Abstract
The thermal tolerance ranges of fish and the temperatures at which a fish performs
optimally are established by the evolutionary history and recent thermal exposure of the
organism. Some species of fish are highly eurythermal; they tolerate a wide range of
temperatures and, typically, exhibit a significant capacity to alter thermal tolerance limits and
thermal optima for physiological function as a result of acclimation. An excellent example of a
eurythermal fish is the goby Gillichthys mirabilis, commonly known as the long-jaw mudsucker,
which is found in waters whose temperatures range between approximately 9°C and 40°C. In
this experiment, I used G. mirabilis acclimated for four weeks to four different temperatures (98
14°, 19° and 26°C) to study the effects of acute decreases in ambient temperature on heart rate, a
physiological trait known to be important in setting thermal tolerance limits and metabolic
capacity. The average heart rate (n-3) of each acclimation group was plotted against the acute
change in temperature to examine the pattern by which heart rate decreased with decreasing
temperature. The respective heart rates are compared at shared temperatures to identify the
ability of the fish to meet the aerobic demand posed at each temperature. Heart rate decreased
with temperature at a constant rate in the fish acclimated at 9°C and 14°C. The heart rate of the
fish acclimated at 19°C and 26°C decreased at a constant rate until reaching 20 beats per minute,
which occurred at temperatures near 9°C for the 19°C fish and 15°C for the 26°C fish. A 20 beat
per minute heart rate was maintained until the temperature dropped below 5°C, at which point
the heart rate declined exponentially. At 1°C the 9° and 14°C acclimated fish maintain a heart
rate of over twice that of the 19° and 26°C acclimated fish, suggesting that temperature
acclimation shifts the thermal window of animals. This experiment could be used in conjunction
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with an experiment raising the temperature of G. mirabilis acclimated at the same temperatures
so that both the upper and lower thermal limits of cardiac function of the fish are discovered.
Introduction
As predominantly ectothermic animals, fish performance is greatly influenced by ambient
water temperature (Gerlach et al. 1990). In order for a species to survive, fish must maintain an
aerobic metabolism that supports growth, reproduction and foraging for food (Pörtner and
Farrell, 2008). Fish frequently select temperatures that allow them to most efficiently meet their
aerobic needs, and temperatures above and below this optimal temperature result in a decrease of
aerobic capacity lowering their fitness. While reduced fitness is observed at temperatures
outside of the optimal temperature range, fish may endure sub-optimal temperatures for limited
periods of time (Pörtner and Farrell, 2008). The overall temperature spectrum that an animal can
endure is known as the thermal window. This range of temperatures is based on how well an
animal can meet the oxygen demands posed by the metabolic demand at a given temperature.
Metabolism of ectotherms like fish increases with rising temperature (termed Qio effects) and
often approximately doubles for a 10°C rise in temperature. Temperature-dependence in whole
organism respiration is reflected in similar changes in the rate of cardiac function (Moffitt and
Crawshaw, 1983), which is to be expected due to the heart's central role in providing oxygen to
respiring tissues (Pörtner, Mark and Bock, 2004).
Since heart rate is coupled to both temperature and performance, the ability of the heart to
sustain adequate activity over a range of temperatures can greatly influence the ability of a fish to
survive over this thermal range. Cardiac physiology is therefore a key factor in niche expansion
and fish diversity (Gamperl and Farrell, 2004). Adaptation to living in a new temperature can
result in the evolution of a new species or reflect extreme cardiac plasticity within a species.
I hypothesized that thermal acclimation would have significant effects on heart rate and
the ability to sustain cardiac function at decreasing temperatures. More specifically, I predicted
that the fish acclimated at 26°C would have the most difficulty sustaining an aerobic metabolism
at low temperatures and that the 9°C acclimated fish will have the least difficulty
Materials and Methods
Animals
Gillichthys mirabilis were obtained from a lagoon on the University of California, Santa
Barbara campus. Prior to the experiment, the fish were acclimated to 9°C, 19°C, 26°C or ambient
seawater (-14°C) for one month in temperature controlled tanks. The tanks were equipped with
continuous water flow and filtration and the fish were fed pellet food once daily.
Pre-Operation
Three fish from a single acclimation group were tested at a time. Fresh seawater was put
into an experimental tank containing a chiller, heater, thermometer and continuous flow pump to
circulate the water. The seawater was heated or cooled to reach the respective temperature. To
anesthetize the fish prior to the heart monitor insertion, each goby was placed in a bucket and
covered with ice. The 26°C fish were anesthetized on ice for 5 minutes, the 19°C for 20 minutes.
the seawater specimens for 30 minutes and the 9°C for 45 minutes. These times were based on
empirical evidence and represent the minimal time needed to immobilize the fish long enough to
insert the electrodes. To make the electrode wires, Teflon coated stainless steel wires were cut
into three-foot segments. The ends were striped and one end was threaded through a small
surgical needle. The top 2mm of wire were bent into a hook over the needle that was used to
insert the electrode (see "Operation").
Operation
Once the fish were anesthetized, they were placed ventral side up on a solid surface.
Wires were inserted on either side of the heart and hooked onto the pericardial cavity via a
needle. The needle was inserted just beneath the pelvic girdle almost parallel to the fish on either
side of the heart. Once the wire was hooked in place, the needle was removed. The wires were
crossed near the anus of the fish and secured in place by suturing to the skin. Veterinary
adhesive was applied over the wire holes and the suture area
Electrocardiogram (ECG)
Following the electrode implantation procedure, the fish was placed into the experimental
tank and held upright until movement was restored. The ends of the wires were placed into a
voltage amplifier (P55 AC Pre-Amplifier, Grass Instrument Co.), which was connected to the
Powerlab system. The thermometers in the tank were calibrated to the correct temperature using
a two point calibration and configured into Powerlab. Aster an hour of recovery in the
experimental tank during which the temperature matched the acclimation temperature, the chiller
was switched on. The water was cooled at a rate of 4°C per hour until the temperature reached
1°C. Data were recorded continuously using the Powerlab system at a rate of 100 readings per
second.
Analysis of Results
Since the temperature decreased 4°C per hour, a degree change occurred roughly every
15 minutes. The ECG results were analyzed by counting the amount of cardiac contractions in a
20 second span every 15 minutes during the course of the experiment. The number of beats per
20 second interval was then multiplied by three to show beats per minute. The heart rates of the
three fish were averaged. The temperatures of the two submerged thermometers at each 15
minute interval were recorded as well and this average was used as the internal temperature of
the fish at the time of measurement of cardiac activity.
Results
Contraction of the pericardial cavity was amplified and converted into a P-wave and
ORS-complex to represent the depolarization of the atrium and contraction of the ventricle (Fig
1). The average heart rate from each acclimated temperature (n-3) was plotted against water
temperature (= fish body temperature) to determine how cardiac activity responded to acute
decreases in temperature (Fig 2). The heart rate of the 19°C and 26°C acclimated fish decreased
steadily with temperature until it fell to approximately 20 beats per minute, at which point the
heart rate remained stable. The plateau began near 15°C for 26°C fish and 9°C for the 19°C
specimens. Subsequently, heart rate fell significantly as temperature decreased below 5°C.
The heart rates of 26°C acclimated fish decreased more rapidly than those of fish
acclimated to cooler temperatures (Fig. 3). Heart rates of the 19°C fish decreased at the same
rate as the 9°C and seawater acclimated fish until the seawater reached 9°C. There is no
significant difference between the heart rate of the 9°C and the seawater acclimated fish. At 1°C,
both maintain a heart rate over double the rate of the 19°C and 26°C fish.
Behavioral differences were also noted. At 1°C, the 9°C and seawater acclimated fish
were responsive to stimuli whereas the 19°C and 26°C fish were not. All of the fish survived the
temperature decrease to 1°C except for two of the 26°C acclimated fish.
Discussion
All of the fish except for the 26°C acclimated specimens demonstrated a typical Qio
relationship for cardiac activity as temperature was decreased from the acclimation temperature
to approximately 5C Since the 9°C, 14°C and 19°C heart rates are very similar at the shared
temperatures, the three acclimations are equally able to meet the aerobic demands of the fish
until temperatures fall below 5°C. Heart rates of the 26°C declined at a faster rate than those of
the other acclimation groups, reaching approximately 20 beats per minute near 15°C. The
inability of the 26°C fish to maintain the same cardiac performance as the other acclimation
groups reveals that the 26°C fish already suffer from a decrease in cardiac performance at 19°(
and 14°C resulting in inadequate oxygen supply and a potential loss of fitness at those
temperatures. At 9°C and 5°C, the 26°C acclimated fish are able to maintain the same heart beat
as the other acclimated fish, reflecting a similar oxygen supply and possibly demand among all
groups. The ability of the 26°C fish to maintain the same heart rate as the other fish at these
temperatures while maintaining a lower heart rate at higher temperatures could be a reflection of
a drop in the fitness levels of the other acclimated fish and the ability of the 26°C fish to maintain
a steady heart rate despite a decrease in temperature.
5°C seems to be a pivotal temperature at which the acclimation effects are most evident
Decreasing the temperature past this point results in a dramatic decline in heart rate by the 19°C
and 26°C fish, which is not observed in the 9°C and 14°C fish. By 1°C, the colder acclimated
fish maintained a heart rate over twice that of the warmer acclimated fish. Thus, the 9°C and
19°C acclimated fish are able to maintain more of an aerobic capacity at temperatures only
slightly above 0°C. The large discrepancy between heart rates in the warmer and cooler
acclimated fish at 1°C provides compelling evidence that temperature acclimation has direct
effects on both the cardiac function and lower thermal limits of the fish. The 9°C and 14°C fish
are able to more effectively circulate blood throughout their bodies at low temperatures, which
demonstrates that acclimation has led to a lowering of the thermal window over which adequate
cardiac function can be maintained.
While the 9°C and 14°C fish maintain a higher heart rate at 1°C, relative to the 19°C and
26°C acclimated fish, this does not necessarily imply that the 9°C and 14°C acclimated fish are
capable of surviving for long periods of time in very cold environments. At this temperature the
fish suffer a loss of fitness and fecundity. In unpublished experiments conducted at the
McMurdo station in Antarctica, Somero, Jayasundara and Healy measured the heart rate of T.
pennellii and T. hansoni. These are polar fish, comparable in size to G. mirabilis, adapted to
live at-1.9°C. When heated to 1°C using similar experimental methods as used in this
experiment, the Antarctic fish displayed a heart rate near 20 beats per minute. As stenothermic
fish, T. pennellii and T. hansoni approximately represent what the heart rate would need to be in
order for a fish to survive and reproduce in a polar environment. The G. mirabilis acclimated at
a 9°C and 14°C maintained a heart rate of around 10 beats per minute at 1°C, only half the heart
rate displayed by the polar species at the same temperature. While the lower temperature
acclimated G. mirabilis have a better acute response to the decreasing temperature, it is clear that
the fish have minimal cardiac activity near the lower limit of their thermal window
The acclimation effects observed in this experiment represent change in the heart
physiology of the fish at different temperatures. By living in different temperatures for a month
long period, the acclimated fish likely remodeled their physiology by changing the types or
amount of enzymes and stress response proteins in the heart in order to increase cardiac output in
their respective environments. Metabolic enzyme activity studies could be performed on the fish
heart tissues to identify the changes in heart physiology. I conjecture that, while acclimating, the
19°C and 26'C expend energy making an increased number of heat shock proteins to protect the
heart from high temperatures and may down-regulate synthesis of other proteins, such as the
metabolic enzymes that generate ATP to support cardiac performance. Conversely, the 9°C and
14°C fish probably expend energy making only enzymes that can function well in cooler
temperatures and produce more enzymes to increase the rate of catalysis at low temperatures.
The results of this study show that acclimation results in a shift in the lower thermal limit
of G. mirabilis, however, it would be equally as interesting to see the effects of the same
acclimation on the upper thermal limits of the fish. If a true physiological change occurs in the
acclimated fish that directly affects ability to survive during acute temperature flux, then
temperature acclimation could result in a shift or a narrowing of the thermal window. By
monitoring the heart rate of the same fish as the ambient water temperature is increased, a more
complete thermal window could be created. I expect that the fish acclimated at 9°C would not
perform as well in high temperatures as the 26°C fish and therefore suffer from cardiac failure at
a lower temperature during acute heat stress.
Other studies of temperature effects on cardiac activity have demonstrated that sustaining
heart function at extremes of high temperature may be critical in the context of global warming
(Somero, 2010; Pörtner and Farrell, 2008). Notably, cardiac function in warm-adapted species
such as tropical congeners is in greatest jeopardy from global warming because critical
temperatures for heart failure lie close to current extremes of high temperature and acclimatory
capacity is much lower in warm-adapted species. Thus, for G. mirabilis, it will be important to
determine whether the species has sufficient phenotypic plasticity to increase the heat tolerance
of heart function over thermal ranges likely to result from on-going climate change.
Acknowledgments
I would like to thank George Somero for initially getting me excited about this research
and then advising me throughout the process. His knowledge on the subject is extensive and his
interest in the long-jaw mudsucker goby is unrivaled. I would also like to thank Nishad
Jayasundara for his generous support and guidance without which I would not have been able to
complete my research. Finally, I would like to thank Barb Block for teaching her Comparative
Animal Physiology class which taught me the importance of studying heart function in animals.
Appendix
Figure 1: Example of P-wave and ORS-complex from experiment
tt

r
Figure 2: Heart rate vs temperature in temperature acclimated fish (n-3). Both heart rate and
temperature are plotted as a function of time. A. 9°C B. 14°C C. 19°C D. 26°C
A.
9C Acclimated Fish n=3
40
12
35
10

30
8
§ 25
20

—2—Average
15
Temp
10
0:000:150:300:451:001:151:301:452:002:15
Time (HH:MM)
B.
C.
40
30
20
50
40
Seawater Acclimated Fish n-3



0:000:150:300:451:001:151:301:452:002:152:302:453:003:153:30
Time (HH:MM)
19C Acclimated Fish n=3



8
87

39
aaaaaaaaaaa-
Time (HH:MM)
14
12
10
8
15
10
——Average
Temp
-Average
—Temperature
D.
26C acclimated fish n=3
120
—9—260 Average
100
—— Temperature


- 80
20
0

15
10


1:15
0:00
2:30
3:45
5:00
6:15
Time (HH:MM)
Figure 3: Figure representing the average heart rate (n-3) of fish acclimated at 9°, 14°, 19° and
26°C at shared temperatures
120
100
80
190
60
140
40
E190
20
EI
1260
5
9
14 19 26
Temperature (C)
15
References
Dietz, T.J. and Somero, G.N., 1992. The threshold induction temperature of the 90-kDa heat
shock protein is subject to acclimatization in eurythermal goby fishes (genus Gillichthys)
Proc. Natl. Acad. Sci. USA 89, 3389-3393
Gamperl, A.K and Farrell, A.P., 2004. Cardiac plasticity in fishes: environmental influences and
intraspecific differences. J. Exp. Biol. 207, 2539-2550.
Gerlach, G.F., Turay, L., Malik, KT.A., Lidia, J., Scutt, A., Goldspink, G., 1990. Mechanisms of
temperature acclimation in carp: a molecular biology approach. Am. J. Physiol. 259,
R237-R244.
Moffitt, B. P. and Crawshaw, L.I., 1983. Effects of acute temperature changes on metabolism,
heart rate, and ventilation frequency in carp Cyprinus carpio. Physiol. Zool. 56, 397-403.
Morita, A. and Tsukuda, H., 1994. The effect of thermal acclimation on the electrocardiogram of
goldfish. J. therm. Biol. 19, 343-348.
Pörtner, H.O. and Farrell, A.P., 2008. Physiology and Climate Change. Science. 322, 290-292.
Pörtner, H.O., Mark, F.C. and Bock, C., 2004. Oxygen limited thermal tolerance in fish?:
Answers obtained by nuclear magnetic resonance techniques. Respir. Physiol. 141, 243-
260.
Somero, G.N., 2010. The physiology of climate change: how potentials for acclimatization and
genetic adaptation will determine winners' and 'losers'. J. Exp. Biol. 213, 912-920