The present protocol illustrates the use of commercially available components to generate a stable and linear thermal gradient. Such gradient can then be used to determine the upper thermal limit of planktonic organisms, particularly invertebrate larvae.
Thermal limits and breadth have been widely used to predict species distribution. As the global temperature continues to rise, understanding how thermal limit changes with acclimation and how it varies between life stages and populations are vital for determining the vulnerability of species to future warming. Most marine organisms have complex life cycles that include early planktonic stages. While quantifying the thermal limit of these small early developmental stages (tens to hundreds of microns) helps identify developmental bottlenecks, this process can be challenging due to the small size of target organisms, large bench space requirement, and high initial fabrication cost. Here, a setup that is geared toward small volumes (mL to tens of mL) is presented. This setup combines commercially available components to generate a stable and linear thermal gradient. Production specifications of the setup, as well as procedures to introduce and enumerate live versus dead individuals and compute lethal temperature, are also presented.
Thermal tolerance is key to organisms' survival and function1,2. As the planet continues to warm due to anthropogenic carbon emissions, increasing attention is being paid to the determination and application of thermal limits3. Various endpoints, such as mortality, failure to develop, and loss of mobility, have been used to determine both upper and lower thermal limits4. These thermal limits are often considered a proxy for an organism's thermal niche. This information is in turn used to identify species that are more vulnerable to global warming, as well as predict future species distribution and the resulting species interactions3,5,6,7. However, determining thermal limits, especially for small planktonic organisms, can be challenging.
For planktonic organisms, particularly the larval stages of marine invertebrates, the thermal limit can be determined through chronic exposure. Chronic exposure is achieved by rearing larvae at several temperatures over days to weeks and determining the temperature at which larval survivorship and/or developmental rate reduces8,9,10. However, this approach is rather time-consuming and requires large incubators and experience in larval husbandry (see reference11 for a good introduction to culturing marine invertebrate larvae).
Alternatively, acute exposure to thermal stress can be used to determine thermal limits. Often, this determination approach involves placing small vials with larvae in temperature-controlled dry baths12,13,14, leveraging thermal gradient functions in PCR thermal cyclers15,16, or putting glass vials/microcentrifuge tubes along a thermal gradient generated by applied heating and cooling on the ends of large aluminum blocks with holes in which the vials fit snuggly17,18,19. Typical dry baths generate a single temperature; hence, multiple units must be operated simultaneously to assess performance across a range of temperatures. Thermal cyclers generate a gradient but only accommodate a small sample volume (120 µL) and require careful manipulations. Similar to thermal cyclers, large aluminum blocks create linear and stable temperature gradients. Both approaches can be coupled with logistic or probit regression to compute the lethal temperature for 50% percent of the population (LT50)12,20,21. However, the aluminum blocks used were ~100 cm long; this size demands a large lab space and access to specialized computer numerical control milling machines to drill the holes. Together with using two research-grade water baths to maintain the target temperature, the financial cost of assembling the setup is high.
Therefore, this work aims to develop an alternative means to generate a stable, linear temperature gradient with commercially available parts. Such a product must have a small footprint and should be able to be easily used for acute thermal stress exposure experiments for planktonic organisms. This protocol is developed with zooplankton that is <1 mm in size as target organisms, and thus, it was optimized for the use of a 1.5 or 2 mL microcentrifuge tube. Larger study organisms will require containers larger than the 1.5 mL microcentrifuge tubes used and enlarged holes in the aluminum blocks.
In addition to making the experimental apparatus more accessible, this work aims to simplify the data processing pipeline. While commercial statistical software provides routines to compute LT50 using logistic or probit regression, the licensing cost is non-trivial. Therefore, an easy-to-use script that relies on the open-source statistical program R22 would make data analysis more accessible.
This protocol shows how a compact heat block can be fabricated with commercially available parts and be applied to exposing zooplankton (larvae of the sand dollar Dendraster excentricus) to acute heat stress to determine their upper thermal limit.
1. Fabrication of the heat block
2. Determination of thermal gradient settings
3. Thermal exposure and live:dead enumeration
NOTE: Step 2 can be omitted once the desired settings for the temperature gradient are determined.
4. Computation of LT50
The goal of this protocol is to determine the upper thermal limit of zooplankton. To do so, a stable and linear thermal gradient is needed. The proposed setup was able to generate a thermal gradient ranging from 14 °C to 40 °C by setting the water bath temperature to 8 °C and the heater to 39 °C (Figure 2A). The temperature gradient can be narrowed and shifted by changing the endpoint values. A thermal gradient with a narrower range (19 °C to 37 °C) was also generated by setting the heater to 37 °C and the water bath to 15 °C. The temperature in the block stabilizes within 45 min to 1 h of setup (Figure 2B).
To illustrate the application of this protocol to zooplankton, the change in the upper thermal limit, indicated by LT50, through ontogeny in the larvae of the sand dollars (Dendraster excentricus) was examined. Gravid sand dollars were obtained commercially (see Table of Materials). The release of gametes was induced by injecting 0.5-1 mL of 0.35 M potassium chloride. The eggs collected were rinsed through 63 µm nylon mesh with 0.45 µm filtered seawater. The sperm was collected dry and kept on ice. The eggs were fertilized at ~104 sperms per mL. Common garden cultures were created with gametes from three males and three females at five individuals per mL. These larval cultures were kept in filtered seawater with a salinity of 32 psu at 18 °C under a 12:12 light:dark cycle with complete water change every other day.
As larval sand dollars developed, the upper thermal limit increased from 28.6 °C (± 0.02 °C S.E) at 2 days post-fertilization to 28.8 °C (± 0.02 °C S.E) at 4 days post-fertilization and 29.3 °C (± 0.02 °C S.E) at 6 days post-fertilization (Figure 3). These upper thermal limits suggest sand dollars live within their thermal limit during the average summer sea surface temperature of ~20 °C or lower along the Pacific coast. However, with increasing frequency and intensity of marine heatwaves, the maximum temperature continues to rise. A peak temperature of 26.4 °C was recorded in Southern California Bight in August 2018 (Fumo et al.24). Given that this species reproduce in spring and summer, the survivorship of their early life stage is likely to diminish during these extreme events. The predicted survivorship would decrease by 10% when the temperature reaches 26.5 °C.
Pair-wise comparisons using the ratio test developed by Wheeler et al.25 suggest the median lethal temperature was significantly different between the three age groups (p < 0.001). Earlier stages (gastrula and early prisms that were 2 days old) were more sensitive to thermal stress than older larvae. This observation suggests that the thermal limit deduced from a single time point of development is not representative of that species throughout its life history.
Figure 1: Labeled diagram of the heat block. (A) Top view of the setup with all components connected. (B,D) Placement and connections for the heater terminals. (C,E) Placement of the heat exchanger (cooling elemenet) and the associated tubings to the water bath. Please click here to view a larger version of this figure.
Figure 2: Temperature changes in the heat block over 1 h with endpoints set to 15 and 37 °C. (A) A linear gradient was achieved within 1 h. Changing in the endpoint settings varies the temperature range, and the largest range was from 14 °C to 40 °C. (B) The temperature difference between replicate rows was negligible (<0.8 °C); data from two replicate rows were plotted for each setting in (B). Please click here to view a larger version of this figure.
Figure 3: Survivorship of larval sand dollars (Dendraster excentricus) across a temperature range of 19 to 37 °C through ontogeny (2, 4, and 6 days post-fertilization [dpf]). Each datum represents the proportion of larvae that survived a 2 h incubation at the specific temperature followed by a 1 h recovery period. A logistic regression was performed using the generalized linear model with binomial distribution in the statistical software R. Please click here to view a larger version of this figure.
Supplementary Coding File 1: An R script to generate logistic curves for the data set with a step-by-step example. Please click here to download this File.
Supplementary Coding File 2: An R script to generate LT50 estimates. Please click here to download this File.
This protocol provides an accessible and customizable approach to determine the thermal limits of small plankton organisms through acute thermal exposure. The 10-hole design and flexible temperature endpoints, controlled by the water bath at the lower end and the heater at the upper end, enable one to determine LT50 with precision. Using this approach, a difference in the thermal limit that is <1 °C could be detected (Figure 3). This approach provides a rapid determination of thermal limits (in hours) for a variety of species, and the resulting values have been applied to multiple species distribution models2,21. However, it is important to note that acute exposure likely provides a different thermal tolerance estimate when compared to chronic exposure8,26.
One major advantage of the current design is that 10 temperature treatments and six replicates are included within a small footprint (20.3 cm x 15.2 cm x 5 cm). Previous publications using a similar thermal gradient approach to determine thermal limits used aluminum bars that were bigger (180 cm x 10 cm x 6 cm in27, 91 cm × 25 cm × 15 cm in10, and 60 cm x 20 cm in17). While dry baths that hold a single temperature are smaller (e.g., 18.5 cm x 18.5 cm x 2.5 cm) and offer multiple replicates, several units (more than four) are required to generate a performance curve that includes multiple temperatures, or the experiments need to be repeated over time which could introduce confounding factors. The heat block design reduces both the fabrication cost and space requirements. The fabrication can be completed with a drill press, or researchers without immediate access to a milling machine could opt for commercial CNC machining services. The use of commercially available parts further controls the fabrication cost. If one can use an existing heating/cooling water bath or aquarium chillers, the remaining cost of the parts totals to less than $350. Otherwise, aquarium chillers for a 10 gallon (~35 L) fish tank can be purchased for <$150.
The current design may be modified to fit the researcher’s needs. If the target organisms are bigger in size, scintillation vials are good alternative containers, and larger holes would be required. That said, the aluminum block is removable in the current design, so multiple blocks can be made and swapped out to fit the experimental needs. If the goal of the experiment is to determine a lower thermal limit or focus on polar organisms, placing chilling water blocks on both ends of the main aluminum block is more appropriate.
Similar to other studies on zooplankton, the current protocol does not include a gradual cooling phase20,27. Researchers can consider removing the microcentrifuge tubes in pairs and shifting them down the temperature gradient (i.e., reversing steps 3.9-3.12) to achieve gradual cooling if their study organisms are sensitive to a sudden temperature decrease.
The utility of this setup can be diminished by several factors, namely the choice of (1) the endpoint temperature settings, (2) the exposure and recovery duration, and 3) the metric used to determine the binomial state (live vs. dead; developed vs. non-developed). To address these potential limitations, preliminary testing is highly recommended.
Since the logistic regression assumes a binomial distribution, endpoints with 100% survival and mortality are preferred. For marine organisms, a reasonable starting range would be the mean annual sea surface temperature of the collection site plus 10-15 °C. One can then narrow the temperature range investigated after such an initial trial, as the smaller the temperature difference between holes, the more fine-tuned the LT50 estimate.
The duration of exposure and recovery are species-specific. For instance, Kuo et al.27 allowed juvenile whelks (Nucella canaliculata) to recover for 24 h, while Hammond et al.28 allowed larval purple urchins (Stronglylocentrotus purprtaus) 1 h for recovery. One could perform a short experiment to determine if the live:dead count differs between recovery periods. Depending on the definition of the binomial state chosen (e.g., live vs. dead), recovery time may not be necessary. If the goal of the experiment is to test if developmental processes, such as cleavage and gastrulation, occur across a range of temperatures. In other words, the binomial state used in the model would be developed versus not developed8,19,21. Fixatives such as 4% paraformaldehyde must be added to the samples at the thermal exposure period without any recovery time.
To ensure accurate count and determination of binomial state (live vs. dead; developed vs. non-developed), it is advisable to count the samples after the recovery time in random order to avoid potential observer biases. If there is sufficient personnel, different researchers could count replicate rows and compare their results. Alternatively, individuals can repeatedly count a small subset of the samples and check if the numbers are consistent.
Another potential limitation is the lack of error estimation of the LT50 from independent samples29. The current data analysis method provides a 95% confidence interval along the fitted logistic curve (Supplementary Coding File 1) and a standard error of the LT50 (Supplementary Coding File 2). These error bounds are generated from the curve fitting process, not through multiple measurements of individuals from the sample population. Given the current heatblock design has six rows, one can fit data from each row to generate six LT50 estimates and obtain the observation-based error estimates.
In summary, an accessible approach to determining acute thermal limits that can be applied to a wide variety of zooplankton is presented. This setup can be used to determine the thermal limits of various organisms and to pinpoint development stages that are vulnerable. This information can help improve the prediction of organismal performance and potential community interactions in the face of global climate change.
The authors have nothing to disclose.
This work is supported by the Faculty Research Fund of the Swarthmore College [KC] and the Robert Reynolds and Lucinda Lewis '70 Summer Research Fellowship for BJ.
0.45 µm membrane filter | VWR | 74300-042 | |
½” Acrylic sheet | McMaster-Carr | 8560K266 | Used to construct a ridged case with sufficient insulation. |
1 mL syringe | VWR | 76290-420 | |
2 Channel 7 Thermocouple Types Datalogger | Omega Engineering | HH506A | Can be replaced with any thermometer that will fit inside a microcentrifuge tube |
Automatic pipette | Ranin | ||
Bolt- and Clamp-Mount Strip Heater with 430 Stainless Steel Sheath, 120V AC, 1-1/2" Wide, 100W |
McMaster-Carr | 3619K32 | |
Crystal Sea Bioassay Mix | Pentair | CM2B | Use to make aritifical seawater |
Denraster excentricus | M-Rep | Sand dollars from California | |
Dissecting microscope | Nikon | SMZ645 | |
DIYhz Aluminum Water Cooling Block, Liquid Water Cooler Heat Sink System for PC Computer CPU Graphics Radiator Heatsink Endothermic Head Silver(40 mm x 120 mm x 12 mm) | Amazon | Connects to water bath and used to cool one end of the block. | |
Easy-to-Machine MIC6 Cast Aluminum Sheet 2" thick 8" x 8" | McMaster-Carr | 86825K953 | Machined to 2" x 6" x 8" with 60 equally spaced holes (11 mm dia., 42 mm depth) with two addition holes drilled in one side for thermostat probes. |
Economical Flexible Polyethylene Foam Pipe Insulation | McMaster-Carr | 4530K121 | Covers the plastic tubing between chiller and block to reduce heat loss. Can be omitted if temperature range is close to room temperature |
EVERSECU 72w 110-240v Aquarium Water Chiller Warmer/Cooler Temperature Controller for Fish Shrimp Tank Marine Coral Reef Tank Below 20 L/30 L Aquarium Chiller | Amazon | Can be used in place of the lab-grade water bath | |
Example with larval sand dollar | |||
GENNEL 100 g Silver Silicone Thermal Conductive Compound Grease Paste For GPU CPU IC LED Ovens Cooling | Amazon | Improves the thermal conductance between the block and the heating and cooling elements. | |
Inkbird WiFi Reptile Thermostat Temperature Controller with 2 Probes and 2 Outlets, IPT-2CH Reptiles Heat Mat Thermostat (Max 250 W per Outlet) | Amazon | Monitors hot and cold ends. Maintains hot end in range | |
Lauda Ecoline Silver Air-Cooled Refrigerated Circulators | VWR | 89202-386 | Can be replaced with an aquarium chiller |
Microcentrifuge Tubes | VWR | 76019-014 | If larger animals are used, scanilation vials (VWR 66022-004) is a good alternative |
Nitex mesh filter | Self made | Used hot glue to attached Nitex mesh to 1/2" PVC tubing | |
Pasteur pipette | VWR | 14673-010 | |
Potassium Chloride (0.35 M) | Millpore-Sigma | P3911-500G | |
R statistical software. | The R Project for Statistical Computing | ||
Syringe needle | VWR | 89219-346 | Depending on size of target organism gague 14 and 16 can be used |
Tygon Tubing | McMaster-Carr | 5233K65 | Adjust to match the chiller and block used |
Zoo Med Repti Temp Rheostat | Chewy.com | Rated to 150 W and rewired to feed directly into the heating element. Used to control rate of heat output |