It is a contribution of the Marine Zooplankton Ecology Group (2017 SGR 87), Proper thermal adaptation is key to understanding how species respond to temperature. However, this is seldom considered in protozooplankton and mixoplankton experiments. In this work, we studied how two heterotrophic dinoflagellates (Gyrodinium dominans and Oxyrrhis marina), one heterotrophic ciliate (Strombidium arenicola), and one mixotrophic dinoflagellate (Karlodinium armiger) responded to warming, comparing strains adapted at 16, 19 and 22 °C and those adapted at 16 °C and exposed for 3 days at 19 and 22 °C (acclimated treatments). Neither CNP contents nor the corresponding elemental ratios showed straightforward changes with temperature, except for a modest increase in P contents with temperature in some grazers. In general, the performance of both acclimated and adapted grazers increased from 16 to 19 °C and then dropped at 22 °C, with a few exceptions. Therefore, our organisms followed the “hotter is better” hypothesis from 16 to 19 °C; above 19 °C, however, the results were variable. Despite the disparity in the responses between species and physiological rates, in general, it seems that 19 °C-adapted organisms performed better than acclimated-only organisms. However, at 22 °C, most species were at the limit of their metabolisms and were unable to fully adapt. Nevertheless, adaptation to higher temperatures conferred some advantages prior to sudden increases in temperature (up to 25 °C) that simulated a heatwave episode. In summary, adaptation to temperature seems to confer a selective advantage to protistan grazers within a narrow range (i.e., ca. 3 °C). Adaptation to much higher temperatures (i.e., 6 °C) does not confer any clear physiological advantage (with few exceptions; e.g., the mixotroph K. armiger), at least within the time frame of our experiments, This research was funded by Grant CTM2017-84288-R by Fondo Europeo de Desarrollo Regional (FEDER)/ Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación (AEI), and by Grant PID2020-118645RB-I00 by Ministerio de Ciencia e innovación (MCIN)/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”. With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Para 4 especies: Volumen (µm3/depredador), Tasa crecimiento (µ 1/d), Tasa de ingestion (cells/ind/d), Eficiencia bruta de crecimiento (GGE, %), Peer reviewed

We aimed at studying the mechanisms underneath the ascending and descending sections of the thermal performance curves in marine protozoans. To do so, we exposed Gyrodinium dominans and two strains of Oxyrrhis marina from different origins to three temperatures representative of each section of the thermal response curve (12ºC, ascending section; 18ºC, top; 25ºC, descending section). As variables, we measured growth, ingestion, and respiration rates (this latter with and without food). The growth rates of O. marina strains plotted as a function of temperature showed a triangular response with maximum values at the intermediate temperature. However, G. dominans showed similar growth rates at 12 and 18ºC, and even if showed a marked decrease in growth rates at 25ºC, this was not significant. Ingestion rates were higher at 18ºC for all the strains. The respiration rates of G. dominans were unaffected by temperature, but the respiration rates of both O. marina strains increased with temperature. The specific dynamic action produced by feeding activity ranged from 2 to 20% of the daily carbon ingestion for all organisms investigated. The calculated energetic budget indicated that the responses to temperature were diverse, even within strains of the same species. G. dominans maintained similar growth at all temperatures by balancing anabolism and catabolism functions. In O. marina strains, on the other hand, the decrease in growth rates at the lowest temperature was driven mainly by reduced ingestion rates. However, increased respiration seemed the primary factor affecting the decrease in growth rates at the highest temperature. These results are discussed in the light of previous studies and on its suitability to understand the response of wild organisms to fluctuations in temperature, This research was funded by Grant PID2020-118645RB-I00 by Ministerio de Ciencia e innovación (MCIN)/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”. R.A.M. was funded by a PhD fellowship from the National Commission of Science (CONICYT), Ministry of Education, Chile. It is a contribution of the Marine Zooplankton Ecology Group (2017 SGR 87). With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Para 3 especies de protozoos marino: Tasa de respiración con y sin comida (µmol O2/ind/h), Tasa crecimiento (µ 1/d), Tasa de ingestion (cells/ind/d), Eficiencia bruta de crecimiento (GGE, %), Peer reviewed

The temperature-size rule hypothesized that there is a negative relationship between the size (volume) of an organism and the temperature. This applies to both unicellular and pluricellular organisms. Here, we question this hypothesis for the particular case of protozoans, because in these organisms the volume is directly related to the consumption of prey, and on most of the occasions the true volume of the cell is unknown. To prove our arguments, we designed a series of experiments with the heterotrophic dinoflagellate O. marina, including functional and numerical responses, time-dependent acclimation responses, and estimation of the protozoan volume during long periods of starvation. Our data showed that, in fact, the observed temperature-size rule in unicellular grazers results from anabolic and catabolic imbalances, and that the relationship between size and temperature weakens after proper thermal adaptation. We also showed that once prey are fully digested, the protozoan’ size is the same irrespectively of the temperature. Finally, we set the basis for proper acclimation during short-term temperature experiments, which specifies that at least 3 days should be allowed for proper temperature acclimation. We also suggest that, for trustable experiments, the grazer should be incubated at the target prey concentration for at least 24h before conducting the experiments. The ecological implications of a lack of correlation between microzooplankton size and temperature are also discussed, This research was funded by Grant PID2020-118645RB-I00 by Ministerio de Ciencia e innovación (MCIN)/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”. It is a contribution of the Marine Zooplankton Ecology Group (2017 SGR 87). With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Para Oxyrrhis marina: Tasas de ingestion a diferentes concentraciones de alimento (presa/ind/d), Tasa crecimiento (µ 1/d), Volumen (µm3); para la presa (Rhodomonas salina): volume (µm3), Peer reviewed

We exposed the calanoid copepod Paracartia grani, reared in the laboratory at 19°C, to warmer conditions (22°C and 25°C) for 11 generations and examined the direct effects of temperature on feeding, fecundity, development, population sex ratio and somatic traits (adult and egg sizes and adult carbon content), This research was funded by Grants [CTM2017-84288-R and PID2020-118645RB-I00] by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. CJ was supported by Grant [PRE2018-084738] funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Rearing temperature, generation, prosome length, carbon content, ingestion rate, egg production rate, egg diameter, gross-growth efficiency, hatching success, Peer reviewed

The temperature-size rule states that as the temperature increases, the body size of organisms decreases. This rule has been observed in a wide range of organisms, including marine phytoplankton and it is particularly important to model and predict the effects of global warming on the structure and functioning of marine ecosystems. This is so because the size of marine phytoplankton is a critical trait that determines their ecological and biogeochemical roles in marine ecosystems. The size of marine phytoplankton is also affected by resource availability, which relates to growth rates. Here, we compare the effects of a long-term exposure to several temperatures on the cell size of three marine microalgae during their growth curves. The species chosen were the cryptophyte Rhodomonas salina, the dinoflagellate Heterocapsa niei, and the diatom Conticribra (previously Thalassiosira) weissflogii. All algae conformed the temperature-size rule during all the phases of the growth curve. However, the size variations of the microalgae in each of the phases were species-specific. R. salina and H. niei showed higher volumes in the exponential growth phase than during the decline of growth and early stationary phases. Contrarily, the diatom showed smaller volumes during the exponential phase of growth than during the other phases. Overall, the effects of growth rate on cell size exceeded those of temperature for the expected ocean warming by the end of the century. These results also partially support the higher relevance of resource supply than temperature in explaining the variability of phytoplankton size structure in marine ecosystems, This research was funded by Grant PID2020-118645RB-I00 by Ministerio de Ciencia e innovación (MCIN)/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”. With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Para las tres microalgas, Rhodomonas salina, Heterocapsa niei, Conticribra weissflogii, abundancias, y volumen a diferentes temperaturas, Peer reviewed

We have studied the response of the marine calanoid copepod Paracartia grani to dietary elemental imbalances, This research was supported by Grant [PID2020-118645RB-I00] funded by MCIN/AEI/10.13039/501100011033, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Elemental content, C:N ratios, C:P ratios, N:P ratios, copepod ingestion rate, copepod egg production rate, hatching success, gross-growth efficiency of egg production, egg size, nauplius body size, threshold elemental ratios for C:N and C:P, Peer reviewed

The study of a species' thermal tolerance and vital rate responses provides useful metrics to characterize its vulnerability to ocean warming. Under prolonged thermal stress, plastic and adaptive processes can adjust the physiology of organisms. Yet it is uncertain whether the species can expand their upper thermal limits to cope with rapid and extreme changes in environmental temperature. In this study, we reared the marine copepod Paracartia grani at control (19°C) and warmer conditions (25°C) for >18 generations and assessed their survival and fecundity under short-term exposure to a range of temperatures (11-34°C). After multigenerational warming, the upper tolerance to acute exposure (24 hours) increased by 1-1.3°C, although this enhancement decreased to 0.3-0.8°C after longer thermal stress (7 days). Warm-reared copepods were smaller and produced significantly fewer offspring at the optimum temperature. No shift in the thermal breadth of the reproductive response was observed. Yet the fecundity rates of the warm-reared copepods in the upper thermal range were up to 21-fold higher than the control. Our results show that chronic warming improved tolerance to stress temperatures and fecundity of P. grani, therefore enhancing its chances to persist under extreme heat events, This research was funded by Grants CTM2017-84288-R and PID2020-118645RB-I00 by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. “ERDF A way of making Europe” C. J. was supported by Grant [PRE2018-084738] funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Rearing temperature, generation, exposure temperature, survival 24h, survival 7d, egg production rate, fitness index, egg diameter, female prosome length, Peer reviewed

Description of methods used for collection/generation/processing of data: Described in the Materials and Methods section from “García-Martínez, M., Saiz, E. & Calbet, A. (2025). Thermal performance of the heterotrophic dinoflagellate Oxyrrhis marina under long-term warming and acute heat stress. Journal of Plankton Research, 47(5), fbaf013. https://doi.org/10.1093/plankt/fbaf013”, This research was funded by Grants PID2020-118645RB-I00 and PID2023-150548NB-I00 by MICIU/AEI/10.13039/501100011033 and “ERDF/EU”. M. G.-M. was supported by Grant PRE2021-096866 funded by MICIU/AEI/10.13039/501100011033 and FSE+, This work contributes to the Institut de Ciències del Mar "Severo Ochoa Centre of Excellence" accreditation CEX2024-001494-S funded by AEI 10.13039/501100011033 of the Spanish Ministry of Science and Innovation, 1. Number of variables: “Thermal performance curves” sheet – 3; “Functional responses” sheet – 30; “Growth, ingestion and doubling” sheet – 5. // 2. Number of cases/rows: “Thermal performance curves” sheet – 21; “Functional responses” sheet – 14; “Growth, ingestion and doubling” sheet – 12. // 3. Variable List: “Thermal performance curves” sheet – Oxyrrhis marina line, Thermal performance curve temperature (ºC), Growth rate (µ; d-1), SE. “Functional responses” sheet – 16ºC line - 16ºC: Prey concentration (prey mL-1), Prey concentration (pg Cprey mL-1), Clearance rate (µL ind-1 d-1), Ingestion rate (prey ind-1 d-1), Ingestion rate (pg Cprey ind-1 d-1); 16ºC line - HS+3ºC: Prey concentration (prey mL-1), Prey concentration (pg Cprey mL-1), Clearance rate (µL ind-1 d-1), Ingestion rate (prey ind-1 d-1), Ingestion rate (pg Cprey ind-1 d-1); 19ºC line - 19ºC: Prey concentration (prey mL-1), Prey concentration (pg Cprey mL-1), Clearance rate (µL ind-1 d-1), Ingestion rate (prey ind-1 d-1), Ingestion rate (pg Cprey ind-1 d-1); 19ºC line - HS+3ºC: Prey concentration (prey mL-1), Prey concentration (pg Cprey mL-1), Clearance rate (µL ind-1 d-1), Ingestion rate (prey ind-1 d-1), Ingestion rate (pg Cprey ind-1 d-1); 22ºC line - 22ºC: Prey concentration (prey mL-1), Prey concentration (pg Cprey mL-1), Clearance rate (µL ind-1 d-1), Ingestion rate (prey ind-1 d-1), Ingestion rate (pg Cprey ind-1 d-1); 22ºC line - HS+3ºC: Prey concentration (prey mL-1), Prey concentration (pg Cprey mL-1), Clearance rate (µL ind-1 d-1), Ingestion rate (prey ind-1 d-1), Ingestion rate (pg Cprey ind-1 d-1). “Growth, ingestion and doubling” sheet – Oxyrrhis marina line, Temperature stress, Growth rate (µ; d-1), Ingestion rate (µg Cprey ind-1 d-1), Doubling efficiency (doublings day-1 ingestion rate-1), Peer reviewed

We studied the impact of sublethal thermal stress (28ºC, 7 days) on the physiological rates and metabolic balance of three populations of the calanoid copepod Paracartia grani, reared at 19, 22 and 25ºC for multiple generations, This research was funded by Grants CTM2017-84288-R, PID2020-118645RB-I00 and PID2023-150548NB-I00 by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” and ERDF/EU. C. J. was supported by Grant [PRE2018-084738] funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. The open-access publication fee was covered by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Peer reviewed