Measures of CTmax and gene expression in <em>Daphnia</em> clones acclimated to three temperatures

The theory of optimal cell size postulates that cell size imposes constraints on oxygen delivery in larger cells due to their smaller surface-to-volume ratios. Smaller cells, with a higher surface to volume ratio, have an increased capacity to take up oxygen, which might result in an increased heat tolerance (CTmax). However, the precise mechanisms linking cell size, body size, and heat tolerance remain unclear. We obtained contrasts in cell size by raising size-matched diploid and triploid juvenile Daphnia at different temperatures. Daphnia raised at lower temperatures had larger cells than those raised at higher temperatures. Triploid clones had larger cells than diploid clones at 16°C, 20°C, and 24°C. CTmax increased with acclimation temperature and was negatively correlated with both cell and body size. Triploid clones had lower CTmax values than both subarctic and temperate diploid clones at 16°C and 20°C. Heat shock significantly increased the expression of Hsp70 and catalase, but these were not correlated with CTmax or ploidy, suggesting that heat tolerance was not directly linked to heat-shock proteins or oxidative stress responses. These findings highlight the role of cell size and polyploidy in shaping the heat tolerance and geographic distribution of ectotherms. Methods 17 Daphnia clones were acclimated at 16°C, 20°C, and 24°C. A total of 153 jars were used (3 jars × 3 temperatures × 17 clones). Heat tolerance was assessed using the critical thermal maximum (CTmax), which corresponds to the temperature at which an animal loses motor functions and faints. CTmax was performed using a thermocycler (Biometra®) with a heating block adapted to fit 48 × 0.5 mL tubes. Individual Daphnia were placed in 0.5 mL tubes containing a solution of 0.75 mg/mL bovine serum albumin in FLAMES culture medium at the rearing temperature. The CTmax protocol was fast ramping, with the temperature rising one degree per minute from the rearing temperature until 45°C. Each run was performed using Daphnia from the same rearing temperature. We obtained 5–25 measures per clone for each temperature. To measure the heating in real time, a temperature probe was placed in a tube containing only the solution. The design was filmed using an HD Camera (Fujifilm FinePix HS30EXR) to record each run. The CTmax of individual Daphnia was scored by watching these video recordings using video editing software (Movavi Video Editor 14). Body and cell sizes were measured for each Daphnia individual using a microscope with a digital microscope camera A35100U3 (OMAX) and ToupView software (OMAX, v.3.7). The total length of Daphnia was measured at 2.5X from the top of the compound eye to the base of the caudal spine. The mean vertical length of ten regular-shaped prints on the carapace was used as a cell size proxy for each Daphnia. A total of 702 individual Daphnia (1–12 individuals × jar × clone × temperature) were used for heat tolerance measurements, body size, and average cell size. The average cell size per Daphnia was calculated using ten measurements (SE of each individual varied from 0.11 to 2.21 µm). To induce the expression of Hsp70 and catalase, Daphnia were individually placed in 10 mL tubes filled with FLAMES medium and heat-shocked in a 30°C water bath for an hour. We used three replicates of four individual Daphnia for each clone (except for A5-7, which only had two individuals per replicate). An untreated control group comprising Daphnia unexposed to heat shock was included to measure the basal expression levels of Hsp70 and catalase. Quantitative real-time PCR was performed on a LightCycler 480 (Roche) using the SensiFAST SYBR® No-ROX mix (98020, Bioline) under the following conditions: 95°C for 10 min and 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s.The mean normalized fold change of target gene expression between heat-shocked and untreated individuals was calculated using the 2-ΔΔCT method described by Livak & Schmittgen (2001), where ∆∆CT (CT target - CT reference genes)Treatment - (CT target - CT reference genes)Control. Technical duplicates were included to test the quality of the manipulations and pipetting errors.