Thermal performance of planktonic ciliates differs between marine and freshwaters: A case study providing guidance for climate change studies

Abstract

Predicting the performance of aquatic organisms in a future warmer climate depends critically on understanding how current temperature regimes affect the organisms' growth rates. Using a meta-analysis for the published experimental data, we calculated the activation energy (E_a) to parameterize the thermal sensitivity of marine and freshwater ciliates, major players in marine and freshwater food webs. We hypothesized that their growth rates increase with temperature but that ciliates dwelling in the immense, thermally stable ocean are closely adapted to their ambient temperature and have lower E_a than ciliates living in smaller, thermally more variable freshwater environments. The E_a was in the range known from other taxa but significantly lower for marine ciliates (0.390 ± 0.105 eV) than for freshwater ciliates (0.633 ± 0.060 eV), supporting our hypothesis. Accordingly, models aiming to predict the ciliate response to increasing water temperature should apply the environment-specific activation energies provided in this study.

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Scientific Significance Statement

On the global average, the temperature increase in the ocean is lower than in lakes. Moreover, most freshwater organisms must cope with wider temperature fluctuations than marine organisms. Knowing if the organisms' thermal sensitivity differs in the two realms is crucial for predicting the respective climate-related changes at community and ecosystem levels. We investigated the thermal sensitivity of planktonic ciliates, which are of tremendous significance for biogeochemical cycling in all aquatic ecosystems. Marine and freshwater ciliates differ in their thermal performance; therefore, system-specific activation energies should be applied in models predicting ciliate responses to altered temperatures. This work may serve as a model study for other taxa and be of interest to many marine and freshwater ecologists.

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Temperature is key for all physiological processes on Earth including respiration, cell growth, (primary) productivity, and reproduction. Accordingly, temperature is an important and useful metric for investigating evolutionary responses (Knies et al. 2009). Furthermore, temperature is arguably the most crucial driver of the ongoing global change (Thomas et al. 2012; IPCC 2013, 2019). Consequently, recognizing and understanding how organisms respond to different thermal regimes is of utmost significance.

Organisms realize three principal alternatives to respond to global environmental change: (i) by evolutionary adaptation (microevolution), (ii) by phenotypic and behavioral plasticity, and (iii) by spatial or temporal shifts in occurrence (Merilä and Hendry 2014; Merilä and Hoffmann 2016; Coulson et al. 2017). The latter response to environmental change can be neglected from a global perspective. Although there is some evidence that fluctuating environments promote evolutionary adaptation (Scott et al. 2018), phenotypic plasticity seems to be more important for organisms to survive in fluctuating environments (Hoffmann and Sgrò 2011; Reed et al. 2011; Crozier and Hutchings 2014). Importantly, these two alternative strategies are not mutually exclusive because phenotypic plasticity itself is an adaptative trait subject to selection (DeWitt et al. 1998; West-Eberhard 2003; Hallsson and Björklund 2012). Empirical evidence and models suggest that the evolution of adaptive phenotypic plasticity (Ghalambor et al. 2007) will be promoted in spatially or temporally fluctuating environments (Agrawal 2001; Fragata et al. 2016; Bitter et al. 2021; and references therein). Furthermore, the “hotter is better” hypothesis (Huey and Kingsolver 1989) posits that taxa adapted to warmer temperatures will have higher maximum growth rates than those adapted to low temperatures (Knies et al. 2009; Wang et al. 2019).

Aquatic ecosystems offer an excellent opportunity to test the hypothesis that the extent of environmental fluctuations affects the organisms' thermal performance. Mainly due to their much smaller volume and lower heat content, most lakes and ponds exhibit larger seasonal, diel, and spatial temperature fluctuations than the sea. Fluctuating temperatures necessitate a greater breadth in performance (i.e., generalist genotypes with wide temperature reaction norms) and hence retard adaptation to specific conditions (specialist genotypes with narrow temperature reaction norms) (Kassen 2002; Duncan et al. 2011; Ketola et al. 2013). For instance, species living in moderately warm environments will occasionally experience hot temperatures. Therefore, they will maintain an ability to grow rapidly at hot temperatures (Ketola et al. 2013, 2014).

We have tested the prediction that fluctuating environments promote higher growth following the “hotter is better” response, using planktonic ciliates. These free-living protists are widespread and important components of aquatic food webs in both freshwater and marine ecosystems (Weisse and Montagnes 2021). Ciliates are morphologically and physiologically diverse (~ 8000 morphospecies; Foissner 1999; Warren et al. 2017), have short generation times, and can be easily manipulated in experimental work. They, therefore, lend themselves to assess cross-taxa trends in adaption. Furthermore, as ciliates reproduce asexually by cell division, their specific growth rate is a useful proxy for fitness. In short, ciliates are models that have been used intensively in cell biology, genetics, evolutionary biology, and ecology (Hausmann and Bradbury 1996; Weisse 2006; Montagnes et al. 2012) and are a suitable target group to compare thermal responses between freshwater and marine environments.

Our meta-analysis of the specific growth rates in aquatic ciliates allows for testing if marine planktonic ciliates, in the course of stabilizing selection, have adapted to their relatively constant thermal environment. If this assumption holds, marine ciliates should be less thermally sensitive than their freshwater counterparts. As the temperature sensitivity of organisms is best parameterized by their activation energy (E_a; the energy needed to start a biochemical reaction) (López-Urrutia et al. 2006; Chen et al. 2012; Chen and Laws 2017; Wang et al. 2019), we have calculated the cross-taxa E_a of ciliate-specific growth and compared it between the two distinct aquatic systems. Likewise, we tested if the general rule that metabolic rates are mass dependent (Brown et al. 2004) applies to ciliates, that is, whether ciliate cell volume affects their growth rate (Montagnes 1996). Since this was not the case, our analyses considered only temperature as a factor for ciliate growth.

Methods Data collection

Maximal (i.e., at food saturation) ciliate-specific growth rates (r, d⁻¹) and cell volumes at a given temperature were obtained from experimental studies. Since field studies may suffer from uncontrolled biases, we excluded them from our analyses. Occasionally, growth rate and volume data had to be extracted from figures, using DataThief III (Tummers 2006), and—when needed and possible—we sought clarification directly from the authors. In cases where the volume was not measured in a study or provided by the authors, estimates were made based upon similar work (Supporting Information Table S1). In tintinnid ciliates, the cell volume was assumed to be 30% of the lorica volume when the former measurement was missing (Gilron and Lynn 1989; Rychert 2011). We used the conversion factor provided by Menden-Deuer and Lessard (2000) to calculate the cell volume from the ciliate carbon content. The analyses covered a temperature range from 5°C to 30°C, for which data from both marine and freshwater ciliate taxa were available. Eventually, these selection criteria yielded 57 studies: 40 marine and 17 freshwater, which provided data on 58 taxa (42 from marine water, 16 from freshwater).

Analysis and model selection

All analyses were performed in R software v. 4.0.2 (R Core Team 2020). We first tested the commonly postulated relation between cell size (volume) and growth rate (Brown et al. 2004) by assessing the allometric relationship between ln specific growth rate (r, d⁻¹) and ln volume (V, μm³). The analysis yielded no significant correlation between volume and growth rate for neither freshwater nor marine ciliates. Consequently, the relation between temperature and growth rate was assessed independently from volume. Moreover, we applied the Wilcoxon rank-sum test (α = 0.05) to compare ciliate cell volumes from freshwater and marine systems as the (ln-transformed) cell volume data were not normally distributed.

To test if cross-taxa thermal performance, determined as activation energy (E_a, eV) associated with growth rate (r, d⁻¹), differed between freshwater and marine habitats, Arrhenius plots were employed; ln r was regressed against 1/kT where, k is the Boltzmann constant (8.6 × 10⁻⁵ eV K⁻¹, and T is the temperature [K]), with the negative slope of the regression being E_a. Differences in E_a were tested with ANOVA (α = 0.05). To account for the non-independence of data points from the same study and/or taxon, “study” and “taxon” were included as random factors. Then, to determine which model was most appropriate for this analysis, multiple linear regression and linear mixed-effects models were used (“lme4” package, Bates et al. 2015; Supporting Information Table S3), and the model with the lowest corrected Akaike information criterion (AICc; “AICcmodavg” package; Mazerolle 2020) was chosen (Supporting Information Table S3). The best-fitting model—the one used in the following steps—accounted for the effects of temperature and habitat and their interaction as fixed factors and ciliate species and study as random factors (Supporting Information Table S3). The percentage of variance explained by the fixed effects (marginal R²) and both fixed and random effects (conditional R²) in the final model were determined following Nakagawa and Schielzeth (2013).

Results

Marine ciliates used in the studies (Supporting Information Table S1) were significantly larger than those from freshwater (Wilcoxon rank-sum test: W = 3519; p < 0.001; Supporting Information Table S4). However, no effect of cell volume on growth rates emerged (Fig. 1). Similarly, no interacting effect of the cell volume and habitat on growth rates could be detected (Supporting Information Tables S3).

Fig. 1. Partial regressions of the effect of ln cell volume (V) on ln growth rate (r) for ciliates from marine (blue, n = 89, slope ± SE: 0.012 ± 0.049) and freshwater habitats (yellow, n = 132, slope ± SE: −0.073 ± 0.087).

Temperature had a significant positive effect on the growth rates of ciliates from marine and freshwater habitats. The E_a (±SE) of freshwater ciliates was significantly higher than that of marine species (0.633 ± 0.060 eV vs. 0.390 ± 0.105 eV; Fig. 2, Table 1). The standard deviation of random factors (i.e., within-taxon or within-study variance) was relatively high compared to the residual values (i.e., between taxon or study variance), supporting the need to account for the random factors in the analysis (Table 1). The fixed effects explained 33% of the variance, and the full model explained 59% of the variance.

Fig. 2. (a) The effect of temperature on ciliate growth rates from freshwater (nf = 132) and marine origin (nm = 89) based on partial regression plots; r is the growth rate (d−1). (b) Arrhenius plot for comparison of activation energies (Ea) in freshwater and marine planktonic ciliates based on partial regression plots; r denotes the growth rate (d−1), k is the Boltzmann constant (8.6 × 10−5 eV K−1), T is the temperature [K], and the negative slope of the line is Ea (eV K−1). The shaded areas bounded by the thin lines represent the 95% confidence intervals.

Table 1 Regression estimates (estimate and standard error) and ANOVA (F and P values) of the linear mixed model testing for differences in E_a. Random factors included ciliate species (i.e., “taxon”) and study (see Supporting Information Table S3). Significant p-values are represented in bold.

[Table omitted. See PDF]

In summary, our cross-taxa meta-analysis yielded that (i) temperature has a strong positive effect on growth rates and (ii) ciliates from freshwater plankton have a higher activation energy than their marine counterparts.

Discussion

This study supports the hypothesis that the cross-taxa activation energy (E_a) differs between freshwater and marine ciliates. In more general terms, the prediction that thermally variable environments (lakes) produce cross-taxa higher responses with high E_a is corroborated, while more thermally-stable environments (the oceans) produce cross-taxa responses that are characterized by lower thermal responses (i.e., lower E_a). Moreover, planktonic ciliates from more thermally variable environments express higher growth rates than their counterparts from more stable environments (the oceans) at the upper end of the temperature range but not at the lower end, which is only in partial accordance with the “hotter is better” hypothesis.

Surprisingly, the data do not support the expected trend that ciliate growth rates decrease with cell volume (Montagnes 1996). Traditional sampling (i.e., plankton nets) and investigation methods (i.e., light microscopy) favored the detection of larger ciliates in remote marine habitats. Accordingly, marine ciliates studied thus far were significantly larger than the freshwater species, but the ciliate volume did not affect the ciliate growth rates. Although this is the exception rather than the rule, similar results have been reported for marine phytoplankton by Chen and Laws (2017), who also found a low allometric exponent of −0.09 for the volume effect on E_a of heterotrophic protists, which is well below the “universal” exponent of −0.25 (Fenchel 1974; Gillooly et al. 2001). Thus, we conclude that (i) the observed difference in cell volumes between the two habitats likely reflects a bias (see above) and (ii), most likely, the lack of an allometric relationship between ciliate cell volumes and growth rates results from the relatively narrow size range of the ciliates studied.

In contrast to cell volume, the data support the assumption that growth rates increase with temperature across taxa and the E_a values are in the expected range (0.2–1.2 eV, average ~ 0.6 eV: (Gillooly et al. 2001; Brown et al. 2004; Chen and Laws 2017). Unfortunately, compared to phytoplankton (Thomas et al. 2012; Edwards et al. 2015, 2016), the available experimental data are scarce and do not allow for inferring thermal responses from the organisms' temperature reaction norm and growth rates at the respective species-specific temperature optima (Supporting Information Table S2). Furthermore, the dataset is limited regarding temperature responses of marine ciliates at both ends of the temperature range (Fig. 2). Nevertheless, the data suggest that environmental stability could be a driving force in adaptation resulting in the wide range of the observed E_a. Our findings, therefore, may have important theoretical and practical implications.

Currently, global summer surface temperatures of lakes are increasing at a rate of 0.34°C per decade with strong regional variations ranging from −0.7°C to 1.3°C per decade (O'Reilly et al. 2015). In 10 European lakes, the maximum lake surface temperature has increased above average during the past 50 years with 0.58°C per decade (Dokulil et al. 2021). The crucial question is: have freshwater ciliates adapted to the larger temperature fluctuations that they are generally exposed to, relative to marine species? The temperature increase in marine waters varies regionally, being much higher in the Arctic Ocean, in which the summer temperatures of the upper mixed layer increased at around 0.5°C per decade during 1982–2017 (IPCC 2019). In the course of global warming, adaptation to elevated temperatures is key for the survival of ciliates and other organisms. If most freshwater ciliates have developed a “thermal safety margin” known from Paramecium (Krenek et al. 2012), then they should be able to tolerate and grow at temperatures above their current temperature environment. Due to the paucity of evidence discussed above, it is an open question if this also applies to marine ciliates. For ciliates isolated from temperate tidal marshes, Martinez (1980) reported a widely distributed tolerance against temperatures even above 30°C. However, the temperature regime of this coastal area and its inhabitants are not representative of oceanic conditions.

While several freshwater and a few marine ciliate species not only tolerate but also grow at temperatures above 25°C (Wang et al. 2019; references in Supporting Information Table S2), we can merely speculate how common temperature-sensitive species (e.g., Histiobalantium bodamicum and Balanion planctonicum; Weisse et al. 2001; Lu et al. 2021) cope with summer temperature maxima exceeding the ciliates' upper-temperature tolerance limits. Migrating to greater depths (Hampton et al. 2014) and the formation of resting cysts have been identified as ciliate strategies to survive unfavorable conditions (Müller 2000). Resting cysts are mainly known from oligotrich and choreotrich ciliates (Kamiyama 2012), the dominant planktonic taxa in the ocean, but cyst formation has been studied in fewer than 30 marine species (mainly tintinnids) and even fewer freshwater species (Agatha 2011; Kamiyama 2012). Both marine and freshwater ciliates follow a seasonal encystment–excystment cycle (Müller 2000; Agatha 2011). Temperature is probably the most important proximate factor inducing excystment (Müller 2000; Kamiyama 2012) and declining temperatures and food supply may also trigger encystment (Müller 2000; Foissner et al. 2005; Kamiyama 2012). However, it remains unknown if high temperatures in the vicinity of the upper temperature tolerance limit are also a cue for encystment of temperature-sensitive species. In conclusion, there is no evidence that cyst formation allows aquatic ciliates to cope with warming, nor do we currently know if the formation of resting cysts is more widespread among freshwater or marine taxa. Another process that could affect thermal adaptation is sexual recombination (conjugation), which is favorable for adaptation to (rapidly) changing and heterogeneous environments (Becks and Agrawal 2010, 2012; Lachapelle and Bell 2012). However, data to evaluate the importance of this process in natural ciliate populations are lacking.

With the recognition that climate change is impending, modelers are applying estimates of thermal performance to many taxa in food web models, including ciliates (Atkinson et al. 2003; Chen and Laws 2017; Wang et al. 2019). For aquatic environments, the applicability of a uniform thermal performance to organisms dwelling in the oceans and freshwater is questionable. Here, we reject this assumption for marine and freshwater planktonic ciliates. Models aiming to predict ciliate responses to increasing temperature (corresponding climate change scenarios) should consider the different activation energies that we provide for marine and freshwater species. However, the reasoning that the thermal stability of (aquatic) ecosystems affects the thermal sensitivity of the resident organisms needs to be further addressed by testing additional taxa representing different trophic levels.

Acknowledgments

The authors thank Jie Huang for her help with searching and translating references from Chinese. This study was supported by the Austrian Science Fund, project number FWF P 32714.