Interacting effects of simulated eutrophication, temperature increase, and microplastic exposure on Daphnia

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/ Interacting effects of simulated eutrophication, temperature increase, and microplastic exposure on Daphnia © 2020 Elsevier Inc. All rights reserved. Accepted version (Final draft) Hiltunen, Minna; Vehniäinen, Eeva-Riikka; Kukkonen, Jussi V. K. Hiltunen, M., Vehniäinen, E.-R., & Kukkonen, J. V. K. (2021). Interacting effects of simulated eutrophication, temperature increase, and microplastic exposure on Daphnia. Environmental Research, 192, Article 110304. https://doi.org/10.1016/j.envres.2020.110304 2021 1 Interacting effects of simulated eutrophication, temperature increase, and microplastic exposure on Daphnia Minna Hiltunena*, Eeva-Riikka Vehniäinenb, and Jussi V. K. Kukkonenc a Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä, Finland, [email protected] b Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä, Finland, eev[email protected] c Department of Environmental and Biological Sciences, University of Eastern Finland, Kuopio, Finland, [email protected]i * Address correspondence to Minna Hiltunen, minna.m.hilt[email protected], Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, FI-40014, Jyväskylä, Finland Running head: Daphnia exposed to multiple stressors 2 ABSTRACT The effects of multiple stressors are difficult to separate in field studies, and their interactions may be hard to predict if studied in isolation. We studied the effects of decreasing food quality (increase in cyanobacteria from 5 to 95% simulating eutrophication), temperature increase (by 3°C), and microplastic exposure (1% of the diet) on survival, size, reproduction, and fatty acid composition of the model freshwater cladoceran Daphnia magna. We found that food quality was the major driver of Daphnia responses. When the amount of cyanobacteria increased from 5 to 95% of the diet, there was a drastic decrease in Daphnia survival (from 81 ± 15% to 24 ± 21%), juvenile size (from 1.8 ± 0.2 mm to 1.0 ± 0.1 mm), adult size (from 2.7 ± 0.1 mm to 1.1 ± 0.1 mm), and reproduction (from 13 ± 5 neonates per surviving adult to 0), but the decrease was not always linear. This was most likely due to lower availability of lipids, eicosapentaenoic acid (EPA), and sterols from the diet. Microplastic exposure did not affect Daphnia survival, size, or reproduction. Food quality had an interactive effect with temperature on fatty acid content of Daphnia. Total fatty acid content of Daphnia was almost 2-fold higher at 20°C than at 23°C when fed 50% cyanobacteria. This may have implications for higher trophic level consumers, such as fish, that depend on zooplankton for energy and essential lipids. Our findings suggest that as proportions of cyanobacteria increase, in tandem with water temperatures due to climate change, fish may encounter fewer and smaller Daphnia with lower lipid and EPA content. Key words: cyanobacteria, fatty acids, climate change, food quality Daphnia magna 3 FUNDING: This study was funded by the Academy of Finland grant (315163) to Minna Hiltunen. The salary of Eeva-Riikka Vehniäinen was covered by the Academy of Finland grant (285296) to ERV. Abbreviations: EPA = eicosapentaenoic acid PUFA = polyunsaturated fatty acid MP = microplastic cyano = cyanobacteria PET = polyethylene terephthalate PS = polystyrene ABS = acrylonitrile butadiene styrene PERMANOVA = Permutational multivariate analysis of variance DistLM = distance-based linear modeling 4 GRAPHICAL ABSTRACT Daphnia magna were grown along a gradient in cyanobacteria (5-95%, mimicking eutrophication) in ambient and +3°C temperature with and without exposure to a mix of secondary microplastics (MPs). Proportion of cyanobacteria in diet was the major driver of Daphnia fitness, and had minor interactions with temperature, while we found no effects of MPs. 5% 5% 25% 50% 75% 95% 95% 5% 5% 25% 50% 75% 95% 95% Gradient in cyanobacteria 20 C23 C +MPs +MPs Size Fatty acid content 14 d 5 1. INTRODUCTION Eutrophication, climate change induced temperature increase, and microplastic contamination are among the biggest challenges that aquatic organisms face today. Excessive nutrient loading results in high phytoplankton biomass consisting mainly of cyanobacteria, which in addition to potentially being toxic, are poor quality resources for zooplankton (Lampert 1987; Paerl and Paul 2012; Huisman et al. 2018). Surface water temperatures are increasing (O’Reilly et al. 2015), which has a fundamental effect on small- and large-scale processes in aquatic ecosystems including biochemical reactions inside the cells, life-history traits of organisms, and water movement and stratification patterns (Adrian et al. 2009). During recent years, microplastics (often defined as <5 mm) have been found in all aquatic systems, ranging from freshwater lakes and streams to the ocean abyss, and their effects on organisms are under intensive investigation (Li et al. 2018; de Sa et al. 2018). These stressors act in unison, and studying them separately may not reveal all the potential impacts they may have on organisms that are facing multiple stressors in the natural environment. Food quality may be more important in explaining variation in zooplankton fitness than food quantity (Müller-Navarra et al. 2000). Fatty acids and sterols in diet influence Daphnia growth, reproduction, and lipid composition (Becker & Boersma 2005; Brett et al. 2006; Martin- Creuzburg et al. 2009; Peltomaa et al. 2017). Lipids are key components of all cells – their composition influences the fluidity, permeability, and protein action of biological membranes, and additionally, lipids act as energy storage material and function as precursors for hormonelike signaling molecules such as eicosanoids (Arts and Kohler 2009). The production of polyunsaturated fatty acids (PUFAs, with two or more double bonds in carbon chain) is mostly restricted to plants, and animals need to acquire PUFAs from their diet for optimal growth and 6 reproduction (Arts and Kohler 2009). Phytoplankton produce phytosterols, which consumers transform to cholesterol (Martin-Creuzburg and von Elert 2009). Cholesterol together with fatty acids regulate cell membrane structure and function (Stillwell & Wassal 2003). Sterols are also needed in production of vitamin D precursors and ecdysteroids, hormones, which are involved in for example molting of arthropods (Martin-Creuzburg and von Elert 2009). Cyanobacteria are a poor quality resource for zooplankton due to their tendency to form large colonies and filaments, production of harmful secondary metabolites (Paerl and Paul 2012), and lack of sterols and longchain PUFAs (≥ C20) (Martin-Creuzburg et al. 2008, 2009). Lake phosphorus concentrations are negatively correlated with the availability of essential PUFAs in the seston, resulting in reduced growth rates and reproduction of zooplankton in eutrophic lakes (Müller-Navarra et al. 2004). Furthermore, increased abundance of cyanobacteria restricts energy and PUFA transfer in the food web and ultimately results in fish being lower quality resources for human nutrition (Taipale et al. 2016b). In contrast to cyanobacteria, diatoms and cryptophytes have abundant long-chain PUFAs and sterols to support high growth rates in zooplankton (Galloway and Winder 2015; Taipale et al. 2016a; Peltomaa et al. 2017). Green algae lack long-chain PUFAs, but contain sterols, and their ingestion generally results in intermediate growth and reproduction of Daphnia (Hiltunen et al. in 2017; Peltomaa et al. 2017). Nutritional requirements of animals may change in response to temperature shifts. Climate scenarios predict that by 2100 air temperatures in the temperate zone will likely rise 0.3 to 4.8°C compared to the 1986-2005 levels due to anthropogenic climate change (IPCC 2014). More severe increases in temperature are expected in the polar regions. Additionally, extreme weather events, such as heat waves and droughts are expected to become more common, resulting in organisms facing episodic thermal stress. Faster metabolic rates of ectothermic animals in higher 7 temperatures impose higher metabolic costs that can only be met if there is enough food available (Gillooly et al. 2001). Furthermore, Daphnia require more PUFAs in colder temperatures, and more sterols in higher temperatures, presumably to maintain membrane stability (Sperfeld and Wacker 2009, 2012). Thus, increasing temperature may either enhance organism fitness by increasing growth rates, or act as an additional stress factor if the animal is already in the upper thermal tolerance limit or in poor nutritional state. Consumers may increase feeding rates at high temperatures, exposing them to higher levels of contaminants present in water (Folt et al. 1999). The interactions of nutrition, temperature and contaminants have been very rarely studied, and to our knowledge, only in the context of food quantity, not quality (Folt et al. 1999; Heugens et al. 2006). Plastic contamination is ubiquitous in all aquatic environments, but the occurrence of microplastics in freshwater systems and their effects on biota are less extensively studied compared to marine systems (Li et al. 2018; de Sa et al. 2018). The limited number of studies done so far have found that the abundance of microplastics in freshwater habitats reaches up to 187 particles L-1 (reviewed by Li et al. 2018). Lenz et al. (2016) highlighted that exposure concentrations in many laboratory studies have been several magnitudes higher (up to 1012 particles L-1) than those actually measured in the environment, which restricts the applicability of their results in the real world. For example, high levels of nanoplastics or secondary microplastics in Daphnia diet caused elevated mortality and decreased reproduction (Besseling et al. 2014; Ogonowski et al. 2016). Imhof et al (2017) found no effects of secondary microplastics on Daphnia survival when using a more realistic exposure concentration, although still higher than found in the field (1% of food particles or 290,000 particles L-1), but gene expression data indicated small increases in stress responses together with inconsistent 8 differences in morphological traits of offspring. Furthermore, Ogonowski et al. (2016) found irregularly-shaped secondary microplastics with broad size distribution to be more harmful to Daphnia than primary microplastics, suggesting that results from exposure studies using pristine microplastics (mainly polystyrene beads) might underestimate the impacts in the environment where secondary microplastics are more common. Moreover, microplastic particles in nature become rapidly coated with biofilm (Rummel et al. 2017), which might further affect their uptake or effects on organisms. Very few studies have so far investigated the effects of microplastics together with other environmental stressors (but see Ferreira et al. 2015; Aljaibachi and Callaghan 2018; Jaikumar et al. 2018). We conducted a laboratory study to investigate how Daphnia magna, a model freshwater zooplankton species, reacts to multiple stressors of eutrophication (increase in cyanobacteria), climate change (increase in temperature), and microplastic exposure. To our knowledge, the interactions of food quality, temperature, and contaminants have not been previously studied. Although the study was done in a laboratory setting, the aim was to keep the levels environmentally relevant to better predict outcomes in the field. We used a gradient in the proportion of cyanobacteria in diet from 5 to 95% to mimic the transition in food quality that takes place from oligotrophic to hyper-eutrophic systems. The chosen temperature increase was 3°C, corresponding to Representative Concentration Pathway 6.0 (RCP 6.0) projections of IPCC for northern Europe by the end of the century (IPCC 2014). Temperature in lake surface waters is increasing at 0.34 °C decade-1 (O’Reilly et al. 2015), potentially leading to 3°C increase in a 100-year time scale. Microplastic exposure was kept at 1% of total food concentration, which at 0.03 mg C L-1 (or 307,000 particles L-1) is higher than recorded for freshwater systems, nevertheless representing only a small fraction of the diet. To better mimic natural conditions, we 15 Supplemental Table S1. The statistical analysis was run with IBM SPSS 24 or Primer 6 and PERMANOVA+ add-on. 3. RESULTS 3.1 Survival Daphnia survival was on average 81 ± 15% (mean ± SD) when fed 5% cyanobacteria (both temperatures and microplastic exposures pooled). Survival of Daphnia decreased when cyanobacteria contributed 50% or more to their diet (Generalized linear model, p < 0.001, Figure 2A, Table 3) and was 24 ± 21% when fed 95% cyanobacteria. The main test indicated differences in Daphnia survival when fed 95% cyanobacteria with temperature (Kruskal- Wallis, χ2 = 10.81, p = 0.012, Figure 2B), but the pair-wise differences were not significant (Bonferroni-corrected Dunn test, p > 0.1). Daphnia survival was not affected by temperature or microplastics when fed 5% cyanobacteria (Kruskal-Wallis, χ2 = 3.80, p = 0.284). 3.2 Juvenile and adult size Daphnia juvenile size was highly influenced by the amount of cyanobacteria in diet (general linear model, p < 0.001, Table 4) but not by temperature (p = 0.173), with high proportions of cyanobacteria leading to smaller Daphnia (linear regression, R2 = 0.817, F = 169.417, p < 0.001, Figure 3A). Daphnia juvenile size decreased from 1.8 ± 0.2 mm to 1.0 ± 0.1 mm when proportion of cyanobacteria in diet increased from 5 to 95%, respectively. Microplastic exposure or temperature had no effect on Daphnia juvenile size when fed 5% or 95% cyanobacteria (Kruskal Wallis, χ2 = 1.217, p = 0.749, and H = 2.377, p = 0.498, respectively) (Figure 3B). Similarly Daphnia final length (measured after 14 days) was highly influenced 16 by the amount of cyanobacteria in the diet (p < 0.001, Table 4) but not by temperature, and there was no interaction between diet and temperature (Figure 4A). Daphnia adult size decreased from 2.7 ± 0.1 mm to 1.1 ± 0.1 mm when proportion of cyanobacteria in diet increased from 5 to 95%, respectively. Microplastic exposure or temperature had no effect on Daphnia final length when fed 5% or 95% cyanobacteria (χ2 = 1.150, p = 0.765, and χ2 = 4.303, p = 0.231, respectively) (Figure 4B). After being fed 5% cyanobacteria (with temperatures and microplastic exposures pooled) for 14 days Daphnia were on average 2.7 ± 0.1 mm long, and when fed 95% cyanobacteria only 1.1 ± 0.1 mm long. 3.3 Reproduction The first neonates were observed on day 10 of the experiment in treatments receiving 5% cyanobacteria, on day 12 in 25% cyanobacteria treatments, and on day 14 in 50% cyanobacteria treatments (except in one replicate on day 12), while Daphnia receiving 75% or 95% cyanobacteria did not produce offspring during the experiment. Reproductive output of Daphnia decreased when the proportion of cyanobacteria in their diet increased (Generalized linear model, p < 0.001, Table 3, Figure 5A), while temperature had no effect (p = 0.662). Microplastic exposure or temperature did not affect the reproductive output when fed 5% cyanobacteria (Kruskal-Wallis, χ2 = 4.260, p = 0.235, Figure 5B). Daphnia produced on average 13 ± 5 neonates per surviving adult when fed 5% cyanobacteria (both temperatures and MP exposures pooled), 7 ± 2 when fed 25% cyanobacteria, and 1 ± 1 when fed 50% cyanobacteria. 3.4 Fatty acid content and composition of Daphnia and phytoplankton The total fatty acid content (µg mg DW-1) was lower in Daphnia receiving high proportion of cyanobacteria in diet, but there were differences between the temperature treatments (general linear model, p = 0.002, Table 4) (Figure 6A). Total fatty acid content decreased linearly with 17 increasing cyanobacteria in Daphnia reared at 23°C (linear regression, F = 40.977, R2 = 0.872, p = 0.001), while Daphnia sustained a high fatty acid content when fed up to 50% cyanobacteria at 20°C, resulting in a quadratic relationship (2nd order polynomial regression, F = 10.588, R2 = 0.779, p = 0.011). The total fatty acid content when fed 50% cyanobacteria was almost two-fold higher in Daphnia reared at 20°C than at 23°C. Furthermore, the fatty acid percent composition of Daphnia was influenced by the amount of cyanobacteria in their diet (DistLM, F = 26.12, p < 0.001, R2 = 0.620). With increasing amount of cyanobacteria, Daphnia had more saturated fatty acids and 18:3ω6 (indicative of Microcystis), and less C16PUFAs and 18:3ω3 indicative of Nitzschia and Acutodesmus (Supplemental Table 1, Figure 7). The ω-3:ω-6 ratio in Daphnia was lower when reared in the higher temperature and decreased with increasing cyanobacteria in the diet (Figure 6B, Table 4). We found no differences in fatty acid composition or total content in Daphnia with or without microplastic exposure at 5% cyanobacteria. However, we needed to pool the exposed and non-exposed Daphnia that had been fed 95% cyanobacteria for fatty acids due to very low amounts of sample material. Total fatty acid content differed among the phytoplankton taxa (general linear model, F = 14.701, p < 0.001), but not between temperatures (F = 0.001, p = 0.981), and there were no interactions between the factors (F = 1.210, p = 0.321). Microcystis had a lower total fatty acid content (51 ± 12 µg mg DW-1) than Acutodesmus or Nitzschia (102 ± 24 and 86 ± 20 µg mg DW-1, respectively) (Bonferroni-corrected pair-wise comparisons, p < 0.01). Fatty acid percent composition differed greatly between the three phytoplankton taxa with taxonomic identity explaining 95% of the variation in the fatty acid data (PERMANOVA, F2,18 = 433.72, p < 0.001). There also were differences in algal fatty acid composition between the temperatures (F1,18 = 170.70, p = 0.006), and an interaction between taxa and temperature (F2,18 = 351.78, p = 18 0.001). The fatty acid composition of Microcystis differed between the temperatures (t = 6.173, p(MC) < 0.001) but there was no difference in Acutodesmus (t = 1.239, p(MC) = 0.233) or Nitzschia (t = 1.823, p(MC) = 0.117). Microcystis was rich in the saturated fatty acid (SAFA) 16:0, which made up 46.8 ± 0.5% of its fatty acids (Figure 7). Microcystis lacked long-chain PUFAs, except for a small quantity of 20:3ω6 (0.1 ± 0.1%), but had abundant ω-3 C18PUFAs and ω-6 C18PUFAs. The share of ω-6 C18PUFAs increased from 15.5 ± 0.7% to 24.3 ± 1.2% and ω-3 C18PUFAs decreased from 26.9 ± 1.4% to 17.4 ± 0.6% with the increased temperature. Acutodesmus also lacked long-chain PUFAs, but was very rich in 18:3ω3 (44.5 ± 1.2%) and 16:4ω3 (17.3 ± 1.0%). Nitzschia had a high proportion of the monounsaturated fatty acid (MUFA) 16:1ω7 (27.9 ± 6.6%) and C16 and C18 ω-4 PUFA (15.1 ± 4.2%). In contrast to Microcystis and Acutodesmus, Nitzschia lacked ω-3 C18PUFAs almost entirely, but was very rich in the long-chain PUFA eicosapentaenoic acid (EPA, 20:5ω3; 23.9 ± 5.9%). We found that the Nitzschia bottle used for feeding the Daphnia grown at 23°C on day 10 was contaminated with Microcystis cells. The fatty acid data shows slightly elevated levels of 18:3ω6 (1.8%) in that sample compared to other Nitzschia samples (≤ 0.5%), but fatty acids characteristic of Nitzschia still formed a majority of fatty acids in this sample, indicating only minor contamination. Nevertheless, the Daphnia in 23°C treatments received slightly higher proportions of cyanobacteria than the Daphnia in 20°C on this single feeding day. This bottle was previously used on day 4 with no signs of contamination, and was not used after the contamination was discovered. 4. DISCUSSION 19 We conducted a laboratory experiment to investigate the interacting effects of eutrophication, climate change, and microplastic exposure on the model freshwater cladoceran Daphnia magna. Daphnia were reared on a gradient of food quality (proportion of cyanobacteria) in two temperatures with and without microplastic exposure, and we found that food quality was the most important factor determining the fitness of Daphnia. Cyanobacteria generally contain neither long-chain PUFAs nor sterols (Galloway and Winder 2015; Taipale et al. 2016a), although we found very small amounts of 20:3ω6 in our Microcystis aeruginosa strain. The lack of sterols is the predominant reason for low somatic growth in Daphnia fed cyanobacteria, while the lack of PUFAs has a strong influence on reproduction and population growth (Martin-Creuzburg et al. 2008). The green algae Acutodesmus sp. also lacked EPA, in contrast to the diatom Nitzschia, which was rich in EPA. Both green algae and diatoms contain sterols (Taipale et al. 2016a; Peltomaa et al. 2017). Thus, in our experiment, Daphnia receiving increasing amounts of cyanobacteria had lower amounts of EPA and sterols available in their diet, which likely explains the drastic effects on survival, juvenile and adult size, reproduction, and fatty acid content. We found that the response of cyanobacteria on Daphnia reproduction was non-linear, while the size of Daphnia decreased linearly when exposed to increasing cyanobacterial abundances. Reproduction was high when cyanobacteria formed 5% of the diet and decreased rapidly after that. Thus, from the parameters we measured, reproduction exhibited the strongest response to increasing cyanobacterial abundances. In contrast, Martin-Creuzburg et al. (2005) found the responses of growth and reproduction of D. magna to be similar, with decrease in both when cyanobacteria formed ≥50% of the diet, while 20% of cyanobacteria did not cause and effect. Temperature is a crucial factor affecting the physiological processes of ectothermic organisms in the aquatic environment. D. magna can tolerate a wide range of temperatures 20 with highest growth rates observed in >25°C for many clones (Mitchell and Lampert 2000). Daphnia also experience seasonal variation in temperature, which may drive reproductive output (George et al. 1990), however, even small deviations (<2°C) in temperature during critical periods in the seasonal cycle may have drastic effects on Daphnia populations (Wagner & Benndorf 2007). In the present study, temperature did not affect size or reproduction of Daphnia, but it had an interactive effect with food quality on Daphnia fatty acid content. Previous research has highlighted that Daphnia fitness is strongly affected by sterols and PUFAs (especially EPA) in their diet (Müller-Navarra et al. 2000; Becker & Boersma 2005; Martin-Creuzburg et al. 2009; Peltomaa et al. 2017). EPA functions as an important component of cell membranes and is also linked to eicosanoid production in Daphnia (Farkas 1979; Schlotz et al. 2012). Maintaining membrane properties is vital when acclimating to temperature (Guschina and Harwood 2006), and Daphnia has been found to increase the unsaturation of membrane lipids in colder temperatures (Farkas 1979). This results in a higher dietary demand of PUFA in lower temperatures, and Daphnia growth is more strongly limited by EPA in 10 to 15°C than 20 to 25°C (Masclaux et al. 2009; Sperfeld and Wacker 2011, 2012; Martin-Creuzburg et al. 2012; von Elert and Fink 2018), which is the range where our experiment was conducted. However, at higher temperatures (25°C) cholesterol seems to be important for Daphnia growth and reproduction (Sperfeld and Wacker 2009; Martin-Creuzburg et al. 2012). In contrast to our hypothesis, we did not observe an increase in growth of Daphnia at the higher temperature, even when the food quality was high and microplastics absent. It is possible that the temperatures we chose were so close to each other, and also close to the optimum value for our Daphnia magna strain that it did not cause large differences. Cyanobacteria blooms often coincide with high temperatures, and it has been predicted that climate change will result in an increase in the prevalence and intensity of cyanobacterial 21 blooms (Paerl and Paul 2012; Huisman et al. 2018). Thus, consumers in future lakes may face decreased food quality with higher temperatures. A previous study found that the effect of temperature and poor food quality together on Daphnia pulex were greater than their effects separately (Przytulska et al. 2015). In the present study, Daphnia survival seemed to actually be higher with the higher temperature when the food quality was low, in contrast to our hypothesis, but this effect was not statistically significant. However, Daphnia reared at 23°C with 95% cyanobacteria were just as small as the Daphnia at 20°C, and did not produce any offspring, indicating that higher temperature would not prevent the population from collapsing under hyper-eutrophic conditions. We also found that the lipid accumulation patterns in Daphnia were driven by temperature when exposed to a gradient in food quality. When the proportion of cyanobacteria in the diet was low (5%), Daphnia total fatty acid content (a proxy for lipid content) was similar in both temperatures. With an increase in cyanobacteria, the total fatty acid content of Daphnia decreased linearly at 23°C, while at 20°C the total fatty acid content remained high until cyanobacteria formed >50% of the diet, decreasing after that. This resulted in almost two-fold higher total fatty acid content in Daphnia at 20°C compared to 23°C when fed 50% cyanobacteria. Higher temperature resulted in lower fatty acid content of cladocerans in other studies using a much wider temperature gradient 6 to 8°C (Przytulska et al. 2015; Masclaux et al. 2012). This may have important implications for the consumers in higher trophic levels as well. Our results indicate that eutrophication combined with temperature increase may result in a drastically lower Daphnia total fatty acid content, PUFA content, and ω-3:ω-6 ratio., which are also important in diets of fish (Sargent et al. 1999, Glencross 2009). Consistent with our results, low ω-3:ω- 6 ratio in Daphnia indicated poor nutritional status in a previous study (Taipale et al. 2015). The differences in phytoplankton fatty acid profiles are largely driven by phylogeny, but environmental conditions also have a minor effect (Galloway and Winder 2015). In our data 22 there are large differences among the algal species in their fatty acid composition, even though small temperature-related variation is also apparent in Microcystis. Despite these compositional differences, the total fatty acid content of the algae did not differ among the temperatures, and likely did not cause the lower fatty acid content in Daphnia reared at 23°C. Previous studies have found that ω-6 PUFA content in green algae and cyanobacteria increase in lower temperatures (Suschik et al. 2003; von Elert and Fink 2018). Von Elert and Fink (2018) found Daphnia growth to increase from 20°C to 25°C, with greater enhancement of growth when the dietary green alga was grown at 25°C, indicating improved food quality of algae grown at a higher temperature. We did not see differences in Acutodesmus fatty acids, but the ω-6 PUFA content increased and ω-3 PUFA content decreased in Microcystis with temperature, leading to lower ω-3:ω-6 ratios both in the algae and in Daphnia feeding on the algae. It is possible that the lack of temperature effects on Daphnia size and reproduction could be driven by these changes in algal fatty acids, however, it does not explain why survival of Daphnia seemed to increase with higher temperature, although the effect was not statistically significant. Previous studies have found that ω-6 fatty acids may have both positive and negative effects on Daphnia growth and reproduction (Becker & Boersma 2005; Martin-Creuzburg et al. 2012; Peltomaa et al. 2017). We investigated the effect of microplastics on Daphnia fitness with low (5%) and high (95%) proportion of cyanobacteria in diet under ambient and elevated temperature. In contrast to our hypothesis, we did not find any effects of the secondary microplastics on Daphnia survival, size, or reproduction, even though we used a rather high number of particles (307 000 particles L-1 or 0.03 mg C L-1) and stressed the Daphnia with low quality food. We were not able to determine the effects of microplastics on fatty acid content when cyanobacteria formed 95% of the diet because of low survival and size of Daphnia in those treatments, however, microplastic exposure did not affect total fatty acid content when fed 5% 23 cyanobacteria. The ingestion of various shapes and sizes of micro- and nanoplastics by Daphnia has been confirmed in multiple studies (e.g. Rosenkranz et al. 2009; Besseling et al. 2014; Ogonowski et a. 2016; Jemec et al. 2016; Imhof et al 2017; Rist et al. 2017). We chose to use a mix of secondary microplastics made from household items in our experiment, and thus could not rely on fluorescence for easy quantification of ingestion as other studies have done (e.g. Rosenkranz et al. 2009; Ogonowski et a. 2016; Rist et al. 2017). However, we see no reason why Daphnia would not ingest the plastic particles in our study, since they were of similar size to the plastic particles used in other studies. Similar to our results, Imhof et al (2017) found no effects on Daphnia survival when they were exposed to a diet consisting of 1% (290,000 particles L-1) secondary microplastics. Several studies have found negative effects of microplastics on Daphnia, but only at much higher concentrations/particle numbers than used in our study (Besseling et al. 2014; Ogonowski et al. 2016; Jemec et al. 2016; Martins and Guilhermino 2018). Rist et al. (2017) found no differences in Daphnia size or reproduction with an exposure of 1 mg L-1 micro- or nanoplastics, and even an exposure concentration of 100 mg L-1 did not affect Daphnia survival or reproduction in a study by Cannif and Hoang (2018). On the other hand, Sussarellu et al. (2016) found oyster reproduction to decrease when exposed to polystyrene beads at a similar concentration as used in our study (ca. 0.023 mg L-1 or 2,000,000 particles L-1 or 0.21% of algal biovolume), potentially reflecting differences in the sensitivity between species. Food quantity also affects the uptake of particles and Daphnia performance when exposed to microplastics (Jemec et al. 2016; Rist et al. 2017; Aljaibachi and Callaghan 2018), but we found no interactive effects between food quality and microplastics exposure in Daphnia. Furthermore, we found no interactions between temperature and microplastics on Daphnia, in contrast to an earlier study that found Daphnia sensitivity to primary and secondary microplastics to increase with temperature (Jaikumar et al. 2018). A recent paper highlighted 24 how the effects of microplastics on organisms are analogous to those caused by other refractory material, such as resuspended sediments (Ogonowski et al. 2018). Thus, the lack of effects is not surprising, since organisms facing other recalcitrant material might have the mechanisms to cope with microplastics, as long as other food – even of poor quality – is also available. Our findings from this laboratory experiment together with a previous study (Przytulska et al. 2015) imply that increasing proportions of cyanobacteria coupled with a temperature increase due to climate change, may result in fish encountering fewer and smaller Daphnia, which have lower fatty acid and PUFA content and produce fewer offspring. However, one should be cautious when predicting outcomes in the field based on small-scale laboratory experiments. Although we aimed at conducting the experiment in an environmentally realistic way, it is impossible to capture the complexity of natural environments in the laboratory. The responses of Daphnia to climate change will be caused by dynamic interactions of both abiotic (e.g. temperature, phenology, trophic state) and biotic factors (algal food, predation) (Wojtal-Frankiewicz 2012), and the purpose of the present study was to investigate few of these to isolate their potential effects in a laboratory experiment. Our results are supported by studies in lake Washington and Esthwaite Water, where environmental parameters (i.e. temperature, day length) together with seasonal and interannual differences food quantity and quality (e.g. proportion of edible algae or cyanobacteria) drives the dynamics in Daphnia production (George et al. 1990, Scheuerell et al. 2002). However, a mesocosm study investigating how eutrophication (nutrient enrichment) and temperature increase affect plankton community did not find any effects on Cladocera (Özen et al. 2013). In conclusion, we found that the eutrophication-driven decrease in food quality was more important in determining Daphnia fitness than a temperature increase or microplastic exposure. Consistent with other studies using similar exposure concentrations, microplastic 31 Ogonowski M, Schür C, Jarsén Å, Gorokhova,E. 2016. The effects of natural and anthropogenic microparticles on individual fitness in Daphnia magna. PloS one 11: e0155063, DOI:10.1371/journal.pone.0155063 O'Reilly CM, Sharma S, Gray DK, Hampton SE, Read JS, Rowley RJ, Schneider P, Lenters JD, McIntyre PB, Kraemer BM, Weyhenmeyer GA. 2015. Rapid and highly variable warming of lake surface waters around the globe. Geophys Res Lett 42: 10-773. Paerl HW, Paul VJ. 2012. Climate change: links to global expansion of harmful cyanobacteria. 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Aquat Ecol 46: 37-53. 34 Tables Table 1. The average size (mean ± SD) of phytoplankton cells, the mean diameter of microplastic particles, carbon content of phytoplankton and microplastics, and number of particles in microplastic suspensions Length/ Stock suspensionsa The feeding mix Daphnia exposure diameter Width C Number Concentration Number Concentration Number Concentration (µm) (µm) (% DW) (particles mL-1) (mg C mL-1) (particles mL-1) (mg C mL-1) (particles L-1) (mg C L-1) Phytoplankton Acutodesmus sp. 13.3 ± 1.8 6.3 ± 2.2 52 Nitzschia sp. 19.2 ± 2.4 5.4 ± 1.3 28 Microcystis aeruginosa 3.9 ± 1.0 46 Microplastics ABS 3.2 84 1 844 000 0.155 218 000 0.018 119 000 0.010 PS 3.7 90 4 525 000 0.054 134 000 0.018 73 000 0.010 PET 3.7 61 398 900 0.392 211 000 0.018 115 000 0.010 a The numbers of microplastic particles in stock suspensions was measured with a particle counter (rounded to nearest 1000), and carbon concentration was calculated based on literature values of density and measured values of carbon content (see methods for details). 35 Table 2. Daphnia diet treatments were run in 20°C and 23°C and consisted of 4 replicates with 10 Daphnia neonates in each. Daphnia were exposed to gradient of cyanobacteria from 5 to 95% of the diet simulating eutrophication in two temperatures (20 and 23°C). The treatments with 5 and 95% cyanobacteria were conducted with and without microplastic exposure (1% of the diet). Proportion of diet (%) Treatment Microcystis Nitzschia Acutodesmus MPs 5% cyano 5.0 47.5 47.5 - 25% cyano 25.0 37.5 37.5 - 50% cyano 50.0 25.0 25.0 - 75% cyano 75.0 12.5 12.5 - 95% cyano 95.0 2.5 2.5 - 5% cyano + MPs 5.0 47.0 47.0 1.0 95% cyano + MPs 95.0 2.0 2.0 1.0 36 Table 3. Results from generalized linear model testing for differences in Daphnia survival and reproduction in relation to proportion of cyanobacteria in diet and temperature. Daphnia were exposed to gradient of cyanobacteria from 5 to 95% of the diet simulating eutrophication in two temperatures (20 and 23°C). Model Wald χ2 df Survival Cyano in diet (%) 25.691** 1 Temp 1.373 1 Cyano in diet (%) * temp 2.949 1 Neonates per surviving adult Cyano in diet (%) 580.66** 1 Temp 0.191 1 Cyano in diet (%) * temp 2.927 1 * < 0.01 ** < 0.001 37 Table 4. Results from a general linear model testing for differences in Daphnia fitness parameters in relation to proportion of cyanobacteria in diet and temperature. Daphnia were exposed to gradient of cyanobacteria from 5 to 95% of the diet simulating eutrophication in two temperatures (20 and 23°C). Model SS df F Length at 6 daysa Cyano in diet (%) 0.125 4 62.783** Temp 0.001 1 1.945 Cyano in diet (%) * temp 0.000 4 0.189 Length at 14 days Cyano in diet (%) 13.645 4 124.603** Temp 0.018 1 0.647 Cyano in diet (%) * temp 0.051 4 0.466 Total FA content Cyano in diet (%) 3264.568 3 10.994* Temp 1423.997 1 14.386* Cyano in diet (%) * temp 3355.509 3 11.300* ω-3:ω-6 ratio Cyano in diet (%) 80.108 3 160.357** Temp 31.438 1 188.793** Cyano in diet (%) * temp 2.508 3 5.020* a length at 6 days was log (x+1) transformed prior analysis. * < 0.01 ** < 0.001 38 Figures Figure 1. A schematic diagram of the experimental design. Daphnia were exposed to gradient of cyanobacteria from 5 to 95% of the diet simulating eutrophication in two temperatures (20 and 23°C). The treatments with 5 and 95% cyanobacteria were conducted with and without microplastic exposure (1% of the diet). 39 Figure 2. Survival of Daphnia A) in relation to proportion of cyanobacteria in the diet and temperature, B) survival with and without microplastic (MP) exposure when fed 5% or 95% cyanobacteria. The box represents the 25th and the top 75th quartile, while the line is the median. The whiskers represent the maximum and minimum values. Daphnia were exposed to gradient of cyanobacteria from 5 to 95% of the diet simulating eutrophication in two temperatures (20 and 23°C). The treatments with 5 and 95% cyanobacteria were conducted with and without microplastic exposure (1% of the diet). 40 Figure 3. Length of six-day-old Daphnia at 20°C or 23°C A) with diets varying in proportion of cyanobacteria. Line represents linear regression with 95% confidence intervals (y = - 0.009x +1.701, R2=0.817). B) with and without microplastic (MP) exposure in 20°C and 23°C with diets of 5% and 95% cyanobacteria. Daphnia were exposed to gradient of cyanobacteria from 5 to 95% of the diet simulating eutrophication in two temperatures (20 and 23°C). The treatments with 5 and 95% cyanobacteria were conducted with and without microplastic exposure (1% of the diet).