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.

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).
     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).
 

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