Thermal Limits Determination for Zooplankton Using a Heat Block

Summary

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.

Abstract

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.

Introduction

Thermal tolerance is key to organisms' survival and function^1,2. 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....

Protocol

1. Fabrication of the heat block

1. Wire the 120 V, 100 W strip heater to the rheostat (see Table of Materials).
2. Prepare the 20.3 cm x 15.2 cm x 5 cm (8 in x 5 in x 2 in) block of aluminum by drilling 60 holes in a 6 x 10 grid (see Table of Materials). Ensure that the holes are spaced 2 cm from center to center in both directions. Each should be 1.1 cm in diameter and 4.2 cm deep (Figure 1).
   NOTE: Perform the drilling on a milling machine or drill press with high-speed steel drill bits. The heating element and cooling element were both chosen to cover as much of ....

Results

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.......

Discussion

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.......

Materials

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