Principles

     Temperature is one of the most critical environmental factors influencing the ecology, behavior, and physiology of ectothermic animals. Characteristically, ectotherms produce metabolic heat at very low rates with a high percentage of activity metabolism from anaerobic respiration.  They have a high thermal conductance and thus derive body temperature mainly from external sources. On land, temperatures routinely vary widely both daily and seasonally. Terrestrial ectotherms frequently use behavioral mechanisms such as basking, hibernation, torpor, and nocturnal rhythms to thermoregulate.

    In many aquatic systems, however, ectotherms have different concerns. Body temperature is largely determined by conductive equilibration with the surrounding water. Temperatures in aquatic ecosystems are relatively stable when compared to terrestrial systems, but aquatic animals have fewer behavioral mechanisms with which they can respond to temperature changes when they do occur. For example, it is less likely that aquatic ectotherms can find the desired thermal microclimate like lizards do when they bask or seek shade. Aquatic ectotherms frequently respond to environmental temperature changes through physiological responses and acclimation rather than behavioral responses. Since most aquatic ectotherms are poikilothermic, having the same temperature as the environment, how are physiological processes affected by environmental temperature?

The Aquatic Ectotherm's Dilemma

 There is a positive relationship between ambient temperature and metabolic rate for ectotherms as measured by changing oxygen consumption rates.  The effect of temperature on physiological processes can be expressed using a convention called Q10.

Ql0 = rate at temperature X + 10 C / rate at temperature X

 If Q10 = 1, then the process is not temperature dependent. Most enzymes (and therefore enzyme mediated processes such as metabolism) have Q10s ranging from 2 to 3.

 If a physiological process has a Q10 = 2 then with every 10  C increase in temperature, there will be a doubling of the rate of that process (or 10  C decrease leading to a halving of the process). Similarly, if a different physiological process has a Q10 = 3, then there is a tripling of the rate of the process with every 10  C increase. In general, chemical reactions have a Q10 value of approximately 2-3 while purely physical processes like diffusion have a lower temperature sensitivity (closer to 1.5).

For temperature changes different from 10 C, the Q10 is calculated from the van't Hoff Equation :

Q10 = (k2/k1) exp10/ (t2 - t1)  where:

k1 = the reaction rate of the physiological process at temperature 1
k2 = the reaction rate of the physiological process at temperature 2
t1 = temperature 1
t2 = temperature 2

 Q10 is used as a convenient comparative measure.  The Q10 for a reaction varies depending upon the temperature range used in the experiment i.e., it is not constant over the entire temperature range, and thus you must state the range of temperatures over which the Q10 was calculated.  eg. Q10(7-24 C).

    In many species, naturally occuring stress from environmental heat or cold induces compensatory changes in physiology or morphology that help an animal cope with this stress. For example, a fish that experiences winter ice over its pond will gradually undergo, over several weeks, a variety of biochemical changes to the low temperature, declining oxygen supply and photoperiod. This process is termed acclimatization.  A species' ability, developed over evolutionary time, to 'adjust' their  physiological processes as their environment changes is referred to as an 'adaptation'. Adaptation occurs over evolutionary time.

    This lab will investigate the effect of three experimental temperatures on the metabolic rates of Carassius auratus, the common goldfish.   Goldfish are used because their metabolic rate is independent of environmental O2 concentrations down to values as low as 1mg 02/liter which removes the otherwise confounding variable of O2 levels.  You will also examine the effects of body mass on metabolic rate.

PROCEDURE

1. Each pair of students will have 3 flasks each containing 2 fish for a total of 6 fish/group.

2. Fill 3- 250ml erlynmeyer flasks, one from each experimental temperature tank. The instructor will measure the O2 content of tank water using the YSI O2 probe.

3. Take 2 fish from the aquaria and place them in each flask filled with water.

4. Overflow each flask so there are no air bubbles, then stopper tightly.

5. Note the exact time. Label the flasks with wax pencil.  Note: Clean pencil marks off each flask with acetone when finished.

6. Place flasks containing fish back into each of the experimental tanks; one flask in each tank. This is so water in the flasks remains at a constant temperature.

7. Allow the fish to remain in flask for the designated time period; approx. 30 min at 28 C, 60 min at 18 C, and 90 min at 8 C;  until ready to measure the O2 consummed by the 2 fish.  It is not important that the time lapsed be precisely 30 min. etc., but is important that you know the exact elapsed time e.g. 33 minutes.

8. Meanwhile, using the 500ml flask provided to you, repeat the above steps with a larger fish.  Incubate the larger goldfish along side the smaller fish at 18 C, for 60 min.

****Keep track of the time !!!

9. Unstopper the flask and quickly measure (YSI O2 probe) the O2 content of the flask water.  Remember to note the time when measurement is taken.

10. Return fish and water to their original tank.

CALCULATING METABOLIC RATE

 One can estimate metabolic rates of acclimated fish at 3 different experimental temperatures by determining the amount of O2 consumed by the fish over a certain period of time in each of the 3 temperatures. This is easily done by measuring the water O2 content before and after adding the fish.

1. Amount of O2 consumed by fish (mg/liter)

         Dissolved O2 (DO) before fish  -  DO after fish = Oxygen Consumed  by 2 fish

2. Determine fish mass (grams) assuming 1 ml water = 1 gram of fish. Assume volumetrically speaking fish = water.
This can be determined by the displacement of water by the fish in a graduated cylinder.  Alternatively one can weigh the fish on the top loading electronic balance.  Be careful to tare the beaker and water before adding fish.  Be careful not to add water when adding the goldfish to the beaker.

            This difference in ml equals mass in grams of 2 fish !

3. Determine the experimental flask volume in liters by filling flask with tap water (without fish) and stoppering. Pour contents into a graduated cylinder.  Next calculate the corrected flask volume (CV) for each particular flask used by subtracting the volume of the fish in each flask.

         CV =  Experimental Flask Volume - Volume of the 2 fish

4.  Calculation of Oxygen Consumption (VO2)  / fish

             (total mg O2 cons. / liter)  X CV
VO2 =     ______________________
                      ( 2 )   time (hours)

  Units will be (mg O2 /  hr)

5.  Calculation of Mass Specific Oxygen Consumption (QO2)

    QO2 =   VO2/  (1/2) mass in grams

    Units will be mg O2/ g / hr

6.  Note when using a single fish/flask, omit the "(2)" in the denominator of step 4 above, and the "(1/2)" in the denominator of step 5.

DATA ANALYSIS

1. Compute mean and standard deviation of VO2 and QO2 for each temperature group. Plot the relationship between temperature and QO2 .

2.  Calculate the Q10 for each of the temperature intervals.

2.  Plot the VO2  as a function of body mass for all the fish at 18 C.

3. Plot log VO2  as a function of log body mass for all the fish at 18 C.  What is the slope of the line ?

4. Plot log QO2 as a function of log body mass for all the fish at 18 C.  What is the slope of the line ?
 

REFERENCES

Blazka, P. 1958 The anaerobic metabolism of fish. Phyisol. Zool. 21: 117128.
Brett, J.R. 1956 Some Principles in the thermal requirements of fishes Quarterly Review  of Biology 31:75-87.
Brown, J.H. and Feldmeth, C.R. 1971 Evolution in constant and fluctuating   environments: Thermal tolerances of desert            pupfish (Cyprinodon). Evolution  25:390-398.
Bullock, T.H. 1955 Compensation for temperature in the metabolism and activity of  poikilotherms.  Biol. Rev. 30:311-342.
Crawshaw, L.I. 1975 Attainment of the final thermal preferendum in brown bullheads  acclimated to different temperatures. Comparative Biochem. Physiol. 52A: 171- 173.
Devries, A.L. 1970 Freezing resistance in antarctic fishes. In Antarctic Ecology Vol 1.  (M. Holdgate, ed.) pp 320-328 NY Academic Press.
Fry, F.E.J., and J.S. Hart 1948 The relation of temperature to oxygen consumption in  the goldfish. Biol. Bull. 94: 66-77.
Graham, J.B. 1974 Aquatic respiration in the sea snake Pelamis platurus. Respir.  Physiol. 21: 1-7.
Hart, J.S. 1952 Geographic variations of some physiological and morphological   characters in certain freshwater fish> University of Toronto Studies, Biological  Series, no. 60 Publ. Ontario. Fish. Res. Lab. 72:1-79.
Hemminsen, A.M. 1960 Energy metabolism as related to body size and respiratory  surfaces, and its evolution. Rep. Steno Hosp. 9(2):1-110.
Hoar, W.S. 1956 Photoperiodism and thermal resistance of goldfish. Nature 178: 364- 365.
Hochachka, P.W. 1980 Living without oxygen: Closed and open systems in hypoxia  tolerance. Cambridge, MA Harvard University Press.
Hoff, J.G. and Westman, J.R. 1966 The temperature tolerances of three species of  marine fishes. J. Mar. Res. 24: 131-140.
Kylstra, J.A. 1968 Experiments in water-breathing. Sci. Am. 219: 66-74.
Lawson, S.D. 1971 Warm-bodied fish. Am Zool. 11:137-145.
McLeese, D.W. 1956 Effects of temperature, salinity, and oxygen on the survival of the  American Lobster. J. Fish. Res. Bd. Can. 13:247-272.
Schmidt-Nielsen K. 1972 Locomotion: Energy cost of swimming, flying and running.  Science 177:222-228.
Schmidt-Nielsen K. 1997. Animal Physiology: Adaptation and Environment. Cambridge  University Press. Chapter 6 : Energy Metabolism; Chapter 7 ,  8: Temperature  Effects, Temperature Regulation; Chapter 1: Respiration in Water