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General Biology II

Lab Report II

Title: The Effect of Temperature Change on the Heart Rate of Daphnia magna

Abstract The water flea Daphnia magna plays a central role in the aquatic food chain and serves as food for other freshwater organisms such as fish. Its habitat exhibits acute variations in temperature which affects its metabolic and physiological processes such as heart and ventilation rates. We investigated the effects of temperature change on the metabolic/heart rate of Daphnia magna and determined the temperature sensitivity of its heart rate by calculating its Q10 value. Our results showed a linear increase in heart rate with temperature but no doubling of the heart rate was observed for 10C increments in temperature. Our results also suggest that Daphnia magna, though poikilothermic, its heart rate has a more complex relationship to temperature than is commonly seen in most biological systems.

Introduction Temperature changes affect physiologic and metabolic processes in endothermic and ectothermic animals alike. For a water flea such as Daphnia magna, acute changes in water temperature have a strong effect on its physiological processes such as heart and ventilation rates (Pinkhaus et al. 2007). Such physiological perturbations in turn call for adaptive responses such as acclimatization, which attempt to restore these processes to normalcy (Campbell et al. 2012). The rate of enzyme-catalyzed reactions is influenced by temperature change, with an increase in temperature (up to an optimum) often resulting in an increase in the rate of reaction (Campbell et al. 2012). Since most metabolic processes are enzyme-catalyzed, the rate of a metabolic process will be influenced by temperature change. This effect of temperature on a metabolic activity is expressed in terms of the temperature coefficient (Q10) - the ratio of the rate of activity at one temperature compared to its rate at a temperature 10C higher. Previous studies have shown that metabolic activities such as heart and ventilation rates, as well as the muscular performances of several daphnia species at 10C and 20C are not as different as expected from the Q10 rule. A typical compensatory control (metabolic adaptation, such as change in heart rate) following such changes in environmental temperature as the animal acclimatizes is primarily based on adjustments in enzyme concentration (Schwerin et al. 2009). In this laboratory experiment, we sought to investigate the effects of temperature change on the metabolic rate (heart rate) of Daphnia magna. We hypothesized that the heart rate of Daphnia magna will be faster at higher temperatures and slower at lower temperatures since most metabolic activities will increase at higher temperatures. Thanks to its characteristic translucent body which provides for a ready visibility of its heart in living specimens, daphnia is a highly suitable invertebrate model for the investigation of the effects of temperature change, as well as other parameters, on a living heart (Corotto et al. 2010). We determined daphnias heart rate at varying temperatures and observed the relationship between these two parameters. Calculated Q10 values were used as a means of better visualizing daphnias temperature dependence.

Materials and Methods Determination of Daphnias Heart Rate at Different Temperatures The daphnia specimen provided us by the instructor was properly placed on a special concave glass slide and then observed under the high power objective of a microscope. Daphnias baseline heart rate was first determined by visually counting the number of heart beats per minute at room temperature. In order to determine its heart rate at 5C, ice water in a beaker stabilized at 4C was applied to the daphnia specimen and its heart rate under this condition determined. This was repeated three times and the average of the three values was the number of heart beats per minute at a given temperature. Similarly, the heart rates at 10C, 15C, 20C, 25C, 30C, and 35C were determined by placing water at these different temperatures with the daphnia specimen and then counting the number of heart beats per minute under these different temperature conditions. Determination of Q10 Using the recorded heart rates and their respective temperatures, Q10 values were calculated using the formula: Q10 = (R2/R1)10/ (T2- T1), where R is heart rate and T temperature. Q10 calculations were determined at three temperature intervals; 5C - 15C, 15C - 25C, and 25C - 35C. Data Analysis The mean heart rate at each temperature for the class data calculated and used to determine Q10. Also, the standard deviation for the class data at each temperature interval was determined. A graph was also used to provide a visual representation of the effect of temperature change on the heart rate of Daphnia magna in our experiment.

Results It was observed that the heart rate of Daphnia magna increased with increasing temperature as shown on table 1.0 and Figure 1. Computed Q10 values (Table 2.0) were found not to be constant for the different 10C increments used (although very similar). A mean Q10 value of 1.10 was obtained in this experiment. Table 1.0: Mean Daphnia Heart Rates at Different Temperature Intervals
Temperaturure (C) 5 Mean Heart rates (beats/min)(S.D) 174.9 58.5 183.8 57.1 194.1 63.5 207.1 73.8 214.2 87.1 229.2 92.9 240.3 99.9

10 15 20 25 30 35

250 240 230 220 210 Mean Heart Rate (bpm) 200 190 180 170 160 150 0 5 10 15 20 Temperature (C) 25 30

y = 2.1932x + 162.37

35

40

Figure 1: Mean Daphnia Heart Rate versus Temperature (Graph shows a proportionate increase in heart rate with temperature)

Table 2.0: Q10 Values at Different Temperature Intervals. Temperature intervals 5C - 15C 15C - 25C 25C - 35C Mean Q10 1.11 1.10 1.12 1.10

Discussion The planktonic crustacean Daphnia magna plays a central role in the aquatic food chain where it also serves as food for other freshwater organisms such as fish. Its habitat exhibits variations in temperature which affects the physiology and metabolism of this freshwater organism. It has been shown that acute changes in water temperature have a strong effect on physiological processes such as heart and ventilation rate of daphnia species (Pinkhaus et al. 2007). Since the effects of temperature on a metabolic rate may be expressed in terms of the Q10, in this experiment, we studied the effects of a change in temperature on daphnia metabolic or heart rate. Our results showed there was an increase in daphnia heart rate with increasing temperature. A mean Q10 value of 1.10 (> 1.0) was associated with this increase indicating that daphnia heart rate is temperature-sensitive (although very minimally). This confirms our initial hypothesis. Also, Daphnia magna is both ectothermic and poikilothermic, which means that its body temperature and therefore its metabolic rate are affected directly by the temperature of the environment. The change in metabolic rate is reflected in the rate at which the heart beats. For most endothermic and ectothermic biological systems involving enzyme-controlled biochemical reactions, and physiological processes such as heart rate or metabolic rate, the Q10 is usually 2 or higher. Our results however did not show such a Q10 relationship between daphnias heart rate and temperature, suggesting that daphnias heart rate has a more complex relationship to temperature than is commonly seen in most biological systems. In fact, daphnia hearts differ from human or mammalian hearts in many respects. In terms of heart rate, the daphnia sinoatrial node is actually a collection of spontaneously active nerves in a body called the cardiac ganglion.

Furthermore, the small Q10 associated with our results might have fallen short of the Q10 = 2 observed in most biological systems, mainly due to erroneous measurements in heart rates during the experiment. This is so because the mean Q10 was computed from mean heart rates of the class data obtained by visually counting the heart beats per minute of different daphnia specimens. This process has an inherent error as measurements from many different sources might be subjective and this could be seen in the larger standard deviation values associated with the class means (table 1.0) indicating a wide variation in heart rate measurements.

Literature cited 1. Pinkhaus O, Schwerin S, Pirow R, Zeis B, Buchen I, Gigengack U, Koch M, Horn W, Paul RJ. 2007. Temporal environmental change, clonal physiology and the genetic structure of a Daphnia assemblage (D. galeata-hyalina hybrid species complex). Freshwater Biol , 52:1537-1554. 2. Campbell, N. A., J. B. Reece, and L. G. Mitchell. (2012) Biology. 9th edition. Pearson Benjamin/Cummings, Menlo Park, CA, USA. Pg. 151-155.

3. Schwerin S, Zeis B, Lamkemeyer T, Paul RJ, Koch M, Madlung J, Fladerer C, Pirow R. 2009. Acclimatory responses of the Daphnia pulex proteome to environmental changes. II. Chronic exposure to different temperatures (10 and 20C) mainly affects protein metabolism BMC Physiology. 9:8, 1472-6793.

4. Corotto, F., Ceballos, D., Lee, A., & Vinson, L. (2010). Making the most of the daphnia heart rate lab: optimizing the use of ethanol, nicotine & caffeine. The american biology Teacher, 72(3), 176-179 .

1. BMC Physiol. 2009; 9: 8. Published online 2009 April 21. doi: 10.1186/1472-6793-9-8 PMCID: PMC2678069 Acclimatory responses of the Daphnia pulex proteome to environmental changes. II. Chronic exposure to different temperatures (10 and 20C) mainly affects protein metabolism Susanne Schwerin,1 Bettina Zeis,1 Tobias Lamkemeyer,2 Rdiger J Paul,1 Marita Koch,1 Johannes Madlung,2 Claudia Fladerer,2 and Ralph Pirow 1 Author information Article notes Copyright and License information This article has been cited by other articles in PMC. Abstract Background Temperature affects essentially every aspect of the biology of poikilothermic animals including the energy and mass budgets, activity, growth, and reproduction. While thermal effects in ecologically important groups such as daphnids have been intensively studied at the ecosystem level and at least partly at the organismic level, much less is known about the molecular mechanisms underlying the acclimation to different temperatures. By using 2D gel electrophoresis and mass spectrometry, the present study identified the major elements of the temperature-induced subset of the proteome from differently acclimated Daphnia pulex. Results Specific sets of proteins were found to be differentially expressed in 10C or 20C acclimated D. pulex. Most cold-repressed proteins comprised secretory enzymes which are involved in protein digestion (trypsins, chymotrypsins, astacin, carboxypeptidases). The cold-induced sets of proteins included several vitellogenin and actin isoforms (cytoplasmic and muscle-specific), and an AAA+ ATPase. Carbohydrate-modifying enzymes were constitutively expressed or downregulated in the cold. Conclusion Specific sets of cold-repressed and cold-induced proteins in D. pulex can be related to changes in the cellular demand for amino acids or to the compensatory control of physiological processes. The increase of proteolytic enzyme concentration and the decrease of vitellogenin, actin and total protein concentration between 10C and 20C acclimated animals reflect the increased aminoacids demand and the reduced protein reserves in the animal's body. Conversely, the increase of

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actin concentration in cold-acclimated animals may contribute to a compensatory mechanism which ensures the relative constancy of muscular performance. The sheer number of peptidase genes (serine-peptidase-like: > 200, astacin-like: 36, carboxypeptidase-like: 30) in the D. pulex genome suggests large-scaled gene family expansions that might reflect specific adaptations to the lifestyle of a planktonic filter feeder in a highly variable aquatic environment.

2. Temporal environmental change, clonal physiology and the genetic structure of a Daphnia assemblage (D. galeata-hyalina hybrid species complex)
PINKHAUS, OLAF SCHWERIN, SUSANNE PIROW, RALPH ZEIS, BETTINA BUCHEN, INA GIGENGACK, ULRIKE KOCH, MARITA HORN, WOLFGANG PAUL, RDIGER J. Published August 2007 Journal Freshwater Biology See all Details Author

Abstract
Summary 1. In a combined field and laboratory study, seasonal relationships between water temperature and oxygen content, genetic structure (composition of MultiLocus Genotypes, MLGs) of a Daphnia assemblage (D. galeata-hyalina hybrid species complex), and the physiological properties of clones of frequent MLGs were studied. In accordance with the oxygen-limited thermal tolerance hypothesis, essential physiological variables of oxygen transport and supply were measured within the tolerable temperature range. 2. A few MLGs (types T1-T4) were frequent during early spring and late autumn at surface temperatures below 10C. Clones of T1-T4 showed a low tolerance towards higher temperatures (above 20C) and a high phenotypic plasticity under thermal acclimation in comparison to clones derived from frequent MLGs from later seasons, and stored high-medium quantities of carbohydrates at 12 and 18C. 3. Another MLG (T6) succeeded the MLGs T1-T4. T6 was frequent over most of the year at temperatures above 10C and below 20C. A clone derived from T6 exhibited a high tolerance towards warm temperatures and a more restricted phenotypic plasticity. It stored high-medium quantities of carbohydrates at 12, 18 and 24C and showed a high capacity for acclimatory adjustments based on haemoglobin expression. 4. During the summer period at temperatures 20C, the MLG T6 was found mainly near to the thermocline, where temperature and oxygen content were distinctly lower, and to a lesser extent in surface water. At the surface, another MLG (T19) was predominant during this period. A clone of this MLG showed a very high tolerance towards warm temperatures, minimal phenotypic plasticity, low

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carbohydrate stores and a high capacity for circulatory adjustments to improve oxygen transport at higher temperatures. 5. This study provides evidence for connections between the spatio-temporal genetic heterogeneity of a Daphnia assemblage and the seasonal changes of water temperature and oxygen content. The data also suggest that not only the actual temperature but also the dynamics of temperature change may influence the genetic structure of Daphnia populations and assemblages.

3.The American Biology Teacher

Published by: National Association of Biology Teachers

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The American Biology Teacher 72(3):176-179. 2010 doi: http://dx.doi.org/10.1525/abt.2010.72.3.9

Making the Most of the Daphnia Heart Rate Lab: Optimizing the Use of Ethanol, Nicotine & Caffeine
Frank Corotto, Darrel Ceballos, Adam Lee, Lindsey Vinson FRANK COROTTO is Professor of Biology at North Georgia College & State University, Dahlonega, GA 30597; e-mail: fcorotto@northgeorgia.edu. LINDSEY VINSON, DARREL CEBALLOS, and ADAM LEE are his students.

ABSTRACT Students commonly test the effects of chemical agents on the heart rate of the crustacean Daphnia magna, but the procedure has never been optimized. We determined the effects of three concentrations of ethanol, nicotine, and caffeine and of a control solution on heart rate in Daphnia. Ethanol at 5% and 10% (v/v) reduced mean heart rate to 50% and 20% of its initial value, respectively. Recovery was rapid after removing 5% ethanol, but recovery from 10% ethanol took 2030 minutes. Nicotine at 100 M reversibly increased mean heart rate by 20%. Higher concentrations produced varied and sometimes irreversible effects. Caffeine at 0.1%, 0.5%, and 2% (w/v) had no convincing effect on heart rate. Of the three agents tested, nicotine's peculiar effects make it the least useful in an educational setting. Caffeine could be used to emphasize the need for blind observers because it does not increase heart rate in Daphnia. If students find that it does, their bias is revealed. Ethanol produces unambiguous effects at 5% and 10%. Heart rates

12 recover quickly after removing 5% ethanol, which allows students to explore reversibility as an alternative to having a separate control group.