Fecal GC: the dashed horizontal line shows the median fecal GC value (2001 ... The vertical red line shows the beginning of the stress test and the vertical gray ...
Comprehensive Endocrine Response to Acute Stress in the Bottlenose Dolphin from Serum, Blubber, and Feces Supplementary Figures: S1: Catecholamine Validation – parallelism with standard curves. S2: Temporal variation of fecal GCm and serum cortisol for individual dolphins throughout the study. S3: Temporal variation of fecal and serum aldosterone for individual dolphins throughout the study. S4: Temporal variation of blubber and serum cortisol for individual dolphins during the stress test. S5: Blubber cortisol analyses including all five study dolphins Supplementary Figure S1
Figure S1. Epinephrine (a) and Norepinephrine (b) enzyme-linked immunoassays (EIAs) were validated for use in bottlenose dolphins in this study. Serial dilutions of pooled plasma from both sexes (“pooled dilutions” shown in gray) were processed along with standards from the assay kits (“standard curve” shown in black). Each serial dilution was parallel with the standard curve. Bottom axes are hormone concentrations (log-scaled), y-axes are response metrics (absorbance at 450 nm wavelength), and the dilutions of the pooled plasma are shown on the top axis (“dilution factor”).
Supplementary Figure S2
Figure S2. Temporal variation in fecal glucocorticoid metabolite (fecal GCm; top row) and serum cortisol (bottom row) concentrations for each subject (TYH, COL, TRO NEH, BLU) during the five-day study. The x-axis scale is the same for all subjects except BLU; her beaching and consequent stress test were delayed one day (stress test was conducted on day 3 but we have 3 days of prior fecal sampling, shown as day “0”; see Methods & Discussion for detail). Fecal GC: the dashed horizontal line shows the median fecal GC value (2001 ng/g across individuals and sample days). The vertical red line shows the beginning of the stress test and the vertical gray line is the time of the fecal sample that day. The y-axis scale is the same for all subjects except NEH, as he had a much higher fecal GC value on the day of the stress test than other subjects. Serum cortisol: The stress test was conducted on day 3 (vertical red line) and associated with increased serum cortisol.
Supplementary Figure S3
Figure S3 Temporal variation in fecal aldosterone metabolites (top row) and serum aldosterone (bottom row) concentrations for each subject (TYH, COL, TRO NEH, BLU) during the five-day study. The x-axis scale is the same for all subjects except BLU; her beaching and consequent stress test were delayed one day (stress test was conducted on day 3 but we have 3 days of prior fecal sampling, shown as day “0”; see Methods & Discussion for detail). Top row: Fecal aldosterone metabolites: The dashed horizontal line shows the median fecal aldosterone metabolite value (7 ng/g across individuals and sample days). The vertical red line shows the beginning of the stress test and the vertical gray line is the time of the fecal sample that day. Bottom row: Serum aldosterone: The stress test was conducted on day 3 (vertical red line) and associated with increased serum aldosterone.
Supplementary Figure S4
Figure S4. Temporal variation in blubber (top row) and serum (bottom row) cortisol concentrations for each subject (TYH, COL, TRO NEH, BLU) during the out-of-water stress test. Top Row: The horizontal grey dashed line shows the median blubber cortisol value (8.2 ng / g lipid) across all individuals. The mean air temperature during the stress test is shown for each subject (mean air temp, in °C); numeric values beneath each sample point are the air temperature deviance at that time (also in °C; see Discussion for further explanation). Bottom Row: Serum cortisol increased in all study dolphins, and was association with concomitant increase in blubber cortisol in all dolphins except BLU (see text for detail).
Supplementary Figure S5
Figure S5. Blubber cortisol analysis included all study subjects. BLU’s blubber cortisol levels were anomalous, and her fecal GC levels indicated she may have experienced an unknown stressor leading up to the stress test and may explain her higher than expected initial blubber cortisol values. We therefore removed her from the blubber cortisol analyses (see Figure 8). In this supplemental figure, we report the statistical analysis if all five dolphins were included: a) Blubber cortisol significantly increased during the stress test (LMM, log-transformed blubber cortisol: F2, 8 = 5.1, p < 0.05). Blubber biopsy samples were collected just after beaching, 60 minutes after beaching, and 120 minutes after beaching (0, 60, and 120 min, samples, respectively). Blubber cortisol level was greater in the 120 min sample than at time 0 (*Dunnett’s test, p < 0.01), but the 60 min sample did not significantly differ from time 0 (p > 0.05). Individuals are shown in different symbols according to the legend. b) Blubber cortisol level showed a marginally significant association with total serum cortisol concentrations (LMM, logtransformed blubber cortisol: F1, 11 = 4.6, p =0.056, mR2 = 0.22). Small numbers by each data point designate sample collection time (0, 60, or 120 min). c) There was a significant influence of air temperature on blubber cortisol levels. We evaluated fixed effects of sample time and air temperature deviance (the difference in air temperature at that time from the mean air temperature at that sample collection time), with dolphin ID included as a random effect in the model. Both fixed-effects significantly influenced blubber cortisol (sample time: F2, 7.2 = 12.1, p = 0.005; temperature deviance: F1, 9.5 = 12.2, p = 0.006). The displayed line of fit and associated mR2 contains both features and thus does not appear as a line-of-best-fit with air temperature deviation. d) There was not a significant association between blubber cortisol and free serum cortisol concentrations.
