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The relationship between heterotrophic feeding and inorganic nutrient availability in the scleractinian coral T . reniformis under a short-term temperature increase

2015; Wiley; Volume: 61; Issue: 1 Linguagem: Inglês

10.1002/lno.10200

1939-5604

Leïla Ezzat, Erica K. Towle, Jean‐Olivier Irisson, Chris Langdon, Christine Ferrier‐Pagès,

Marine and fisheries research

Limnology and OceanographyVolume 61, Issue 1 p. 89-102 ArticleFree Access The relationship between heterotrophic feeding and inorganic nutrient availability in the scleractinian coral T. reniformis under a short-term temperature increase Leïla Ezzat, Corresponding Author Leïla Ezzat Marine Department, Centre Scientifique de Monaco, Monaco, Principality of Monaco Leïla Ezzat and Erica Towle authors contributed equally to this workCorrespondence: leila@centrescientifique.mc or etowle@rsmas.miami.eduSearch for more papers by this authorErica Towle, Corresponding Author Erica Towle Department of Marine Biology and Fisheries, The Rosenstiel School of Marine and Atmospheric Sciences, Miami, Florida, USA Leïla Ezzat and Erica Towle authors contributed equally to this workCorrespondence: leila@centrescientifique.mc or etowle@rsmas.miami.eduSearch for more papers by this authorJean-Olivier Irisson, Jean-Olivier Irisson Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire d'Océanographie de Villefranche (LOV), Villefranche-sur-Mer, FranceSearch for more papers by this authorChris Langdon, Chris Langdon Department of Marine Biology and Fisheries, The Rosenstiel School of Marine and Atmospheric Sciences, Miami, Florida, USASearch for more papers by this authorChristine Ferrier-Pagès, Christine Ferrier-Pagès Marine Department, Centre Scientifique de Monaco, Monaco, Principality of MonacoSearch for more papers by this author Leïla Ezzat, Corresponding Author Leïla Ezzat Marine Department, Centre Scientifique de Monaco, Monaco, Principality of Monaco Leïla Ezzat and Erica Towle authors contributed equally to this workCorrespondence: leila@centrescientifique.mc or etowle@rsmas.miami.eduSearch for more papers by this authorErica Towle, Corresponding Author Erica Towle Department of Marine Biology and Fisheries, The Rosenstiel School of Marine and Atmospheric Sciences, Miami, Florida, USA Leïla Ezzat and Erica Towle authors contributed equally to this workCorrespondence: leila@centrescientifique.mc or etowle@rsmas.miami.eduSearch for more papers by this authorJean-Olivier Irisson, Jean-Olivier Irisson Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire d'Océanographie de Villefranche (LOV), Villefranche-sur-Mer, FranceSearch for more papers by this authorChris Langdon, Chris Langdon Department of Marine Biology and Fisheries, The Rosenstiel School of Marine and Atmospheric Sciences, Miami, Florida, USASearch for more papers by this authorChristine Ferrier-Pagès, Christine Ferrier-Pagès Marine Department, Centre Scientifique de Monaco, Monaco, Principality of MonacoSearch for more papers by this author First published: 30 September 2015 https://doi.org/10.1002/lno.10200Citations: 37 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Worldwide increase in seawater temperature represents one of the major threats affecting corals, which experience bleaching, and thereafter a significant decrease in photosynthesis and calcification. The impact of bleaching on coral physiology may be exacerbated when coupled with eutrophication, i.e., increasing plankton, inorganic nutrient concentrations, sedimentation and turbidity due to coastal urbanization. Whereas zooplankton provision (heterotrophy) may alleviate the negative consequences of thermal stress, inorganic nutrient supply may exacerbate them, which creates a paradox. Our experimental study aims to disentangle the effects of these two components of eutrophication on the physiological response of Turbinaria reniformis subject to normal and to a short-term temperature increase. Additionally, three different inorganic nutrient ratios were tested to assess the influence of nutrient stoichiometry on coral physiology: control (ambient SW 0.5 μM N and 0.1 μM P), N only (ambient + 2 μM N) and N + P (ambient + 2 μM N and + 0.5 μM P). Our results show a deleterious effect of a 2 μM nitrate enrichment alone (N) on coral photosynthetic processes under thermal stress as well as on calcification rates when associated with heterotrophy. On the contrary, a coupled nitrate and phosphorus enrichment (N + P) maintained coral metabolism and calcification during thermal stress and enhanced them when combined with heterotrophy. Broadly, our results shed light on the tight relationship existing between inorganic nutrient availability and heterotrophy. Moreover, it assesses the relevance of N: P stoichiometry as a determining factor for the health of the holobiont that may be adapted to specific nutrient ratios in its surrounding environment. Rising seawater temperature and eutrophication are currently two of the most serious factors threatening coral reefs throughout the world (Fabricius 2011; Fabricius et al. 2011; Fabricius et al. 2013). Over the last 30 yrs, global climate change has indeed increased average oceanic temperature by approximately 0.2°C per decade (Hansen et al. 2006). Therefore, the world's oceans are warmer now than at any point in the last 50 yrs and seawater temperature is expected to continue increasing. Thermal stress has detrimental effects on many marine organisms, some of which have already started to reach the limits of their capacity to persist (Visser 2008). In symbiotic corals, high temperatures induce bleaching (i.e., the loss of the dinoflagellate symbionts of the genus Symbiodinium sp.), thus weakening the symbiotic association (Brown 1997). Symbiodinium, through inorganic nutrient acquisition and photosynthesis, provide 75–100% of the daily metabolic requirements of their host (Grottoli et al. 2006; Tremblay et al. 2012). Therefore, bleaching induces the loss of the coral's major source of nutrition and can cause widespread mortality (Glynn 2012). In addition, human activities such as increased coastal fertilizer use, land clearing, and urbanization contribute to eutrophication (enhanced nutrients, particles, and sedimentation levels) of coastal sites and have modified the nutrient stoichiometry in these ecosystems (Sterner and Elser 2002). Eutrophication alters coral health because it enhances algal growth and induces competition for space and light (Bruno et al. 2009; Norström et al. 2009; De'ath and Fabricius 2010; Graham et al. 2015). It has also been suggested that elevated inorganic nutrient levels directly impact corals by decreasing their thermal thresholds for bleaching. Nutrient addition indeed tends to enhance symbiont growth inside the coral host tissue, increasing oxidative stress during warm seasons (Nordemar et al. 2003; Wooldridge 2009; Wooldridge and Done 2009; Wiedenmann et al. 2013; Vega Thurber et al. 2014). Therefore, there is evidence to suggest that corals living in nutrient-rich environments that are also experiencing thermal stress may be particularly endangered. However, several laboratory experiments have also shown that nutrient supplementation may increase the ability of corals to withstand climate change stress because it can offset the negative effect of a temperature increase by favoring symbiont growth and photosynthesis. Nutrients can be acquired either via heterotrophy, the capture of zooplankton prey and or particulate organic matter (POM) by the coral host, or via autotrophy, via the uptake of inorganic nutrients (e.g., nitrate, ammonium, phosphate) by the symbionts. Aside from improving coral metabolism under nonstressful conditions (Houlbrèque and Ferrier-Pagès 2009), several studies have indeed identified coral heterotrophy as an indicator of resilience to bleaching stress (Grottoli et al. 2006; Rodrigues and Grottoli 2007; Palardy et al. 2008; Connolly et al. 2012). In their model, Anthony et al. (2009) stated that increased heterotrophy was a good predictor of survivorship and reduced mortality risk during a bleaching event, and Tolosa et al. (2011) discovered that zooplankton feeding played an essential role in sustaining Turbinaria reniformis metabolism under thermal stress. Furthermore, moderate inorganic nitrogen supply has been shown to promote coral growth and metabolism (Tanaka et al. 2007; Sawall et al. 2011), especially under elevated pCO2 (Langdon and Atkinson 2005; Holcomb et al. 2010; Chauvin et al. 2011), or thermal stress (Béraud et al. 2013). Based on these studies, there are lines of evidence to suggest that corals thriving in nutrient-rich environments may be more resilient to stress than others, if not in competition with algae. In view of this conflicting evidence, more research is needed to understand the effects of heterotrophy and/or inorganic nutrient supplementation on coral metabolism during environmental stresses. Several hypotheses for the differing results have been put forward. In their meta-analysis of the effects of eutrophication on corals, Shantz and Burkepile (2014) assert that the physiological response of corals to eutrophication is context-dependent. Thus, when comparing studies, several parameters such as coral taxa, coral morphology, nutrient concentration and nitrogen source, and type (i.e., ammonium vs. nitrate, organic vs. inorganic) have to be taken into account. More importantly, the stoichiometry of the N: P ratio needs to be close to the N: P ratio of the coral tissue, i.e., a complete lack of phosphate with nitrate enrichment may be unfavorable for the symbiosis (Wiedenmann et al. 2013). In coastal ecosystems, eutrophication enhances both POM and inorganic nutrient concentrations. Nutrient-rich reefs tend to have higher gross production as well as higher heterotrophic consumption than oligotrophic reefs (Mioche and Cuet 1999; Szmant 2002). To date, only two studies have attempted to decipher the effect of the different components of eutrophication (POM, inorganic nutrient, sedimentation and turbidity) on coral growth and survival (Fabricius 2005; Fabricius et al. 2013). The first study by Fabricius (2005) highlighted sedimentation and turbidity as the two major factors of stress for coral growth. Conversely, their model suggested an increase in growth with POM, and a moderate reduction in growth with dissolved inorganic nutrient enrichment. Fabricius et al. (2013), however, later observed low coral growth under a coupled enrichment in dissolved inorganic nutrients (nitrate) and POM. In view of these contrasting observations on coral growth, more work is needed to improve our knowledge of the effect of eutrophication on the coral physiological response in terms of photosynthesis, calcification, and tissue growth. In this experimental study, we sought to disentangle the combined effect of POM (heterotrophy) and inorganic nutrient enrichment from their individual effects on the physiological response of the scleractinian coral T. reniformis maintained under ambient temperature conditions as well as during short-term thermal stress. In addition, we implemented different N : P ratios (4 : 1, 5 : 1, and 20 : 1) of inorganic nutrients in seawater to assess the effect of nutrient stoichiometry on coral physiology and symbiotic interactions. We hypothesize that (1) heterotrophy has a different effect than inorganic nutrient supplementation on coral metabolism; (2) the inorganic N : P ratio shapes the metabolic response of T. reniformis under both normal and stressful conditions; nitrate enrichment being more detrimental to coral physiology than a combined enrichment in nitrate and phosphate (Wiedenmann et al. 2013); (3) the combined effect of heterotrophy and inorganic nutrient supplementation differs from their individual effects on coral physiology. Materials and procedures Experimental set-up The widely distributed scleractinian coral T. reniformis (Dendrophylliidae) was used in this study due to its ability to thrive in turbid and organically rich environments (Veron 2000; Anthony et al. 2006). Five large colonies were sampled between 5 m and 10 m depth in different locations of the Northern Red Sea, brought back to the laboratory, and tagged with an electronic microchip implant to account for parent colony before being used to produce a total of 456 nubbins of about the same size (4 cm diameter). Nubbins were then evenly distributed into 12 independent 20 L tanks (38 nubbins per tank), maintained under the following controlled conditions: aquaria were continuously supplied with oligotrophic seawater containing a low amount of nutrients (∼ 0.5 μM nitrate (NO3) and ∼ 0.1 μM phosphate, PO4) at a flow rate of 20 L h−1. A constant seawater temperature of 25°C ± 0.5°C was regulated using submersible resistance heaters (Visi-ThermH Deluxe, Aquarium Systems, France) and metal halide lamps (Philips, HPIT 400W, Distrilamp, Bossee, France) provided a constant irradiance of 200 μmol photons m−2 s−1 inside the tanks (12: 12 light: dark). Submersible pumps ensured water mixing in the tanks. Nubbins were kept unfed for four weeks to prevent any impact of heterotrophy on the physiological responses of corals before performing the experiments described below. After the acclimation period, six different nutritional conditions were established (in duplicated tanks) as described in Table 1 and below. Tanks were cleaned weekly to avoid any algal proliferation. Table 1. Experimental conditions for the six different treatments: Control condition (C), Control condition + zooplankton provision (Heterotrophy) (CH), Nitrate enrichment (N), Nitrate enrichment + Heterotrophy (NH), Nitrate and phosphorus enrichment (NP), Nitrate and phosphorus enrichment + Heterotrophy (NPH). Treatments Characteristics C CH N NH NP NPH Natural seawatera x x x x x x Supply of 2000 artemia d−1 x x x 2 μM nitrate addition x x x x 0.5 μM phosphate addition x x a (< 0.05 μM N and < 0.1 μM P). The “x” indicates presence of each of the characteristics for each treatment. Four control tanks were maintained in duplicate under low inorganic nutrient concentrations (as described above), with or without zooplankton (heterotrophy). In four other tanks, supplied with or without zooplankton, nitrate was added to reach a N: P ratio of 20: 1. Finally, in the last four tanks, maintained with or without zooplankton, nitrate and phosphate were added in parallel to reach a N: P ratio of 4 : 1. Zooplankton was provided in the “heterotrophic” tanks every evening for 5 d per week, by means of ca. 2000 Artemia salina (in total) that were left in the tanks until they were eaten or diluted with the seawater renewal. For the nitrate and nitrate-phosphate enriched conditions, tanks were, respectively, supplied with a solution of sodium nitrate (NaNO3) alone or in combination with sodium dihydrogenophosphate (NaH2PO4). Stock solutions were continuously pumped from individual batch tanks via a peristaltic pump and delivered to the experimental tanks at a final concentration of 2.0 μM for NaNO3 and 0.5 μM for NaH2PO4. The stock solutions were renewed every 3 d and added to the tanks at a constant flow rate of 0.3 L h−1. Inorganic nutrient concentrations in the water were monitored in each tank and in triplicate twice a week using an Autoanalyzer (Alliance Instrument, AMS, France) according to Tréguer and Le Corre (1975). After 3 weeks under these experimental conditions (T0), temperature was increased from 25°C ± 0.5°C to reach 30°C ± 0.5°C over a 10 day-period and was kept constant for 7 d (T1). This thermal stress was chosen according to the highest temperature observed in the northern Red Sea (Sawall et al. 2014). After 17 d under thermal stress, all tanks were brought back to 25°C ± 0.5°C over 10 d for two additional weeks to assess recovery (T2). Several measurements (described below) were performed to assess physiology and photophysiology of T. reniformis at the end of each temperature step (T0, T1, and T2). Measurements Photosynthesis and respiration rates, total chlorophyll, symbiont density, and protein determination Rates of net photosynthesis (Pn) and respiration (R) were assessed at 200 and 0 μmol photons m−2 s−1 on six randomly selected nubbins per treatment in 50 mL glass chambers supplied with filtered seawater (FSW). Chambers were equipped with a Unisense optode (oxygen-sensitive minisensor) connected to the Oxy-4 software (Chanel fiber-optic oxygen meter, Presens, Regensburg, Germany). The optodes were calibrated before each experiment against air-saturated and nitrogen-saturated seawater (for the 100% and 0% oxygen, respectively). Stir bars were used to continuously mix the water. Light was provided by a metal halide lamp (Philips, HPIT 400W, Distrilamp, Bossee, France). Pn and R were calculated by regressing oxygen production or consumption against time. Gross photosynthesis was estimated by adding the absolute value of R to Pn. At the end of each incubation, nubbins were frozen overnight (−20°C) before the subsequent determination on the same nubbins of symbiont density, total chlorophyll, and protein concentration according to Hoogenboom et al. (2010). Briefly, tissue was removed from the skeleton of each nubbin with an air-pick, and collected in 10 mL of 0.45 μM FSW. Tissue slurry was then homogenized with a potter grinder and divided into 5 mL subsamples. The first subsample was used for the determination of zooxanthellae density and protein concentration. Density of symbionts was quantified (in 500 μL) using an inverted microscope (Leica, Wetzlar, Germany) and the Histolab 5.2.3 image analysis software (Microvision, Every, France). Protein concentration was assessed according to Smith et al. (1985) using a BCAssay Protein Quantification Kit (Uptima, Interchim) and a Xenius® spectrofluorometer (SAFAS, Monaco). The second 5 mL subsample was centrifuged at 8000 × g for 10 min. Supernatant was removed and the symbionts resuspended in 5 mL acetone for chlorophyll a (Chl a) and c2 extractions. Chlorophyll concentration was then determined according to the method of Jeffrey and Humphrey (1975) with a spectrophotometer. Data were normalized to the upper side surface area (cm2) of each nubbin, using the wax-dipping method from Veal et al. (2010). Relative electron transport rate Measurements were performed on six nubbins per treatment as for photosynthesis. A pulse amplitude modulation (PAM) fluorometer [DIVING-PAM, Walz, Germany (Schreiber et al. 1986)] was used to measure the minimal (F0) and maximal (Fm) Chl a fluorescence after 5 min of dark adaptation by applying a weak pulse red light (max. intensity < 1 mol photon m−2 s−1, width 3μs, frequency 0.6kHz) and a saturating pulse of actinic light (max. intensity 5000 μmol photon m−2 s−1, pulse width 800 ms) on coral nubbins placed under a constant distance from the optical fiber. The rapid light curve function of the PAM assessed the relative electron transport rate (rETR) after exposing nubbins to eight light intensities (from 69 μmol photons s−1 m−2 to 1500 μmol photons s−1 m−2) over 10 s. Feeding rates Feeding rate experiments were conducted as described in Houlbrèque et al. (2004). Each coral nubbin was incubated in individual Plexiglas flumes of 1 L, specially designed for this kind of measurement according to Levy et al. (2001), based on the procedures described by Vogel and LaBarbera (1978). Flow speed was approximately 4 cm s−1 as this was found to allow for maximum feeding in T. reniformis (Ferrier-Pagès et al. 2010), and chambers were placed in a water bath maintaining constant temperature. Measurements were run at T0, T1, and T2 on fed nubbins only (Table 1, n = 6 per treatment). When polyps were fully expanded, the experimental run was started by delivering 1000 A. salina nauplii L−1 according to Gori et al. (2015), previously quantified using a Bogorov counting chamber. Six replicated 10 mL seawater samples were taken from the flow chamber just after the start of the experiment (0 min), then at 15 min, 30 min, 45 min, and 60 min. A. salina nauplii in each sample were counted and immediately replaced in the chambers to avoid any concentration decrease. Three control experiments were also conducted at each temperature step (prey only, without corals) to account for possible concentration decrease due to nauplii becoming trapped in the flume. Feeding rates were calculated as the decrease in prey concentration against time and normalized to number of polyps. Calcification rates Growth rates (n = 10 per treatment) were monitored weekly using the buoyant weight technique (Davies 1989). Growth data are presented as percent weight increase per day, using the equation: [(F-I)/(I × number of days)] × 100, where F is the weight of each nubbin at the end of a given time step (T0, T1, T2) and I is the weight at the beginning of a given time step. Statistical analyses Statistical analyses were processed using the program JMP Version 11.0.0. Data were checked for normality using the Kolmogorov–Smirnov test with Lilliefors correction and for homoscedasticity using Levene's test. When conditions were not fulfilled, a data transformation (natural logarithms) was used. Preliminary partly nested ANOVA tests were used to test for any possible tank effect. This effect was not significant for any of the parameters so this factor was excluded from analyses (Underwood 1997). A general linear model for parametric repeated measure analysis of variance (ANOVA) was performed on the feeding and growth rate response variables using “Temperature” as dependent variable and “Inorganic” enrichment, “Organic” feeding (zooplankton provision) and the corresponding sample as categorical predictors. The assumption of sphericity (independency of the repeated measures) was tested. When not fulfilled, the hypotheses were tested using the multivariate approach (Wilks test) for repeated measurements. A Three-Way ANOVA was then performed on all other studied variables with “Inorganic” enrichment, “Organic” feeding, and “Temperature” as factors. When significant differences appeared, analyses were followed by a posteriori testing (Tukey's HSD test). p-values were considered significant for p < 0.05. Assessment Evolution of the treatments during the experiment During heat-stress, unfed control corals (C) significantly decreased their symbiont density (Table 4, Tukey HSD, p = 0.007) as well as their protein content (Table 4, Tukey HSD, p = 0.035). In addition, the rETR of all control corals (C and CH) was lower during thermal stress (Fig. 2, Tukey HSD, p < 0.0055), while respiration rates and Pg were increased (Fig. 1, Tukey HSD, p < 0.0445 and p < 0.0013, respectively). During the recovery period, Pg of fed corals and R of unfed corals decreased and returned to their basal level measured at 25°C (Tukey HSD, p = 0.028 and p = 0.0002, respectively, compared to 30°C). Figure 1Open in figure viewerPowerPoint Rates of gross photosynthesis and respiration (μmol O2 h−1 cm−2) for the different nutritional treatments according to the temperature steps from left to right (T0, T1, T2). Data are presented as mean ± 1 SE. Letters represent statistical differences based on post hoc Tukey's HSD test (p < 0.05). Dark bars represent zooplankton (heterotrophic) supply. During thermal stress in the nitrate-enriched treatments, rETRmax of unfed colonies (Fig. 2, Tukey HSD, p < 0.0001 and Tukey HSD, p = 0.047, respectively) and calcification rates of fed colonies (Fig. 2, Table 5, Tukey HSD, p = 0.02, respectively) significantly decreased. Pg increased slightly for NH corals (Tukey HSD, p = 0.0115), while no difference was observed for N nubbins compared to control conditions (Tukey HSD, p > 0.05). During the recovery period, calcification rates of fed corals remained very low, while protein content largely increased compared to 25°C and 30°C (Table 5, Tukey HSD, p < 0.012). Symbiont density of unfed corals significantly increased compared to 30°C (Tukey HSD, p = 0.0046). Figure 2Open in figure viewerPowerPoint (a) rETR max values, (b) Rates of calcification (% change in growth per day) and (c) feeding (Nb artemia h−1 polyp−1) for the different nutritional treatments according to the temperature steps from left to right (T0, T1, T2). Data are presented as mean ± 1 SE. Letters represent statistical differences based on post hoc Tukey's HSD test (p < 0.05). Dark bars represent zooplankton (heterotrophic) supply. Figure 3Open in figure viewerPowerPoint Budgets highlighting the major changes in the physiological parameters between 25°C and 30°C. The arrows indicate a significant increase or decrease (up or down arrows, respectively), whereas the equal sign indicates no significant changes. NP-enriched corals (NP and NPH) significantly changed their physiology throughout the entire study. At 30°C, Pn of all colonies, R of unfed colonies, and calcification of fed colonies significantly increased compared to respective rates at 25°C (Figs. 1, 2, Tables 2, 3 and 5 Tukey HSD, p < 0.01, p < 0.0001, and p = 0.0061). During recovery, respiration rates in unfed corals returned to their basal values (Tukey HSD, p < 0.0001) and rETR max values decreased compared to 25°C and 30°C (Tukey HSD, p < 0.0065). Table 2. Changes observed (as %) in physiological parameters at and between the different time steps (25°C, 30°C, and 25°C Recovery). Comparisons Parameter C CH N NH NP NPH % change between enriched treatments and control conditions At 25°C Chlorophyll content — (C) — (C) — (BC) — (BC) +350% (AB) +450% (A) At 30°C Chlorophyll content — (B) — (B) — (AB) — (AB) +280% (A) +311% (A) Symbiont density — (B) — (B) — (B) — (B) — (B) +236% (A) Protein content — (AB) — (A) — (AB) −48% (B) — (AB) — (A) At 25°C recovery Chlorophyll content — (AB) −371% (C) — (BC) — (BC) — (B) +514% (A) Symbiont density — (B) — (B) +285% (A) — (AB) — (AB) — (AB) % change within each treatment Between 25°C and 30°C Symbiont density −52% — — — −47% — Protein content −37% — −38% — — — Between 30°C and −25°C recovery Symbiont density — — +266% — — — Protein content — — — +482% — — Letters indicate significant differences after performing Tukey's HSD post hoc tests. Table 3. Summary of ANOVAs testing the effect of the different time steps (T) (25°, 30°, and 25R °C), Inorganic nutrient enrichment (I), and Organic feeding (zooplankton provision) (O) on T. reniformis main physiological parameters. Bold face numbers indicates p < 0.05. Source of variation df Sum of squares F p Zoox. density (Nb Zoox cm−2) T 2 6044006007 11.1932 <0.0001 I 2 2624173771 4.8598 0.0103 T × I 4 5169596097 4.7869 0.0017 O 1 170779219 0.6325 0.4288 T × O 2 454644507 0.8420 0.4347 I × O 2 1220422046 2.2602 0.1111 T × I × O 4 1115478181 1.0329 0.3957 Chlorophyll (μg Chl (a + c2)) cm−2 T 2 0.0416158 0.4891 0.6150 I 2 1.5730277 18.4890 <0.0001 T × I 4 0.0015235 0.0358 0.8504 O 1 0.1646167 0.9674 0.4302 T × O 2 0.0070177 0.0825 0.9209 I × O 2 0.0610869 0.7180 0.4909 T × I × O 4 0.1435669 0.8437 0.5017 Protein (μg cm−2) T 2 283468.76 20.2754 <0.0001 I 2 621.99 0.0445 0.9565 T × I 4 35659.37 1.2753 0.2869 O 1 152.34 0.0218 0.8830 T × O 2 8686.62 0.6213 0.5399 I × O 2 2773.42 0.1984 0.8205 T × I × O 4 72202.5 2.5822 0.0435 Table 4. Summary of ANOVAs testing the effect of the different time steps (T) (25°, 30°, and 25R °C), Inorganic nutrient enrichment (I), and Organic feeding (zooplankton provision) (O) on T. reniformis main physiological photosynthetic parameters. Bold face numbers indicates p < 0.05. Source of variation df Sum of squares F p ETR T 2 5097.8614 215.5329 <0.0001 I 2 73.4705 3.1063 0.0518 T × I 4 152.2962 3.2195 0.0182 O 1 13.0689 1.1051 0.2972 T × O 2 54.2981 2.2957 0.1092 I × O 2 78.6251 3.3242 0.0425 T × I × O 4 74.5125 1.5752 0.1921 Net photosynthesis (μmol O2 h−1 cm−2) T 2 1.6928 23.6108 <0.0001 I 2 1.5857 22.1175 <0.0001 T × I 4 0.4132 2.8818 0.0270 O 1 0.0405 1.1296 0.2907 T × O 2 0.0637 0.8883 0.4150 I × O 2 0.0203 0.2830 0.7542 T × I × O 4 0.0385 0.2687 0.8974 Respiration (μmol O2 h−1 cm−2) T 2 3.2974 46.3595 <0.0001 I 2 0.2742 3.8546 0.0248 T × I 4 0.5638 3.9634 0.0052 O 1 0.0097 0.2736 0.6022 T × O 2 0.0595 0.8365 0.4366 I × O 2 0.0578 0.8126 0.4469 T × I × O 4 0.1905 1.3390 0.2617 Gross photosynthesis (μmol O2 h−1 cm−2) T 2 12.4146 53.0252 <0.0001 I 2 6.0940 26.0290 <0.0001 T × I 4 1.1381 2.4306 0.0586 O 1 0.0194 0.1658 0.6854 T × O 2 0.1367 0.5841 0.5611 I × O 2 0.5286 2.2581 0.1143 T × I × O 4 1.2939 2.7634 0.0366 Table 5. Summary of repeated-measured ANOVA testing the effect of Inorganic nutrient enrichment (I) and Organic feeding (zooplankton provision) (O) according to the different time steps (T) on Growth and Feeding ratesa. Bold face numbers indicates p < 0.05. Source of variation df F p Growth (% change d−1) T 2 6.1830 0.0039 I 2 15.5391 <0.0001 T × I 4 9.1835 <0.0001 O 1 0.0182 0.8933 T × O 2 1.3379 0.2713 I × O 2 3.6217 0.0336 T × I × O 4 3.4939 0.0102 Feeding (Nb Artemia h−1 polyp−1) T 2 8.0095 0.0071 I 2 7.6711 0.0071 T × I 4 1.2618 0.3148 a The feeding rate parameter was tested with Inorganic nutrient enrichment (I) through the different timesteps (T). Comparison between treatments at each temperature step At 25°C, NPH colonies presented a higher chlorophyll content per su