Surf smelt accelerate usage of endogenous energy reserves under climate change

temperature, pH, and CT, the variability in all three measurements results in relatively high variability in pCO2 estimates. The ambient pCO2 tanks had an average standard deviation of ± 50.58 μatm pCO2, while the elevated pCO2 tanks had considerably higher variability, with an average standard deviation of ± 275.89 μatm (Table 1). Differences in mean pCO2 between temperature levels within pCO2 treatments and were generally less than 150 μatm, consider- ably smaller than the approximately 800 μatm difference between the ambient and elevated pCO2 treatments (Table 1). Therefore, data are presented in the elevated pCO2 and ambient pCO2 treatments in figures for embryo and larval characteristics. Given the need to use elec- trode pH measurements, we acknowledge the limitation of these estimates of pCO2. Embryo yolk usage and heartrate The proportion of embryo yolk size relative to total egg size (Y:E) decreased throughout the duration of the experiment in all treatments with day having a significant negative effect on Y: E (Table 2; Fig 1). Temperature also had a significant, negative effect on Y:E throughout the 13-day experiment (Table 2; Fig 1). The average Y:E decreased by 21.6% after 13 days of incu- bation at 13˚C compared to a 28.2% decrease at 18˚C. This equates to Y:E being 7.3% smaller at 18˚C compared to 13˚C at the final time point. There was also a significant, positive effect of Table 1. Treatment conditions. In-Situ Measurements Discrete Samples Experiment Conditions pH Temperature pCO2 CT Salinity (pCO2 +˚C) (NBS Scale) (˚C) (μatm) (μmol kg�SW-1)

Embryo Yolk ambient +13 7.88 ± 0.01 (36) 13.77 ± 0.24 (36) 793. 85 ± 28.59 (15) 2015.06 ± 10.44 (15) 30.8 ± 0.6 (15) ambient +15 7.87 ± 0.03 (42) 14.89 ± 0.03 (42) 749.37 ± 141.69 (15) 2013.21 ± 16.53 (15) 30.7 ± 0.5 (15) ambient +18 7.83 ± 0.02 (39) 17.94 ± 0.23 (39) 872.25 ± 133.73 (15) 2010.92 ± 14.86 (15) 30.6 ± 0.6 (15) elevated+13 7.56 ± 0.09 (39) 13.44 ± 0.24 (39) 1695.41 ± 337.05 (15) 2049.61 ± 11.56 (15) 31.1 ± 0.9 (15) elevated+15 7.56 ± 0.06 (39) 15.02 ± 0.03 (39) 1617.05 ± 324.02 (15) 2050.43 ± 15.9 (15) 30.9 ± 0.9 (15) elevated+18 7.53± 0.03 (39) 17.93 ± 0.23 (39) 1695.41 ± 368.98 (15) 2047.64 ± 17.34 (15) 30.6 ± 0.6 (15) Larvae Yolk and Oil Globule ambient +13 7.85 ± 0.01 (9) 13.22 ± 0.35 (9) 828.75 ± 25.25 (6) 2032.91 ± 11.06 (6) 31.0 ± 0.6 (6) ambient +14 7.83 ± 0.01 (9) 14.15 ± 0.08 (9) 888.25 ± 25.49 (6) 2028.65 ± 12.21 (6) 30.8 ± 0.4 (6) ambient +18 7.79 ± 0.01 (9) 17.42 ± 0.15 (9) 992.22 ± 39.38 (6) 2022.67 ± 14.04 (6) 30.6 ± 0.6 (6) elevated+13 7.54 ± 0.07 (9) 13.43 ± 0.43 (9) 1759.74 ± 316.37 (6) 2067.31 ± 16.84 (6) 31.2 ± 0.7 (6) elevated+14 7.51 ± 0.06 (9) 14.15 ± 0.23 (9) 1787.97 ± 247.35 (6) 2077.95 ± 17.39 (6) 30.9 ± 0.6 (6) elevated+18 7.51 ± 0.06 (9) 17.32 ± 0.11 (9) 1907.61 ± 309.72 (6) 2065.77 ± 16.79 (6) 31.0 ± 0.6 (6) Average seawater temperature, pH, pCO2, and CT for two experiments. Data for the embryo heartrate experiment are presented in S1 Table. Data are shown as time-

averaged means ± 1 SD of (n) measurements. pH and temperature were measured daily, while CT was measured 3 times per week. pCO2 was derived from temperature, pH, and CT measurements. https://doi.org/10.1371/journal.pone.0270491.t001 Table 2. Generalized linear model results for Y:E. Value Std. Error z-value p-value Intercept 6.5614 0.5062 12.960 <2.00e-16 Day -0.49 0.0181 -27.927 < 2.00e-16 pCO2 0.4460 0.1024 4.356 1.32e-05 Temperature -0.2321 0.0303 -7.654 1.94e-14 Summary of results of a generalized linear mixed effect model (family = binomial, link = logistic) examining the effect of day, temperature, and pCO2 on embryo yolk sac relative to total egg size. https://doi.org/10.1371/journal.pone.0270491.t002 PLOS ONE Surf smelt response to climate co-stressors PLOS ONE | https://doi.org/10.1371/journal.pone.0270491 June 27, 2022 6 / 14