DOI: 10.5061/dryad.2280gb5qw
title: Data from: Are acute and acclimated thermal effects on metabolic rate modulated by cell size? A comparison between diploid and triploid zebrafish larvae

description: Being composed of small cells may carry energetic costs related to
 maintaining ionic gradients across cell membranes as well as benefits
 related to diffusive oxygen uptake. Here we test the hypothesis that these
 costs and benefits of cell size in ectotherms are temperature dependent.
 To study the consequences of cell size for whole-organism metabolic rate
 we compared diploid and triploid zebrafish larvae differing in cell size.
 A fully factorial design was applied combining three different rearing and
 test temperatures that allowed us to distinguish acute from acclimated
 thermal effects. Individual oxygen consumption rates of diploid and
 triploid larvae across declining levels of oxygen availability were
 measured. We found that both acute and acclimated thermal effects affected
 the metabolic response. In comparison to triploids, diploids responded
 more strongly to acute temperatures, especially when reared at the highest
 temperature. These observations support the hypothesis that animals
 composed of smaller cells (i.e. diploids) are less vulnerable to oxygen
 limitation in warm aquatic habitats. Furthermore, we found slightly
 improved hypoxia tolerance in diploids. By contrast, warm-reared triploids
 had higher metabolic rates when they were tested at acute cold
 temperature, suggesting that being composed of larger cells may provide
 metabolic advantages in the cold. We offer two mechanisms as a potential
 explanation of this result, related to homeoviscous adaptation of membrane
 function and the mitigation of developmental noise. Our results suggest
 that being composed of larger cells provides metabolic advantages in cold
 water, while being composed of smaller cells provides metabolic advantages
 in warm water.
description: Oxygen consumption was measured in a closed respirometry system
 using a 24-wells glass microplate equipped with oxygen sensor spots glued
 onto the bottom of 200 µL wells (Loligo Systems, Denmark) integrated with
 a 24-channel fluorescence-based oxygen reading device (SDR SensorDish®
 Reader, PreSens, Germany). The sensor spots measure the partial pressure
 of oxygen (kPa), which we combined with the temperature dependent
 solubility to calculate the oxygen concentrations at different
 temperatures. Depletion of oxygen over time was used to calculate oxygen
 consumption rates per individual (in nmol O2 per h) as well as the
 critical value in PO2 (in kPa).<br>      In each run, diploid and
 triploid larvae without morphological abnormalities were transferred into
 the 24-wells microplate with E2 medium, and these wells were then sealed
 using an adhesive optical PCR sealing film (Microseal 'B' PCR
 Plate Sealing Film, Bio-Rad, Hercules, California, USA) making sure to
 avoid air bubbles inside the wells. In most cases, 10 diploids and 10
 triploids were tested in one run, leaving four wells of our 24-well
 microplate empty, which were used as a control. The microplate with
 animals was placed in a flow-through water bath connected to a
 cooling/heating circulating bath (Grant LT ecocool 150, UK) which allowed
 for temperature stabilization at 23.5°C, 26.5°C and 29.5°C (±0.1°C,
 respectively). Oxygen concentrations inside the wells were recorded every
 30 seconds with automatic temperature and pressure correction using
 MicroResp™ v1.0.4 software (Loligo Systems, Denmark). All measurements on
 metabolic rate were carried out in darkness due to sensitivity of the
 sensor spots to UV light. Spontaneous activity of larvae was possible
 during measurements, so their metabolic rate in normoxia was defined as
 the routine metabolic rate. In each run, at least four randomly selected
 wells with E2 medium but without animals were used the assess background
 respiration. The entire trial lasted at least 16 hours (between 4 p.m. and
 8 a.m.), during which all larvae completely depleted the available oxygen
 from the wells (Fig. S1). After each experimental run the dead larvae were
 removed from the wells. To prevent accumulation of bacteria in the wells,
 the microplate was bleached using a 35% H2O2 solution and rinsed with demi
 water. We conducted a total of 24 runs (8 rounds × 3 temperatures),
 measuring oxygen levels in the wells until they were fully depleted in 422
 larvae (208 diploids and 214 triploids). The first 20 minutes of each run
 was discarded from further analysis as the temperature equilibrated during
 this period and the larvae adjusted to being transferred to the
 microplate. Rates of oxygen consumption were calculated from the declines
 in oxygen levels over time as the slope of a linear regression using a
 moving time window of 5 minutes. We also evaluated in separate trials the
 extent of oxygen ingress for each test temperature by incubating the
 microplate sensors under hypoxic conditions and measuring how fast oxygen
 levels increased over time. The larval oxygen consumption rates were
 adjusted for both background respiration and oxygen ingress although both
 influences were negligible compared to the respiration rates of the larvae
 (e.g. background respiration rates as assessed with blanks never exceed 3%
 of the measured respiration rates in larvae).<br>  
description: see readme.txt