The effect of temperature on the Heart rate and Respiration
of Daphnia
Introduction
A lot of research has been conducted on the influence of temperature on physiological
processes in animal models, results typically show an increase in the process rate with
increasing temperature. The aim of this study is to further investigate the effects of
temperature on heart rate for action potential generation, and oxygen consumption in
Daphnia.
Understanding the effects of temperature on physiological processes is of great value. As a
temperature reduced from its optimum results in a decreased rate of metabolic processes,
this may be used to improve neurological outcomes in patients who have suffered a brain
trauma such as stroke. Target temperature management (TTM) involves inducing
hypothermia in a patient to decrease metabolic rate and re-establish appropriate oxygen
levels. Many investigations show that in patients with perinatal asphyxia, TTM resulted in
improved neurological outcomes (CSZ Medical, 2015). Evidence also suggests that TTM may
be used to treat neurological injuries, such as those seen in stroke patients. One piece of
research showed that moderate TTM resulted in an average reduction of 44% of the
magnitude of the infarction and improved neurological outcome of patients who suffered
an Ischemic stroke (Andresen et al., 2016).
This presents the importance of research regarding the effects of temperature on metabolic
processes.
As ethical reasons prohibit the use of vertebrate animals in this study it was performed on
Daphnia. Daphnia, also known as ‘water-flea’, are freshwater invertebrate animals which
respire aerobically, they have an optimum temperature of 18-22 0C but can survive in
temperatures between 2-300C (Goss and Bunting, 1983). Daphnia’s internal organs are
easily seen under a light microscope due to their transparency, their heart is also an
excitable tissue which is dependent on oxidative metabolism and is stimulated by action
potentials. As Daphnia is an ectoderm, it’s internal body temperature can be changed by
changing its environment, making it a suitable model for investigating the effects of
temperature on heart rate and rate of oxygen consumption.
The Q10 values for heart rate and OCR from this experiment will be calculated and compared
to those deduced in previous experiments. Q10 is the ratio between two reaction rates or a
physiological process at two temperatures which are 100C apart, it is used to mathematically
describe temperature sensitivity. The Q10 for metabolic processes are usually 2-3, this is the
range expected from this experiment. The activation energy for heart rate and OCR will also
be produced by plotting an Arrhenius Plot of Ln(k) (rate process) against 1/T. The slope of
the line of best fit on this graph is equal to -Ea/R.
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Hypothesis:
1) There will be a significant increase in heart rate with increasing water temperature.
2) There will be a significant increase in the rate of oxygen consumption with increasing
water temperature.
Methods
A total of 220 Daphnia were used by 11 groups, in each group 20 Daphnia from Sciento
(Manchester), which were maintained in accordance with the suppliers’ instructions were
required, 15 were used to investigate oxygen consumption and 5 were used to investigate
heart rate. The experiment was conducted under the same conditions in each group and a
mean was calculated for heart rate and oxygen consumption at temperatures of ~100C
~150C, ~200C, ~250C and ~300C.
Equipment used
-
Perfusion Reservoir
Hot water bath (at 600C)
Crushed ice
Electrical thermometer (error of ±0.050C)
Light microscope
Microscope stage
Petroleum Jelly
Water Jacket
Oxygen Electrode (Clark, Cambridge)
Electrode controller
Digital Voltmeter
Plunger
Stirrer controller
Daphnia (Sceinto, Manchester)
Pipette
Equipment set up
The equipment to determine heart rate was set up by adding a small amount of petroleum
jelly to the bottom of the inner Perspex chamber, 5 Daphnia were then carefully placed on
to the jelly to ensure they remained static before the medium was added to the chamber,
which was illuminated with a bench lamp. A thermometer was then placed in the chamber
to monitor the temperature and the Daphnia were positioned so that the heart could be
viewed and the rate recorded. Different Daphnia were used to calculate heart rate for each
of the 5 temperatures.
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The equipment to determine oxygen consumption rate was set up using an O2 electrode to
measure the concentration of oxygen (O2(aq)) in the fluid of the closed system. The electrode
contained 4ml of oxygen rich pond water. In order to obtain accurate oxygen recordings the
stirring bar was adjusted so that it was continuously mixing the pond water at a slow rate,
the electrode controller was then adjusted so that a reading of ~350mV was displayed on
the voltmeter. To ensure the oxygen meter was working effectively the stirring was
switched off, resulting in a decrease in oxygen levels recorded. When the stirring was
switched back on the previous reading of oxygen levels recovered close to the original value.
A pipette was then used to transfer 15 Daphnia into the Perspex chamber of the oxygen
electrode. The plunger was then slowly twisted down into the chamber and the system was
checked to ensure there were no air bubbles, any excess solution was removed using the
pipette.
The required water temperature was calculated using the following equations:
Vc = ( Vf x (Th-Tf) ) / (Th-Tc) and Vh = Vf – Vc
Where Tf= final temperature required, Th = hot water temperature (60 0C), Tc = cold water
temperature (00C), Vf = final volume (500ml) , Vh = volume of hot water and Vc = volume of
cold water. The temperature was measured in Degrees Celsius(0C) and the volume was
measured in millilitres(ml).
For example for the temperature of 100C
Volume of cold water required = ( 500 x (60-10) ) / (60 – 0) = 417ml
Volume of hot water required = 500 – 417 = 83ml
The water was then prepared for final temperatures of ~100C ~150C, ~200C, ~250C and ~300C
just before the experiment at the required temperature began.
Experiment
The perfusion reservoir was filled with water at ~100C, the clamp was then adjusted to allow
a steady flow of water through the outer chamber. The temperature within the chamber
was then measured with the thermometer and maintained at ~100C whilst measurements
of heart rate and oxygen consumption were recorded simultaneously. The number of
heartbeats were counted and recorded by one individual for 60 seconds, this was repeated
to obtain 5 results for heart rate at ~100C. During this time a different individual recorded
the concentration of oxygen displayed on the voltmeter every 60 seconds for 5 minutes.
This was then repeated to deduce heart rate and oxygen concentrations at ~150C, ~200C,
~250C and ~300C by preparing and maintaining the temperature using the equation
previously described with hot water and ice for each new temperature.
The experiment for OCR was conducted in a closed system, which prevented oxygen levels
from being effected by factors other than the aerobic respiration of the Daphnia,
consequently the decrease in oxygen(mV) in the system measured by the digital voltmeter,
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was directly proportional to oxygen metabolised by the Daphnia. The oxygen concentration
recorded in millivolts is equivalent to the concentration in micro molar.
Calculations for Heart Rate
In each group the mean heart rate for the varying temperatures were calculated and
recorded in GraphPad Prism for comparison. The results for each temperature were used to
calculate a mean heart rate and SEM from the classes data.
This was then used to plot a graph of heart rate against temperature to visualise the effects
of temperature on the OCR of Daphnia (see figure 1).
Q10 Calculations:
The mean heart rates at temperatures 100C apart were used to calculate the Q10
value using the equation:
Q10 = (HR at T) / (HR at t)
Where ‘HR at t’ is the classes mean heart rate at a chosen temperature.
‘HR at T’ is the heart rate calculated at the temperature 100C higher than the
previously chosen temperature.
Example: Heart rate at 100C= 81, Heart rate at 200C= 146
Q10 = 146 / 81 = 1.80
As there were slight variations in Q10 values at different temperatures the mean
Q10 value was also calculated from these results (see table 1).
Activation Energy Calculations:
1/T was then calculated by converting the temperature in Degrees Celsius to Kelvin
by addition of 273. (eg. 100C = 283K), then diving 1 by the temperature (eg.
1/283=353.4x10-5).
Ln(heart rate) was then calculated and plotted against 1/Temperature to produce an
Arrhenius Plot (see figure 3).
As the slope of the Arrhenius Plot is equal to -Ea/R, this was then used to calculate
the activation energy of this process. The equation was rearranged to Ea = - Slope X R
(where Ea is the activation energy(KJ mol-1), and R is the gas constant (8.314x10-3).
For example, the activation energy for an Arrhenius Plot with a slope of -5012 was
deduced as follows.
Ea = - - 5012 X 8.314x10-3 = +41.67KJ mol-1.
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Calculations for Oxygen Consumption Rate (OCR)
In each group the oxygen consumption was calculated for each temperature by subtracting
the start oxygen concentration at 0 minutes by the end oxygen concentration at 5 minutes
(eg. 336-282=54mM). This gave the change in concentration of oxygen in the Perspex
chamber over 5 minutes, this figure was then divided by 5 to deduce the change in oxygen
concentration over 1 minute. (e.g. (336-282)/5=10.8mM min-1).
As there were 15 Daphnia in the Perspex chamber, this figure was divided by 15 to show the
OCR of 1 Daphnia (e.g. 10.8/15 = 0.72 mM min-1 Daphnia-1). This value was then converted
to nmoles by multiplying it by x103 (e.g. 0.72 x 103 = 720nM min-1 Daphnia-1).
Each group then entered their mean OCR value for each temperature onto GraphPad Prism
for comparison. Miscalculated results were identified and removed by Dr Paul Smith. The
remaining results for each temperature were used to calculate a mean OCR and SEM for the
classes data.
A graph of Mean OCR against Temperature was plotted to visualise the effects of
temperature on the OCR of Daphnia (see figure 2).
Q10 Calculations:
The mean OCR at temperatures 100C apart were then used to calculate the Q10
value using the equation:
Q10 = (OCR at T) / (OCR at t)
Where ‘OCR at t’ is the classes mean oxygen consumption rate at a chosen
temperature.
‘OCR at T’ is the oxygen consumption rate calculated at the temperature 10 0C higher
than the previously chosen temperature.
Example: OCR at 100C= 572, OCR at 200C= 1150)
Q10 = 1150 / 572 = 2.01
As there was a variation between Q10 values at different temperatures, the mean
Q10 value was also calculated from these results (see table 2).
Activation Energy Calculations:
Ln(OCR) was then calculated and plotted against 1/Temperature to produce an
Arrhenius Plot (see figure 4), using the 1/T values previously obtained.
The Arrhenius Plot was used to deduce the Activation Energy for Oxygen
Consumption as previously described for heart rate(see table 3).
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Results
The results show a steady increase in mean heart rate with increasing temperature,
statistical analysis also shows a significant positive association between temperature and
mean heart rate of Daphnia. The SEM for mean heart rate also increases with temperature.
This can be seen in figure 1.
Figure 1
(Figure 1 gives a visual representation of the change in mean heart rate and SEM calculated
from the classes results with increasing temperature (n=11 at each temperature). The
Pearsons test performed on Prism produced a correlation coefficient value of r=0.987, and
p=0.0007).
The results show an increase in mean OCR with increasing temperature. Statistical analysis
tests showed a significant positive association between temperature and mean OCR of
Daphnia. The SEM for mean OCR also increases with temperature increase. This can be seen
in figure 2.
Figure 2
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(Figure 2 gives a visual representation of the mean OCR and SEM, calculated from the
classes results as a function of temperature (n=11 before, after n=6 at 100C, n=6 at 150C,
n=7 at 200C, n=8 at 250C). Some results were removed due to miscalculations. The Pearsons
test performed on Prism produced a correlation coefficient value of r=-0.918, and
p=0.0409).
The Q10 value for heart rate was found to slightly increase from 1.80 to 1.81 between the
temperature intervals of 10-200C and 15-250C. The Q10 value is highest between 15-250C,
before it decreases to 1.73 between 20-300C. The mean Q10 value calculated for heart rate
in Daphnia was found to be 1.78. This can be seen in table 1.
Table 1
Temperature Intervals / 0C
10 – 20
15 – 25
20 – 30
Q10 Value
1.80
1.81
1.73
Mean: 1.78
(Table 1 shows the temperatures from which the mean Q10 Value for Heart Rate were
deduced. SD±0.036 and SEM±0.021).
The Q10 value for Oxygen Consumption Rate was found to increase from 2.01 to 3.25
between the temperature intervals of 10-200C and 15-250C. The mean Q10 value calculated
for OCR in Daphnia was found to be 2.63. This can be seen in table 2.
Table 2
Temperature Intervals / 0C
10 – 20
15 – 25
Q10 Value
2.01
3.25
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Mean: 2.63
(Table 2 shows the temperatures from which the mean Q10 Value for OCR were deduced.
SD±0.620 and SEM±0.438).
A significant linear association was found between mean ln heart rate and 1/temperature,
as seen in figure 3.
Figure 3
6 .0
M e a n L n H e a rt R a te
5 .5
5 .0
4 .5
4 .0
330
350
340
T e m p e ra tu re ( 1 /K x 1 0
5
360
)
(Figure 3 gives a visual representation of the association between the mean Ln heart rates
and 1/T. The Pearsons test performed on Prism produced a correlation coefficient value of
r=-0.999, and p=<0.0001. The gradient of the graph was found to be -5012 ± 127.9).
A significant linear associations was found between mean ln OCR and 1/temperature, as
seen in figure 4.
Figure 4
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(Figure 4 gives a visual representation of the association between the mean Ln OCR and 1/T.
The Pearsons test performed on Prism produced a correlation coefficient value of r=-0.963,
and p=0.0183. The gradient of the graph was found to be -8306 ± 1636).
The activation energy deduced for both Heart rate and oxygen consumption were positive,
with oxygen consumption having a higher activation energy than heart rate, this can be seen
in table 3.
Table 3
Slope of the
Arrhenius Plot
Activation
Energy (KJ mol1)
41.67
69.06
Q10
Value
Results for Heart Rate
-5012
1.78
Results for Oxygen
-8306
2.63
Consumption
(Table 3 shows the activation energy and mean Q10 Values for heart rate and OCR).
Discussion
For the one-tailed Pearsons test it was assumed that the data are parametric for both heart
rate and OCR. The results for mean heart rate against temperature and OCR against
temperature show a strong significantly positive relationship, supported by the Pearsons
statistical test (heart rate: r=0.987, p=0.0007) (for OCR: r=0.918, p=0.0409), therefore
hypothesis 1 and hypothesis 2 have been accepted and their retrospective null hypothesis
rejected.
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As Daphnia are ectotherms increasing the temperature of the water (their external
environment) increases their internal body temperature. This rise in temperature results in
an increased metabolic rate, creating a thermal reaction norm in response to change in
environment (Gajic, 2013). The increased temperature within the Daphnia resulted in
increased kinetic energy, therefore more particles had the activation energy to initiate a
chemical reaction. These metabolic reactions require ATP, therefore to supply the increasing
demand there is an increase in oxygen consumption for respiration, as oxygen and glucose
are converted to carbon dioxide, water and ATP. There is also an increase in heart rate to
supply the respiring cells with oxygen for respiration to occur, and remove the carbon
dioxide waste product. As temperature was further increased the metabolic rate also
increased. If the temperature had continued to increase past 30 0C it would have been
expected to start to decrease the metabolic rate as a result of denaturing the enzymes and
eventually killing the Daphnia.
The Q10 results show how temperature dependant the rate of reaction is, a value of 1 would
suggest that the rate of reaction is completely independent of temperature, the higher the
Q10 value is the more temperature dependant the rate of reaction is. The Q10 results for
both heart rate(1.78 (SEM±0.021)) and OCR(2.63 (SEM±0.438)) were >1, showing that these
processes are temperature dependant. The mean Q10 value for heart rate in this experiment
was found to be lower than the mean Q10 value for OCR, suggesting that respiration is a
more temperature dependant process than action potential generation for heart
contraction in Daphnia.
The Q10 value for OCR is in the expected range of 2-3 in most biological systems.
The Q10 value for heart rate lies below the expected range of 2-3 for most biological
systems, this may be due to human error as the mean heart rate recordings that the Q10
value was calculated for was from the class data, which was obtained by 11 different
individuals visually counting heart beats over a 60 second period. This could be improved by
having the same individual count the heart beats throughout as individual differences may
have occurred across the group on what a definite heart beat looked like.
Also an issue with Q10 is that it is limited over a temperature range of 100C, but as seen in
table 1 and table 2 the ratio between reaction rates vary across temperatures with a larger
range than 100C, reducing its accuracy. An Arrhenius Plot of Ln(rate of process) against 1/T
was therefore produced, the slope of this graph was used to calculate the activation energy
for heart rate(41.67KJ mol-1) and OCR(69.06KJ mol-1).
A limitation to this experiment was that metabolism is influenced by factors which were not
controlled, such as body size and food abundance. As these factors weren’t taken into
account the results produced were less reliable, therefore the experiment should be
repeated taking these factors into account.
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Overall this study shows that the rates of metabolism are slower at lower temperatures as a
result of the induced hypothermia. As heart rate is reduced by cooling the Daphnia, this
shows that decreasing temperature also decreases metabolic reactions in the brain as the
rate of action potential generation has decreased to result in the decreased heart rate. This
could reduce neurological damage in the brain tissue of stroke patients. In the acute stages
of stroke there’s a decrease in cerebral blood flow causing an ionic imbalance. Metabolic
rate has been found to decrease by 6-7% for a 10C decrease in an individual’s temperature
(Polderman and Herold, 2009). Induced hypothermia can therefore be used to reduce
respiration and preserve oxygen and energy supplies. Although hypothermia has been
effective at treating stroke in laboratories it hasn’t been successful in clinical trials,
therefore more studies need to be performed on the effectiveness of hypothermia as a
treatment for stokes.
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