnia to hypoxic conditions? (1 points) Since HIF is the protein that allows them to survive in hypoxic conditions and subsequently turn pink, the lack of it would cause them to die. 6. What types of changes did you see in gene expression of HIF, Hemoglobin, and Beta tubulin? What other genes may have their expression modulated in response to hypoxic conditions? (1 points) In the control, actin and HIF were not expressed, but GAPDH and HB were with a faint band. For the experiment both actin and HIF were expressed with a strong band, and the other two were not.

7. If you were continuing this research project, where would you go from here? What are the future directions you could explore with this project? (1 points) In the future, more research could be done on ways to create hypoxic conditions. Experiments could be done to create hypoxic conditions of varying degrees and compare the results. A variety of methods could also be used so that there is more data than just the nitrification of water. 8. What went well with your experiment? What do you wish you could improve if you were to do it again? (1 points) The survival rate of hypoxic daphnia went well. We also had four bands from the gel electrophoresis. Next time, the amount of nitrogen added to the water should be increased by increasing the time it is treated. Also, conditions should be improved so that all the control daphnia should not die. References: 1. Kring RL, O’Brien WJ. 1976. Effects of Varying Oxygen Concentrations on the Filtering Rate of Daphnia pulex. Ecology 57(4):808-814). 2. Gorr TA, Cahn JD, Yamagata H, Bunn HF. 2004. Hypoxia-induced Synthesis of Hemoglobin in the Crustacean Daphnia magna is Hypoxia-inducible Factor-dependent. The Journal of Biological Chemistry 279(34):36038-36047. 3. Zhang H, Ludsin S, et al. 2009. Hypoxia-driven changes in the behavior and spatial distribution of pelagic fish and mesozooplankton in the northern Gulf of Mexico. Journal of Experimental Marine Biology and Ecology 381(1):S80-S91. 4. Andrewartha SJ, Burggren WW. 2012. Transgenerational Variation in Metabolism and Life-History Traits Induced by Maternal Hypoxia in Daphnia magna. Physiological and Biochemical Zoology 85(6):625-634.

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