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 STROM Daphnia magna Hypoxia 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. 2 STROM Daphnia magna Hypoxia 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kindti can handle dissolved oxygen concentrations as low as 0.75-1.25 mg/L (UMD Graduate Student Mike Sorensen, unpublished data).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 3 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). 4 STROM Daphnia magna Hypoxia 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). 5 STROM Daphnia magna Hypoxia 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).