Organism and Culturing
Tetraselmis suecica (UTEX LB 2286) was cultured in 250 mL Erlenmeyer flasks containing sterilized natural seawater. We used natural seawater as artificial seawater such as Instant Ocean has been shown to sometimes affect experimental outcomes (Pechenik et al. 2019). Seawater was sterilized using vacuum filtration to remove particulate matter and sediment, and subsequently autoclaved at 121ºC for 60 minutes. Non-experimental lineages were created at least every 10 days, and Micro Algae Grow nutrient media (Florida Aqua Farms Inc., Dade City, FL) was added every day beginning on day 0 to prevent nutrient limitation. These cultures were used to seed the experiments. Stock cultures were maintained in Darwin environmental chambers (Darwin Chambers Company, St. Louis, MO) at a constant temperature of 20ºC under continuous light provided by LED light strips. Stock cultures were gently swirled for 10 seconds daily to ensure homogeneity and proper aeration.
Experimental Setup
The experimental design involved acclimating cultures to eight different base temperatures (10ºC, 15ºC, 18ºC, 20ºC, 25ºC, 28ºC, 30ºC, 35ºC) over the course of 5 days in a Darwin environmental chamber. At the beginning of the experiment, cultures were placed on Peltier devices (Adafruit, New York, NY) housed in plastic egg-crate cages to prevent spillage. White LED strip lights were fastened inside the cages. Light was measured using a PAR sensor to verify that each cage was receiving at least 150 µmol m-2 s-2 (saturating irradiance (Bernhardt et al., 2018), and under 200 µmol m-2 s-2 to maintain consistent light conditions between cages.
Temperature Control and Manipulation
To control temperature during the experiment, we used TEC (thermoelectric cooling) Controllers (Meerstetter Engineering, Rubigen, Switzerland) to control the Peltier devices. Cultures were exposed to one of 5 fluctuating temperature treatments +/- 5ºC around the base temperature, or the constant temperature (n=18 per base temperature, n=3 per treatment within each base temperature). The five fluctuating temperature treatments for each mean temperature were characterized by the following frequencies: (i) frequency of 1 or period of 144 hours, (ii) frequency of 3 or period of 48 hours, (iii) frequency of 6 or period of 24 hours, (iv) frequency of 18 or period of 8 hours, and (v) frequency of 24 or period of 6 hours.
In order to ensure that cultures were in the exponential growth phase for the duration of the experiment, we used cultures at a known concentration of between 1,000,000 and 2,000,000 cells/mL on Day 1 of the experiment. To achieve this, we inoculated cultures the previous week at a lower concentration (i.e. 25,000 cells/mL) such that they would reach 1,000,000 by the end of their pre-experimental acclimation period. This lower concentration depended on the temperature to which they were being acclimated, as cultures further away from their TOPT would grow more slowly.
Cells were counted daily for each of the five days using a Beckman Coulter Z2 Particle Counter (Beckman Coulter, Indianapolis, IN) set to count particles between 5–16 µm. This size range was experimentally determined by photographing cells on a Hemacytometer and analyzing size with ImageJ (version 1.54). Samples were diluted in ISOTON II Diluent (Beckman Coulter, Indianapolis, IN), with a dilution factor such that cell counts ranged from 10,000 to 200,000 before factoring in a dilution factor. Three samples were taken per culture, and three counts were run on each sample to ensure consistency.
