Mass-specific rate of ammonia production and oxygen production of ​Orconectes

virilis ​under different temperature conditions

Ishira Bharadwaj 20010234
Ariel Elman-Walker 20015488

Workplan

Calibrate Qubit electrode

Set volume of chamber to 1ml
Calibrate lower limit (0mg/L): fill chamber with N2 bubbled water, turn on stir bar and wait for voltage to
stabilize

**remove N2 bubbled water and flush chamber with oxygenated water several times before next step**

Calibrate upper limit (9.1mg/L): fill chamber with oxygenated water, turn on stir bar and wait for voltage
to stabilize

Drain chamber

Crayfish Trials

Remove 3ml of water out of control and experimental crayfish chambers at t = 0, 20, 40, and 60
*replenish chamber with fresh water for exact volume of sample water removed*

Measure dissolved O2: fill Qubit electrode vessel with 1ml water from sample and "Collect" when
readings stabilize (repeat for each sample collected)

Volume of animal chambers: control _______ml experimental: _______ml

Weigh crayfish: control: mg = ________ experimental: mg = ________

Prepare spectrophotometric ammonia assay

***For NH4Cl use 10 or 100µL pipette, for H20, use 1000µL pipette***
1mL each of:
0µM: 0µL NH4CL + 1000µL H20
5µM: 5µL NH4CL + 995µL H20
10µM: 10µL NH4CL + 990µL H20
20µM: 20µL NH4CL + 980µL H20
40µM: 40µL NH4CL + 960µL H20
80µM: 80µL NH4CL + 920µL H20

Make Reagent 5: 1mL of Reagent 1 + 5mL of Reagent 2

Dispense 200µL of each standard and unknown (t= 0, 20, 40, 60) in triplicate into plate
+ 25µL of Reagent 3, then mix
+ 25µL of Reagent 4, then mix
+ 25 µL of Reagent 5, then mix

Place in dark for 40min then read in SoftMax Pro

equation of standard curve: ____________

Analyze Data
Calculate mass-specific rate of ammonia production (M​N​)

Calculate mass-specific rate of oxygen production (MO2) and convert to µmol/min/g

Calculate nitrogen quotient (NQ)

Hypothesis
We hypothesize that warmer water temperature induces a crayfish’s metabolic rate to
increase due to the higher energy availability for metabolic reactions to occur. We predict that
the metabolic rate (assessed using measures of nitrogen excretion and dissolved oxygen) of ​O.
virilis​ will be higher in 30°C water than in 21°C control condition.

Materials and Methods

For this experiment we measured the rates of ammonia production and levels of dissolved
oxygen in the chambers of two ​O. virilis​ specimens, one in a room temperature (control)
condition and one in a 30°C condition at four time points (t = 0, 20, 40, and 60 minutes). Each
animal was enclosed in a glass jar roughly 250ml in volume and was either placed in a 30°C
water bath (experimental condition) or placed on the lab bench (control) for the length of the
experiment (1 hour). Each animal chamber was airtight and had two syringe plungers sealed to
the lid, one with a short (~1cm) and one with a long (~6cm) piece of PE90 tubing attached, to
allow 3ml of water to be drawn from the chamber using one syringe (long tubing) while 3ml of
oxygenated water is replenished by the other (short tubing) to keep the total volume of water the
same.
Every 20 minutes a 3ml sample was drawn from each of the two animal chambers and
kept in an airtight syringe plunger and any air bubbles remaining were removed. Each sample
was analyzed for ammonia levels using a spectrophotometric ammonia assay and dissolved
oxygen levels using a Qubit electrode.
The ammonia assay was created using a working stock of 1mM ammonium chloride to
create six standard solutions (0µm, 5µm, 10µm, 20µm, 40µm, 80µm) with additional reagents:
salicylate, sodium nitroprusside, sodium hypochlorite, and alkaline citrate (details in
supplemental information). 200µl of each standard solution and each trial sample was pipetted
into a 96-well microplate in triplicate and analyzed in SoftMax.
To measure dissolved oxygen with the Qubit electrode, the system was set to hold 1ml of
liquid and then calibrated using N​2​ bubbled water containing chemical zero sodium sulphate to
obtain the lower limit (0mg/L dissolved O​2​) and 100% air-saturated water to obtain the upper
limit (9.1mg/L dissolved O​2​). For calibration and between each measurement of the sample
trials, the Qubit electrode was flushed with oxygenated water to ensure that no residue from the
previous sample remained. The stir bar inside the Qubit electrode was turned on during each
measurement and turned off in between trials. Dissolved oxygen concentrations for each trial
sample were calculated as the mean of five diffused oxygen readings surrounding the 1-minute
mark after the sample was injected into the Qubit electrode.
Mass-specific rate of ammonia production (M​N​) (in µmol/min/g) was calculated for each
sample interval using the following equation:

Mass-specific rate of oxygen production (MO​2​) was calculated using another equation,
which differs slightly from M​N​ by using dissolved oxygen concentrations instead of ammonia
concentrations:

Finally, the nitrogen quotient was determined by the ratio of M​N ​/ MO​2​, using the average
mass-specific rates of ammonia and oxygen production across all time intervals. This value,
when divided by 0.27, is used to indicate how much of ​O. virilis​’ aerobic respiration is fueled by
protein.
Results

Figure 1. ​a​. Mass-specific oxygen consumption rates (MO​2​) of ​O. virilis,​ measured in µmol/min/g, across three time
points (t0-t20, t20-t40, and t40-t60) between the room temperature (control) and 30°C (experimental)
conditions. ​b​. Mass-specific ammonia production rates (M​N​) of ​O. virilis​, measured in µmol/min/g, across
three time points between the room temperature and 30°C conditions. Values are displayed in
Supplementary Table 1.

Mass-specific oxygen consumption rates (MO​2​) and mass-specific ammonia production

rates (M​N​) were calculated for each of three time intervals and compared between conditions
(Figure 1). MO​2​ and M​N​ were both negative values at interval t20-t40, and also at t40-t60 for M​N​.
This result was unexpected and may be reflective of experimental error. Levels of diffused
oxygen are seen to increase slightly in the room temperature condition and decrease in the
experimental condition. Ammonia levels remain relatively stable in the room temperature
condition and decrease drastically in the experimental condition.

Figure 2. Comparison of mean mass-specific oxygen consumption rate and mean mass-specific ammonia production
rate of ​O. virilis,​ both measured in µmol/min/g, between room temperature and 30°C conditions.
 Oxygen consumption and ammonia production rates were averaged across the time
intervals and compared between conditions (Figure 2). Negative values were not included in the
calculations to eliminate results due to experimental error. For both oxygen and ammonia, mean
metabolite production rates were higher in the high-temperature experimental condition
(MO​2exp.ave.​: 1.18µmol/min/g, M​Nexp.ave.​: 16.31µmol/min/g) than in the room temperature condition
(MO​2cont.ave.​: 0.32µmol/min/g, M​Ncont.ave.​: 1.66µmol/min/g).

Figure 3. ​a. ​Comparison of nitrogen quotient (NQ) of ​O. virilis​, calculated by ratio of M​N​/MO​2​, between room
temperature and 30°C conditions. ​b. ​Comparison of respiratory quotient (RQ) of ​O. virilis,​ calculated by
NQ/0.27 between room temperature and 30°C conditions.

Both the nitrogen quotient (NQ) and the respiratory quotient are higher in the 30°C
condition (NQ​exp.​: 13.83, RQ​exp.​: 51.23) compared to the control condition (NQ​cont.​: 5.24, RQ​cont.​:
19.39) (Figure 3). The mean values of oxygen and ammonia production rates shown in Figure 2
were used to calculate NQ which was subsequently used to calculate RQ, by dividing by 0.27,
which signifies the proportion of aerobic respiration of ​O. virilis​ that is fueled by protein.

Interpretations

Our results suggest a general proportional trend in higher temperatures coinciding with a
higher metabolic rate seen through our elevated nitrogen excretion and oxygen consumption
rates. This data is consistent with our hypothesis that higher temperatures are associated with
higher metabolic rates.
It is known that aquatic animals usually excrete ammonia when breaking down proteins
in the process of metabolism. The mechanisms underlying metabolism require sufficient
molecular energy in order to fuel cellular processes. It is commonly proposed that high
temperature conditions require more ATP to fuel faster processes as a result of higher kinetic
cellular energy [1]. When cellular components of an organism, in this case an aquatic animal,
encounter increased temperature conditions, their kinetic energy increases as a result, causing the
metabolic process of aerobic respiration such as oxidative phosphorylation to speed up. This
increases O2 consumption and waste production, seen in increasing O2 consumption and
ammonia production respectively in Figure 2.
It is evident that numerous discrepancies in our data may be the result of contamination
in our experimental results, causing inconsistent trends such as the low, high, low pattern for
control conditions in Supplementary Table 1. It is also crucial to mention that during this
experiment, movement and transportation of crayfish may have elicited a stress response due to
abnormal movement and noise, leading to changes in metabolic processes that may not be
indicative of temperature changes seen in nature without interference. Additionally, experimental
error such as leaks or air bubbles in the animal chamber may have affected the results. There was
also a large size difference between the crayfish in the experimental and control conditions, with
the control condition crayfish being nearly twice as large. The larger size may have contributed
to the reliable results obtained from the control condition samples.

References

1. Clarke, A. and K. P. P. Fraser. 2004. Why does metabolism scale with temperature?
Functional Ecology​ 18:243-251.
https://besjournals.onlinelibrary.wiley.com/doi/full/10.1111/j.0269-8463.2004.00841.x
Supplemental Information

Spectrophotometric Ammonia Assay

Standard Solutions:
1mL each of:
0µM: 0µL 1mM NH4CL + 1000µL H20
5µM: 5µL 1mM NH4CL + 995µL H20
10µM: 10µL 1mM NH4CL + 990µL H20
20µM: 20µL 1mM NH4CL + 980µL H20
40µM: 40µL 1mM NH4CL + 960µL H20
80µM: 80µL 1mM NH4CL + 920µL H20

200µL of each standard and unknown (t= 0, 20, 40, 60) in triplicate into plate, then add:
• 25µL of 40% sodium salicylate, then mix
• 25µL of 0.02/100ml sodium nitroprusside, then mix
• 25 µL of Reagent 5*, then mix

*Reagent 5: 1ml 6% sodium hypochlorite + 5mL 35% alkaline citrate

Place in dark for 40min then read in SoftMax Pro

M​N MO​2

Condition 0-20min 20-40min 40-60min 0-20min 20-40min 40-60min

Control (RT) 0.959 2.730 1.382 0.0646 0.320 0.569

Experimental 16.31 -6.578 -0.739 1.635 -1.055 0.723

Supplementary Table 1. Mass-specific ammonia production and mass-specific oxygen production rates in response
to either control (room temperature) or experimental (30°C) conditions.
Supplementary Figure 1. Dissolved oxygen measurement output from LoggerPro. Dissolved oxygen data points
were calculated as the average of 5 surrounding recorded measurements, demonstrated by the highlighted
points above.

Calculations

Raw data:
Ammonia Assay (µmol) Diffused Oxygen (mg/L)

Time (min) Control Experimental Control Experimental

t0 6.442 4.055 5.241 6.318

t20 7.100 10.009 5.952 15.8.70

t40 8.978 7.607 9.478 9.704

t60 9.931 7.337 15.74 13.93

Crayfish Mass:
Control: 8.514g Experimental: 4.590g

Volume of animal chamber: 256ml

Volume of water in chamber containing animal:

Control: 247.49ml Experimental: 251.41ml
Sample calculation for t20-t40 interval in control condition:
M​N​:

M​N​ ​= (((8.978-7.100)÷(40-20))*247.49)÷8.514 = ​2.730µmol/min/g

MO​2​:

MO​2​ ​= (((9.478-5.952)÷(40-20))*0.24749 = 0.436mg/min

0.0436mg/min ÷ 16 = 0.00273mmol/min

0.00273mmol/min * 1000 = 2.73µmol/min

2.73µmol/min ÷ 8.514 = ​0.320µmol/min/g

NQ:

Mean control M​N​ = 0.318 µmol/min/g

Mean control MO​2​ = 1.66 µmol/min/g

NQ of control condition = 0.318 ÷ 1.66 = ​5.24

RQ:

RQ of control condition = 5.24/0.27 = 19.39