Animal culture and medium
Five different clonal lineages of the water flea Daphnia magna were sampled from two ponds on agricultural land in Belgium (Vleteren: 50°55′06.7″ N, 2°43′27.0″ E and De Haan 51°13′53.8″ N, 3°01′49.2″). They were cultured separately in 210 ml glass jars under optimized laboratory conditions (20 ± 1 °C, 14:10 h light:dark cycle). Seed shrimp and rotifer resting eggs were obtained from a commercial supplier (MicroBioTests Inc., H. incongruens strain MBT/1999/10, product code TB36; B. calyciflorus, product code TK21, Belgium) and represent laboratory cultured, single clonal lineages. More details on animal culture are reported in the online supplementary methods (Appendix 3).
Natural pond water was used as medium both in animal cultures and the experiment. It was extracted from a Belgian region (50°59′00.92″ N, 5°19′55.85″ E, Zonhoven) with soft, poorly buffered water (Alkalinity 3–8°d; pH 6.5–8.5) which is likely to be susceptible to acidification under elevated pCO2. More information on medium and mineral composition is reported in the online supplementary information (Appendix 3; Table S3, Appendix 1).
Experimental set-up
Organisms were exposed to three pCO2 treatments, an ambient control (C; 1,520 ppm ± 702 SD), an elevated (T1; 25,609 ppm ± 4,541 SD) and an extreme pCO2 level (T2; 83,201 ppm ± 15,533 SD). The control pCO2 level represents the current global mean that is measured in lentic freshwaters considering most ponds and lakes are already supersaturated10,12. The T1 level is currently only observed in more extreme cases11. However, it reflects a pCO2 level that could be encountered more commonly in the field in the future. The T2 treatment represents an extreme test of the tolerance limits of extant species. These treatments are a necessary simplification of reality since pCO2 can experience strong fluctuations in ponds and lakes. An overview of freshwater pCO2 concentrations from literature can be found in Table S1 (Appendix 1).
The elevated pCO2 concentrations were manipulated in the water by injecting pure CO2 (99.998% pure, ALPHAGAZ CO2 SFC * B50-N48, Airliquide, Belgium) from gas cylinders into the water (cf.49) at a constant flowrate, using a high-pressure regulator (HBS 200–10.2,5; AirLiquide, Belgium) and a flow controller (Sho-rate model 1350G, Brooks Instruments, USA). In the control treatment, ambient air was supplied at a similar rate as the CO2 to ensure equal perturbation levels across all containers. Water of all experimental containers (including control) were also injected with ambient air to keep the water oxygenated. A relatively constant pCO2 was ensured by continuously monitoring pH and kept between a range of ~ 20,000–30,000 ppm (pH 6.9–6.7) for T1 and ~ 70,000–120,000 ppm (pH 6.4–6.1) for T2 (Figure S2, Appendix 2).
Each treatment included 13 replicate 210 mL glass jars per species, resulting in a total of 117 experimental units. Per replicate, one mature water flea (8–11 days old) was inoculated in a jar containing aerated pond water. The five clonal lineages were distributed evenly over the experimental conditions so that each condition had the same number of replicates per clone. Seed shrimp replicates each contained one newly hatched (< 24 h old) individual. An autoclaved pebble was provided in each jar as a substrate for oviposition. Rotifer replicates contained one newly hatched individual (< 24 h old) to start a clonal population. For the water flea and the seed shrimp, juveniles were counted and removed. Since these rotifers only have a lifespan of 4–7 days at 20 °C50, clonal rotifer populations were allowed to grow during the entire experiment. The experimental containers were kept under standardized laboratory conditions in an incubator (20 ± 1 °C; 14:10 h light:dark cycle; 6000 K warm white LEDs, ± 3700 lx). The animals were fed ad libitum with a solution of dead green algae (Acutodesmus obliquus, 100 × 106 cells/ml) reconstituted from thawed aliquots stored at −20 °C.
Throughout the experiment, standard water quality variables were measured (Table 1). pCO2 and DIC was calculated using pH, temperature and alkalinity measurements according to Fasching et al. (2014)51 (formulas in Appendix 4; variability in Figure S2, Appendix 2). The CO2SYS program52 was also used to calculate pCO2 for reproducibility purposes, which resulted in similar values as reported here (Table S4, Appendix 1).
Measured responses
Each experimental replicate was monitored at fixed intervals over 24 days (water flea: 3 × /week, seed shrimp/rotifer: 2 × /week) to determine life history traits and mortality (more details in Table S5, Appendix 1). As water fleas and seed shrimps were kept individually, mortality reflects individual survival. In the short-lived rotifers, mortality was assessed as survival of the clonal population in each jar.
Body size of water fleas (from eye to spine base) and seed shrimp (longest straight line along the longitudinal axis) was measured up to the nearest µm under a stereomicroscope. Somatic growth rate was calculated for each of three intervals during the experiment. Mean daily fecundity was calculated as the average number of neonates per female per day in the water flea (released from brood chamber) and seed shrimp (hatched from eggs). The cumulative number of neonates produced per female at the end was calculated as a measure of lifetime fecundity. Seed shrimp age and size at maturity were determined from the moment the first eggs were found in the jars.
Population size of the rotifers was determined using a stereomicroscope, a Sedwick-Rafter counting chamber and Lugol’s solution for staining. Population growth rate was calculated as the intrinsic rate of population increase r = ln(F2) − ln(F1)/t2 − t1 53 for each of three experimental intervals. The maximum population size of each clonal population and the timepoint at max. pop. size were also calculated. Mean daily fecundity was determined as the average number of individuals with eggs per day as a proxy for the fecundity of the clonal population50. The cumulative number of females with eggs at the end was determined as a measure of lifetime fecundity.
Statistical analysis
All analyses were performed in R Studio version 1.0.143 (R version 3.6.1). The survival probability of each model species under the different treatments was estimated using Kaplan–Meier survival curves (ggsurvplot, ggfortify package). Differences in survival probability between treatments and clones and between species within treatments were tested using log rank tests (survdiff, survival package) and pairwise comparisons with Holm corrections (pairwise_survdiff, survminer package).
To test for the overall impact of the pCO2 treatments on water flea growth rate and body size, linear mixed models were used (LMM, lmer, lme4 package). Treatment (C, T1, T2), time and their interaction were included as fixed predictors and the identity of the experimental jars (ID) as a random factor to correct for repeated measurements on the same individual. Clone and a treatment × clone interaction were also included to test for differences in the overall sensitivity of clones, or differences in the responses of clones to the treatments, respectively. To investigate the effect of the treatments at specific time points, separate LMMs were constructed for growth rate for each of three intervals (int. 1, 2, 3), body size on day one (since this is the only data point in T2) and body size for four time points (day 6, 13, 20, 24). Treatment was included as a fixed predictor and clonal identity as a random factor to correct for the dependency of measurements within the same clonal lineage. LMMs (lmer, lme4 package) were constructed to investigate the effect of the treatments on water flea mean daily fecundity and lifetime fecundity, including treatment as fixed and clone as random factor.
LMMs were constructed to investigate the overall effect of the pCO2 treatments on seed shrimp growth rate and body size and rotifer population growth rate and size. In each model, ID was included as a random factor to account for repeated measurements. Analysis of variance (ANOVA, aov, car package) tests were used to test for the effect of the treatments at specific time points (growth rate: int. 1, 2, 3; body size: day 7, 14, 21, 24; population growth rate: int. 1, 2, 3; population size: day 6, 13, 20, 23) and for the effect on mean daily fecundity, lifetime fecundity, seed shrimp size at maturity and rotifer maximum population size. There was no variation in the age at maturity so no data analysis could be performed. The maximum population size timepoint was analyzed using a generalized linear model with a Poisson error distribution (GLMs, glm, stats package). The GLM was tested and corrected for overdispersion with the quasibinomial distribution.
Two out of the five inoculated water flea clones did not survive long enough in the T1 treatment to gather sufficient life history data. Therefore, these were excluded from analyses. In case of sometimes very high mortalities in T2, insufficient individuals remained for statistical analysis of certain life history traits. In that case, the T2 treatment was removed and t tests were used instead to test for differences between C and T1 (t-test, stats package). Normality of residuals and homogeneity of variances was verified using Shapiro–Wilk and Levene’s tests, respectively, for all linear models when applicable. In case of non-normal distribution of residuals, log transformation or non-parametric equivalents were used (Kruskall Wallis and Mann Witney U tests, stats package). Tukey post-hoc tests were performed (glht, multcomp package). Significance was always interpreted at p < 0.05.
Source: Ecology - nature.com
