Volume 28, Issue 2, Pages 327-332.e3 (January 2018) Rising pCO2 in Freshwater Ecosystems Has the Potential to Negatively Affect Predator- Induced Defenses in Daphnia  Linda C. Weiss, Leonie Pötter, Annika Steiger, Sebastian Kruppert, Uwe Frost, Ralph Tollrian  Current Biology  Volume 28, Issue 2, Pages 327-332.e3 (January 2018) DOI: 10.1016/j.cub.2017.12.022 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2018 28, 327-332.e3DOI: (10.1016/j.cub.2017.12.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Change of pCO2 and pH with Time for All Four Investigated Reservoirs (A) Mean pCO2 values increase significantly with time. (B) pH values of all reservoirs decrease significantly. There is a significant correlation between the decrease in pH and the increase in pCO2 in each reservoir (see also Table S1). Additional background information on the reservoirs is given in Table S7. Current Biology 2018 28, 327-332.e3DOI: (10.1016/j.cub.2017.12.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Increased Levels of pCO2 Do Not Result from Changes in Chlorophyll-a or Total Phosphorous (A–D) Anthrophogenic environmental health measures, reduced total phosphorus from the four freshwater reservoirs (A) Möhne, (B) Sorpe, (C) Henne, and (D) Lister to a minimum in the year 1999 (Möhne, Lister) and 2000 (Sorpe, Henne), respectively. Removal of total phosphorus (TP) can have a direct impact on the phytoplankton population density so that chlorophyll-a (Chl-a) is reduced. This is the case in the Sorpe and the Lister but not the Möhne and the Henne. A decrease in phytoplankton population density could affect overall CO2 level due to a decrease in CO2-fixation rates (see also Tables S2 and S3 for statistical analysis). (E–H) In all four reservoirs (E) Möhne, (F) Sorpe, (G) Henne, and (H) Lister, pCO2 levels increased even after Chl-a and TP have stabilized. Also see Tables S2 and S3. Current Biology 2018 28, 327-332.e3DOI: (10.1016/j.cub.2017.12.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Effect of pCO2-Dependent Acidification on the Development of Morphological Defenses in Daphnia (A) Chaoborus-induced neckteeth expression in D. pulex. Under control conditions in the absence of predator cues, only minute neckteeth are developed. Predator cues induce neckteeth significantly at control conditions. This expression is significantly reduced under elevated pCO2 conditions (mean pCO2 16,154.13 μatm ± 2,344.1 with pH 6.7 and mean pCO2 11,406.2 μatm ± 1,484.8 with pH 6.9) compared with control conditions (2,203.6 μatm ± 778.9; pH 7.5; Table S4). (B) Notonecta-induced crest development in D. longicephala. Under control conditions in the absence of predator cues, no crests are developed. Predator cues induce crests significantly at control conditions. This development is significantly reduced under elevated pCO2 conditions (mean pCO2 16,154.13 μatm ± 2,344.1 with pH 6.7 and mean pCO2 11,406.2 μatm ± 1,484.8 with pH 6.9) compared with control conditions (2,203.6 μatm ± 778.9; pH 7.5; also see Tables S5 and S6). (C and D) Daphnia growth in the absence of kairomones in control and pCO2-acidified conditions. Neither D. pulex in the second juvenile instar ([C] ANOVA, F2,509 = 0.457, p = 0.63) nor D. longicephala in the fifth juvenile instar ([D] F2,162 = 0.47; p = 0.62) showed significant differences between treatments. Therefore, differences in pCO2 do not interfere with Daphnia growth. (A–D) Boxes show interquartile ranges with median (square); whiskers display 25% data ranges with outliers displayed as circles. ∗∗∗p ≤ 0.001. (E) Stability of CO2-dependent pH conditions throughout the experimental period (from start to end). Bars show mean and whiskers the standard deviation. Information on water parameters for pCO2 calculation is given in Table S8. Current Biology 2018 28, 327-332.e3DOI: (10.1016/j.cub.2017.12.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Effect of pH-Dependent Acidification on the Development of Inducible Morphological Defenses in Daphnia (A) Chaoborus-induced neckteeth expression in D. pulex. In the absence of predator cues, no neckteeth are developed regardless of pH. In the presence of kairomone, neckteeth are significantly expressed, but HCl-dependent acidification from control to pH 6.9 and pH 6.7 does not impact neckteeth development. (Kruskal–Wallis test, H [2, N = 177] = 0.611, p = 0.74). (B) Notonecta-induced crest expression in D. longicephala. Under control conditions crests are significantly developed in response to Notonecta kairomone (ANOVA, F2,214 = 67.194, p ≤ 0.001). HCl-dependent acidification from control to pH 6.9 and pH 6.7 does not impact crest development (ANOVA, F2,214 = 0.123, p = 0.8). pCO2 in all treatments was 2,2203.6 μatm ± 778.9 μatm. (A and B) Boxes show interquartile ranges with median (square); whiskers display 25% data ranges with outliers displayed as circles. Current Biology 2018 28, 327-332.e3DOI: (10.1016/j.cub.2017.12.022) Copyright © 2017 Elsevier Ltd Terms and Conditions