DOI: 10.5061/dryad.5hqbzkhk6
title: Measures of CTmax and gene expression in <em>Daphnia</em> clones acclimated to three temperatures

description: 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.
description: 17 <em>Daphnia</em> 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.
description: # Measures of CTmax and gene expression in *Daphnia* clones acclimated to
 three temperatures Dataset DOI:
 [10.5061/dryad.5hqbzkhk6](https://doi.org/10.5061/dryad.5hqbzkhk6) ##
 Description of the data and file structure Measures of CTmax and gene
 expression in Daphnia clones exposed to different temperatures. ### Files
 and variables #### File: Daphnia_CTmax2.xlsx **Description:** File that
 includes the CTmax measurements as well as cell size and body size for
 different Daphnia clones acclimated to three temperatures. Date, Time-
 date and time taken for study Acc.Temperature (Acclimation temperature
 were 16 degree C, 20 degree C, and 24 degree C) Clone (Daphnia genotype)
 Jar (in which the animals were raised) Rep (Individual replicate) BodySize
 (mm) Ctmax (Temperature at which individuals dropped the bottom of the
 tube) CellSize (average of 10 cells in micrometers) Cell1 (micrometers)
 Cell2 (micrometers) Cell3 (micrometers) Cell4 (micrometers)
 Cell5(micrometers) Cell6(micrometers) Cell7(micrometers)
 Cell8(micrometers) Cell9 (micrometers) Cell10(micrometers) Ploidy (2n or
 3n) Climate (temperate or subarctic) Missing data are left blank for
 proper functioning of the script. #### File: Daphnia_HSP70_Catalase.xlsx
 **Description:** Quantification of heatshock protein 70 gene and catalase
 in Daphnia exposed to heat shock and no heat shock. Clone (Daphnia
 genotype) Code (NE for non-exposed and E for exposed to heat shock)
 Treatment (Heat shock or control) Rep (replicate jar) Tech_Rep (technical
 replicate) HSP (2deltaCT for heat shock protein) CAT (2deltaCT for
 catalase) Ploidy (2n or 3n) Climate (Subarctic or temperate) #### File:
 Script_Stats_Analysis_v2.R **Description:** This R script imports
 experimental data on Daphnia, cleans and transforms it, then fits multiple
 linear mixed-effects models (lmer) to test how cell size, body size,
 ploidy, climate, and temperature affect thermal tolerance (CTmax) and
 stress protein expression (HSP70 and catalase). It compares models using
 AIC/BIC and examines relationships among traits via residuals and
 estimated marginal means (emmeans). ## Code/software All statistical
 analyses were performed using R 4.5.1 (R Core Team, 2025) with the “lme4 ”
 (Bates et al., 2015) and “lmerTest ” (Kuznetsova et al., 2017) packages.
 The degree of freedom of the dominator was approximated using the
 Satterthwaite method and type III Anova were used. We visually checked
 normality, homoscedasticity, and independence of residuals with the
 package “performance” (Lüdecke et al., 2021). Tukey post-hoc tests and
 linear contrasts were performed using the “emmeans” package (Lenth, 2018).