Warming alters the metabolic rates and life-history parameters of Ceriodaphnia silvestrii (Cladocera)

: Temperature rise has effects on the metabolic process of organisms, population structure, and ecosystem functioning. Here, we tested the effects of warming on the metabolic rates and life-history parameters of the widespread cladoceran Ceriodaphnia silvestrii . Two scenarios of global warming were established, an increase of 2 °C and an increase of 4 °C; the control temperature was 22°C. Our results showed that warming altered C. silvestrii metabolic rates, by increasing the rates of assimilation and secondary production, and decreasing the rates of fi ltration and ingestion. Warming also increased C. silvestrii fecundity and the body size of neonates and juveniles, and decreased the embryonic and post-embryonic time of development. C. silvestrii might be an important food resource at intermediary temperature as it had higher assimilation rates, even fi ltering fewer algae. At the highest temperature, we observed a substantial decrease in assimilation and secondary production, which could be a sign of stress starting. The increase in temperature by global warming will affect the cladocerans’ metabolic processes and the population survival, even a small increase (2°C) might induce drastic fl uctuations in such processes and affect the


INTRODUCTION
The expected temperature increase in the forecasts for future climate scenarios (IPCC 2014) has concerned the scientifi c community in recent years, due to the strong effects of temperature on organisms, populations, and ecosystems (Yurista 1999, Jeppesen et al. 2010, Shurin et al. 2012, Šorf et al. 2015).Warming may lead to gradual losses of populations, reducing their growth and survival (Brown et al. 2004), by exceeding their tolerance limits (Loreau et al. 2001, Alcaraz et al. 2014).Or even, change species composition in natural environments, by favoring species with high thermal preferences and/or phenotypic plasticity (De-Meester et al. 2018).
In an organism level, temperature fluctuations affect the energy budget, which alters metabolic rates such as assimilation (Lampert 1977), fi ltration (Burns 1969, Geller 1975), respiration (Kobayashi 1974), and excretion rates (Yurista 1999(Yurista , 2004)).Such alterations affect the balance of energy distribution inside organisms, and consequently, alter their rates of growth and reproduction (secondary production, body size, fecundity, and development time).All these changes negatively affect the ecological interactions in which such organisms are involved and may also affect the population's persistence in that thermal environment (Yurista 1999, Kooijman 2000).The metabolic rates mentioned affect the flow of energy and matter in aquatic ecosystems (Cabral & Marques 1999, Traill et al. 2010), as these processes are linked to energy contend of organisms (Gama-Flores et al. 2015), and energy transfer between trophic levels.Therefore, analyze the changes in life-history and the rates of secondary production, respiration, excretion, assimilation, and ingestion may help to better understand the response of populations to warming and evaluate the implications for ecosystem functioning (Sosnová & Klimešová 2009, Hébert et al. 2017).
In this context, subtropical freshwater environments are characterized by high biodiversity providing several ecosystem services, but it is predicted that these environments will be highly influenced by global warming due to the limited adaptations generated by the minor variations of the climate in the evolutionary time (Pörtner & Knust 2007).In these systems, among all communities, ectotherms such as zooplankton are more likely to be affected by this unpredictable warming, as these animals are not able to regulate their body temperature actively (Verberk et al. 2016).Each zooplanktonic species has its thermal history (thermal preference, phenotypic plasticity, and genetic responses) and will respond differently to changes in temperature (Loreau et al. 2001, De-Meester et al. 2018).For some species, a small increase of 2 °C leads to significant declines in growth, reproduction, and ingestion rates or can lead to the populations' extinctions due to high metabolic demands, as observed for Daphnia magna Straus, 1820 (Kooijman et al. 1989, Kooijman 2000).Such shifts alter the ecosystem functioning, as the changes in cladocerans' biomass alter also the structure of their prey and predators (O'Connor et al. 2009, Abo-Taleb 2019), and modify the nutrient's availability (Saba et al. 2009).
Subtropical freshwater environments have the predominance of small-bodied zooplankton, among them the cladocerans of the genus Ceriodaphnia, which present high abundances in these environments (Choueri et al. 2007, Lansac-Tôha et al. 2009, Brito et al. 2013, 2016).The filtration, assimilation, and excretion rates of these cladocerans directly affect the cycles of carbon and organic matter in subtropical aquatic environments (Hébert et al. 2017).Thus, studies on energy budget components, taking into account forecasted climate change (IPCC 2014), can highlight how warming affects the flux of energy and matter in freshwater ecosystems.Also, the use of native zooplankton species might show a more realistic response of the natural environments -and as far as we know, this is the first study investigating the metabolic rates of the cladoceran C. silvestrii regarding global warming scenarios.
Here, we analyze the effects of warming on the metabolic rates and life-history parameters of the cladoceran Ceriodaphnia silvestrii Daday, 1902.For that, we tested two scenarios, an increase of 2 °C and an increase of 4 °C in temperature.These scenarios represent, respectively, the optimistic and pessimistic forecasts of future global temperatures, according to the Intergovernmental Panel on Climate Change (IPCC 2014).As warming increases metabolic demands, we predict that with the increase of 2°C in the temperature C. silvestrii would present I) higher consumption rates (filtration and ingestion), higher respiration rates, higher assimilation rates, higher investment in secondary production (faster population growth through fecundity and time of development), and higher metabolic losses (excretion); and the increase of 4°C in the temperature would result in II) lower rates of filtration, ingestion, secondary production, and assimilation, but we expect the highest metabolic losses (excretion) and respiration rates due to temperature stress.

Alga and cladoceran stock cultures
Cells of Raphidocelis subcapitata (Korshikov) F. Hindák 1990 were obtained from cultures maintained at the Plankton Laboratory of the Federal University of São Carlos (SP, Brazil), where all the experiments were conducted.The Chlorophyceae R. subcapitata was weekly cultivated in Erlenmeyer flasks of 2 L filled with 1 L of CHU-12 as medium (Müller 1972).The algae were initially inoculated at 1 x 10 5 cells mL -1 and maintained at 25 ± 2 °C, under a 12:12 h (light/ dark) photoperiod until reaching the stage of exponential growth.After that, the algal cultures were centrifuged to remove the CHU-12 medium (which can eventually become toxic to the zooplankton) and were subsequently stored at 4°C for up to one week, and this procedure was repeated until the end of the experiments.
Populations of C. silvestrii were sampled from different regions of two subtropical shallow lakes with similar characteristics (area of approximately 800 m 2 and 2.0 m of depth each one), localized around the city of São Carlos/São Paulo/Brazil.The individuals of C. silvestrii were acclimated and cultured for many generations (parthenogenetic reproduction), during approximately three months, in incubator chambers under controlled conditions of temperature (22°C = control, +2°C, and +4 °C) and photoperiod (12:12 h light: dark cycle).C. silvestrii were kept in 2 L beakers filled with 1.5 L of reconstituted water as the culture medium, with 50 adults per liter.This culture medium (reconstituted water) was prepared in the laboratory in agreement with standards described by the ABNT (2017), which include hardness from 40-48 mg CaCO 3 L -1 , pH from 7.0-7.6,and conductivity around 160 μS cm -1 .The culture medium was completely renewed three times a week, with new food added every time, which consisted of 1 x 10 5 cells mL -1 of R. subcapitata and a food supplement made from fermented fish food and yeasts (ABNT 2017).

Metabolic rates and secondary production experiment
To evaluate the effects of warming on metabolic rates and secondary production of C. silvestrii we first set up three temperatures (22°C = control, +2, and +4°C), including three replicates per temperature.In this first stage, 20 synchronized ovate females were put in each replicate (beaker) (based on previous experiments).To calculate biomass, we counted the number of eggs of each female and measured their body size (before the experiment started), corresponding to the distance between the superior extremity of the head and the end of the carapace (Hardy 1989).The experimental medium was completely renewed twice a week, by filtering the old medium with cladocerans in a net with mesh openings of 45 µm.The retained C. silvestrii were carefully transferred to the new medium, where pH, temperature, and food concentration was previously adjusted.The cladocerans were fed every two days during the experiment.The experiment was set up in incubator chambers under controlled conditions of temperature (cited above) and photoperiod (12:12 h light: dark cycle).C. silvestrii was kept in 2 L beakers filled with 1 L of reconstituted water as the experimental medium, the experiment was run for 15 days.The experimental period of 15 days was selected based on the life cycle of cladocerans (Allan 1976); this time is sufficient to analyze the zooplankton population's responses to the different treatments.
After 15 days we started the second stage: the fi ltration and ingestion experiment, which was conducted at the three temperatures (control, +2°C and +4°C), with three replicates per temperature.All individuals contained inside the beakers were placed into other 2 L beakers with 1 L of reconstituted water and 1 x 10 5 cells mL -1 of R. subcapitata (alga/food); three subsamples of each replicate were taken at 0 and 2 h to quantify the initial and final algal concentrations.Moreover, a control (no animals added) was incubated under the same experimental conditions, to evaluate only the algal growth after 2 h.All subsamples were fi xed with 1% formaldehyde buffered with sodium borate, frozen in liquid nitrogen, and stored (-20 °C) until analysis.Defrosted samples (500 µL) were analyzed in a FACSCalibur cytometer (Becton and Dickinson Franklin Lakes, NJ, U.S.A.) equipped with a 15 mW Argon-ion laser (488 nm emission) using the FL3-H (red fluorescence) and the SSC-H (lateral dispersion) channels, following Sarmento et al.'s (2008) procedures.Fluorescent beads (6 μm, Fluoresbrite® carboxylate microspheres, Polysciences Inc., Warrington, PA, U.S.A.) were added to the samples, as an internal standard.The cytometry data were analyzed using FlowJo software, version 10.0 (Treestar.com,USA)." With these data, we calculated the fi ltration rates (F) (µL Ceriodaphnia -1 h -1 ) and ingestion rates (I) (cells Ceriodaphnia -1 h -1 ).Filtration refers to the particles of the water that are fi ltrated by the cladocerans; while, the particles (i.e. the algae) that are taken to the cladoceran's mouth and digestive system for subsequent ingestion refers to ingestion (Bownik 2020).These rates were calculated according to the modified equation of Gauld (1951), with a correction factor (A): F= where C0 and Ct are, respectively, the initial and fi nal algal concentration (cells mL -1 ), t is the experimental time (hours) and n is the number of individuals in the volume V (mL).A refers to a correction factor for changes in the control with algal fi nal concentration C't after the time t.The expression represents the geometric mean of food concentration (algal cells) during the time t.
In the third stage to estimate the respiration rates, also at the 15 th day, we removed 18 C. silvestrii adults from each temperature (three replicates* six individuals * three temperature = 54 individuals) and placed in respirometric chambers (2 mL) containing sterile reconstituted water, at control, +2°C and +4°C.The decrease in the oxygen concentration was recorded for approximately 30 minutes using the Unisense micro-respiration system (Arhus, Denmark).The sensor signal was previously calibrated at 24 °C (mean of all treatments) using sterile and aerated water (100% of O 2 saturation) and a solution of 10 g/L sodium sulfi te (0% O 2 saturation).Oxygen consumption rates R (µmol O 2 ind -1 h -1 ) were calculated according to the equation described by Massarin et al. (2010): where [O 2 ]0 was the oxygen concentration (µmol L −1 ) measured at t = 0, V = the volume (L) of the respiration chamber, = the incubation time, and k = the consumption coeffi cient (h -1 ) obtained for the exponential models suitable for the oxygen concentration observed: An Acad Bras Cienc (2022) 94(2) e20200604 5 | 14 Finally, to estimate the secondary production after 15 days, the cladocerans were fixed in 4% formaldehyde buffered with borate and glucose, for better conservation and subsequent quantification.In fixed samples, we counted the number of individuals (Ceriodaphnia L -1 ) for each size class (neonates, juvenile, adults, and ovate females) and the number of eggs inside each female' brood chamber.We calculated the dry weight (over 48 h/70°C) for each stage of development (five replicates -neonates, juvenile, adults, and ovate adults).We applied the length and weight measures in a regression formula ( The assimilation and excretion rates were calculated from the modified equation of Petrusewicz (1967): Assimilation = Production + Respiration; and, Excretion = Ingestion -Assimilation

Life-history experiment
The experiment to study the life history parameters of C. silvestrii was conducted at the three temperatures (control, +2°C and +4°C) and photoperiod (12:12 h light: dark cycle).For this, neonates (< 24h old) were placed in 50 ml beakers (10 replicates per temperature, totalizing 30 replicates, with one neonate each) filled with reconstituted water as the experimental medium and 1 x 10 5 cells mL -1 of R. subcapitata (as food), the experimental medium and food were renewed every day.The bionomic parameters such as body length for all life stages (neonates, juveniles, and adults), the presence of exuviae (cladocerans' exoskeleton remaining from molt), egg-laying, and the number of eggs were daily observed in a stereomicroscope (Leica MZ6, Germany) until the third clutch (reproduction by parthenogenesis).From these observations were obtained the embryonic development time (EDT -the time from egg-laying to hatching), postembryonic development time (PDT I -neonate to juvenile, PDT II -juvenile to adult, and primiparous-from neonate to the first clutch) (Kotov & Boikova 1998, Güntzel et al. 2003), and the body size mean of each development stage.The individuals of C. silvestrii were considered as neonates from birth until the first exuviae (cladocerans' exoskeleton remaining from molt); they were considered as juveniles from the first exuviae until before the first egg production; at the moment that they produced eggs for the first time they were considered as adults, and all these characteristics were observed daily under a stereomicroscope (Leica MZ6, Germany).

Statistical analysis
To evaluate the effect of warming on C. silvestrii metabolic rates, we performed a multivariate analysis of variance (MANOVA, to avoid type I error) (Gotelli & Ellison 2004), followed by analyses of variance (ANOVAs) and post-hoc Tukey analyses.For the mentioned analyses, temperature (control, +2 °C and +4 °C) was used as the categorical predictor variable, and the rates of filtration, ingestion, secondary production, respiration, assimilation, and excretion were used as response variables.
Regarding the secondary production rates in the analyses of variance, the difference between the final and initial experimental values was considered.The response variables were logtransformed to achieve the assumption of normality.The significance of MANOVA was reported using Pillai's trace, and the p-values.All the assumptions were tested and, the significance level adopted was p ≤ 0.05.Analyses of variance (ANOVAs) applying Bonferroni correction (0.05/6 = 0.008, adjusted p-values for multiple comparisons; Gotelli & Ellison 2004) were performed to verify which response variables differed between treatments, and post-hoc Tukey analyses were used to verify which treatments differed from each other.
Due to the absence of normality assumption, Kruskal-Wallis tests were performed to analyze the effect of warming (control, +2 °C and +4 °Ccategorical predictor variable) on the body-size of the development stages (neonate, juvenile and adult), fecundity, and development time stages (embryonic development time -EDT; postembryonic age -PDT I and PDT II; primiparous age).Post-hoc analyses (Wilcoxon test) were performed to analyze at which temperatures these response variables differ.All analyses were performed using the package "stats" in R (R Core team 2019).

DISCUSSION
Warming has effects on several ecological process and organization levels, here we observed that the small-bodied cladoceran C. silvestrii decreased progressively its filtration and ingestion rates with the increase in temperature, while increased the assimilation and secondary production with slight warming and decreased again at the highest temperature.Warming also increased C. silvestrii neonates and juveniles body size, and adults' fecundity; and accelerated the time of development.An unbalance of energy production and transfer is reveal by the alteration of metabolic rates and life-history parameters analyzed -due to warming.These alterations affect food-web dynamics in freshwater ecosystems (O'Connor et al. 2009, Gama-Flores et al. 2015, Hébert et al. 2017, Abo-Taleb 2019).
Different than expected, C. silvestrii reduced progressively its filtration and respiration rates with warming from 22°C to 26°C.Some Daphiniidae species such as Daphnia magna, D. galeata, D. ambigua, and D. pulicaria present their grazing effectiveness around 25°C (Burns 1969, West & Post 2016), whereas D. schodleri and D. pulex at lower temperatures as 20°C (Burns 1969).Cladoceran species may also present contrasting responses in the relationship warming-respiration rates (Duval & Geen 1976, Goss & Bunting 1980, Forster et al. 2011a, Gerke et al. 2011).The different responses among species are possibly related to their geographical distribution and phenotypic plasticity (Gerke et al. 2011, De-Meester et al. 2018).Every species has a thermal window where their molecular, cellular, and systemic processes are optimized (Pörtner & Farrell 2008).Thus, high temperatures are linked to high metabolic demands, which might decrease: the carrying capacity of the zooplankton species (Allen et al. 2002), the rates of attack and manipulation on preys (Dell et al. 2014, West & Post 2016), and the predation effectiveness (i.e.filtration).Slight warming as 2 °C is sufficient to induce such decreases, as observed for D. magna by Kooijman et al. (1989), in agreement with our finds.Also, at high temperatures the organism's demand for oxygen increase in the same way that oxygen turns rarefied, limiting the species reaction and survival in aquatic environments (Verberk et al. 2016).
As we observed, the organisms' metabolic processes respond to warming in a correlated way due to the metabolic demands, i.e. higher respiration rates are linked to higher filtration rates (Kooijman 2000).Also, warming might lead to incomplete digestion by reducing the time that the nutrient remains inside the body (Kooijman 2000), reflecting in higher excretion values and lower assimilation.Although C. silvestrii had ingested the highest number of algal cells at the lowest temperature it also had the highest metabolic losses at this temperature (higher excretion and respiration and lower assimilation), and smaller-bodied neonates and juveniles.The opposite occurred at +2°C, where C. silvestrii presented larger-bodied neonates and juveniles and higher assimilation rates.
Other factors as age-depending responses and availability of high-quality fatty acids at high temperatures might also play a role in C. silvestrii responses.The quantity and quality of lipids reserves affect the growth and reproduction of cladocerans species, and it is related to temperature variations (Brett et al. 2006, Masclaux et al. 2012).Gama-Flores et al. (2015) observed crescent percentages of fatty acids in adult Moina macrocopa with warming up to 25°C, in contrast, in this same study neonates contained much higher proportions of these reserves, regardless of temperature regimes.These energy reserves in neonates promote reproductive maturity and offspring production, and in all stages might allow adaptation in natural populations to climate variation (Masclaux et al. 2012, Gama-Flores et al. 2015).Cladocerans fatty acids are assimilated from food -algae -, which in turn adjust them in their cellular membranes at high temperatures: producing more saturated fatty acids (Gladyshev et al. 2014).So, the high secondary production, assimilation, and fecundity of C. silvestrii at +2°C might represent an indirect effect of algae quality-contend versus temperature, through the filtration process.
We observed that at +2°C, C. silvestrii presented the fastest maturation, greatest fecundity, largest neonates and juveniles body-size, and greatest secondary productivity, contrasting the temperature size rule -which predicts that ectotherms grow fast at high temperatures and present smaller body sizes (biomass) (Atkinson 1994, Forster et al. 2011b, Hoefnagel et al. 2018).Furthermore, these responses observed for C. silvestrii represent tradeoffs between temperature changes and estimators of energetic gains and losses, showing efficiency in converting food into biomass at intermediate temperature.At the lowest and highest temperature, C. silvestrii reduced fecundity, neonates, and juveniles body-size, and delay maturation because at these same temperatures this species also presented high metabolic losses, and possibly had lower lipids reserves.Even though there was not a significant difference between secondary production at +2°C and +4°C, we observed lower values at the highest temperature, which might indicate that if the temperature continued to increase, this rate could decay even further, because of physiological stress (Savage et al. 2004).
Finally, the alteration of metabolic rates of C. silvestrii by warming has ecological implications on aquatic food-webs dynamics.The highest assimilation values, even consuming a lower number of algal cells, turn this species a food resource with high energy content for smallbodied fishes, natural predators of C. silvestrii in subtropical systems (Lazzaro 1987), and increase the amount of energy transferred to higher levels (Lang et al. 2017).Also, warming might at the same time: reduce the cladocerans density, and increase algal growth -prevailing nonedible algae (Visser et al. 2016).This could alter the trophic status of freshwater environments, causing unpleasant consequences for water quality and human well-being (Brooks et al. 2016).

CONCLUSIONS
Besides C. silvestrii is a subtropical species, it has a short thermal window, with thermal preferences around 24°C (i.e.greater food assimilation and biomass production).The increase in temperature by global warming (IPCC scenarios tested) will affect the cladocerans metabolic processes and the population survival, even a small increase (2°C) might induce drastic fluctuations in such processes and affect the carbon and energy availability inside aquatic food-webs, altering the entire ecosystem functioning.

Figure 1 .
Figure 1.Box plot showing C. silvestrii metabolic rates, a) Secondary productivity, b) Filtration rates, c) Ingestion rates and d) Assimilation rates at three temperatures (control, +2 and +4°C), p-value from ANOVA analyses is shown.Symbols(-,*) above the columns indicate significant differences in post-hoc analyses -treatments that share a symbol do not differ significantly, p >0.05.• indicates the mean, ± standard error bars are shown.

Figure 2 .
Figure 2. Box plot showing C. silvestrii fecundity (average of the number of eggs/ female from the three clutches) at three temperatures (control, +2, and +4°C), Kruskal-Wallis significant analyses (p = 0.022) are shown.Symbols (-,*) above the columns indicate significant differences (posthoc analyses) -treatments that share a symbol do not differ significantly, p >0.05.• indicates the mean, ± standard error bars are shown.

Figure 3 .
Figure 3. Box plot showing C. silvestrii body size (mm) in different development stages: neonate, juvenile, and adult (average from the three clutches) at three temperatures (control, +2, and +4°C), p-value from Kruskal-Wallis analyses are shown.Symbols(-,*) above the columns indicate significant differences in post-hoc analysestreatments that share a symbol do not differ significantly, p >0.05.• indicates the mean, ± standard error bars are shown.

Figure 4 .
Figure 4. Box plot showing C. silvestrii development time in days (EDT = embryonic development time; Primiparous = from neonate to the first clutch; PDT I = neonate to juvenile; PDT II = juvenile to adult) at three temperatures (control, +2 and +4°C), p-value from Kruskal-Wallis analyses is shown.Symbols (-,*,+) above the columns indicate significant differences in post-hoc analyses -treatments that share a symbol do not differ significantly, p >0.05.• indicates the mean, ± standard error bars are shown.

Table I .
Initial and final average of C. silvestrii population rates, respiration, and excretion rates in the different temperatures.Note: ind.= individual.DW = dry weight.SE = standard error and SD = standard deviation.*Nonsignificant results.