Thermal Tolerance of Planktonic Copepods

Plankton samples were collected with surface tows at field sites in Groton, Connecticut, and Punta Gorda, Florida (table 1), during July and August 2017 using a 250 µm mesh plankton net and non-filtering cod end. Sea surface temperature data for both sites (table 1) were obtained from the AQUA-MODIS satellite database [38 (link)]. Both sampling locations were in shallow water (less than 2 m); thus, surface temperature data are likely a good representation of temperature throughout the water column. Connecticut represents a cool, more variable thermal environment compared with Florida, which is characterized by warm and stable temperatures. Daily temperature variation at each site is minor compared with the inter-site differences [39 (link)]. Initial laboratory populations of more than 1500 mature adults were established from collected animals. Cultures were maintained in 0.6 µm filtered seawater under common garden conditions (salinity: 30 practical salinity units, 12 h : 12 h light : dark, 18°C) for several generations. During this time, copepods were fed ad libitum a diet of the microalgae Tetraselmis sp., Rhodomonas sp. and Thalassiosira weissflogii, which were semi-continuously cultured in F/2 medium (F/2 – silicate for Tetraselmis sp. and Rhodomonas sp.) under the same conditions. Cultures were maintained under these conditions for several generations before the experiments, thus minimizing the effects of previous environmental acclimation (i.e. differences in food abundance/quality and temperature) in the field.
Table 1.

Site name, geographical coordinates, mean annual temperature, mean annual maximum and mean annual temperature range for all collection locations.

population	coordinates (latitude, longitude)	mean annual temperature (°C)	mean annual maximum temperature (°C)	mean annual temperature range (°C)
Connecticut (CT)	41.320591 N, −72.001564 W	13.3	22.7	22.5
Florida (FL)	26.940398 N, −82.051036 W	24.9	31.4	15.3

Body size measurements were taken for individuals from the laboratory cultures (n = 30 males and 30 females for both sexes from both populations). Individuals were isolated in a drop of filtered seawater and photographed using a camera attached to an inverted microscope after the water had been removed. Body lengths were measured as the length of the prosome using Image-J (https://imagej.nih.gov/ij/).
To test for the effect of developmental temperature, a fraction of the eggs from the 18°C culture were moved to 22°C to develop. All other variables were held constant. Once mature, individuals from both developmental conditions (18 and 22°C) were exposed to a 24 h acute heat stress. Individuals were carefully transferred to a microcentrifuge tube filled with 1.5 ml of filtered seawater, then transferred to heat blocks set to a constant temperature (18–38°C at 1°C intervals). Each individual experienced a single temperature. Individual survivorship was recorded after 24 h as binary data (1, survival; 0, mortality). Survivorship was determined during examination under a dissection microscope by response to stimuli or visible gut-passage movement. A total of 1717 individuals were used throughout the experiments (727 CT individuals and 990 FL individuals). Initial heat stresses were performed across the entire range of temperatures (18–38°C) in order to determine where additional heat stresses were needed for each of the populations. Therefore, different numbers of individuals were used for the two populations as the two temperature ranges differed between the populations.
All analyses were performed using the software package R v. 3.5.1 [40 (link)]. Body size measurements were analysed using a three-way ANOVA (body size ∼ population * developmental temperature * sex). A Levene's test was used to test the assumption of homogeneity of variance. A Tukey post hoc test was then used to examine pairwise differences between the various groups. To analyse the survivorship data, an initial ANOVA was run for all data (survivorship ∼ stress temperature + sex + developmental temperature + population, and all two-way interactions). Three-way and four-way interactions were excluded. ANOVAs were also run for each population separately (survivorship ∼ stress temperature * sex * developmental temperature). Thermal performance curves were estimated using logistic regressions on the data from both developmental temperatures from both populations. Because of the common garden design, differences in the performance curves between developmental conditions within a population can be attributed to developmental phenotypic plasticity, whereas differences between populations should reflect the effects of genetic differentiation. LD₅₀ (the temperature with 50% mortality) was calculated for each performance curve. The change in LD₅₀ between the two developmental conditions (ΔLD₅₀) was used as a measure of the magnitude of the plastic response.