Measuring heart rate during a thermal challenge provides insight into physiological responses of organisms as a consequence of acute environmental change. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography as a relatively noninvasive and nonlethal approach to measure heart rate in late stage invertebrates.
Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by environmental temperature, this has the potential to alter thermal physiological performance in a variety of marine organisms. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography to understand how cardiac performance in late stage invertebrates changes under acute thermal stress. The protocol presents a minimally invasive technique that allows for real-time collection of heart rate during a temperature ramping experiment. Data are easily manipulated to generate an Arrhenius plot that is used to calculate Arrhenius break temperature (ABT), the temperature at which heart rate begins to decline with increasing temperatures. This technique can be used in a variety of late stage invertebrates (i.e., crabs, mussels, or shrimps). Although the protocol focuses solely on the impact of temperature on cardiac performance, it can be modified to understand the potential for additional stressors (e.g., hypoxia or hypercapnia) to interact with temperature to influence physiological performance. Thus, the method has potential for wide-ranging applications to further understand how marine invertebrates respond to acute changes in the environment.
In recent decades, increased input of greenhouse gases (i.e., carbon dioxide, methane, and nitrous oxide) into the atmosphere has resulted in widespread patterns of environmental change1. The world’s oceans are rapidly warming2,3, a trend that may have severe impacts on organismal physiology. Temperature heavily influences physiological rates, and organisms have an optimal temperature range for performance4,5,6. As such, individuals may encounter difficulties in maintaining proper oxygen delivery to tissues as temperatures stray outside of this range. This has the potential to lead to declines in aerobic performance in the face of warming ocean temperatures5,7.
In a laboratory setting, a method to understand the physiological impacts of environmental change is to examine cardiac performance in the context of thermal stress. This provides insight into how exposure to predicted warming conditions may alter performance curves5,6 as well as the potential for acclimation plasticity8. A variety of methods have been successfully implemented to previously measure heart rate in marine invertebrates. However, many of these techniques involve surgical removal or major manipulation of the exoskeleton and prolonged implantation of measurement devices9,10,11, which introduces additional stress to the test subject and increases the time needed for a successful recovery prior to experimentation. Moreover, less invasive techniques (e.g., visual observation, videography) may be restricted to early life history stages when organisms may be fully or semi-transparent12. Furthermore, additional challenges may be presented to researchers who are not well-versed in more technologically advanced methodologies (e.g., observations via infrared transducers or Doppler perfusion8,11).
This protocol uses the American lobster (Homarus americanus) as a late stage marine invertebrate model to demonstrate the use of impedance pneumography for assessing changes in heart rate during a temperature ramping experiment. Impedance pneumography involves passing of an oscillating electrical current (AC) across two electrodes positioned on either side of the pericardium to measure changes in voltage as the heart contracts and relaxes13,14. This technique is minimally invasive, as it employs the use of small electrodes (i.e., 0.10–0.12 mm diameter) that are gently implanted just beneath the exoskeleton. Finally, it provides real-time assessments of both heart rate and water temperature during the ramp through the use of a data logger.
The protocol also provides instructions for calculating Arrhenius break temperature (ABT), the temperature at which heart rate begins to decrease with increasing temperatures13,15. The ABT serves as a nonlethal indicator of the thermal limit of capacity in test subjects that may be favored over measuring the critical thermal maximum (CTmax, the upper limit of cardiac function5,6), as lethal limits are often extreme and rarely encountered in the natural environment5.
1. Equipment setup
2. Implantation of electrodes
3. Temperature ramping
4. Data conversion
5. Calculation of Arrhenius break temperature
This protocol describes the use of impedance pneumography to obtain real-time data for heart rate (in voltage) and temperature during a temperature-ramping experiment. When perforing this technique, the amplitude of the voltages and temperatures recorded will vary based on experimental design and focal species. However, the voltage output displayed in real-time follows a generic sine distribution when the protocol is implemented correctly (Figure 1A). As the temperature in the arena is increased, the real-time distribution of voltage changes to reflect an increased frequency of voltage peaks (i.e., heart beats; Figure 1B). As the arena temperature continues to increase to levels outside of the test subject’s optimal performance window, the distribution changes to depict a reduced frequency of voltage peaks with a sine-like shape interrupted by sporadic peaks and/or moments of “flat-lining” (Figure 1C).
Once raw data are converted using the Data Pad component of the LabChart software, the resulting distribution of heart rate (in beats per minute) over the course of the temperature ramp follows a parabolic distribution if the experiment is successful (Figure 2). As the temperature in the arena is increased, the heart rate of the test subject also increases to meet elevated energetic demands associated with warmer temperatures. However, as temperature continues to increase and the test subject begins to experience moderate to extreme thermal stress, heart rate begins to decline or becomes erratic as the subject begins to exhibit passive thermal tolerance (e.g., onset of anaerobic respiration, metabolic rate suppression, and reduced activity5,7). When heart rate and temperature data are transformed and an Arrhenius plot is generated, the point at which the heart rate begins to decline (ABT) can be calculated (Figure 3). The Arrhenius plot is then fit with a piecewise regression using statistical software in which the intersection of the two lines represents the ABT.
Figure 1: Representative output from LabChart data logger. Real-time change in voltage across electrodes of the test subject is displayed in red, and the concomitant real-time output of the arena temperature (°C) is displayed in blue. In the beginning of the experiment under cooler temperatures (e.g., 13.1 °C), voltage should follow a generic sine-like distribution (A). As temperature is increased (e.g., 23 °C), the frequency of voltage peaks should increase, but the distribution should remain sine-like (B). Finally, as the test subject is pushed outside of its optimal thermal performance window (e.g., 28.5 °C), the voltage peaks should become erratic as the frequency decreases (C). Please click here to view a larger version of this figure.
Figure 2: Expected distribution of heart rate over the temperature ramp course. Voltage data collected by the data logger are converted to heart rate in beats per minute (BPM) using the Data Pad component of the software. When the ramp is conducted correctly, a parabolic distribution of heart rate over the temperature range tested is displayed. Please click here to view a larger version of this figure.
Figure 3: Example of an Arrhenius plot. Once data have been converted in the Data Pad and exported, they are transformed to generate an Arrhenius plot. In this example, data are fit with a piecewise nonlinear regression in SigmaPlot, generating equations for the left- and right-hand segments (region 1 and region 2, respectively) of the regression line, as well as goodness-of-fit metrics. The intersection of the two regression lines is solved as the ABT (red star). Please click here to view a larger version of this figure.
This protocol describes the use of impedance pneumography to measure changes in heart rate of late stage invertebrates during a temperature ramping experiment. The primary benefit of this technique compared to other laboratory-based approaches9,10,11 is that it is minimally invasive and does not involve major surgical manipulation of the exoskeleton, thus reducing the amount of recovery time needed prior to experimentation. Moreover, the equipment is easy to use, and resulting data can be simply manipulated and interpreted in the suggested software program. While the American lobster is used here as a model subject, this technique has been successfully implemented in blue mussels (Mytilus spp.14) and can be easily modified for use in other late stage invertebrates (i.e., crabs, shrimps, and other bivalves).
An additional benefit of the protocol is that it focuses on calculating the ABT as a nonlethal indicator of thermal limits. Although numerous studies present the CTmax as the significant endpoint when determining thermal physiological performance5,8,20,21,22,23, organisms rarely encounter temperatures in this range in the natural environment5. Moreover, as the CTmax is often a lethal temperature, using this metric as the preferred endpoint precludes the use of test subjects in additional or follow-on experimentation post-thermal stress23. When aiming to calculate the ABT using this protocol, it is crucial to increase the temperature in the experimental arena to the point of pushing the test subject to its physiological limit without inducing death. Therefore, it is recommended to determine the potential thermal limits of the focal species via a pilot study (when possible) prior to determining the full range of the experimental temperature ramp.
It is also recommended that researchers determine and observe natural variations in basal heart rate of a focal species when temperature in the experimental arena is maintained at a constant and non-stressful level prior to the ramping experiment. This is particularly helpful for focal species in which resting heart rate information is not available in the published literature. It also serves as ample practice of electrode implantation techniques. This may also help researchers determine the appropriate acclimation time required to ensure that no false spikes in heart rate are due to handling stress at the beginning of an experiment.
Although the protocol discusses the use of impedance pneumography in the context of thermal stress alone, it can also be utilized to explore the potential interactive effects of other stressors on thermal physiology. Organismal performance may be reduced in the presence of environmental stressors (i.e., hypoxia, hypercapnia, pollutants, and/or changes in salinity), which may also compress optimal temperature ranges for performance7,24,25,26. As such, this protocol can be modified to explore how exposure to various stressors prior to temperature ramping may impact performance.
For example, Harrington and Hamlin27 exposed juvenile H. americanus to current or predicted end-century pH conditions (8.0 and 7.6, respectively) for 2 months prior to assessing cardiac performance during a temperature ramp. Lobsters pre-exposed to more acidic environments exhibited a significant reduction in mean ABT compared to those held under current pH conditions. This suggests that a low pH environment reduces thermal performance and may increase the risk of cellular damage due to heat stress at lower temperatures27. Future efforts could expand on the method presented here to include pre-exposure to any combination of environmental stressors prior to following this protocol. Moreover, this protocol can be modified to measure changes in cardiac performance during exposure to biotic stressors as well as how thermal limits can change according to ontogeny4,5.
A major limitation of this protocol is that the equipment as described is restricted for use in a laboratory setting, potentially limiting its applicability for field-based experiments that require more specialized equipment8. This technique also requires the restraint of highly motile test subjects (e.g., lobsters and crabs) to reduce the production of false data points resulting from non-cardiac muscle movements. Although this may restrict natural behaviors during a temperature ramp, the impact of restraints are consistent across all test subjects. Most importantly, there is the potential for tissue damage or death in test subjects if aggressive or careless drilling during electrode implantation is implemented. This contrasts sharply with infrared photoplethysmography, a truly noninvasive technique that utilizes an external infrared transducer to pass light through the pericardium and record heart function by converting reflected light energy to voltage8,28.
Although infrared photoplethysmography reduces the risk of handling stress compared to impedance pneumography, correctly implanting electrodes using the described method results in minimal trauma, allows for a quick acclimation time, and leads to rapid recovery without inducing mortality in test subjects following the ramping experiment27. As there is no significant difference in the cardiac output recorded by both methods28, it is concluded that impedance pneumography is a reliable and minimally invasive technique for assessing cardiac performance. Finally, the numerous benefits and flexibility of the protocol have the potential to elucidate how various environmental factors interact with temperature to impact physiological performance in late stage crustaceans.
The authors have nothing to disclose.
The authors thank Paul Rawson for laboratory assistance and the National Science Foundation award IIA-1355457 to Maine EPSCoR at the University of Maine for funds to purchase equipment. This project was supported by the USDA National Institute of Food and Agriculture, Hatch project number MEO-21811 through the Maine Agricultural and Forest Experiment Station, as well as NOAA National Marine Fisheries Service Saltonstall Kennedy Grant #18GAR039-136. The authors also thank three anonymous reviewers for their comments on a previous version of this manuscript. Maine Agricultural and Forest Experiment Station Publication Number 3733.
1.6 mm (1/16 in) drill bit | Milwaukee Tool at Home Depot | 1001294900 | This is for a 1.6 mm (1/16 in) diameter drill bit. This item can be found at most home-improvement stores. |
38 AWG Copper Magnet Wire | TEMCo | MW0093 | This wire is used to make the wire electrode leads that are implanted into the test subjects. This listing is for a 4 oz coil of 38-gauge magnetic wire. TemCo also has 36-gauge magnetic wire that is also suitable for use in constructing wire electrodes. |
Cyanoacrylate glue | Loctite | 852882 | This item includes a brush tip, which makes it easier to control the amount of glue used to secure electrodes to the carapace. |
Ethanol, 70% Solution, Molecular Biology Grade | Fisher BioReagents | BP82931GAL | This reagent is used in combination with the sterile cotton balls to disinfect the carapace prior to electrode implantation. |
Excel | Microsoft | N/A | This program is used in the protocol for organizing, manipulating, and analyzing data. It is compatible with both PC and Mac operating systems. |
Fisherbrand 8-Piece Dissection Kit | Fisher Scientific | 08-855 | This kit includes the forceps, scissors, dissecting knife (and blades), and dissecting needle needed to accomplish the electrode implantation steps in the protocol. |
Fisherbrand Isotemp Refrigerated/Heated Bath Circulators: 5.4-6.5L, 115V/60Hz | Fisher Scientific | 13-874-180 | This is a complete system that consists of an immersion circulator and a bath. It can be used as a temperature controlled bath or to circulate fluid externally to an application. Temperature range of this water bath is -20 to +100 °C, and the unit heats/cools rapidly and is easy to drain upon conclusion of use. |
Fisherbrand Sterile Cotton Balls | Fisher Scientific | 22-456-885 | These swabs should be soaked in 70% ethanol before being used to disinfect the carapace prior to electrode implantation. |
Fork Terminal, Red Vinyl, Butted Seam, 22 to 16 AWG, 100 PK | Grainger | 5WHE6 | Terminals are soldered to the magnetic wire to construct the wire electrodes. These can be purchased from a variety of home-improvement vendors. |
Impedance converter | UFI | Model 2991 | Measures impedance changes correlated with very small voltage changes, ranging from 0.2 ohm to over 5 ohms. This model can convert impedance changes that stem from resistance, capacitance, or inductance variations, as well as a combination of all three. |
LabChart software | ADInstruments | N/A | Purchase of the PowerLab datalogger includes the LabChart software, but a license for the software can also be directly downloaded online. LabChart allows the user to record data, open and read LabChart files, analyze data, as well as save and export files. There is a free version of the software, LabChart Reader, but users can only open and read LabChart files and analyze them (i.e., it cannot be used to record, save, or export data files). One also has the option of selecting LabChart Pro, which includes LabChart teaching modules that can be used for educational purposes. |
LED Soldering Iron | Grainger | 28EA35 | This is a generic soldering iron that can be used to solder the magnetic wire to the fork terminals to create the wire electrodes. |
PowerLab datalogger | ADInstruments | ML826 | There are a variety of models of the PowerLab. This catalog number is for the 2/26 model that is a 2 channel, 16 bit resolution recorder with two analog input channels, independently selectable input sensitivities, two independent analog outputs for stimulation or pulse generation and a trigger input. The PowerLab features a wide range of low-pass filters, AC or DC coupling and adaptive mains filter. This unit has a USB interface for connection to Windows or Mac OS computers and a sampling rate of 100,000 samples/s per channel. |
Prism8 | GraphPad | N/A | This program provides an additional option for calculating the Arrhenius Break Temperature through its “Segmental linear regression” data analysis option. This program does not require any programming and is compatible with both Mac and Windows operating systems. |
R | R Project | N/A | This is free software for statistical computing that is compatible with UNIX platforms, as well as Windows and Mac operating systems. This program can also be used to calculate the Arrhenius Break Temperature using the “segmented” package. There are a number of tutorials and user guides available online through the r-project.org website. |
Rosin Core Solder | Grainger | 331856 | This product has a diameter of 0.031 in (0.76 mm) and is ideal for use in soldering speaker wire (similar gauge as magnetic wire used for electrodes). |
SAS | SAS Institute | N/A | This program provides an additional option for calculating the Arrhenius Break Temperature. However, it does require programming and is not compatible with Mac operating systems. |
SigmaPlot | Systat Software, Inc. | N/A | This is the authors’ preferred program for statistical determination of the Arrhenius Break Temperature. The “Regression Wizard” is easy to use and does not require any programming. One can obtain a free 30-day trial license before purchase. However, it is compatible only with PC computers. |
T-type Pod | ADInstruments | ML312 | Suitable for measurement of temperatures from 0-50 °C using T-type thermocouples. |
T-type Thermocouple Probe | ADInstruments | MLT1401 | Compatible with the T-type Pod for connection. Measures temperature up to 150 °C, and is suitable for immersion in various solutions, semi-solids, and tissue (includes a needle for implantation). This product is a 0.6 mm diameter isolated probe that is sheathed in chemical-resistant Teflon and a lead length of 1.0 m. |
UV Cable Tie, Black | Home Depot | 295813 | This is for a 100-pack of 8-inch (20.32 cm), black cable ties. However, based on the size of test subjects, smaller or larger cable ties may be needed. This item, and others like it, can be purchased at any home-improvement store. |