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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 gen...
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...
The authors have no conflict of interest to declare.
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.
Name | Company | Catalog Number | Comments |
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 |
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