Data Availability StatementAll relevant data are inside the paper. were always submerged below water surface, so chicks inevitably had to ingest water while feeding (similar to wild conditions). Shorebirds inhabiting hypersaline habitats rely on hypo-osmotic prey such as brine shrimp and diptera larvae and adults, whose body water content is about 78C87% of body mass [11]. The water content of fly larvae provided in our experiment was 73.3 0.5% [17]. The food and water were replaced three times a day, and salinity levels of residual and fresh water were measured using a conductivity meter (HI 98402). Open in a separate window Fig 2 Resting metabolic rate (RMR) and body mass responses in relation to salinity levels.Body mass, whole RMR, and mass-corrected RMR of captive-reared chicks at 0 (n = 9), 20 (n = 8), and 60 (n = 9) salinity (squares), and wild fledglings from hypersaline pans (n = 8) and freshwater reservoirs (n = 5; circles) (means SE). Whole RMR and mass-corrected RMR are presented as least square and adjusted means from the respective ANOVA and ANCOVA models. Data subject to log-transformation are shown as back-log-transformed least-square means. There were no significant differences among treatments (see text for further details). Morphological and Physiological Measurements All chicks were weighed (0.1-precision g) and measured (bill length and tarsus-plus-toe; 0.01-precision mm) daily Imatinib Mesylate pontent inhibitor by the same person (AR) around 14:15 h. For every treatment group, we described chick growth price (mmd-1) as the coefficient of the regression of mean body size (tarsus-plus-toe size) on chick age group [26]. After three weeks, experimental fledglings had been Imatinib Mesylate pontent inhibitor transported in past due afternoon towards the laboratory from the College or university of Extremadura for over night RMR measurements, with regards to air consumption, using regular flow-through respirometry (discover full information on the task in [16,17]). We performed RMR measurements through the nocturnal period (relaxing amount of the fledglings circadian routine) and in post-absortive state (fledglings were placed in outdoor cages without food for about 3 h but with water ? salinity according to treatment). Fledglings were individually placed in metabolic chambers (15 L) LEP in darkness and housed in a temperature-controlled cabinet at a constant temperature (27C; within the thermoneutral zone of precocial shorebird chicks Imatinib Mesylate pontent inhibitor [27]). The metabolic chambers received atmospheric air at a rate of 1 1,000 mlminC1 from calibrated mass flow controllers (MFS-5; Sable Systems, Las Vegas, NV, USA). Water vapour was removed from the air stream immediately downstream from the metabolic chambers using desiccant columns (Drierite?), followed by a multiplexer (TR-RM4; Sable Systems), which allowed automatic switching between four channels. A subsample of the air was taken at 150 mlminC1 using a subsampler mass flow meter unit (SS-3; Sable Systems), and the oxygen concentration was determined using a gas analyzer (FC-10 Oxygen Analyzer; Sable Systems). The latter was calibrated regularly using pure nitrogen and a certified mixture of 21% O2 as the low and the high reference, respectively. The oxygen concentration was logged at a 1 Hz sampling rate on a computer using ExpeData software (v. 1.1.25; Sable Systems) and a UI2 converter. Each sampling sequence started with logging ambient baseline air for 10 min, followed by sampling each chamber for 10 min, with the system being flushed for 2 min between samples to remove latent gases. This sequence was repeated four times, so that there were four records per bird per night. Birds were weighed prior to and after RMR measurements, and their mean body mass was used in the analyses (see below). The metabolic rate was calculated using an energy equivalent of 20 Jml O2 [28]. In addition to captive-reared individuals, we also measured RMR in wild fledglings captured in hypersaline (Samouco saltpans; n = 8) and freshwater (reservoirs from Caia, Portugal, 3900N, 712W; n = 5) habitats. These birds were captured in the field in late afternoon and then immediately transported to the University of Extremadura for metabolic measurements at night. The wild fledglings from saltpans were captured in hypersaline pans (67 ), while wild fledglings from reservoirs only had available freshwater. The procedure for metabolic measurements was identical as described above for captive-reared fledglings. After RMR measurements, we collected a blood sample (about 70 l) from the.