This protocol uses juvenile American lobsters as a model to demonstrate the use of impedance pneumography to assess physiological performance during a thermal stress. This technique passes a small oscillating electrical current across two electrodes implanted on either side of the heart and measures the change in voltage as the muscle contracts and relaxes. This voltage can then be easily converted to beats per minute to plot heart rate across the temperature range assessed to determine thermal performance windows.
This technique is minimally invasive and allows for real time data collection in late stage organisms in which it may be difficult to directly visually observe cardiac performance. This method also focuses on calculating the Arrhenius break temperature or ABT and uses this as a non lethal endpoint to determine thermal limits. Wrap clear malleable tubing around itself to create a heat exchanging coil that is approximately eight to 10 centimetres in diameter and has extensions that are 40 to 70 centimetres long.
Secure the coil using electrical tape. Attach the heat exchanging coil to the external supply and return fittings of the circulating water bath. Ensure the connection is secure using hose clamps.
Fill the well of the circulating water bath with reverse osmosis water and plug the power cord into an outlet. Turn the water bath on and make sure there are no leaks in its connection to the heat exchanging coil. Set up the impedance converter by plugging in the black BNC cable to the AC output on the unit and connecting it to the power lab data logger using the channel one port.
Plug the thermistor into the T type pod then plug the T type pod into the channel two port of the power lab data logger. Plug the power cord of the power lab data logger into a power supply and connect the data logger to a PC computer using the USB cable connector. Fill the acclimation chamber in the experimental arena with 7.5 litres of artificial seawater.
Place the lobster on a plastic grid. Carefully secure the lobsters claws and abdomen to the plastic grid using small cable ties. Dry off the carapace with a paper towel and clean it with a cotton swab soaked in 70%ethanol.
Carefully hand drill two small holes nearly through the carapace on either side of the pericardium. Finish each hole by gently inserting a sterile dissecting needle. Scrape off a small bit of the wires insulation using a razor blade.
Carefully bend the tip of each wire using forceps and insert one into each of the newly drilled holes. Secure each wire lead using a small drop of superglue and allow it to dry for five to 10 minutes. Once the glue is dry, attach the wire leads to the impedance converter and turn it on.
Place the lobster into the acclimation chamber and allow it to acclimate to the implanted electrodes for 15 to 20 minutes. Turn the power lab on and open the lab chart software on the computer. Click New experiment and leave the chart view screen open.
In chart view, locate the channel function menu for Channel One. Choose input amplifier from the menu and select AC coupling. On the impedance converter, adjust the gain and balance until a strong signal is observed on the power lab output, aiming to keep the balance near zero.
On channel two select T-type pod to record real time temperature data. Click the Start button and the power lab will begin logging data. Place the plastic grid with the attached lobster carefully onto the experimental arena and set the heat exchanging coil on top of the grid.
Place the thermistor near the lobster, before placing the lid on the experimental arena to reduce visual stress on the test subject. Adjust the balance as needed and place a comment on the output stating that the trial has begun. The output should be saved periodically throughout the experiment.
Click File and select Save As to initially save the output to the computer. Increase the water temperature of the experimental arena at a rate of approximately 1.5 degrees Celsius every 15 minutes to achieve a ramp from 12 to 30 degrees Celsius over a two hour and 30 minute period by adjusting the temperature of the recirculating water bath. When the ramp is completed, remove the lobster from the experimental arena and place it into a recovery bath for approximately 20 minutes.
After 20 minutes, hit the stop button on the power lab output and save the file. Carefully remove the electrodes and cut the cable ties before returning the test subject to its holding tank. Open the data pad by clicking on the Window menu and selecting data pad.
Set column A to be time by double clicking on column A and clicking on selection and Active Point, select time, and close the window by clicking OK.Set column B to be the average temperature over the selected portion of data by double clicking on column B and selecting Statistics. Select mean from the right hand side of the menu and channel two as the calculation source at the bottom of the Menu's window. Double click on column C and select selection and Active Point.
Select selection duration, click OK to close the window. Double click on column D and select cyclic measurements. Select event count, select channel one as the calculation source, click OK to close the window.
This will count the peaks of the data to determine heart rate across a selected portion of data. Double click on column E and select cyclic measurements. Select average cyclic rate and channel one as the calculation source at the bottom of the menus window.
Click OK to close the window and this provides the final estimation of heart rate as beats per minute over a selected portion of data. Go back to the data file and highlight the desired sections of the output. Select commands and multiple add to the data pad.
Select time, and pull data every 30 seconds by checking the every box and entering 30 under the Select menu. Click current selection and click Add. Return to the data pad screen and select File and Save As to save the output as an Excel file.
Open the data file in Excel. Convert temperature from degrees Celsius to the inverse of Kelvin. Take the natural log of heart rate, generate an Arrhenius plot by plotting heart rate as a function of temperature.
Fit these data with a piecewise regression and determine the intersection point. This is the Arrhenius brake temperature or ABT. This is an example of expected output using the power lab data logger.
The voltage is shown in red and the arena temperature in blue. These data show an expected distribution of heart rate over the course of a temperature ramp. Finally, this panel shows an expected Arrhenius plot from an experimental animal.
Fit with a piecewise regression, the red star highlights their intersection which is the Arrhenius break temperature. When using this technique, it's important to allow your test subject proper acclamation and recovery time periods to ensure that you're accurately recording cardiac performance. It is also important to determine the appropriate rate of warming for your experiment prior to running this on a test subject.
Although we focus solely on temperature in this video, this technique can be broadly applied to understand environmental impacts on organismal physiology by pre exposing test subjects to additional stressors such as ocean acidification, or reduced oxygen availability prior to running them through the temperature ramp.
