Juvenile and Adult Daphnia Magna Survival in Response to Hypoxia

Juvenile and adult Daphnia magna survival in response to hypoxia

Erica Strom University of Minnesota Duluth

University Honors – Senior Capstone Spring 2015 UROP Project

Faculty Advisor: Dr. Donn Branstrator

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Abstract This study was undertaken to determine survival of juvenile and adult Daphnia magna under hypoxic conditions in comparison to its predators. Because D. magna perform diel vertical migration to avoid visually-oriented predators, they spend a significant portion of the day at depth where light and oxygen levels are low. In this series of experiments, Daphnia magna were exposed to low dissolved oxygen concentrations and assessed for survival. Juvenile D. magna were hypothesized to tolerate a lower dissolved oxygen concentration than adults because of their smaller size and presumed lower oxygen consumption. The dissolved oxygen concentration lethal to 50 percent of both juvenile and adult D. magna was found to be 0.47 mg/L, which is significantly lower than the minimum dissolved oxygen limits experienced by its predators.

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Introduction Daphnia magna (Crustacea: Cladocera) is a species of zooplankton that resides in freshwater lakes, mainly in northeastern North America (EPA 1985). At night, D. magna feed on algae near the surface, but during the day they migrate lower into the water column to avoid predation by visually-oriented fish – a behavioral pattern called diel vertical migration, DVM (Lampert 1989). Going deeper into a lake can expose D. magna to a hypoxic (low-oxygen) environment, as well as a lower light and temperature levels (Wetzel 2001). Prolonged exposure to hypoxia can induce hemoglobin production, causing some Daphnia to turn red (Gorr et al. 2004). D. magna and other similar species are an important food source for small fish and carnivorous zooplankton such as the invasive spiny water flea, Bythotrephes longimanus (Pangle and Peacor 2006). In a series of hypoxia studies it was shown that Bythotrephes longimanus can survive dissolved oxygen concentrations as low as 1.75-2.25 mg/L and Leptodora kindti can handle dissolved oxygen concentrations as low as 0.75-1.25 mg/L (UMD Graduate Student Mike Sorensen, unpublished data). In comparison, most fish species cannot withstand dissolved oxygen levels below 2 mg/L (Vanderploeg et. al. 2009). A better understanding of D. magna and other zooplankton hypoxia tolerances could help to develop better management techniques for aquatic invasive species. Because D. magna is not native to Minnesota, the results of this study could be used in assessing the risk of impact D. magna may have if it were to spread to Minnesota lakes. Daphnia magna was chosen for this study for a number of reasons. Collecting organisms from the field was not feasible for this project since it was conducted during winter months, but D. magna clones can be cultured in a laboratory setting throughout the year. D. magna is a relatively large species of zooplankton, which makes viewing and transferring them easier than with smaller species, such as Ceriodaphnia dubia (Christine Polkinghorne, personal communication). The Daphnia species most common in Minnesota are Daphnia mendotae, Daphnia pulicaria, and Daphnia retrocurva (Hirsch 2014). Daphnia magna are studied as a surrogate species when other Daphnia species are unavailable, and have been the subject of a wide variety of studies (Goto et. al. 2012). D. magna are commonly used in toxicity testing and other environmental experiments for similar reasons (Adema 1978, Seda and Petrusek 2011). The purpose of this study was to determine the LC50 of juvenile and adult D. magna under conditions of fish-based hypoxia (≤ 2 mg/L). The LC50 (lethal concentration) is the exposure treatment at which 50% of the test organisms die. It was hypothesized that juvenile and adult D. magna would display different hypoxia tolerance limits. Juvenile D. magna are much smaller than adult D. magna, so they presumably respire at a slower rate, consuming less oxygen. Juveniles were expected to tolerate a lower

STROM Daphnia magna Hypoxia dissolved oxygen concentration than adults. Overall, D. magna was hypothesized to tolerate a lower dissolved oxygen concentration than its predators.

Materials and Methods Study organism Daphnia magna were provided by the Lake Superior Research Institute (LSRI) at the University of Wisconsin - Superior. Organisms were cultured at 23°C ± 2°C, under a 16 hour light: 8 hour dark photoperiod, in hard reconstituted water prepared following US EPA standard operating procedures for use in amphipod and cladoceran culturing (Christine Polkinghorne, personal communication). At LSRI, D. magna cultures were fed algae (Selenastrum 8 capriconutum; 1.0 x 10 cells/mL) and YCT (yeast, Cerophyll leaves and trout chow; 1800 mg/L), each at a concentration of 7 ml/L.

Water source Water used for the experiments was collected from Pike Lake, Duluth, Minnesota because of the lake’s easy winter access (experiments were conducted January through April 2015) and its previous use in Mike Sorensen’s hypoxia study. To collect the water, an auger was used to drill a hole in the ice and 1000-ml beakers were lowered into the hole to fill with lake water. Water was poured through 65 μm mesh filters into three 20-L carboys for transport to the lab. Water was collected 1-2 days before the start of a trial, and was stored in an incubator at 10°C. Because Daphnia magna cultures were raised in hard reconstituted water, it was necessary to gradually acclimate them to Pike Lake water and the lower temperature. Once cultures were obtained from LSRI in hard reconstituted water, culture water was diluted (50:50) with filtered Pike Lake water and the cultures were put into the incubator at 10°C to acclimate for at least 24 hours.

Heart rate measurements The metabolic rate of D. magna was measured here through measurement of heart rate. The heart rates of 1-, 2-, 5-, and 7-day old Daphnia magna were measured at room temperature, 20°C. Using an eye dropper, one organism at a time was placed on a damp concavity slide and viewed using a Leica MZ125 dissecting microscope. The number of heart beats per 10 seconds was tallied, and the value was recorded. The value was multiplied by 6 to obtain the heart rate in beats per minute. The microscope light was turned off prior to counting, since intense light can be a cause of stress in daphnids (Goto et. al 2012).

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Reducing dissolved oxygen in the water The concentration of dissolved oxygen in water can be quickly lowered by sparging with nitrogen gas. To accomplish this, nitrogen gas (Praxair Distribution Inc.) was streamed from a tank through plastic tubing to an air stone submerged in a 10-L carboy of filtered Pike Lake water in an incubator (Percival Intellus environmental controller) at 10 °C. An oxygen meter (YSI 5000) was used to monitor dissolved oxygen concentration about 5 cm below the water surface until the desired concentration was reached. The sparging process took from 15 to 35 minutes, depending on the targeted dissolved oxygen concentration.

Exposing Daphnia magna to low dissolved oxygen Plastic tubing was used to siphon sparged water into 300-mL glass BOD bottles. Bottles were filled from the bottom to avoid introducing excess oxygen via air mixing. Each bottle was allowed to overflow before stoppering. Each trial consisted of four dissolved oxygen treatments: saturation (about 10 mg/L), 0.6 mg/L, 0.4 mg/L, and 0.2 mg/L. Ten 300-mL bottles were assigned to each treatment. Daphnia were divided into culture wells so that 5 individuals could quickly be pipetted into each bottle. The number of individuals per test bottle was chosen for ease of transfer and because the density (5 individuals per bottle) was calculated to be very low relative to natural densities. Daphnia can reach population densities up to 200 to 500 individuals per liter in its natural environment (Pennak 1987), which translates to 60 to 100 individuals per 300-mL BOD bottle. Bottles containing D. magna at four levels of dissolved oxygen were left in the incubator overnight for 12 hours at 10°C. The duration of 12 hours was chosen to simulate the average number of hours each day that Daphnia spends at depth to stay out of sunlight and avoid predators. The trial was performed on juvenile D. magna (approximately 2 days old) and adult D. magna (approximately 2 weeks old).

Pre- and post-trial comparisons, Methods comparisons Pre- and post-trial dissolved oxygen measurements were taken in order to determine if dissolved oxygen concentration in the BOD bottles remained steady over the 12-hour trial period. Comparisons were also made between the oxygen meter (YSI 5000), a field probe (YSI 85 Oxygen, Conductivity, Salinity and Temperature), and the Winkler method. Six BOD bottles were filled with sparged water and measured first with the field probe, then with the YSI 5000 DO meter, and finally with the Winkler method. The Winkler method is a very accurate, wet chemistry approach for measuring dissolved oxygen concentration in lake water. It was performed in accordance with methods listed for a 300-mL BOD bottle in the Azide Modification of Winkler Method document (Hach Company 2014).

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A comparison of different dissolved oxygen measurement techniques was done in order to verify that subsequent DO measurements were accurate and consistent. Inconsistent DO values would have produced inaccurate results when reporting the LC50. It was important to calibrate the YSI 5000 meter to be accurate since it was the main method of DO measurement used for the experiment.

Survival analysis and LC50 At the end of each 12 hour trial, each individual was assessed for survival using a dissecting microscope (Leica MZ125). An individual was determined to be a survivor if it was actively swimming or if a heartbeat could be observed. Post-trial dissolved oxygen levels were recorded in half of the tested bottles. After assessment, individuals were preserved in 70% ethanol for further study. All data were entered into Microsoft Excel for analysis. Survival percentages were calculated for each bottle. The dissolved oxygen concentration lethal to 50 percent of the population (LC50) was calculated using the EPA WET Analysis Spreadsheet (EPA 2000). The level of oxygen at which water at 10°C is saturated was calculated to be 10.59 mg/L via a publicly-available Excel spreadsheet published by YSI. For each treatment of ten bottles, the measured post-trial dissolved oxygen concentration was averaged, and this value was subtracted from the saturation DO level to obtain a standardized dissolved oxygen concentration for use in the WET model. Example: Saturation DO at 10°C: 10.59 mg/L Average post-trial DO: 10.03 mg/L Saturation – Average= 10.59 mg/L – 10.03 mg/L = 0.56 mg/L This was done since the WET model interprets the lowest concentration to be the control, while in this experiment the control was the highest concentration. The number of organisms per bottle and number of survivors per bottle were entered into the WET model for each standardized DO concentration, and the LC50 and upper and lower 95% confidence intervals were calculated. The calculated LC50 was then converted to actual LC50 by subtracting the calculated LC50 from the saturation DO. Confidence intervals were converted as well. Example: Saturation DO at 10°C: 10.59 mg/L Calculated LC50: 10.12 mg/L Actual LC50: 10.59 mg/L – 10.12 mg/L = 0.47 mg/L

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Daphnia magna length to weight conversion Half of the ethanol-preserved D. magna were measured to estimate average body mass. Length was measured from the base of the tail spine to the top of the helmet. Using a regression equation for D. magna from Burns (1969), Daphnia length (mm) was converted to weight (mg). Length and weight data were compared between juvenile and adult D. magna.

Results Heart rate Daphnia magna heart rate was similar at different ages, ranging from 266 to 326 beats per minute at room temperature, 20°C (Table 1).

Dissolved oxygen Figure 1 shows the change in dissolved oxygen concentration over the 12-hour trial period for juvenile and adult D. magna. Bottles that began at saturation (about 10 mg/L) showed less change and a loss in DO over the 12-hour trial than bottles that began with lower pre-trial DO concentration and showed a gain in DO. The amount of oxygen introduced to low- DO treatments varied, but on average was between 0.1 and 0.3 mg/L. Figure 2 shows the comparison between three DO measurement techniques. Generally, the YSI 5000 meter provide the highest estimate, followed by the Winkler method.

DO concentration lethal to 50 percent of the population Survival responses to hypoxia exposure for juvenile and adult D. magna are summarized in Table 2. Survival percentages declined gradually at lower dissolved oxygen concentrations. The 50 percent lethal concentration of dissolved oxygen for both juvenile and adult D. magna is 0.47 mg/L (Table 3). Figures 3 and 4 represent the survival percentages of juvenile and adult D. magna as a function of dissolved oxygen for the data at the low dissolved oxygen concentrations only. Confidence intervals are slightly different, but do overlap. The confidence interval for juveniles was 0.45 to 0.49 mg/L, while the confidence interval for adults was 0.43 to 0.49 mg/L (Table 3). The confidence interval is tighter around the juvenile LC50 estimation.

Daphnia magna size Weight as a function of length for juvenile and adult D. magna used in this experiment, displayed in Figure 5, was estimated by the equation: Weight=0.009*Length2.63 (Burns 1969).

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Discussion Since adult Daphnia magna are significantly larger than juveniles, they are much more vulnerable to sight-based predation and thus may need to inhabit deeper, darker areas of the lake to avoid predation. Adult D. magna, however, do not seem to have a higher tolerance of hypoxia at the resolution achieved by this series of experiments. It is possible that adult D. magna may produce more hemoglobin, allowing them to persist under more intense hypoxia, if acclimated to hypoxia slowly (Weider and Lampert 1985). While there was some variation in heart rate with D. magna age, it was not significant. Observed heart rates were consistent with literature values (Yu 1969), but temperature is more important in determining heart rate than age. Literature values for a closely related species, Daphnia pulex, cite an average adult heart rate at 25°C of 256 to 288 beats per minute (Table 1), with slower heart rates of 88 to 104 beats per minute at 4°C (Yu 1969). A lack of difference in heart rate is consistent with a similar LC50 between juveniles and adults, and may indicate that juvenile and adult D. magna have a similar respiratory demand. Measuring dissolved oxygen concentration via different methods demonstrated the importance of calibrating the DO meter (YSI 5000) to match the more accurate readings of the Winkler method. Increases in dissolved oxygen concentration in low-DO bottles over the course of the 12- hour trials (as shown in Figure 2) probably happened either while bottles were filled, while dissolved oxygen was measured with the DO meter, and/or while animals added. In each of these cases, the bottle stopper was removed from the BOD bottle briefly exposing the sparged water to atmospheric oxygen. It is unlikely that the cause for oxygen increase was a non- airtight bottle. Decreases in dissolved oxygen concentration over the course of the 12-hour trials (only seen in bottles filled with oxygen-saturated water) were likely caused by D. magna respiration, as well as respiration by any bacteria or other microorganisms that passed through the 65 μm mesh filter (Donn Branstrator, personal communication). The indicated LC50 for each of the age groups (Figures 3 and 4) was 0.47 mg/L. For Daphnia, a dissolved oxygen LC50 of 0.47 mg/L is very low compared to the LC50’s of its predators. Most fish cannot withstand a DO lower than 2 mg/L (Vanderploeg et. al. 2009), and the spiny water flea Bythotrephes can only withstand a DO as low as 1.75 to 2.25 mg/L (UMD Graduate Student Mike Sorensen, unpublished data). This difference in hypoxia tolerance between predators and prey indicates that Daphnia may be able to escape predation by migrating lower into the water column, even when predator densities are high. D. magna’s ability to withstand such low oxygen levels indicates that it could easily establish itself in low- oxygen Minnesota lakes if it were to spread to this region. Daphnia may also be able to survive an infestation of invasive spiny water fleas by inhabiting hypoxic regions of the lake. However, extended periods of time spent in a hypoxic environment could impact both the health of individuals and the population structure.

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Acknowledgements I thank Donn Branstrator, my faculty advisor, for helpful discussions and guidance throughout the course of the project. Matt TenEyck and Christine Polkinghorne at LSRI proved vital to the success of this project by providing me with test animals and information about Daphnia manga. I am especially indebted to UMD graduate student Mike Sorensen for volunteering his time and resources to support me through the entire research process. UMD student Ben Block assisted with laboratory work. Funding was provided by the University of Minnesota Undergraduate Research Opportunities Program (UROP).

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Literature Cited

Adema, D.M.M. 1978. Daphnia magna as a test animal in acute and chronic toxicity tests. Hydrobiologia 59(2): 125-134.

Burns, C.W. 1969. Relation between filtering rate, temperature, and body size in four species of Daphnia. Limnology and Oceanography. 14(5): 693-700.

Environmental Protection Agency. 2000. Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR Part 136) (EPA publication number 821-B-00-004). US Environmental Protection Agency, Office of Water.

Environmental Protection Agency. 1985. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms (Third Edition). (EPA publication number 600/4-85-013). US Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH.

Gorr, T. A., Cahn, J. D., Yamagata, H., Bunn, H. F. 2004. Hypoxia-induced synthesis of hemoglobin in the crustacean Daphnia magna is hypoxia-inducible factor-dependent. Journal of Biological Chemistry. 279: 36038- 36047.

Goto, D., Lindelof, K., Fanslow, D., Ludsin, S. A., Pothoven, S. A., Roberts, J. J., Vanderploeg, H. A., Wilson, A. E., Hook, T. O. 2012. Indirect consequences of hypolimnetic hypoxia on zooplankton growth in a large eutrophic lake. Aquatic Biology. 16: 217-227.

Hach Company. 2014. Azide Modification of Winkler Method (Method 8215). Edition 8. Hach Company World Headquarters.

Hirsch, J. 2014. National Lakes Assessment 2012: Zooplankton Communities in Minnesota Lakes. Minnesota Department of Natural Resources.

Lampert, W. 1989. The adaptive significance of diel vertical migration of zooplankton. Functional Ecology (3): 21- 27.

Pangle, K.L. and Peacor, S.D. 2006. Non-lethal effect of the invasive predator Bythotrephes longimanus on Daphnia mendotae. Freshwater Biology (51): 1070-1078.

Pennak, R.W. 1987. Fresh-water Invertebrates of the United States. John Wiley & Sons, Inc. New York, NY: 369-384.

Seda, J., Petrusek, A. 2011. Daphnia as a model organism in limnology and aquatic biology: some aspects of its reproduction and development. Journal of Limnology. 70(2) Special Insert: 337-344.

Vanderploeg, H.A., Ludsin, S.A., Cavaletto, J.F., Höök, T.O., Pothoven, S.A., Brandt, S.B., Liebig, J.R., and Lang, G.A. 2009. Hypoxic zones as habitat for zooplankton in Lake Erie: Refuges from predation or exclusion zones? Journal of Experimental Marine Biology. 381: S108-S120.

Weider, L.J. and Lampert, W. 1985. Differential response of Daphnia genotypes to oxygen stress: respiration rates, hemoglobin content and low-oxygen tolerance. Oecologia. 65: 487-491.

Wetzel, R.G. 2001. Limnology: Lake and River Ecosystems, Vol. 3. Academic Press, San Diego, USA.

Yu, M.L. 1969. Response of the Heartbeat of Daphnia pulex to Variations in Physical and Chemical Surroundings. Biological Bulletin of National Taiwan Normal University: 34-47.

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Table 1: Daphnia heart rates for Daphnia magna of different ages

Daphnia magna age (days) 1 2 5 7 Measured heart rate (beats/minute) at 20°C 192 234 308 306 200 270 336 324 228 286 324 338 252 318 324 312 252 278 336 320 238 290 344 264 248 320 290 290 332 266 364 300 268 286 330 290 278 294 340 290 268 270 320 248 336 294 320 320 210 274 322 262 284 296 326 322 312 250 306 314 252 262 258 302 264 278 340 316 0 296 328 282 254 326 346 290 254 326 344 224 240 340 352 300 318 334 310 282 326 288 336 308 320 290 330 314 Average measured heart rate (beats/ minute) 266 290 326 297

Average literature heart rate (beats/minute) of adult D. pulex at 25°C 256 to 288

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Table 2: Hypoxia survival of juvenile and adult D. magna

Median post-trial DO (mg/L) Average percent survival Juvenile 10.03 95.50 0.55 75.67 0.38 13.67 0.16 0.00 Adult 9.78 100.00 0.65 84.98 0.50 63.08 0.39 38.85

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Table 3: LC50 and Confidence Intervals based on the EPA WET Analysis Spreadsheet

LC50 (mg/L) Lower 95% Upper 95% Confidence Interval Confidence Interval Juvenile 0.47 0.45 0.49 Adult 0.47 0.43 0.53

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0.6 Figure 1: Change in Dissolved Oxygen Juvenile 0.5 Concentration Adult 0.4 0.3 0.2 0.1 0.0 0 2 4 6 8 10 12 -0.1 Change in DO (mg/L)DO in Change -0.2 -0.3 -0.4 Pre-trial Dissolved Oxygen (mg/L)

Figure 1: Dissolved oxygen levels were measured before and after a 12-hour trial. Bottles that began at saturation (about 10 mg/L) generally decreased in DO concentration over the 12-hour trial. Bottles with a lower pre-trial DO showed an increase in dissolved oxygen concentration at the end of the trial.

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Figure 2: Comparison of DO measurement 1.2 methods

0.8

0.6

0.4

Dissolved Oxygen (mg/L) Oxygen Dissolved 0.2

0 1 2 3 4 5 6 Bottle number Field Probe YSI 5000 DO meter Winkler titration

Figure 2: Dissolved oxygen measurements from three methods. Field probe measurements were taken while sparging; YSI measurement taken after bottles were filled with sparged water and were immediately followed by the Winkler method.

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Figure 3: The concentration of dissolved oxygen lethal to 50% of juvenile D. magna in this study was 0.47 mg/L, with a 95% confidence interval of 0.45 to 0.49 mg/L. Each point represents one 300-ml BOD bottle treated with low-oxygen water (n= 4-6 juveniles per bottle).

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Figure 4: The concentration of dissolved oxygen lethal to 50% of adult D. magna in this study was 0.47 mg/L, with a 95% confidence interval of 0.43 to 0.53 mg/L. Each point represents one 300- ml BOD bottle treated with low-oxygen water (n= 4-6 adults per bottle).

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Figure 5: Estimated dry weight (Burns 1969) as a function of body length (measured in this study) for D. magna.