Concerns about increasing atmospheric CO 2 concentrations and global warming have initiated studies on the consequences of multiple-stressor interactions on marine organisms and ecosystems. We present a fully-crossed factorial mesocosm study and assess how warming and acidification affect the abundance, body size, and fatty acid composition of copepods as a measure of nutritional quality. The experimental set-up allowed us to determine whether the effects of warming and acidification act additively, synergistically, or antagonistically on the abundance, body size, and fatty acid content of copepods, a major group of lower level consumers in marine food webs. Copepodite (developmental stages 1–5) and nauplii abundance were antagonistically affected by warming and acidification. Higher temperature decreased copepodite and nauplii abundance, while acidification partially compensated for the temperature effect. The abundance of adult copepods was negatively affected by warming. The prosome length of copepods was significantly reduced by warming, and the interaction of warming and CO 2 antagonistically affected prosome length. Fatty acid composition was also significantly affected by warming. The content of saturated fatty acids increased, and the ratios of the polyunsaturated essential fatty acids docosahexaenoic- (DHA) and arachidonic acid (ARA) to total fatty acid content increased with higher temperatures. Additionally, here was a significant additive interaction effect of both parameters on arachidonic acid. Our results indicate that in a future ocean scenario, acidification might partially counteract some observed effects of increased temperature on zooplankton, while adding to others. These may be results of a fertilizing effect on phytoplankton as a copepod food source. In summary, copepod populations will be more strongly affected by warming rather than by acidifying oceans, but ocean acidification effects can modify some temperature impacts.

We conducted a mesocosm experiment in the Kiel Fjord, Western Baltic Sea, an area where plankton is already exposed to strong short-term and seasonal variations in pCO 2 (summer and autumn variation 375–2300 μatm; summer and autumn means: 700 μatm [ 41 ]). Therefore, we expected direct detrimental effects of CO 2 on copepods, as well as indirect food web effects; and interactive effects with rising temperatures could not be excluded a priori, and need to be tested. Populations from highly pH fluctuating ecosystems might be adapted and therefore less sensitive than other populations of the same species of different ecosystem locations [ 10 ]. Based on previous single stressor studies of warming [ 22 ] and acidification effects [ 42 ], we hypothesized that warming and acidification would mainly lead to antagonistic responses in copepod abundance, body size and fatty acid composition.

Calanoid copepods constitute approximately 80% of the global zooplankton and are the dominant trophic link between phytoplankton and fish [ 35 ]. It is known that warming decreases body size, survival and reproductive success [ 22 ], studies have shown the opposite for other organisms [ 11 ]. Additionally, warming leads to changes in fatty acid composition [ 35 ], but to-date it remains unclear how ocean acidification will affect fatty acid composition [ 11 ]. It is unknown whether the effects of warming and acidification mutually enhance or reciprocally dampen the responses of marine copepods. Short-term studies have shown no effects on eggs, larvae and adults at up to 2000 μatm pCO2 [ 36 – 40 ].

Metabolic energy, in form of lipids (hereafter called fatty acids (FA)) is one important metabolic trait that can adapt to changing environmental conditions (i.e. nutrient availability temperature and CO 2 concentration). FAs consist of hydrocarbon chains of different length and grade of saturation (identified by number of double bounds). Generally, FAs are classified into saturated (SFA, no double bonds), monounsaturated (MUFA, one double bond), and polyunsaturated fatty acids (PUFA, with two or more double bonds). Among these fatty acids, some PUFAs are essential and cannot be synthesised de novo and have to be taken up via diet. PUFAs are documented to be crucial for copepod egg production, hatching, growth and development [ 33 ]. Also higher trophic levels, fish and their larvae, depend on the fatty acid composition in their food for successful recruitment and reproduction (e.g. [ 34 ]). The fatty acid composition in marine micro-algae, the major food source of copepods, differs between taxonomic groups and therefore fatty acids can be used as trophic markes, e.g 22:6(n-3) (DHA; docosahexaenoic) and 20:5(n-3) (EPA; eicosapentaenoic acid) for flagellates and diatoms respectively.

An increasing number of studies suggest that the combined effects of temperature and pCO 2 prevail on diverse marine (e.g. [ 14 , 15 ]) and freshwater taxa (e.g. [ 26 ]) by affecting their survival, calcification, growth and abundance [ 14 , 15 , 27 , 28 ]. Interacting stressors can affect organisms in three ways: (1) through additive effects (sum of the individual effects), (2) synergistic effects (combined effects are greater than the sum of the individual effect), and (3) antagonistic effects (stressor offsets the effect of the other)[ 15 ]. Meta-analyses highlight that responses to more than one stressor are trait-, taxa-, life stage- and habitat specific, reflecting local adaptations. Studies that address effects of OA and warming on marine organisms, especially primary producers, show that these factors can lead to reductions in quality in terms of macromolecular composition [ 7 , 11 , 29 ] and consequently impact the nutritional value for higher trophic levels that depend upon these primary producers as a source of essential biomolecules to gain energy for growth, reproduction and development. [ 11 , 30 , 31 ]. OA and warming effects on marine organisms are found to alter energy budgets due to reduced performance curves under interacting stressors [ 32 ].

Anthropogenic activities have almost doubled the atmospheric carbon dioxide (CO 2 ) concentration, and have driven both global warming and ocean acidification (OA) due to the greenhouse effect. The uptake of CO 2 by the surface ocean has caused profound changes in marine carbonate chemistry: increased aqueous CO 2 , bicarbonate (HCO 3 - ), and hydrogen ion (H + ) concentrations, while the concentration of carbonate ions (CO 3 2- ) declined [ 1 ]. Contemporary surface ocean pH has declined by 0.1 units since pre-industrial time [ 2 ]. Simultaneously ocean sea surface temperature is predicted to increase up to 3–5°C by the year 2100 [ 3 ]. The consequences of ocean warming and OA for planktonic organisms remain unclear as only a few studies to-date have experimentally tested their combined effects on natural plankton communities [ 4 – 8 ]. Particularly the effects on plankton phenology (directly modifying food quantity) and physiology (modifying food quality) remain unresolved.

Temperature and pCO 2 levels were set as categorical explanatory variables, in separate, 2-way ANOVAs with abundance, mean developmental index, and fatty acid ratios as respective continuous response variables. Phytoplankton biomass was analysed by cross-correlation through time within mesocosms and followed by ANOVA with cross-correlation coefficients for temperature and pCO2 effects. 3-way ANOVAs were used to identify prosome length differences in response to temperature, pCO 2 and both stressors in respect to species (differences in adult prosome length) and developmental stages (differences in Paracalanus sp.). Tukey’s honest significant difference test was used as the post hoc test for all ANOVAs. To test homogeneity of variance, Fligner-Killeen tests were applied in all cases. All statistical tests were conducted at a significance threshold of α = 0.05. All statistical analyses were conducted in R, Version 0.97.551, R Inc. using the packages stats, multcomp, and car.

We tested for the combined effects of temperature and pCO 2 on the: (1) abundance of copepodits (stage C1-C5), adults, and nauplii of all occurring taxa, (2) prosome length of adult copepods, (3) stage-specific prosome length of Paracalanus sp., (4) mean developmental index, and (5) fatty acid ratios. Normal distribution of all response variables was tested by using Kolmogorov-Smirnov analyses for temperature and pCO 2 effects on all response variables (α = 0.05), data were normally distributed and no transformation was needed.

The observed combined impact of both stressors was compared with their expected net additive effect [(stressor1 warm/low – control cold/low )+(stressor2 cold/high – control cold/low )], which was based on the sum of their individual effects. If the observed combined response of both stressors exceeded their expected additive response then interaction was identified as being synergistic. In contrast, if the observed response was less than the additive response, the interaction was denoted as antagonism [ 26 ]. For illustration of interaction response types, we show graphically the difference between observed [(stressor warm/high – control cold/low )] and predicted additive effects, which indicates the direction and magnitude of the interaction.

To analyze copepod total fatty acid content and fatty acid composition, 30 adult Paracalanus sp. individuals were pooled in tin cups and extracted in chloroform / dichlormethane / methanol (1:1:1 v/v/v) following Arndt and Sommer [ 53 ]. Prior to extraction two internal standards, heneicosanoic acid (C21) and FAME mix (C19) were added. Methyl esters were prepared by esterification with toluene and H 2 SO 4 (1%) in methanol heated up to 50°C for 12 hours. After extraction with n-hexane, the fatty acid methyl esters were analyzed with a gas chromatograph (Thermo Scientific Trace GC Ultra with autosampler AS 3000), comparing peaks against the standards FAME Mix C4-C24 SUPELCO, Sigma-Aldrich, Germany.

Body size of taxonomically identified copepods was measured digitally via photographs and digital software (ZEISS AxioVision 4.8 and AxioCam MRc) with a precision to the nearest μm [ 22 ]. Means were calculated stage-specifically for copepods of each genus found in each mesocosm (see S1 Table ). The mean developmental index was calculated following Ismar et al. [ 52 ]: Where N = number of copepods at certain stage, S = assigned stage value, N tot = total number of copepods staged. Stages were scored as: C1 = 1, C2 = 2, C3 = 3, C4 = 4, C5 = 5, adult = 6.

Water temperature, salinity and pH were measured daily ( S1 Fig ). Phytoplankton was sampled three times per week (Monday, Wednesday, and Friday), and fixed with Lugol’s iodine, and subsequently identified to species level. To estimate phytoplankton biomass, density and cell size were measured and converted to carbon biomass following Hillebrand et al. [ 51 ]. Zooplankton was sampled weekly by three vertical net hauls, with a hand-held plankton net (64 μm mesh size, 12 cm diameter, and from 150 cm depth) and fixed with Lugol’s iodine. Each net haul sampled a volume of approximately 5.1 L. The total zooplankton catch was divided in a sample splitter (HydroBios, Germany), so that ¼ of the total catch volume was counted and identified, and copepod developmental stages and sexes could be distinguished accurately [ 22 ]. The body length constancy between moults enables a clear assignment of size to a given stage. All copepods were identified to genus level by using a ZEISS Discovery V.8 microscope with the magnification between 25x and 40x [ 22 ].

Total dissolved inorganic carbon (DIC) samples (10 mL) were taken on each sampling day with glass vials (Resteck, Germany) filled using a peristaltic pump (flow rate 6 mL min -1 ) and an intake tube containing a single syringe filter (0.2 μm, Sartorius). Filtered samples were fixed with saturated HgCl 2 solution (20 μL), crimped with a headspace of less than 1% and stored in the dark at 4°C. DIC was measured following Hansen et al. [ 46 ] using a SRI-8610C (Torrence, USA) gas chromatograph. For total alkalinity (TA), 25 mL samples were filtered (Whatman GF/F filter 0.2 μm) and titrated at 20°C with 0.05M HCl-solution in an automated titration device (Metrohm Swiss mode). The remaining carbonate parameter pCO 2 was calculated using CO2SYS [ 47 ] and the constants supplied by Hansson [ 48 ] and Mehrbach et al. [ 49 ], that were refitted by Dickson and Millero [ 50 ].

Over the course of the experiment, light supply and day length were adjusted according to the seasonal patterns expected at this latitude and season. Light was supplied by computer controlled light units (GHL Groß Hard- und Softwarelösungen, Lampunit HL3700 and ProfiluxII). Above each of the mesocosms, one unit consisted of 5 HIBay-LED spotlights (purpose-built item of Econlux, each 100 W) was installed. Daily irradiance patterns were set to follow the pattern for a cloudless 21st September at Kiel (according to Brock [ 45 ]) and reduced to 50% to account for moderate under water light attenuation. The light-dark cycle was 11h50 min: 12h10 min. The daily maximum light intensity in the middle of the water column was 252 μmol m -2 s -1 PAR.

CO 2 manipulation was monitored during the experimental period using a flow of 30–60 L h -1 CO 2 -enriched air (low: 560 μatm; high: 1400 μatm CO 2 ) through the headspace of the mesocosms. To balance the natural draw-down of CO 2 by phytoplankton production that occurred during the course of the experiment, CO 2 enriched water was added to the high CO 2 mesocosms three times (October 29th, November 2nd and 9th) [ 44 ]. For this purpose, water taken from the mesocosms was filtered (0.2 μm pore size), CO 2 -saturated by bubbling, and again transferred into the mesocosms. The required volumes were calculated on the basis of dissolved inorganic carbon (DIC) and total alkalinity ( Fig 1 and S1 Fig ).

A 24-day mesocosm experiment was conducted October 19th to November 12th 2012. Unfiltered seawater from Kiel Fjord (54° 20′ N, 10° 8′ E) was used to fill twelve indoor mesocosms, each with a volume of 1400 L. Mesocosms contained the natural early autumn plankton community composition of algae, bacteria and protozoa. To minimize between-mesocosm differences in the initial community compositions and densities of phytoplankton, seawater was pumped from approximately 2 m depth into a mixing chamber by a rotary pump. From this mixing chamber the water was simultaneously filled into the mesocosms. Mesozooplankton from net catches (Kiel Bight) were added at target concentrations of 20 individuals L -1 , to mimic natural densities of copepods during this season. The plankton was gently stirred by a propeller to homogeneously mix the water column without incurring mesozooplankton mortality. A full-factorial replicated experiment (n = 3 per treatment) was used with two temperature regimes (9°C and 15°C, ∆3°C of ambient temperature) and two pCO 2 levels (560 μatm, hereafter called “low”, and 1400 μatm pCO 2 , hereafter called “high”). The target levels mimic the extent of warming and acidification predicted for this season (October 19th to November 12th 2012) and region of the IPCC prediction (Scenario IS92a, atmospheric CO 2 : 788μatm) for the year 2100, when the surface seawater CO2 in the Baltic Sea is predicted to reach 1400μatm and higher [ 41 , 43 ]. The resulting set-up of twelve mesocosms was installed in four temperature-controlled culture rooms. Target temperatures and levels of pCO 2 were reached in all treatments three days after filling (19 October 2012), which will henceforth be called day -3 ( Fig 1 and S1 Fig ). Temperature deviation in a mesocosm between day 0 and day 21 was a maximum of ± 0.3°C. Maximal temperature deviation between mesocosm in the same temperature treatment was 0.3°C (for the warm) and 0.4°C (for the cold treatment).

The ratio of EPA/TFA (eicosapentaenoic acid (20:5(n-3)) was neither affected by temperature, pCO 2 or their interaction (Tables 6 and 7 , Fig 6E ). ARA/TFA ratios (arachidonic acid (20:4(n-4))) were significantly affected by the interaction of temperature and OA, and by both single factors, respectively (Tables 6 and 7 ). The lowest ARA/TFA ratio was found under high temperature and low pCO 2 conditions, and the highest ratio under high temperature and high pCO 2 ( Fig 6F ); warming and OA additively affected ARA/TFA ratio ( Fig 3 ), and their interaction significantly increased the ratio by 167.57% ( S2 Table ).

The ratio of polyunsaturated-to-TFA content (PUFA/TFA) was neither significantly affected by temperature, pCO 2 nor their interaction (Tables 6 and 7 ). But even without significant effects of all treatments, lower PUFA/TFA ratios were measured at higher temperature treatments compared to ambient temperature treatments ( Fig 6C ). Overall, warming decreased PUFA/TFA ratios by 24.89% ( S2 Table ). The responses in polyunsaturated essential fatty acids, which cannot be synthesised de novo by heterotrophic organisms like copepods, were analysed for temperature, and OA effects, and their interactions. The DHA/TFA ratio (docosahexaenoic acid (22:6(n-3)) was significantly affected by temperature, but neither by OA, or the interaction of both (Tables 6 and 7 ). The lowest proportion of DHA/TFA was found in the high temperature and low pCO 2 treatment ( Fig 6D ) and warming decreased the DHA/TFA by 35.73% ( S2 Table ).

The total amount of fatty acids per adult individual of Paracalanus sp. was not significantly influenced by temperature, OA or their interaction (Tables 6 and 7 , Fig 6A ). The ratio of saturated fatty acids-to-total fatty acids (SFA/TFA) was significantly affected by the interaction of temperature and OA, with the lowest amount of SFA/TFA under low temperature/low pCO 2 and the highest amount at higher temperature/high pCO 2 ( Fig 6B ), resulting in an antagonistic interaction effect ( Fig 3 ). SFA/TFA ratio increased by 45.25% under OA and warming compared to low temperature/low pCO 2 , but the differences between both OA treatments at higher temperatures were not significant ( Table 7 and Fig 6B , S2 Table ). Warming increased SFA/TFA by 47.90% and OA by 17.90% compared to low temperature and pCO 2 treatments, respectively, and therefore combined factors of OA and warming affected SFA/TFA antagonistically ( Fig 3 , S2 Table ); the temperature effect dominated the response of SFA/TFA ratio more strongly than the effect of high pCO 2 .

Paracalanus sp. was abundant enough for an analysis across most developmental stages. Prosome size changes with temperature and OA were specifically analysed for stage-specific changes ( Fig 5B ). Warming only significantly affected mean stage-specific prosome lengths; neither OA nor the interaction of both factors affected the mean prosome lengths significantly (Tables 4 and 5 , Fig 5B ). Overall, warming decreased the mean prosome lengths in all developmental stages compared to low temperature (C1: -1.33%; C2: -26.86%; C3: -14.53%; C4: -8.94%; C5: +0.03%; adults: -18.47%)( S2 Table ). Even if OA did not affect significantly mean prosome lengths of Paracalanus stages; prosome lengths increased with higher pCO 2 compared to the low pCO 2 treatments (see S2 Table ). On average, adult Paracalanus sp. were 21.29 μm larger at lower temperature compared to high temperature, and 9.82 μm smaller at low pCO 2 compared to high pCO 2 treatments.

The mean prosome length of adult copepods of all occurring species was significantly affected by the interaction of temperature and pCO 2 , as well as temperature and pCO 2 alone (Tables 4 and 5 , Fig 5A ). The interaction of OA and warming antagonistically affected adult prosome length of all species ( Fig 3 ), with the smallest prosome lengths at high temperature/low pCO 2 compared to low temperature/high pCO 2 conditions ( Fig 5A ). Overall high temperature decreased prosome lengths of adults and high pCO 2 increased mean prosome lengths, irrespective of the copepod species (Tables 4 and 5 , Fig 5A ), resulting in an antagonistic interaction of warming and OA on adult copepod prosome length ( Fig 3 ).

The cross-correlation coefficients for warming and acidification impacts on edible phytoplankton biomass as a copepod food source were negative at the beginning of the experiment, and positive during the mid-phase and the end of the experiment ( S3 Table and S2 Fig ). The ANOVA analysis of all correlation coefficients over the experimental time in all tanks indicated that food biomass was significantly affected by pCO 2 ( S4A and S4B Table ). Higher food biomass was found under high pCO 2 and was neither affected by temperature nor by the interaction of both stressors. Consequently, high pCO 2 had a significantly positively affected copepod biomass ( S4A and S4B Table ).

The copepod community included the genera Paracalanus, Pseudocalanus, Oithona, Acartia, Temora, and Calanus. Oithona sp. dominated in cold treatments, while Paracalanus sp. in warm treatments ( Fig 4 ). The mean developmental index (MDI) was significantly affected by temperature with the highest MDI in high temperature/high pCO2 treatments and the lowest under low temperature/high pCO2 conditions (Tables 1 and 2 ). At higher temperatures, the MDI indicated a dominance of the copepodite stages C2 and C3, whereas copepods at lower temperature had on average to between copepodite stages C1 and C2, indicating that copepodites developed faster in warmer treatments ( Table 3 ). The differences in MDI indicated a phenological shift between temperature treatments. The MDI showed neither a response to pCO2 nor to the interaction of both stressors (Tables 1 and 2 ).

Temperature and pCO 2 interacted significantly and affected nauplii abundance antagonistically with the lowest abundance under high temperature/high pCO 2 ( Table 1 , Figs 2 and 3 ). OA and warming decreased nauplii abundance by 30.90% compared to the low temperature/low pCO 2 treatment ( S2 Table ), although the nauplii abundance was lowest under OA and warming ( Fig 2 ). Warming decreased the abundance significantly by 27.96% ( Table 1 and S2 Table ). Overall, nauplii abundance responses to OA differed significantly between the two temperature treatments, and OA thus had no significant first-order effect on the abundance of nauplii (Tables 1 and 2 ).

Discussion

Our results indicate that in a future ocean scenario, temperature effects will dominate zooplankton responses, with acidification partially counteracting some observed effects of increased temperature, whilst adding to others. The abundance of adult copepods in our study was negatively affected by warming, while OA impacts could modify temperature effects on the abundance of younger life stages. Warming significantly reduced the prosome length of copepods and the interaction of warming CO 2 antagonistically affected prosome length, but not significantly. Fatty acid composition was also significantly affected by warming. The content of saturated fatty acids increased, and the ratios of the polyunsaturated essential fatty acids docosahexaenoic- (DHA), and arachidonic acid (ARA) to total fatty acid content increased with higher temperatures. Additionally, arachidonic acid content significantly increased with higher pCO 2 , and there was a significant additive interaction effect of both parameters. In summary, copepod populations will likely be affected more strongly by warming than by acidifying oceans, but ocean acidification effects can modify temperature impacts on zooplankton. Our results indicate that body size will be impacted by OA and temperature antagonistically, while showing additive OA interactions with rising temperatures on aspects zooplankton nutritional composition. Each of these responses is discussed in more detail below.

Abundance The observed interactive effect of warming and OA on nauplii abundance suggests that the reproductive success of copepods during the experiment was sensitive to higher temperatures and higher pCO 2 ; nauplii were more abundant in treatments that were exposed to only higher temperature or only higher pCO 2 . Studies observed that nauplii stages seem to be more vulnerable to environmental changes than others (i.e. CO 2 concentration [54,55], invertebrates [27,56], and other marine organisms [27,28]. Several factors can lead to the lower number of nauplii under warming and OA: 1) females might have produced a lower number of eggs [40,55], 2) hatching success may have been reduced due to a higher number of unfertilized eggs, non-viable fecund eggs or viable fecund eggs in a quiescent state [57], or 3) higher mortality rates [58]. Copepodite stages (C1-C5) were also significantly affected by the interaction of warming and OA, but the antagonistic effect was lower than within the nauplii. Especially OA positively affected copepodites, whereas warming decreased the copepodite abundance. Younger developmental stages seem to be more vulnerable to OA than older ones, especially adults seem to be highest tolerant to OA effects as our study has shown [59,60]. Temperature seemed to be the more important driver within older developmental stages of marine copepods. Higher temperatures alone affected the stage composition and shifted the MDI from a copepodite stage mixture of 1 and 2 to dominance in copepodite stage mixture of 2 and 3 in warmer treatments; no effect of CO 2 was detected. This can be explained by faster maturation of copepods at higher temperatures. At higher temperatures, copepods developed faster to the reproductive stage and can reproduce earlier [23,61]. Higher daily mortality and lower stage-specific survival rate [22] at higher temperatures may have resulted in the lower abundance of copepods observed in our samples. A positive effect of OA on Baltic Sea copepods was experimentally identified by Rossoll et al. [42], they concluded that this is most likely the response to a higher food biomass. Thus the findings of Rossoll et al. [42] provide an experimental explanation for our results of higher copepod abundances at high pCO 2 treatments, where phytoplankton biomass was also higher [6]. During this mesocosm experiment temperature mostly negative affected phytoplankton biomass during the bloom, Paul et al. [7] observed a 50% decline in bloom phytoplankton biomass and an increase of carbon-to-phosphorus ratio by 5–8%. The stoichiometry (C:P) of food resources are important for copepod developmental rates and less balanced nutrient ratios can lead to slower development, growth or increased respiration rates [62,63]. Increased energetic costs to persist environmental changes lead to altered metabolic allocation to accommodate increased energetic expenses. Imbalances in metabolism can decrease lifetime fitness du to re-allocation of energy [32,64,65]. Yet, food limitation can be excluded as a sole direct causal factor for the observed changes in mesozooplankton abundance. Edible phytoplankton biomass was not significantly correlated with copepod biomass. The significant temperature-acidification interaction term suggested that acidification partially reversed the negative influence of warming on abundance. These antagonistic effects on zooplankton abundance were also observed in a limnic multi-stressor study of Christensen et al. [26]. Calanoid copepods like Acartia sp. might be able to compensate higher metabolic costs at higher temperature by increased consumption rates and higher food availability in acidification treatments. Additionally, copepods originating from the Kiel Fjord seem to be acid-tolerant due to pre-adaptation in the high pCO 2 fluctuation environment [43]. Christensen et al. [26] argued that the limnic species Daphnia catawba, an acid-tolerant herbivore, benefits more from acidification than acid-sensitive competitors due to the positive effects of warming on feeding rates and growth.

Body size This study shows that warming and OA interactively affected adult copepod body size; warming decreased and higher pCO 2 concentration increased adult body sizes. These antagonistic results suggest that the smaller adult body size at higher temperatures can be partially compensated by the positive effect of OA. Indirect positive CO 2 effects on copepod size via the effect on food availability are indicated from our cross-correlation results. Phytoplankton biomass data, used as a proxy for available food biomass in our experiment, demonstrated that acidification acted as a fertilizer for phytoplankton but that higher temperature treatments had lower phytoplankton biomass compared to the low temperature/low pCO 2 , which was also experimentally shown in other studies [11,66]. The results of a negative effect of higher temperature are in line with results of a meta-analysis of Daufresne et al. [20] and an experimental study of Garzke et al. [22]. The Temperature-Size Rule (TSR) [61] describes the growth response of ectothermic organisms to temperature by which individual organisms grow faster at higher temperatures, but attain smaller sizes at maturity than at lower temperatures. Size at a defined stage is a product of growth rate (increase in biomass per time) and developmental rate (increase in life stage per time, and the TSR signals that both rates are decoupled [67]. Forster et al. [68] showed that the developmental rate in marine pelagic copepods has a greater temperature dependence across all life stages than growth rates. We could show that the relative size change increased with higher life history stage resulting in greater difference of adult sizes between the climate change scenarios (warming, OA, and warming x OA) compared to the low temperature/low pCO 2 . Body size changes with OA in copepods were not investigated directly within on species between the different developmental stages. Only a few studies have investigated the effects of OA or the interaction of warming and OA on body mass. Hildebrand et al. [69] experimentally showed that body mass (here body carbon) decreased with increasing pCO 2 as well as a decrease of dry weight of the arctic copepod species Calanus hyperboreus. Hildebrand et al. [69] argued that the decrease in body carbon resulted due higher energetic costs under OA for the acid-base regulation. Our results suggest that ocean acidification may have weaker effects on copepod body size compared to ocean warming. Havenhand [70] suggested that the ecologically most important groups of the Baltic Sea food web (phytoplankton, zooplankton, macrozoobenthos, cod, and sprat) seem to be more or less well adapted to future acidification but more vulnerable to higher temperatures