BIO A02S Winter 2012 - LAB 4

LAB 4
Physiological Responses to Temperature using Daphnia spp.
OVERVIEW
In this lab, you will examine the impact of an environmental factor, namely temperature on the physiological functioning of a small invertebrate. The organism is a crustacean zooplankton species belonging to the genus Daphnia; this organism is commonly known as the water flea. You will be using changes in heart rate as a measure of the effect of temperature on this organisms physiological behaviour.

Objectives:
On completion of Lab 4: 1. 2. 3. you should appreciate the impact that even a single environmental factor can have on the physiological responses of an organism. you should be familiar with the concept underlying the use of Q10 values. you should be able to define all terms in bold print in the Background Information for this Lab. Use your lecture notes and textbook to supplement your understanding. This will help you prepare for the QUIZ at the beginning of Lab 4.

Lab 4 is based on a protocol designed by BGY A02 / BIO A02 faculty from Dept. of Biological Sciences, UTSC with modifications by Mary Olaveson, Dept. of Biological Sciences, UTSC 71

BIO A02S Winter 2012 - LAB 4

CHECKLIST
BEFORE COMING TO YOUR LAB 4 SESSION:
Read the Lab 4 material carefully before coming to the lab. Review lecture material and relevant sections in Chapter 32-Introduction to Animal Organization and Physiology (pages 547-576) in your textbook (Russell et al. 2010). This will give you the necessary background for the Quiz and to effectively organize your time and carry out this Lab. Complete the Question, Research Hypothesis and Predictions in LAB REPORT # 4 before coming to your Lab. Review Appendix V on the use of the dissecting microscope. Bring a calculator that can calculate logs and antilogs.

DURING YOUR LAB 4 SESSION:

Make sure you have all the materials required to carry out the lab exercises. Make sure you have your Lab Manual, rulers and pencils for your lab. You MUST have your lab coat and goggles. Budget your time carefully in order to complete all that is required. Use proper laboratory practices throughout the lab session and follow any instructions given by your TA. Remember that you must have your Lab Manual and wear your lab coat in all Biology labs. You must have your goggles for the labs indicated. Food and drinks (including water bottles) are not allowed in labs. Students will be asked to leave the lab if they do not comply with Lab Safety Protocols; Students asked to leave because they are unprepared to do the Lab

will receive a mark of 0 for the lab work involved and will not be allowed to do a make-up Lab.

BEFORE LEAVING YOUR LAB 4 SESSION:

Complete LAB REPORT # 4 and submit it to your TA before leaving the Lab. If the Lab Report is not handed in, it will be considered as a late Report and treated accordingly. Clean your lab bench according to the instructions listed on page 78. Remove all of your personal belongings. Your TA will check your lab bench before you are allowed to leave the lab. 72

BIO A02S Winter 2012 - LAB 4

LAB 4
Physiological Responses to Temperature using Daphnia spp.
BACKGROUND INFORMATION
Temperatures over the face of the Earth vary tremendously, ranging from the boiling springs of Yellowstone Park down to the -80C of a winter day in Antarctica. While a few amazing creatures have adapted to the extremes of temperature seen on Earth, most living cells can only function over a much narrower range of temperatures (see Figure 4.1). Ice crystals form and damage cellular components when the cells cool below 0C. Proteins in the cell begin to denature as temperatures approach 45C. Thus, while a few organisms have developed adaptations, which allow them to survive at extreme temperatures, the vast majority of Earth's species must maintain internal temperatures between 0C and 45C to survive. Even within the range of 0 to 45C, temperature changes create problems for animals. This is because most physiological processes are temperature sensitive, going faster (up to a point) as the temperature rises. But not all processes are equally sensitive to temperature changes. Some physiological processes will have their rates changed more than other physiological processes for a given temperature change. This can cause a problem if the two physiological processes need to work together in a coordinated fashion.

Temperature

Figure 1.1. The tolerance range of an organism to a temperature gradient indicating that this organism has an optimum temperature range over which it survives and grows. Above and below of this optimal range, the organism suffers with reduced growth and survival. 73

BIO A02S Winter 2012 - LAB 4

Scientists have developed the term Q10 to express a reaction's temperature sensitivity. The Q10 describes the relationship between the rate of a reaction and temperature (Formula 1).

Formula 1
log Q10 = 10 (log Rhigher - log R1ower ) (thigher tlower)
In this equation:

Rhigher = rate of reaction at higher temperature R1ower = rate of reaction at lower temperature thigher = higher temperature (in C) tlower = lower temperature (in C)

Example: If Rhigher = 100, R1ower = 50, thigher = 20C and tlower = 10C then log Q10 = 10 X (log 100 - log 50) (20 - 10) log Q10 = 10 X (2 1.699) (10) log Q10 = 10 X (0.301) (10) log Q10 = 0.301

Q10 = antilog 0.301 Q10 = 2

When the thigher and tlower are exactly 10C apart, Formula 1 can be simplified to Formula 2.

Formula 2
Q10
In this equation:

RT R T-10

RT = rate of the reaction at a certain temperature RT-10 = rate of the same reaction at a temperature 10C lower than the initial temperature

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BIO A02S Winter 2012 - LAB 4

LAB 4
Physiological Responses to Temperature using Daphnia spp.
INSTRUCTIONS FOR LAB EXERCISE

The Q10 varies over different parts of the 0 to 45C temperature range, especially in biological systems. Since most vital processes become very slow at temperatures close to 0C, the Q10 for the lower ranges of temperature is often high while, as the upper limits of thermal tolerance are approached, the Q10 drops. Thus the Q10 for a given reaction is not a constant and the range over which a given Q10 has been determined must be stated. Furthermore, the Q10 values of different processes in the same animal may not be the same. In this Lab, you will have the chance to measure Q10 values for the heart rate in the poikilothermic invertebrate, Daphnia, commonly known as the water flea. This will be done by measuring the heart rate at over a range of temperatures. At each specific temperature, it will be necessary to keep the temperature as constant as possible for a period sufficient to allow the heart to come to temperature equilibrium. You will also become familiar with the use of the dissecting microscope while performing this experiment. You will complete and submit LAB REPORT # 4 at the end of Lab 4.

Materials
beaker calculator (BRING YOUR OWN!) culture dish Daphnia dissecting microscope ice bucket ice pipettes (and bulbs) stopwatch thermometer vaseline water bath

Work in groups as instructed by your TA either 1 group per bench (groups of 5 students) or as 2 groups per bench (groups of 2 or 3 students). The group arrangements may vary depending on the number of Daphnia available.

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Procedure
1. Put a small smear of vaseline on the bottom of a dry culture dish and place a Daphnia on the vaseline smear (Figure 4.2), in a position that allows the contraction of the heart to be seen clearly. A side view of Daphnia is given in Figure 4.3. culture dish water level vaseline smear Daphnia specimen

Figure 4.2. Positioning of a Daphnia specimen on smear of vaseline in bottom of dry culture dish.

2nd antenna 1st antenna

compound eye mouth trunk appendage ovary carapace anus brood chamber brain heart ostium

Figure 4.3. Lateral view of Daphnia sp. 2. Add aged water that has been held at room temperature to the dish. Wait five minutes to allow for the animal to reach equilibrium at the temperature of the tap water, then measure and record the temperature of the tap water.

Before your Lab - Review Working with a Dissecting Microscope in APPENDIX V

in your Lab Manual. 76

BIO A02S Winter 2012 - LAB 4

3. Locate the Daphnia using the lowest magnification lens of a dissecting microscope. Once you have located the Daphnia, move the culture dish such that the Daphnia is centered in the field and swing in the 4X lens. Locate the heart of the Daphnia. If the beating heart is not clearly visible, move the Daphnia so that the contraction of the heart can be clearly seen. (You may find that using the dark-field plate helps seeing the internal organs of the transparent Daphnia). 4. Once you have identified the heart, count the number of beats over a 10 second period. Wait 10 seconds, and then count the heartbeats for another 10 second period. Repeat this process over a total of 3 minutes. You should have 9 measurements in total giving you an estimate of the heart rate. Determine the mean (= average) heart rate, and record the value in Table 1 in LAB REPORT # 4. NOTE: WHEN NOT OBSERVING THE Daphnia, MAKE SURE THE MICROSCOPE LIGHT IS TURNED OFF. KEEPING THE LIGHT TURNED ON WILL INCREASE WATER TEMPERATURE. 5. In a beaker, use ice and water to make a water mixture with a temperature between 5C and 10C. Once the ice is melted (or removed) and you have chilled water, you are ready to proceed to the next step. Use a pipette to take the tap water from the culture dish and replace it with chilled water. Wait three to five minutes to allow for the Daphnia to reach equilibrium. Measure and record nine successive counts of the number of contractions, using the technique described in Step 4 above. Calculate the mean, and record the average heart rate on Table 1 in LAB REPORT # 4. Now begin increasing the temperature in 5 degree increments. For this part of the experiment, get a beaker, and get some warm water from the container in the water bath. Using a Pasteur pipette, slowly replace a portion of the colder water with the warmer water, until the water temperature in the culture dish is the desired temperature. Do this slowly; exchange small amounts of colder water from your dish with warm water. You must be patient as you do this. If you add too much too quickly, you may overshoot your target temperature or worse, and you may cause heat damage to the Daphnia. Once you achieve each 5 degree increase, let the Daphnia equilibrate for 5 minutes, and then do another set of heart beat measurements. Measure and record nine successive counts of the number of contractions, using the technique described in Step 4 above. Calculate the mean, and record the average heart rate in Table 1 in the LAB REPORT # 4.

6. 7.

8.

9.

10. Repeat this process until you have reached a maximum water temperature of 35C. For each temperature increment, be sure to let the Daphnia specimen reach equilibrium at that temperature (wait 5 minutes). Also remember to take 9 estimates of the heart rate and calculate the mean heart rate. Record the mean rate in Table 1 in LAB REPORT # 4. . NOTE: You may not be able to get exactly 5C increments, record the temperatures you achieve. At high heart rates, push the counter only every 10th beat.

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LAB CLEAN-UP
Procedure
1. 2. 3. 4. Return your dissecting microscope to proper location in the microscope cabinet. Leave all equipment on your Lab Bench as set out at the beginning of your Lab. Follow your TAs instructions for cleaning up after this Lab. Discard other wastes in GARBAGE CONTAINER - DO NOT DISCARD garbage in the sinks in the lab.

5. Make sure your lab bench and sinks are clean. 6. Remove all of your personal belongings.

Bench Checked - TA Initials _________________

REFERENCES
Sadava D, Heller HC, Orians GH, Purves WK, Hillis D. 2008. Life: the science of biology. 8th edition. Sinauer Associates Inc. Sunderland Massachusetts. 1252 pp.

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BIO A02S Winter 2012 - LAB 4

LAB REPORT # 4 Physiological Responses to Temperature using Daphnia spp.

COMPLETE LAB REPORT # 4 AND SUBMIT TO YOUR TA AT THE END OF LAB 4 NAME: __________________________________________ LAB SECTION: ________ Student #: _______________________ Bench _____ Seat #: _____

1. Complete the following sections Question, Research Hypothesis and Predictions before coming to your Lab. (a) Question:

_________________________________________________________________

(b) Research Hypothesis: ____________________________________________________________________________ ____________________________________________________________________________ ____________________________________________________________________________

(c) Predictions: ____________________________________________________________________________ ____________________________________________________________________________ ____________________________________________________________________________ ____________________________________________________________________________ 79

BIO A02S Winter 2012 - LAB 4

2. Summarize your Results in Table 1 below. Table 1. Effect of temperature on the heart rate of Daphnia. TREATMENT Tap Water (initial) Temp. ______C WATER TEMPERATURE (C) MEAN HEART RATE (beats/10 sec)

Coldest Water Temperature Temp. ______C

Temp. ______C

Temp. ______C

Temp. ______C

Temp. ______C

Temp. ______C

Temp. ______C

Warmest Water Temperature Temp. ______C

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NAME: __________________________________________ LAB SECTION: ________

Results:
3. Consider your Results by comparing the original room temperature heart rate measurement with the heart rate taken at room temperature after the Daphnia specimen had been cooled down. Did the rate of contraction return to the original value at room temperature? Explain your observation. _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ 4. Plot a graph of the mean heart rate against temperature in C using the graph provided. Be sure to give your graph an appropriate caption and label the axes. 5. Calculate the Q10 values over the ranges of temperatures you tested. Start with your lowest temperature reading and go to the next reading that is approximately 10 degrees higher for your first Q10 value. Proceed from that temperature upward in 10 degree increments.
For example, if you took readings at 6C, 11C, 15C, 24C, 30C, and 35C, you should calculate the Q10 values for the 6C to 15C interval, for the 15C to 24C interval, and for the 24C to 35C interval.

If you managed to get exactly 10 degree intervals (when rounded to the nearest degree), use Formula 2 given above. If not, in order to calculate the Q10 values where the difference between the two temperatures was not exactly 10C, use the log version of the formula (Formula 1). 5. Record your Q10 values in Table 2. Remember to give your Table an appropriate caption.

Table 2. INTERVAL #
1 2 3 4 5 81

TEMPERATURE INTERVAL (C)

Q10 VALUES

BIO A02S Winter 2012 - LAB 4

6. Were the Q10 values the same or different over the observed temperature range? _______________________________________________________________________ 7. If some of the Q10 values were different: (a) Which temperature interval had the largest Q10 value? _______________________ (b) The Daphnia heart rate is most sensitive to temperature in which temperature interval? _______________________________________________________________________

Submit your LAB REPORT # 4 to your TA at the end of your Lab!!

MARK=

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