Thermal Limits Determination for Zooplankton Using a Heat Block

As global temperatures continue to rise, quantifying thermal limits and the relationship with acclimation and ontogeny are vital for determining the vulnerability of species to future warming. For marine organisms with complex life histories, determining thermal limits can be logistically challenging. This protocol introduces a small footprint, easy to build method to estimate critical temperatures of small planktonic organisms.

While the method was developed for small, less than a millimeter sized plankton, it can be adopted for larger marine organisms that would fit into scintillation vials of approximately 50 milliliters volume. Begin by wiring the strip heater to the rheostat. Drill 60 holes in a grid of six by 10 size to prepare the aluminum block of a size of 20.3 by 15.2 by five centimeter, and ensure that the holes are spaced two centimeters from center to center in both directions.

Drill two additional holes between the first and second column and the ninth and 10th column, matching the size of the temperature controller probes. To hold the elements in place and insulate the completed heat block, construct a case from clear acrylic sheets, ensuring to apply two layers at the backside of the heating element. In the final assembly, apply thermal paste to maximize heat conductance from the heating element into the block and from the block to the cooling element.

Connect the water bath with Tygon tubing and insert the thermostat probe into the holes on the side of the aluminum block. In all the milled holes, place 1.5 milliliter micro-centrifuge tubes, filled with tap water to the brim. Turn on the temperature controller and set the stop heating temperature of probe one to 35 to 37 degrees Celsius, and probe two to 21.5 to 22.5 degrees Celsius.

Rotate the rheostat to turn on the heating element and set it to medium. Turn on the water bath and set the chiller temperature to 15 degrees Celsius. Using a thermocouple with a K-type electrode, check the temperature inside each micro-centrifuge tube every 10 minutes afterward.

Adjust the values of the end points by changing the settings of the temperature controller and the water bath as needed. Turn on the recirculating water bath and heater and set them to 15 degrees Celsius and 37 degrees Celsius respectively, to generate a temperature gradient from 19.5 degrees Celsius to 37 degrees Celsius. Once the micro-centrifuge tubes are placed in milled holes and the temperature of the heat block is reached, check the temperature inside each micro-centrifuge tube using a thermocouple with a K-type electrode.

And note down these temperatures. Fill a 1.5 milliliter micro-centrifuge tube with seawater, filtered through a 0.45 micrometer mesh. Concentrate the study organism's culture with reverse filtering so that the organisms remain at the bottom of the beaker.

Rinse the concentrated culture with filtered seawater, and repeat the reverse filtering once more to concentrate the sample. Count the small planktonic organisms under a dissecting microscope and transfer a known number of organisms into half-filled micro-centrifuge tubes using a glass pasteur pipette. Now add filtered sea water until the final volume in these tubes reaches one milliliter.

Now place these tubes in pairs into the heating block, starting from the cold end to allow the organisms to gradually warm up to the desired experimental temperature. Wait for 10 minutes and move the pairs of micro-centrifuge tubes to the adjacent drilled holes with warmer temperatures. Place additional pairs of micro-centrifuge tubes in each row at the cold end and continue to shift them towards the warmer end in pairs.

Once the entire block is filled, incubate it for two hours at the designated temperature. At the end of the incubation period, measure the temperature in each tube and note it down. Then transfer all tubes to the pre-labeled holders and incubate them at a predetermined temperature for one hour to allow recovery.

For enumerating the alive organism portion, transfer the contents of an individual micro-centrifuge tube in a 35 millimeter petri dish using a glass pipette. Under a dissecting microscope, count the live and dead organisms and note down the numbers. The observed number of organisms should match the originally taken organism.

If it does not, check the side of the micro-centrifuge tubes and petri dish, Generate a data table in CSV format with the headers grouping variable of interest, the temperature of the tube in degrees Celsius, number of individuals alive, and number of individuals dead. To fit the data with a logistic regression, use a generalized linear model with a binomial distribution. To run the model, type source, and use the R file, modelloop.r.

Compute the predictor values at which 50%of the individuals survived, to determine the median upper thermal limit. Using this protocol, the survivorship of larval sand dollars was measured, which was across a temperature range of 19 to 37 degrees Celsius, at two, four and six days post-fertilization. As larval sand dollars developed, the upper thermal limit increased from 28.6 degrees Celsius at two days post-fertilization, to 28.8 degrees Celsius at four days post-fertilization, and approximately 29 degrees Celsius at six days post-fertilization.

Incubation and recovery times are species specific. It is important to conduct a preliminary test to ensure that timing selected yields a reliable live versus dead estimation.