Zooplankton provisioning does not enhance thermotolerance and survival of Favia fragum settlers

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It is yet unclear to what extent this could occur during early life stages of corals, and how this affects survival. We exposed settlers (primary polyps) of the Caribbean Golf ball coral Favia fragum to a lab-controlled heatwave lasting 48 days under two feeding regimes (36 versus 3600 Artemia salina nauplii L − 1 ), starting from 28°C and peaking at 32.4°C with a daily increase of 0.19°C. Thermal stress, measured as declining effective photosystem II yield, became apparent at 31.8°C (or 7.5 Degree Heating Weeks, DHW). Growth of heat-exposed settlers ceased between 31.0-31.8°C (4.1–7.5 DHW), regardless of feeding regime. All heat-exposed settlers had died at 14.4 DHW. Mortality was preceded by a 41–64% loss of symbiont densities and a 46–59% reduction of chlorophyll a fluorescence at 32.4°C (9.4 DHW). No beneficial effect of feeding on thermotolerance was observed, likely because settlers were unable to feed on zooplankton during the heatwave: at 32.4°C (9.4–10.7 DHW), prey capture was reduced by up to 98% as compared to controls. Our findings identify the coupling between symbiont and host performance under thermal stress in F. fragum settlers, and help explain low recruitment on Caribbean reefs. coral bleaching zooplankton climate change reef resilience recruitment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Caribbean reefs are experiencing increasingly stronger heatwaves, leading to dramatic changes in their composition and functioning (Bove, Mudge and Bruno 2022 ). Reef-building scleractinian corals already live close to their upper thermal limit (Baker et al. 2008 ), with temperature anomalies resulting in coral bleaching, disease and mortality (Randall and van Woesik 2015 ). The 2023–2024 El Niño bleaching event wreaked havoc amongst coral reefs worldwide, with unprecedented thermal stress recorded in the Caribbean and beyond (Hoegh-Guldberg et al. 2023 ; Reimer et al. 2024 ; Doherty et al. 2025 ). Record values of degree heating weeks (DHW), a measure of cumulative thermal stress (Liu et al. 2003 , 2006 ), were recently recorded on the Great Barrier Reef, Brazil, the Red Sea and the Caribbean, exceeding 20 DHW (Hoegh-Guldberg et al. 2023 ). This is a sobering fact, as values of 8–12 DHW and beyond are strongly correlated to widespread coral bleaching and mortality (Kleypas et al. 2016 ; Kayanne 2017 ). Although climate-driven loss of coral reefs is reason for grave concern, there is evidence that at least some reef-building corals can temporarily shift from autotrophic to heterotrophic feeding during bleaching episodes for a duration of several months (Mies et al. 2018 ). During temperature-induced bleaching, some coral species enhance zooplankton feeding which may offset the incurred energy cost from autotrophic symbiont loss (Ferrier-Pagès et al. 2010 ; Grottoli et al. 2006 ; Levas et al. 2016 ). This shift towards heterotrophic feeding may have mitigating effects on thermal stress during episodic heatwaves (Ferrier-Pagès et al. 2010 ; Connolly et al. 2012 ), which are increasing in frequency and severity (Grottoli et al. 2014 ). Stable isotope analyses suggest that corals which rely more heavily on heterotrophic feeding have a higher thermotolerance, with more heterotrophic species tolerating up to almost twice the cumulative thermal stress as compared to autotrophic species (Conti-Jerpe et al. 2020 ). It is yet unclear, however, to what extent coral recruits are able to rely on heterotrophy during early life, thus immediately after metamorphosis and settlement. Thus, the potential of coral recruits to cope with thermal stress through a trophic shift currently remains unknown. This poses a critical knowledge gap as it may help explain why recruits on Caribbean reefs show low outgrowth to adult corals, hampering reef recovery (Edmunds 2021 ). Heterotrophic feeding may be a major driver of survival and growth in coral settlers (primary polyps), especially in species having horizontal symbiont transmission (Suzuki et al. 2013 ). Until tissues are sufficiently colonised by dinoflagellate symbionts, utilisation of lipid reserves and heterotrophic feeding may be the only means by which settlers can meet their metabolic demand (Graham et al. 2008 ; Geertsma et al. 2022 ). This heterotrophic dependency would be exacerbated during episodic heatwaves. A notable coral species within this context is the massive Caribbean Golf ball coral, Favia fragum . This species produces offspring capable of capturing zooplankton prey as early as 1 day post metamorphosis (DPM, Geertsma et al. 2022 ). This heterotrophic capacity may be a strategy to rapidly increase energy availability for growth, potentially enhancing benthic competition and survival. It may also offset the loss of autotrophy during episodes of thermal stress and bleaching. Interestingly, F. fragum shows stable recruitment rates across the Florida Keys (Harper et al. 2023 ), which may be explained by its nutritional plasticity. Therefore, the aim of this study was to evaluate the potential mitigating role of heterotrophic feeding on thermotolerance of early life-stage F. fragum . We investigated how zooplankton provisioning affected thermotolerance and performance (as growth and survival) of settlers during a heatwave, and linked these observations to prey capture rates. Settlers were exposed to a heatwave lasting 48 days, starting from 28°C and peaking at 32.4°C, with a daily increase of 0.19°C. Heat-exposed and control settlers were cultured under two feeding regimes, 36 and 3600 Artemia salina nauplii per litre, as these densities are within one order of magnitude as those found under natural and aquaculture conditions, respectively (Roman et al. 1990 ; Yahel et al. 2004; Geertsma et al. 2022 ). We measured thermal stress as changes in effective yield of photosystem II (PSII) and bleaching as changes in symbiont densities and chlorophyll a fluorescence. In addition, we measured survival, areal growth and prey capture rates. The expectation was that settlers showed higher thermotolerance at the high feeding regime, reflected by increased survival and growth during the heatwave, resulting from enhanced prey capture rates and subsequent nutrient intake. Materials and Methods Coral collection To establish a breeding population for coral larvae, 15 adult colonies of the Caribbean Golf ball Coral ( Favia fragum Esper 1797) were collected by SCUBA between 3 and 5 m depth around the Sea Aquarium, Willemstad, Curaçao in July 2019. These corals were hand-picked by removal with a small chisel, labelled, and individually placed in zip lock bags underwater. After collection, corals were transported to CARMABI Institute (Willemstad, Curaçao) and placed in aquaria with running seawater for acclimation. For transport to Wageningen University, corals were individually placed in 1 L Ziplock bags, with 30/70% seawater/air. A secondary zip lock bag was placed around the primary bag, to increase isolation and security. All bags were stacked in a 30 L isolated box and shipped in a heated cargo hold. Collection was permitted by the CARMABI institute, and transport was done under CITES export permit no. 19CW002 and import permit no. 19NL270335/11. After transport, corals were temperature-acclimated over a two-hour period to 28°C in a 300 L aquarium at CARUS Aquatic Research Facility (ARF) of Wageningen University (see Coral husbandry and larval collection for details). Transport water was discarded. Coral husbandry and larval collection Corals were maintained in a 300 L glass aquarium with the following daily-checked target parameters: temperature 28°C, Salinity 36 PSU, alkalinity 2.5 mEq L − 1 , calcium 420 mg L − 1 , nitrate 0–1 mg L − 1 , orthophosphate 0.02–0.1 mg L − 1 . Calcium and alkalinity were maintained using regular additions of calcium chloride dihydrate and sodium bicarbonate, respectively. In addition, 30% of the aquarium water was exchanged three times a week using artificial seawater (ASW) produced from deionised water (reverse osmosis) and Zoo Mix sea salt (Tropic Marin GmbH, Germany), which was aerated for at least two days prior to use. Corals were provided with < 24 h old Artemia nauplii daily at a final density of 30–100 individuals L − 1 . Two LED fixtures (CoralCare generation 1, Philips, The Netherlands) provided a parabolic light regime with 12 h photoperiod (9:30–21:30 h) and a peak irradiance of 270 µmol quanta m − 2 s − 1 (∼400–700 nm). Irradiance was measured in situ with a Li-Cor using a LI-COR 192SA quantum underwater sensor (LI-COR, USA). No lunar light cycle was provided. Water flow was provided using a pump (Turbelle nanostream 6045, Tunze, Germany) with a flow capacity of 4500 L h − 1 . Water filtration consisted of a 20 W UV filter (AquaHolland, The Netherlands) and a protein skimmer (MCE 600, Deltec, Germany). The aquarium was stocked with several fish (1 Zebrasoma flavescens and 3 Ctenochaetus spp.) and invertebrate species (25 Trochus and 25 Turbo spp.) to graze turf algae. After 1 month of acclimation, coral colonies were placed in a larval collection system several times per week around 16:30 h. Coral larvae were collected from adult colonies using a custom-made flow-through system (Geertsma et al., 2022 ), which consisted of 8 one litre measuring beakers each holding an individual adult colony. Aquarium water was provided by a pump (Silence 300–3000 L h − 1 , Tunze, Germany) delivering aquarium seawater into a 20 mm PVC pipe which branched out to each beaker via a 6 mm plastic hose. The seawater flowed over the beaker handles into collection sieves (mesh size of 120 µm) of which the bottom half was submerged. From this water layer, coral larvae which had been released by their parent colonies overnight were collected between 10:30 and 11:00 a.m. The advantage of this setup is that larvae are not pumped through any pipes or tubing thus preventing damage. They also are provided with a continuous gentle supply of seawater of stable salinity and temperature, originating from and draining back into the main aquarium. Larvae were released five to eight days before full moon (Nov 30th –Dec 4th 2022) from adult colonies reared at Carus. Following the protocol of Geertsma et al. ( 2022 ), larvae were collected overnight and kept in closed 550 mL plastic containers in an aquarium (28°C) at a density of 50 larvae per 500 mL filtered artificial seawater (FASW) until the experiment began. FASW was produced as the ASW described above, after which it was filtered over a 0.5 µm membrane (Alapure, Waterfilterexpert.nl, The Netherlands) to remove bacteria. With plastic pipettes, healthy larvae were transferred to clean containers every other day, separating them from early settled and dead larvae. Three weeks prior to the heatwave experiment, PVC tiles (50 x 50 x 3 mm, L x W x H) were placed in the main holding aquarium with adult F. fragum colonies for growth of crustose coralline algae (CCA), which promotes coral larval settlement (Ritson-Williams et al. 2009 ). On December 6th, groups of five larvae were divided over closed 550 mL plastic containers with FASW and a biologically conditioned PVC tile to initiate larval settlement and metamorphosis. On December 7th, which is designated as 0 days post metamorphosis (DPM), N = 60 settlers on separate PVC tiles were obtained. Additional settlers (N = 24) were prepared in the same way for the baseline symbiont and chlorophyll a measurements (see below). Experimental design and setup The experiment was conducted at Carus ARF at Wageningen University and Research, the Netherlands, between December 2022 and January 2023. We used a 2 x 2 factorial design with F. fragum settlers exposed to either a control temperature (28°C) or a heatwave (28°C à 32.4°C) in combination with either a low prey (36 Artemia nauplii L − 1 ) or a high prey density (3600 Artemia nauplii L − 1 ) over a course of 48 days (Figure S1 ). Every other day, settlers were fed with low or high prey densities of < 24 h old Artemia salina (brine shrimp) nauplii. On the other days, the containers were cleaned and the seawater was fully renewed with FASW. Containers were rinsed with a sponge and, if present, algae were carefully removed from the PVC tile around the coral with a brush. Settlers were outside the water for only a few seconds when moved to a clean container. Nauplii cultures were provided by Carus ARF staff and densities were based on Geertsma et al. ( 2022 ). Zooplankton feeding started at 3 DPM, since it has been observed that this is the moment > 50% of F. fragum settlers start capturing prey (Geertsma et al. 2022 ). As the species-specific optimal water flow for prey capture by F. fragum settlers has been shown to be 0 cm s − 1 (Geertsma et al. 2022 ), pumps inside containers were turned off for one hour during feeding to allow settlers to capture nauplii. From 12 DPM onwards, the temperature of half the containers was gradually increased, peaking at 38 DPM (32.4°C) after which the temperature was kept fairly constant (Fig. 1 ). The average daily increase was 0.19°C. For each of the four treatment combinations, N = 15 independently operating 1.6L containers were used, with two randomly selected PVC tiles with a settler placed in each container. The plastic containers were covered by perforated plastic lids to limit evaporation. Temperature and salinity were measured daily with a HQ4100 conductivity meter (Hach, Germany) in eight alternating containers per temperature treatment and adjusted as needed. Out of each container (stocked with two settlers), one settler was used for PSII, growth, symbiont and chl a measurements, whereas the other was used to determine prey capture rates and survival. An additional bare PVC tile was included in each container to prevent settler tiles from moving (Figure S1 ). After allowing settlers a few days to firmly attach to the tiles, one small 7 W pump (170 L h − 1 , Eheim, Germany) was added to each container at 5 DPM for water circulation. Containers were partially submerged in four different aquaria, each equipped with a 300 W heater (Schego, Germany), a digital thermostat (TS125, H-Tronic, Germany) and a 23 W pump (Eheim, Germany) to uniformly distribute heat (Figure S1 ). Thermostat sensors were put in 1.6 L containers with FASW and one small 7 W pump. This was done to correctly measure and control the temperature experienced by the settlers, as the small flow pumps emitted some heat. Settlers were exposed to an irradiance of ~ 50–100 µmol m − 2 s − 1 using eight 170 W LED lamps (CoralCare LED Gen2, Philips Lighting, The Netherlands) with a day/night cycle of 12/12h. By rotating the containers after each cleaning, the average irradiance was calculated to be ~ 75 µmol m − 2 s − 1 . PSII yield Pulse Amplitude Modulation (PAM) fluorometry was used to non-intrusively determine the effective quantum yield of photosystem II within the symbionts using a Diving-PAM-II fluorometer (Heinz Walz GmbH, Germany). Measuring and saturating light were provided by a full spectrum lamp within the fluorometer and delivered to the settlers via a 2 mm diameter fibre optic cable. Effective PSII yield was measured 4 times per settler at approximately 2 mm distance directly from above as soon as the surface area of settlers was large enough (above ~ 3.1 mm 2 ) to retrieve a signal. Although settlers were large enough at 23 DPM, measurements were done from 30 DPM onwards as photography based coral size determination (see below) always trailed several days behind. From 33 DPM onwards, effective PSII yield was measured every other day after the aquarium lights were switched on, between 9:30 − 13:00, until it fell below a threshold of 0.55. This threshold was based on previous work, which showed an effective PSII yield of ~ 0.55 to be the point at which symbionts and chlorophyll a are rapidly lost (i.e. bleaching occurs, Wijgerde et al. 2020 ). For measurements, settlers were outside the water for approximately one minute in a climate-controlled laboratory set at ~ 26°C. Under light conditions (actinic light of the fluorometer was set to OFF), minimum (F) and maximum (Fm′) fluorescence were measured to calculate effective (ΔF/Fm′) PSII yield (Schreiber et al. 1986 ). Growth and survival During the experiment, survival was checked daily. Settlers were declared dead when no live tissue could be identified. Settlers were photographed weekly in a Petri dish filled with filtered 36 PSU artificial seawater (FASW) under an Amscope stereomicroscope (type) with Amscope software (x64, 4.11.19627.20210925) to determine their size over time. A miniature ruler was included for reference (Tool #23, Size 0.50 mm Micro-Scale, Electron Microscopy Sciences, Hatfield, USA). Settler surface area (mm 2 ) was determined by first calibrating photos using the included ruler and subsequently outlining the periphery of live tissue in ImageJ (v1.53t). During handling, settlers were outside of the water for a few seconds, inducing tentacle retraction. This minimised size variations owing to tentacle extension during surface area analysis of these small settlers. Prey capture rates During prey capture rate measurements, settlers were individually placed in a flow chamber (Wijgerde et al. 2012 ) and allowed to acclimate for 15 minutes. The chamber’s water flow rate was set to 5 cm s − 1 , a suitable value for prey capture as previously determined for F. fragum settlers (Geertsma et al. 2022 ). Settlers were fed newly hatched (< 24h) Artemia salina nauplii and filmed for 30 minutes with a camera mounted on a binocular (Amscope, United Kingdom) using imaging software (v4.11.21462, AmScope, United Kingdom) at a resolution of 3840x3040 pixels and 20 frames per second. Capture of nauplii was scored as such when they were attached to the settler for at least ten seconds (Wijgerde et al. 2011 ). After each prey capture measurement, the chamber was rinsed with FSW. Prey capture was measured at two moments: first when around 70% of settlers were able to capture prey (5–7 DPM, Geertsma et al. 2022 ) and second when effective PSII yield fell below the 0.55 threshold (40–42 DPM). As stated above (see PSII yield), this moment was based on falling below the predefined effective PSII yield threshold of 0.55 (Wijgerde et al. 2020 ). Temperature in the flow chamber was set to 28°C during the first round, as the heatwave had not started yet. During the second measurement round, temperature was either 28°C (control) or 32.4°C (heatwave maximum). As one incubation took up approximately one hour, multiple days were used to perform measurements. To account for this, different treatments were tested at random. Measurements took place between 07:31 and 22:20h. During this period, tentacles were extended and settlers were deemed able to capture prey. It is important to note that prey capture of F. fragum does not seem to be influenced by time of day (Geertsma, pers. obs.). Symbiont density and chlorophyll a At 0 DPM, a baseline determination of symbiont density and chlorophyll a fluorescence was established in the additionally collected settlers (N = 24). At 40 DPM, symbiont density and chlorophyll a fluorescence of settlers were measured by sacrificing half the individuals (i.e. one settler from each container where available, N = 12–15 for each of the 4 treatment combinations). As stated above (see PSII yield), this moment of sacrifice was based on falling below the predefined effective PSII yield threshold of 0.55 (Wijgerde et al. 2020 ). Settlers were first individually photographed as described above. Next, they were removed from their PVC tile using a scalpel and transferred to 1.5 mL Eppendorf tubes. Whole settlers were homogenised with a pellet mixer in 50 µL 9 PSU FASW for 1 minute. The pestle was rinsed in the Eppendorf to collect remaining tissue, with a final volume of 500 µL. After pipette homogenising, samples were centrifuged for 30 minutes at 13,000 RPM at room temperature. The supernatant containing fragmented coral tissue was decanted and stored at -20°C. The pellets containing symbionts were resuspended in 500 µL 9 PSU FASW and centrifuged again for 30 minutes at 13,000 RPM at room temperature, after which the supernatant was discarded, resulting in clean symbiont pellets. After homogenising these pellets in 250 µL FASW, total sample volumes were determined. Next, two 15 µL subsamples were transferred to a Fuchs-Rosenthal counting chamber. With a BH2 microscope (Olympus, Japan), pictures were taken at 100x magnification using the software 2nd Look (version 2.0.4.0 64-bit, IO Industries, Canada) and symbionts were counted with ImageJ (version Java 1.8.0_172 64-bit). Counts were corrected for sample volume, normalised for settler surface area and expressed as cells per mm 2 . To measure chlorophyll a fluorescence, a 200 µl sample obtained from each settler was pipetted into a flat-bottom 96-well plate (VWR, The Netherlands). The well plates were analysed for fluorescence on a CLARIOstar Plus plate reader (BMG Labtech, Germany). Sample excitation was done at 435 − 10 nm using the bottom optics, and emission measured at 678 − 20 nm with the dichroic filter set at 554 nm. For all readings, FASW was used in three separate wells to correct for background fluorescence. Readings were corrected for settler surface area. Statistical analysis All data were collected in Microsoft Excel and analysed in SPSS Statistics 28.0.1.0 (IBM Corp., Armonk, USA). To quantify cumulative thermal stress experienced by settlers exposed to the heatwave, we used degree heating weeks (DHW, Liu et al. 2003 , 2006 ). DHW’s were calculated by summing values during the 48-day experiment that were greater than or equal to 1°C above the Maximum Monthly Mean (i.e. SST ≥ MMM + 1°C) of 28°C as determined for our collection site ( https://coralreefwatch.noaa.gov/product/vs/timeseries/caribbean.php#abc_islands ) over the time period of 1985–1990 plus 1993 ( https://coralreefwatch.noaa.gov/product/5km/methodology.php#dhw ; Heron et al. 2015 ). Data of collected end points was checked for normality with a Shapiro-Wilk test and homogeneity of variances with Levene’s test. Data for settler size, prey capture rate and areal symbiont density were log 10 transformed to meet the assumptions for parametric testing, after which analysis of variance (ANOVA) was used. Temperature and feeding regime were treated as between subjects factors, and time as a within subjects factor. As sphericity for settler size data was violated (Mauchly’s Test, p > 0.05), degrees of freedom were Greenhouse-Geisser corrected. For PSII yield, nonparametric signed rank tests were used to compare differences over time (Wilcoxon) and between temperature groups (Mann-Whitney U). Simple effects contrasts were used to follow up significant interactive terms, which were always Bonferroni-adjusted to compensate for family-wise errors. To this end, p-values were automatically multiplied by SPSS with the number of post-hoc tests. P-values below α = 0.05 were considered statistically significant, except for the Mann-Whitney and Wilcoxon tests, for which α was divided by the number of post-hoc tests as automatic p-adjustment was unavailable. Data are presented as means ± 1 standard error of the mean (S.E.M.). Results General observations Seawater temperatures in the control containers were highly stable over time (Figure S2 ). The same was true for salinity, for both temperature regimes (Figure S3 ). After two weeks of acclimation, seawater temperature and cumulative temperature stress (expressed as degree heating weeks) of the heatwave treatments gradually increased as planned (Fig. 1 ). An overview of all measured end points, which will discussed in detail below, can be found in Table 1 . Table 1 Overview of observed effects of temperature and feeding regime on Favia fragum settlers. Arrows indicate direction of the effect (increase ↑, decrease ↓). Variable Temperature Feeding regime Additive/interactive Figure Effective PSII yield ↓ 25% at 8.8 DHW N/A N/A Figure 2 Settler size ↓ growth stagnation between 4.1–7.5 DHW No effect Interactive effect of feeding with time, ↑ 49% at 37 DPM Figure 3 Survival ↓ 0% at 14.4 DHW N/A N/A Figure 4 Prey capture rate ↓ 98%* at 9.4–10.7 DHW ↑ 1371%* Interactive, ↓ heat wave (94–98%**), no feeding effect during heat wave Figure 5 Symbiont density ↓ 54%* at 9.4 DHW No effect Interactive, ↓ heat wave (41–64%**), no feeding effect during heat wave Figure 6 Chlorophyll a ↓ 53%* at 9.4 DHW ↑ 21%* Interactive, ↓ heat wave (46–59%**), no feeding effect during heat wave Figure 7 *See interactive effect, **Percentages refer to feeding regimes of 36 and 3600 Artemia nauplii L − 1 , respectively. N/A: not applicable as the model used did not allow to test for an interactive effect (PSII yield) or because the data was treated as descriptive (survival). PSII yield The effective PSII yield of control settlers (~ 28°C) was stable over time, with a slight decrease between 37 and 39 DPM (days post metamorphosis), regardless of feeding regime (Fig. 2 , Table S1 ). PSII yield of the heatwave treatments declined between 35 and 37 DPM, and further between 37 and 39 DPM (Fig. 2 , Table S1 ). PSII yield of heatwave treatments was significantly lower compared to controls from 37 DPM onwards, corresponding to 31.8°C or 7.5 DHW (degree heating weeks), irrespective of feeding regime (Fig. 2 , Table S2 ). Growth Settler size gradually increased in all treatments (Fig. 3 ). An interactive effect of feeding regime and time on size was found (Fig. 3 , Table S3 ). This was reflected by 49% larger corals provided the higher feeding regime of 3600 prey L − 1 at 37 DPM, regardless of temperature treatment (Fig. 3 , Table S3 ). In addition, an interactive effect of temperature regime and time was found (Table S3 ), with growth of the heatwave treatments stagnating between 30 and 37 DPM, regardless of feeding regime (Fig. 3 , Table S3 ). This growth cessation corresponded to 31.0-31.8°C or 4.1–7.5 DHW. Growth of the control groups continued throughout the experiment, regardless of feeding regime (Fig. 3 , Table S3 ). Settler size of the heatwave treatments was 11% lower compared to controls at 37 DPM, corresponding to 31.8°C or 7.5 DHW, irrespective of feeding regime Fig. 3 , Table S3 ). Survival Settler survival of the controls ranged from 87 to 100% (Fig. 4 ). Two settlers within the high feeding treatment died early in the experiment around 10 DPM, after which survival was stable for the duration of the experiment. During the heatwave, survival decreased rapidly from 100% to 0% between 39 to 48 DPM, at a similar rate between both feeding regimes, corresponding to 32.3–32.4°C and 8.8–14.4 DHW. Bleaching and tissue necrosis was observed for both heatwave groups in this time interval (Figure S4 ). Prey capture rate Feeding regime and temperature affected prey capture rates of settlers over time (Fig. 5 ). Most importantly, the heatwave dramatically reduced prey capture: at 40–42 DPM, feeding rates were reduced by 94 and 98% relative to controls at 36 and 3600 prey L − 1 , respectively (Fig. 5 , Table S4 ). In addition, no effect of feeding regime on prey capture rates was found during the heatwave (Fig. 5 , Table S4 ). In contrast, elevating the prey density at control temperatures resulted in significantly higher prey capture rates, with a 1721 and 1633% increase at 5–7 and 40–42 DPM, respectively (Fig. 5 , Table S4 ). A similar result was found for the heatwave groups at 5–7 DPM before temperatures were elevated, with a 782% higher prey capture rate at 3600 prey L − 1 (Fig. 5 , Table S4 ). Time (i.e. settler size) also had a strong effect; within the control temperature, prey capture rates at 40–42 DPM were 411 and 387% higher as compared to those at 5–7 DPM at 36 and 3600 prey L − 1 , respectively (Fig. 5 , Table S4 ), when the settlers still were 6–9 times smaller. This was not the case within the heatwave treatments, of which prey capture rates during heat stress at 40–42 DPM declined with 88 and 92% at 36 and 3600 prey L − 1 , respectively (Fig. 5 , Table S4 ). Symbiont density and chlorophyll a fluorescence At 40 DPM, corresponding to 32.4°C and 9.4 DHW, half the corals of each treatment (one from each 1.6L container where available) were sacrificed. Control settlers at 36 prey L − 1 showed comparable areal symbiont densities as at the start of the experiment (Fig. 6 ). An interactive effect of feeding regime and temperature on symbiont densities was found (Table S5). This was reflected by a feeding effect for the control settlers only, with 40% higher symbiont densities at 3600 prey L − 1 (Fig. 6 , Table S5). Feeding regime had no effect on the heatwave-exposed settlers (Fig. 6 , Table S5). In addition, the heatwave resulted in a 41–64% loss of symbiont densities at feeding regimes of 36 and 3600 prey L − 1 , respectively (Fig. 6 , Table S5). At 40 DPM, settlers at the control temperature had 194–290% higher chlorophyll a fluorescence compared to the start of the experiment at 36 and 3600 prey L − 1 , respectively (Fig. 7 ). Beyond that, a pattern similar to symbiont densities was found, with an interactive effect of feeding regime and temperature on chlorophyll a fluorescence (Table S6). This was reflected by a feeding effect for the control settlers only, with 33% higher chlorophyll a fluorescence at 3600 prey L − 1 (Fig. 7 , Table S6). Feeding regime had no effect on chlorophyll a fluorescence of heatwave-exposed settlers (Fig. 7 , Table S6). Chlorophyll a fluorescence of the heatwave settlers was 46–59% lower than that of the control settlers at feeding regimes of 36 and 3600 prey L − 1 , respectively (Fig. 7 , Table S6). Discussion The aim of this study was to investigate whether zooplankton provisioning would affect thermotolerance of F. fragum settlers during a heatwave, and to link these observations to prey capture rates. Our expectation was that high prey availability would enhance settler survival and growth during a heatwave, as heterotrophic feeding would compensate for a loss of autotrophic input. However, we found that heavy plankton feeding did not mitigate the impact of a heatwave on F. fragum settlers. Despite promoting growth rates under normal temperatures, feeding Artemia salina nauplii at a high density did not enhance settler survival or growth during a heatwave, or delay a bleaching response. This is most likely due to the fact that settlers were unable to effectively feed during the heatwave, regardless of prey density, which will be discussed in detail below. The lack of enhanced settler thermotolerance despite heavy feeding became apparent from PSII yield, symbiont density and chlorophyll a fluorescence. At 31.8°C (7.5 DHW), settlers exhibited reduced PSII yield, irrespective of feeding regime. This was followed by loss of symbionts and chlorophyll a at 32.4°C (9.4 DHW), where feeding regime did not mitigate this response. Although the high feeding regime increased symbiont density and chlorophyll a fluorescence at normal temperatures, in line with previous research (Houlbrèque and Ferrier-Pagès 2009 ; Titlyanov et al. 2000 ), the heatwave reduced these end points by 64% and 59%, respectively, at the same feeding regime. This observed lack of enhanced thermotolerance was further reflected by settler survival, which rapidly declined between 32.3 and 32.4°C (8.8–14.4 DHW), regardless of prey density. Finally, growth rates further substantiated the lack of any beneficial feeding effect under temperature stress. Although the high feeding regime enhanced settler growth rates at normal temperatures, in agreement with a previous study involving F. fragum (Petersen et al. 2008 ), the heatwave resulted in stagnated growth between 31.0-31.8°C (4.1–7.5 DHW) under both feeding regimes. Despite growing under non-stressful elevated temperatures for almost four weeks, potentially benefiting calcification rates (Clausen and Roth 1975 ; Lough and Barnes 2000 ), settler size of the heatwave treatments generally was 27% lower compared to controls at 31.8°C (37 DPM or 7.5 DHW). The fact that growth already ceases between 31 and 31.8°C, just before PSII yield declines, suggests a direct negative temperature impact on the coral host itself, in addition to symbiosis dysfunction. Coral host heat stress responses include protein misfolding and increased DNA repair (Rädecker et al. 2021 ; reviewed by Helgoe et al. 2024 ), which may compromise growth rates. Another mechanism explaining growth stagnation is reduced photosynthate translocation by symbionts to the host coral under heat stress. Such reduced photosynthate transfer may be due to the incurred cost of cellular repair of heat-damaged DNA or D1 protein (Tremblay et al. 2016 ), or enhanced symbiont growth due to ammonium uptake from the catabolising host (Rädecker et al. 2021 ). The lack of a beneficial feeding effect on coral thermotolerance logically follows the observed impaired settler prey capture rates during the heatwave, which were reduced by 94 to 98% as compared to controls. For reef-building corals, it has been shown that they follow a type II saturating prey capture response under non-stressful conditions (Ferrier-Pages et al. 2003). This also held true for our settlers under control temperatures, with an ~ 18-fold prey capture increase at a 100-fold prey density increase. This high prey capture ability was previously observed by Geertsma et al. ( 2022 ). During the heatwave, however, prey capture rates collapsed. This was likely due to decreased mucus synthesis and energy status as photosynthesis was impaired, reflected by low PSII yield. Corals may secrete up to 50% of their autotrophically acquired carbon as mucus (Davies et al. 1984; Muscatine et al. 1984 ; Naumann et al. 2010a ), which in turn is vital to effective prey capture and thus supporting heterotrophic input (Goldberg 2018 ; Wijgerde et al. 2011 , 2013 ). When photosynthesis is impaired under thermal stress, reduced mucus production will likely negatively affect prey capture. Indeed, a study using adult Stylophora pistillata colonies found that several days of thermal stress dramatically reduced the coral’s mucus layer (Fitt et al. 2009 ). Related to this, Wijgerde et al. ( 2013 ) found that mucus-consuming epizoic flatworms (Naumann et al. 2010b ) reduced tentacle adhesion and prey capture by Galaxea fascicularis polyps. Unfortunately, our recorded videos did not provide sufficient detail to confidently show reduced prey adhesion to F. fragum tentacles under temperature stress. This warrants future study using a microscopic setup which allows to visualise the feeding behaviour of these small polyps in higher detail. In addition, such a setup may show that heat-stressed polyps exhibit reduced tentacle activity, possibly due to reduced energy (ATP) availability. Our videos did suggest such reduced tentacle activity, but we were unable to quantify this due to lack of detail. Although reef-building corals can maintain normal ATP levels under thermal stress (Kochman et al. 2021 ), our prolonged heatwave may have exhausted this capacity by depleting energy reserves (Grottoli et al. 2006 ). The coupling between auto- and heterotrophy is further substantiated by Love et al. ( 2025 ), who found that bleached corals exhibit reduced prey capture rates and lipid content. They suggested there may be an energetic cost to prey capture that normally is provided by the symbiont. Thus, when temperatures continued to increase above 31°C, both energy acquisition pathways became impaired, ultimately resulting in depletion of energy reserves, growth stagnation, tissue necrosis and mortality. This could be further substantiated by measuring lipid and fatty acid content ( sensu Love et al. 2025 ), which should be addressed in a future study. Our results contrast with those reported by Connolly et al. ( 2012 ), who found that feeding increases survival rates of Acropora intermedia during a heatwave and that it may aid in recovery. However, these authors used an acute, very short heatwave (< 1 DHW, https://coralreefwatch.noaa.gov ) rather than a more natural, prolonged one as in our study. A similar finding was reported based on a short-term, acute heatwave conducted by Ferrier-Pagès et al. ( 2010 ), who found that zooplankton supplementation prevented heat-induced PSII damage and bleaching of three coral species. They also found that one species, Stylophora pistillata , reduced its prey capture rates during thermal stress. Levas et al. ( 2016 ) found that Porites astreoides was able to increase its prey capture rate when in a bleached state during a mild heatwave (3–4 DHW compared to controls), but not during a second round a year later, suggesting that cumulative thermal stress disrupts feeding abilities. Furthermore, Grottoli et al. ( 2006 ) found that bleached Montipora capitata was able to meet its full metabolic demand through zooplankton capture, although they determined feeding rates after a two-week recovery period following experimental thermal bleaching. Finally, Borell and Bischof ( 2008 ) found that feeding sustains PSII yield of Stylophora pistillata during thermal stress, although their heat wave lasted only 10 days with a high daily rise of 2–3°C. In addition to the unnatural heat stress experiments outlined above, all these authors used adult colonies, which may behave differently from settlers. Rivera et al. ( 2023 ) arrived at a conclusion similar to ours, with a limited impact of feeding on thermotolerance of the temperate coral Oculina arbuscula . Although we cannot calculate DHW from their data, they applied a significant heatwave with a ~ 9 degree Celsius increase over a period of two weeks. They conclude that, amongst others, coral energy status prior to heat stress and stress duration could limit the beneficial impact of increased prey availability. The energy status of our settlers is likely to have been favourable, as the parent colonies we used were healthy and had been fed year-round under optimal aquaculture conditions. Thermal stress duration and intensity, expressed as DHW, are likely to be the decisive factor when assessing the role of heterotrophy in coral thermotolerance, and may explain discrepancies between studies. Taken together, repeated or prolonged heat stress, such as in our study, may result in a point of no return after which corals are too weakened to effectively feed. A future study should determine prey capture dynamics during a prolonged heatwave in more detail, for both early settlers as well as adult corals. This would identify the temperature/DHW tipping point after which plankton feeding is disrupted to such an extent that it can no longer offset the incurred loss of autotrophic input. Although the cumulative temperature stress applied in our study (14.4 DHW) seems excessive, NOAA data over the 2015–2025 period from our collection site show high values of up to 26 DHW ( https://coralreefwatch.noaa.gov/product/vs/timeseries/caribbean.php#abc_islands ; Muñiz-Castillo et al. 2019 ), owing to the 2023–2024 ENSO event. While this study provides valuable insight into the physiological response of early life-stage corals to temperature stress, it should be taken into account that our corals were fed solely with Artemia salina nauplii, which, while a common aquaculture food source, do not fully represent the nutritional and size complexity of natural reef diets (Palardy et al. 2006 ; Levas et al. 2016 ). Future research should incorporate natural prey types, such as copepods, crab zoea or amphipods, which may alter coral response due to different nutritional composition. It also would be interesting to include molecular or transcriptomic analyses to help clarify the mechanisms underlying the observed responses. For example, carbohydrate, fatty acid and protein levels of settler tissue were likely negatively impacted (Rivera et al. 2023 ; Love et al. 2025 ), which could explain the observed cessation of prey capture during the heatwave. While it is yet to be determined whether our results with F. fragum larvae that vertically inherit symbionts can be generalised to aposymbiotic settlers, this study provides a valuable framework for understanding early-stage coral responses to environmental stress. Notably, the health performance of the control groups, evident by high PSII yield, robust growth and active prey capture, demonstrates the potential of these juvenile corals when provided with species-specific favourable conditions. Our results establish a critical growth baseline for future stress response studies using F. fragum and provide new insight into the limited role of heterotrophy during heat stress of early life-stage corals. Zooplankton grazing, at least in the form of Artemia nauplii, is not a decisive factor in settler survival during realistically prolonged heat stress such as experienced during ENSO temperature anomalies. Identifying temperature thresholds beyond which various coral species, during early or later life stages, can no longer temporarily rely on heterotrophic feeding for survival remains an important avenue of research. Our findings identify a clear coupling between symbiont physiological status and host performance in F. fragum settlers. In addition, they provide an explanation for why recruitment of scleractinian corals on Caribbean reefs, which experience increasingly damaging ENSO events, is limited. Declarations Acknowledgements The authors wish to thank Joost Hamoen and the staff of Carus Aquatic Research Facility for their support. Funding This work was funded by Wageningen University and Research. Ethics declarations Conflict of interest The authors declare that they have no competing interests. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of organisms were followed. Consent to participate Not applicable. Consent to publish All authors consent to the publication of this manuscript. Data Availability Data collected and analyzed during this study are available as electronic supplementary material. Contributions TW designed and coordinated the study, performed data analyses, and wrote the manuscript. 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Near-bottom depletion of zooplankton over coral reefs: I: diurnal dynamics and size distribution. Coral Reefs 24: 75–85 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7584376","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513195841,"identity":"a5f80256-cb2a-4cf8-a376-1bebd1bedc94","order_by":0,"name":"Robbert C Geertsma","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Robbert","middleName":"C","lastName":"Geertsma","suffix":""},{"id":513195842,"identity":"0a4a2c56-625b-48c2-8049-11b8d4a18b30","order_by":1,"name":"Jasmijn Polinder","email":"","orcid":"","institution":"Radboud University Nijmegen","correspondingAuthor":false,"prefix":"","firstName":"Jasmijn","middleName":"","lastName":"Polinder","suffix":""},{"id":513195843,"identity":"116585cc-d961-4523-877d-a9c81649b65a","order_by":2,"name":"Dorien Groen","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Dorien","middleName":"","lastName":"Groen","suffix":""},{"id":513195844,"identity":"5b351191-e539-4709-af51-f3af9524d2a6","order_by":3,"name":"Diederik Padmos","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Diederik","middleName":"","lastName":"Padmos","suffix":""},{"id":513195845,"identity":"c4515b7e-31bb-4dca-8afe-21b7eeb049d5","order_by":4,"name":"Ronald Osinga","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Ronald","middleName":"","lastName":"Osinga","suffix":""},{"id":513195846,"identity":"63c7abd5-4313-41a2-a0f9-87cd586850a2","order_by":5,"name":"Albertinka J Murk","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Albertinka","middleName":"J","lastName":"Murk","suffix":""},{"id":513195847,"identity":"d1b788c4-12d7-4918-87ed-af2aa23b066a","order_by":6,"name":"Tim Wijgerde","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACZhBhwAYmGT5AhBrw6uBB1sI4AyzGSEALEtuAmYcYLfbs3AnMPAV8dv3Shzc+tm07nM8g3UjIYbwbmHkM2JJn9qUVG+e2HbZskDlIhJYcoBaDMzxm0rnbDhswSCQSqcX+DI/5b0tStNgZ8PCYMTMSpeUw74bDfwzYEiTOsBVL9v5LN2AjpIW9/+zGhzP+HLPn72He+OHHGWsDfonkA3i1gABQxTGEyWwE1UNAjT2RCkfBKBgFo2AkAgDQjjwmsAIVqQAAAABJRU5ErkJggg==","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":true,"prefix":"","firstName":"Tim","middleName":"","lastName":"Wijgerde","suffix":""}],"badges":[],"createdAt":"2025-09-10 15:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7584376/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7584376/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00338-026-02821-2","type":"published","date":"2026-02-05T15:59:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91135425,"identity":"32ffecb8-0ec5-445b-b9e6-2d7da8536888","added_by":"auto","created_at":"2025-09-12 03:13:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126603,"visible":true,"origin":"","legend":"\u003cp\u003eDaily temperatures (black line) and degree heating weeks (DHW, grey line) of the heat wave treatments over time. Post metamorphosis was defined as the moment at which coral planula larvae have settled onto substrates and formed settlers (primary polyps). Values are means (N=5 containers per treatment combination). Grey arrow: symbiont and chlorophyll \u003cem\u003ea\u003c/em\u003e baseline determination of newly formed settlers (N=24) at 0 DPM (Figures 6 and 7), black arrow: determination of settler prey capture rates (N=11-15) at 5-7 DPM (Figure 5), gradient arrow: determination of symbiont and chlorophyll \u003cem\u003ea\u003c/em\u003e during the heatwave (N=12-15) at 40 DPM (Figures 6 and 7) and of settler prey capture rates (N=11-15) at 40-42 DPM (Figure 5).\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/a5b23a83691a755a5b2f185d.jpeg"},{"id":91134594,"identity":"105d5189-ebe4-4941-af14-ceda518f6e7f","added_by":"auto","created_at":"2025-09-12 02:57:39","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98942,"visible":true,"origin":"","legend":"\u003cp\u003eEffective photosystem 2 (PSII) yield of the control (28 °C) and heatwave (28 → 32.4 °C) treatments, at prey concentrations of 36 prey L\u003csup\u003e-1\u003c/sup\u003e (black line) or 3600 prey L\u003csup\u003e-1\u003c/sup\u003e (grey line). Values are means ± S.E.M. (N=8-15 per treatment combination). Values with different superscripts and show significant differences over time and between temperature treatments, regardless of feeding regime (Wilcoxon/Mann-Whitney with Bonferroni-correction, p\u0026lt;0.010). Post metamorphosis was defined as the moment at which coral planula larvae have settled onto substrates and formed settlers (primary polyps).\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/bc4ec9c80920e937d62e1cec.jpeg"},{"id":91134597,"identity":"0f23c927-c3d5-4110-9493-cb486a591a24","added_by":"auto","created_at":"2025-09-12 02:57:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126081,"visible":true,"origin":"","legend":"\u003cp\u003eSettler size of the control (28 °C) and heatwave (28 → 32.4 °C) treatments, at prey concentrations of 36 prey L\u003csup\u003e-1\u003c/sup\u003e (black line) or 3600 prey L\u003csup\u003e-1\u003c/sup\u003e (grey line). Values are means ± S.E.M. (N=13-15 per treatment combination). Values with different superscripts show significant effects of temperature and feeding regimes at 37 DPM (p\u0026lt;0.05; simple effects contrasts with Bonferroni-adjusted p-values). For clarity, not all significant differences are shown. Post metamorphosis was defined as the moment at which coral planula larvae have settled onto substrates and formed settlers (primary polyps).\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/83ed9911f4a3587e403fff93.jpeg"},{"id":91135426,"identity":"713054a9-4ed2-4c6a-9d03-0b744764d7b6","added_by":"auto","created_at":"2025-09-12 03:13:39","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":92631,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of the control (28 °C) and heatwave (28 → 32.4 °C) treatments, at prey concentrations of 36 prey L\u003csup\u003e-1\u003c/sup\u003e (black line) or 3600 prey L\u003csup\u003e-1\u003c/sup\u003e (grey line). Values are means ± S.E.M. (N=0-15 per treatment combination). Post metamorphosis was defined as the moment at which coral planula larvae have settled onto substrates and formed settlers (primary polyps).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/4b129687d486e585ece0c9dd.jpeg"},{"id":91134621,"identity":"d17ef721-ea35-4afe-a9e9-a216c8d6b01d","added_by":"auto","created_at":"2025-09-12 02:57:40","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":93798,"visible":true,"origin":"","legend":"\u003cp\u003ePrey Capture Rate of the control (28 °C) and heatwave (28 → 32.4 °C) treatments, at prey concentrations of 36 prey L\u003csup\u003e-1\u003c/sup\u003e (black bars) or 3600 prey L\u003csup\u003e-1\u003c/sup\u003e (grey bars). \u0026nbsp;Values are means ± S.E.M. (N=11-15 per treatment combination), and were obtained between 5-7 DPM (~28°C) and 40-42 DPM (32.4°C or 9.4-10.7 DHW). Values with different superscripts differ significantly (p\u0026lt;0.05; simple effects contrasts with Bonferroni-adjusted p-values). Post metamorphosis was defined as the moment at which coral planula larvae have settled onto substrates and formed settlers (primary polyps).\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/25cf719875f565d3de0dde8a.jpeg"},{"id":91135427,"identity":"a3fda6ab-ed5b-47b3-a5af-8fec3b173f73","added_by":"auto","created_at":"2025-09-12 03:13:39","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":75048,"visible":true,"origin":"","legend":"\u003cp\u003eSymbiont density at the start of the experiment (0 DPM, light grey bar), and of the control (28 °C) and heatwave (28 → 32.4 °C) treatments, at prey concentrations of 36 prey L\u003csup\u003e-1\u003c/sup\u003e (black bars) or 3600 prey L\u003csup\u003e-1\u003c/sup\u003e (grey bars) at 40 DPM (32.4°C or 9.4 DHW). Values are means ± S.E.M. (N=24 for the baseline group, N=12-15 per treatment combination). Values with different superscripts differ significantly (p\u0026lt;0.05; simple effects contrasts with Bonferroni-adjusted p-values). Post metamorphosis was defined as the moment at which coral planula larvae have settled onto substrates and formed settlers (primary polyps).\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/640073a82dffe1aa3b7a0c87.jpeg"},{"id":91134615,"identity":"4d6c0ed8-6cd9-43ca-bc9a-165ce6a5b8a7","added_by":"auto","created_at":"2025-09-12 02:57:39","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":65174,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll\u003cem\u003e a\u003c/em\u003e fluorescence at the start of the experiment (0 DPM, light grey bar), and of the control (28 °C) and heatwave (28 → 32.4 °C) treatments, at prey concentrations of 36 prey L\u003csup\u003e-1\u003c/sup\u003e (black bars) or 3600 prey L\u003csup\u003e-1\u003c/sup\u003e (grey bars) at 40 DPM (32.4°C or 9.4 DHW). Values are means ± S.E.M. (N=24 for the starting group, N=12-15 per treatment combination). Values with different superscripts differ significantly (p\u0026lt;0.05; simple effects contrasts with Bonferroni-adjusted p-values). Post metamorphosis was defined as the moment at which coral planula larvae have settled onto substrates and formed settlers (primary polyps).\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/cdacd6c93de91243a867125d.jpeg"},{"id":102234361,"identity":"05157f1c-aeb2-4c40-9c4d-1a27ba41d4c4","added_by":"auto","created_at":"2026-02-09 16:10:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1479085,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/de59a119-2e44-4df7-a514-3a1941796e3a.pdf"},{"id":91134596,"identity":"2b0161d7-81dd-44c1-b878-1db7d1d4b767","added_by":"auto","created_at":"2025-09-12 02:57:39","extension":"sav","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20711,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation1.sav","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/beec0d4f4f470d04b55a948c.sav"},{"id":91134598,"identity":"d16a58a6-6935-4af7-bd0b-ded4160e5bff","added_by":"auto","created_at":"2025-09-12 02:57:39","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19019,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation2.csv","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/9c0977f3ba7d6107ce047c14.csv"},{"id":91134603,"identity":"2d819ef2-04f5-4016-8a30-d8c93c7db26f","added_by":"auto","created_at":"2025-09-12 02:57:39","extension":"sav","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3102,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation3.sav","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/1726244954bafa3e7fc80140.sav"},{"id":91134622,"identity":"4928bb80-a01a-4b8d-b22d-87675ea7de71","added_by":"auto","created_at":"2025-09-12 02:57:40","extension":"csv","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2257,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation4.csv","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/449a013a6e7b8907dbffce2b.csv"},{"id":91134868,"identity":"c2ffb4c1-b3e1-400b-b20b-a0dd5c60f05e","added_by":"auto","created_at":"2025-09-12 03:05:39","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":699895,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInfo.docx","url":"https://assets-eu.researchsquare.com/files/rs-7584376/v1/d967fa0f7470cec1ad5b8159.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zooplankton provisioning does not enhance thermotolerance and survival of Favia fragum settlers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCaribbean reefs are experiencing increasingly stronger heatwaves, leading to dramatic changes in their composition and functioning (Bove, Mudge and Bruno \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Reef-building scleractinian corals already live close to their upper thermal limit (Baker et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), with temperature anomalies resulting in coral bleaching, disease and mortality (Randall and van Woesik \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The 2023\u0026ndash;2024 El Ni\u0026ntilde;o bleaching event wreaked havoc amongst coral reefs worldwide, with unprecedented thermal stress recorded in the Caribbean and beyond (Hoegh-Guldberg et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Reimer et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Doherty et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Record values of degree heating weeks (DHW), a measure of cumulative thermal stress (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), were recently recorded on the Great Barrier Reef, Brazil, the Red Sea and the Caribbean, exceeding 20 DHW (Hoegh-Guldberg et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This is a sobering fact, as values of 8\u0026ndash;12 DHW and beyond are strongly correlated to widespread coral bleaching and mortality (Kleypas et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kayanne \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough climate-driven loss of coral reefs is reason for grave concern, there is evidence that at least some reef-building corals can temporarily shift from autotrophic to heterotrophic feeding during bleaching episodes for a duration of several months (Mies et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). During temperature-induced bleaching, some coral species enhance zooplankton feeding which may offset the incurred energy cost from autotrophic symbiont loss (Ferrier-Pag\u0026egrave;s et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Grottoli et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Levas et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This shift towards heterotrophic feeding may have mitigating effects on thermal stress during episodic heatwaves (Ferrier-Pag\u0026egrave;s et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Connolly et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which are increasing in frequency and severity (Grottoli et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Stable isotope analyses suggest that corals which rely more heavily on heterotrophic feeding have a higher thermotolerance, with more heterotrophic species tolerating up to almost twice the cumulative thermal stress as compared to autotrophic species (Conti-Jerpe et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is yet unclear, however, to what extent coral recruits are able to rely on heterotrophy during early life, thus immediately after metamorphosis and settlement. Thus, the potential of coral recruits to cope with thermal stress through a trophic shift currently remains unknown. This poses a critical knowledge gap as it may help explain why recruits on Caribbean reefs show low outgrowth to adult corals, hampering reef recovery (Edmunds \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHeterotrophic feeding may be a major driver of survival and growth in coral settlers (primary polyps), especially in species having horizontal symbiont transmission (Suzuki et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Until tissues are sufficiently colonised by dinoflagellate symbionts, utilisation of lipid reserves and heterotrophic feeding may be the only means by which settlers can meet their metabolic demand (Graham et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Geertsma et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This heterotrophic dependency would be exacerbated during episodic heatwaves. A notable coral species within this context is the massive Caribbean Golf ball coral, \u003cem\u003eFavia fragum\u003c/em\u003e. This species produces offspring capable of capturing zooplankton prey as early as 1 day post metamorphosis (DPM, Geertsma et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This heterotrophic capacity may be a strategy to rapidly increase energy availability for growth, potentially enhancing benthic competition and survival. It may also offset the loss of autotrophy during episodes of thermal stress and bleaching. Interestingly, \u003cem\u003eF. fragum\u003c/em\u003e shows stable recruitment rates across the Florida Keys (Harper et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which may be explained by its nutritional plasticity. Therefore, the aim of this study was to evaluate the potential mitigating role of heterotrophic feeding on thermotolerance of early life-stage \u003cem\u003eF. fragum\u003c/em\u003e. We investigated how zooplankton provisioning affected thermotolerance and performance (as growth and survival) of settlers during a heatwave, and linked these observations to prey capture rates. Settlers were exposed to a heatwave lasting 48 days, starting from 28\u0026deg;C and peaking at 32.4\u0026deg;C, with a daily increase of 0.19\u0026deg;C. Heat-exposed and control settlers were cultured under two feeding regimes, 36 and 3600 \u003cem\u003eArtemia salina\u003c/em\u003e nauplii per litre, as these densities are within one order of magnitude as those found under natural and aquaculture conditions, respectively (Roman et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Yahel et al. 2004; Geertsma et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We measured thermal stress as changes in effective yield of photosystem II (PSII) and bleaching as changes in symbiont densities and chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence. In addition, we measured survival, areal growth and prey capture rates. The expectation was that settlers showed higher thermotolerance at the high feeding regime, reflected by increased survival and growth during the heatwave, resulting from enhanced prey capture rates and subsequent nutrient intake.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCoral collection\u003c/h2\u003e\u003cp\u003eTo establish a breeding population for coral larvae, 15 adult colonies of the Caribbean Golf ball Coral (\u003cem\u003eFavia fragum\u003c/em\u003e Esper 1797) were collected by SCUBA between 3 and 5 m depth around the Sea Aquarium, Willemstad, Cura\u0026ccedil;ao in July 2019. These corals were hand-picked by removal with a small chisel, labelled, and individually placed in zip lock bags underwater. After collection, corals were transported to CARMABI Institute (Willemstad, Cura\u0026ccedil;ao) and placed in aquaria with running seawater for acclimation. For transport to Wageningen University, corals were individually placed in 1 L Ziplock bags, with 30/70% seawater/air. A secondary zip lock bag was placed around the primary bag, to increase isolation and security. All bags were stacked in a 30 L isolated box and shipped in a heated cargo hold. Collection was permitted by the CARMABI institute, and transport was done under CITES export permit no. 19CW002 and import permit no. 19NL270335/11. After transport, corals were temperature-acclimated over a two-hour period to 28\u0026deg;C in a 300 L aquarium at CARUS Aquatic Research Facility (ARF) of Wageningen University (see \u003cem\u003eCoral husbandry and larval collection\u003c/em\u003e for details). Transport water was discarded.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCoral husbandry and larval collection\u003c/h3\u003e\n\u003cp\u003eCorals were maintained in a 300 L glass aquarium with the following daily-checked target parameters: temperature 28\u0026deg;C, Salinity 36 PSU, alkalinity 2.5 mEq L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, calcium 420 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, nitrate 0\u0026ndash;1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, orthophosphate 0.02\u0026ndash;0.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Calcium and alkalinity were maintained using regular additions of calcium chloride dihydrate and sodium bicarbonate, respectively. In addition, 30% of the aquarium water was exchanged three times a week using artificial seawater (ASW) produced from deionised water (reverse osmosis) and Zoo Mix sea salt (Tropic Marin GmbH, Germany), which was aerated for at least two days prior to use. Corals were provided with \u0026lt;\u0026thinsp;24 h old \u003cem\u003eArtemia\u003c/em\u003e nauplii daily at a final density of 30\u0026ndash;100 individuals L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Two LED fixtures (CoralCare generation 1, Philips, The Netherlands) provided a parabolic light regime with 12 h photoperiod (9:30\u0026ndash;21:30 h) and a peak irradiance of 270 \u0026micro;mol quanta m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u0026sim;400\u0026ndash;700 nm). Irradiance was measured \u003cem\u003ein situ\u003c/em\u003e with a Li-Cor using a LI-COR 192SA quantum underwater sensor (LI-COR, USA). No lunar light cycle was provided. Water flow was provided using a pump (Turbelle nanostream 6045, Tunze, Germany) with a flow capacity of 4500 L h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Water filtration consisted of a 20 W UV filter (AquaHolland, The Netherlands) and a protein skimmer (MCE 600, Deltec, Germany). The aquarium was stocked with several fish (1 \u003cem\u003eZebrasoma flavescens\u003c/em\u003e and 3 \u003cem\u003eCtenochaetus\u003c/em\u003e spp.) and invertebrate species (25 \u003cem\u003eTrochus\u003c/em\u003e and 25 \u003cem\u003eTurbo\u003c/em\u003e spp.) to graze turf algae. After 1 month of acclimation, coral colonies were placed in a larval collection system several times per week around 16:30 h.\u003c/p\u003e\u003cp\u003eCoral larvae were collected from adult colonies using a custom-made flow-through system (Geertsma et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which consisted of 8 one litre measuring beakers each holding an individual adult colony. Aquarium water was provided by a pump (Silence 300\u0026ndash;3000 L h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Tunze, Germany) delivering aquarium seawater into a 20 mm PVC pipe which branched out to each beaker via a 6 mm plastic hose. The seawater flowed over the beaker handles into collection sieves (mesh size of 120 \u0026micro;m) of which the bottom half was submerged. From this water layer, coral larvae which had been released by their parent colonies overnight were collected between 10:30 and 11:00 a.m. The advantage of this setup is that larvae are not pumped through any pipes or tubing thus preventing damage. They also are provided with a continuous gentle supply of seawater of stable salinity and temperature, originating from and draining back into the main aquarium.\u003c/p\u003e\u003cp\u003eLarvae were released five to eight days before full moon (Nov 30th \u0026ndash;Dec 4th 2022) from adult colonies reared at Carus. Following the protocol of Geertsma et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), larvae were collected overnight and kept in closed 550 mL plastic containers in an aquarium (28\u0026deg;C) at a density of 50 larvae per 500 mL filtered artificial seawater (FASW) until the experiment began. FASW was produced as the ASW described above, after which it was filtered over a 0.5 \u0026micro;m membrane (Alapure, Waterfilterexpert.nl, The Netherlands) to remove bacteria. With plastic pipettes, healthy larvae were transferred to clean containers every other day, separating them from early settled and dead larvae. Three weeks prior to the heatwave experiment, PVC tiles (50 x 50 x 3 mm, L x W x H) were placed in the main holding aquarium with adult \u003cem\u003eF. fragum\u003c/em\u003e colonies for growth of crustose coralline algae (CCA), which promotes coral larval settlement (Ritson-Williams et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). On December 6th, groups of five larvae were divided over closed 550 mL plastic containers with FASW and a biologically conditioned PVC tile to initiate larval settlement and metamorphosis. On December 7th, which is designated as 0 days post metamorphosis (DPM), N\u0026thinsp;=\u0026thinsp;60 settlers on separate PVC tiles were obtained. Additional settlers (N\u0026thinsp;=\u0026thinsp;24) were prepared in the same way for the baseline symbiont and chlorophyll \u003cem\u003ea\u003c/em\u003e measurements (see below).\u003c/p\u003e\n\u003ch3\u003eExperimental design and setup\u003c/h3\u003e\n\u003cp\u003eThe experiment was conducted at Carus ARF at Wageningen University and Research, the Netherlands, between December 2022 and January 2023. We used a 2 x 2 factorial design with \u003cem\u003eF. fragum\u003c/em\u003e settlers exposed to either a control temperature (28\u0026deg;C) or a heatwave (28\u0026deg;C \u0026agrave; 32.4\u0026deg;C) in combination with either a low prey (36 \u003cem\u003eArtemia\u003c/em\u003e nauplii L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) or a high prey density (3600 \u003cem\u003eArtemia\u003c/em\u003e nauplii L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) over a course of 48 days (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Every other day, settlers were fed with low or high prey densities of \u0026lt;\u0026thinsp;24 h old \u003cem\u003eArtemia salina\u003c/em\u003e (brine shrimp) nauplii. On the other days, the containers were cleaned and the seawater was fully renewed with FASW. Containers were rinsed with a sponge and, if present, algae were carefully removed from the PVC tile around the coral with a brush. Settlers were outside the water for only a few seconds when moved to a clean container. Nauplii cultures were provided by Carus ARF staff and densities were based on Geertsma et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Zooplankton feeding started at 3 DPM, since it has been observed that this is the moment\u0026thinsp;\u0026gt;\u0026thinsp;50% of \u003cem\u003eF. fragum\u003c/em\u003e settlers start capturing prey (Geertsma et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As the species-specific optimal water flow for prey capture by \u003cem\u003eF. fragum\u003c/em\u003e settlers has been shown to be 0 cm s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Geertsma et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), pumps inside containers were turned off for one hour during feeding to allow settlers to capture nauplii. From 12 DPM onwards, the temperature of half the containers was gradually increased, peaking at 38 DPM (32.4\u0026deg;C) after which the temperature was kept fairly constant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The average daily increase was 0.19\u0026deg;C. For each of the four treatment combinations, N\u0026thinsp;=\u0026thinsp;15 independently operating 1.6L containers were used, with two randomly selected PVC tiles with a settler placed in each container. The plastic containers were covered by perforated plastic lids to limit evaporation. Temperature and salinity were measured daily with a HQ4100 conductivity meter (Hach, Germany) in eight alternating containers per temperature treatment and adjusted as needed. Out of each container (stocked with two settlers), one settler was used for PSII, growth, symbiont and chl \u003cem\u003ea\u003c/em\u003e measurements, whereas the other was used to determine prey capture rates and survival. An additional bare PVC tile was included in each container to prevent settler tiles from moving (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). After allowing settlers a few days to firmly attach to the tiles, one small 7 W pump (170 L h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Eheim, Germany) was added to each container at 5 DPM for water circulation. Containers were partially submerged in four different aquaria, each equipped with a 300 W heater (Schego, Germany), a digital thermostat (TS125, H-Tronic, Germany) and a 23 W pump (Eheim, Germany) to uniformly distribute heat (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Thermostat sensors were put in 1.6 L containers with FASW and one small 7 W pump. This was done to correctly measure and control the temperature experienced by the settlers, as the small flow pumps emitted some heat. Settlers were exposed to an irradiance of ~\u0026thinsp;50\u0026ndash;100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using eight 170 W LED lamps (CoralCare LED Gen2, Philips Lighting, The Netherlands) with a day/night cycle of 12/12h. By rotating the containers after each cleaning, the average irradiance was calculated to be ~\u0026thinsp;75 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePSII yield\u003c/h3\u003e\n\u003cp\u003ePulse Amplitude Modulation (PAM) fluorometry was used to non-intrusively determine the effective quantum yield of photosystem II within the symbionts using a Diving-PAM-II fluorometer (Heinz Walz GmbH, Germany). Measuring and saturating light were provided by a full spectrum lamp within the fluorometer and delivered to the settlers via a 2 mm diameter fibre optic cable. Effective PSII yield was measured 4 times per settler at approximately 2 mm distance directly from above as soon as the surface area of settlers was large enough (above ~\u0026thinsp;3.1 mm\u003csup\u003e2\u003c/sup\u003e) to retrieve a signal. Although settlers were large enough at 23 DPM, measurements were done from 30 DPM onwards as photography based coral size determination (see below) always trailed several days behind. From 33 DPM onwards, effective PSII yield was measured every other day after the aquarium lights were switched on, between 9:30\u0026thinsp;\u0026minus;\u0026thinsp;13:00, until it fell below a threshold of 0.55. This threshold was based on previous work, which showed an effective PSII yield of ~\u0026thinsp;0.55 to be the point at which symbionts and chlorophyll \u003cem\u003ea\u003c/em\u003e are rapidly lost (i.e. bleaching occurs, Wijgerde et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For measurements, settlers were outside the water for approximately one minute in a climate-controlled laboratory set at ~\u0026thinsp;26\u0026deg;C. Under light conditions (actinic light of the fluorometer was set to OFF), minimum (F) and maximum (Fm\u0026prime;) fluorescence were measured to calculate effective (ΔF/Fm\u0026prime;) PSII yield (Schreiber et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1986\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGrowth and survival\u003c/h3\u003e\n\u003cp\u003eDuring the experiment, survival was checked daily. Settlers were declared dead when no live tissue could be identified. Settlers were photographed weekly in a Petri dish filled with filtered 36 PSU artificial seawater (FASW) under an Amscope stereomicroscope (type) with Amscope software (x64, 4.11.19627.20210925) to determine their size over time. A miniature ruler was included for reference (Tool #23, Size 0.50 mm Micro-Scale, Electron Microscopy Sciences, Hatfield, USA). Settler surface area (mm\u003csup\u003e2\u003c/sup\u003e) was determined by first calibrating photos using the included ruler and subsequently outlining the periphery of live tissue in ImageJ (v1.53t). During handling, settlers were outside of the water for a few seconds, inducing tentacle retraction. This minimised size variations owing to tentacle extension during surface area analysis of these small settlers.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePrey capture rates\u003c/h2\u003e\u003cp\u003eDuring prey capture rate measurements, settlers were individually placed in a flow chamber (Wijgerde et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and allowed to acclimate for 15 minutes. The chamber\u0026rsquo;s water flow rate was set to 5 cm s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a suitable value for prey capture as previously determined for \u003cem\u003eF. fragum\u003c/em\u003e settlers (Geertsma et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Settlers were fed newly hatched (\u0026lt;\u0026thinsp;24h) \u003cem\u003eArtemia salina\u003c/em\u003e nauplii and filmed for 30 minutes with a camera mounted on a binocular (Amscope, United Kingdom) using imaging software (v4.11.21462, AmScope, United Kingdom) at a resolution of 3840x3040 pixels and 20 frames per second. Capture of nauplii was scored as such when they were attached to the settler for at least ten seconds (Wijgerde et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). After each prey capture measurement, the chamber was rinsed with FSW. Prey capture was measured at two moments: first when around 70% of settlers were able to capture prey (5\u0026ndash;7 DPM, Geertsma et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and second when effective PSII yield fell below the 0.55 threshold (40\u0026ndash;42 DPM). As stated above (see PSII yield), this moment was based on falling below the predefined effective PSII yield threshold of 0.55 (Wijgerde et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Temperature in the flow chamber was set to 28\u0026deg;C during the first round, as the heatwave had not started yet. During the second measurement round, temperature was either 28\u0026deg;C (control) or 32.4\u0026deg;C (heatwave maximum). As one incubation took up approximately one hour, multiple days were used to perform measurements. To account for this, different treatments were tested at random. Measurements took place between 07:31 and 22:20h. During this period, tentacles were extended and settlers were deemed able to capture prey. It is important to note that prey capture of \u003cem\u003eF. fragum\u003c/em\u003e does not seem to be influenced by time of day (Geertsma, pers. obs.).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSymbiont density and chlorophyll\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 0 DPM, a baseline determination of symbiont density and chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence was established in the additionally collected settlers (N\u0026thinsp;=\u0026thinsp;24). At 40 DPM, symbiont density and chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence of settlers were measured by sacrificing half the individuals (i.e. one settler from each container where available, N\u0026thinsp;=\u0026thinsp;12\u0026ndash;15 for each of the 4 treatment combinations). As stated above (see PSII yield), this moment of sacrifice was based on falling below the predefined effective PSII yield threshold of 0.55 (Wijgerde et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Settlers were first individually photographed as described above. Next, they were removed from their PVC tile using a scalpel and transferred to 1.5 mL Eppendorf tubes. Whole settlers were homogenised with a pellet mixer in 50 \u0026micro;L 9 PSU FASW for 1 minute. The pestle was rinsed in the Eppendorf to collect remaining tissue, with a final volume of 500 \u0026micro;L. After pipette homogenising, samples were centrifuged for 30 minutes at 13,000 RPM at room temperature. The supernatant containing fragmented coral tissue was decanted and stored at -20\u0026deg;C. The pellets containing symbionts were resuspended in 500 \u0026micro;L 9 PSU FASW and centrifuged again for 30 minutes at 13,000 RPM at room temperature, after which the supernatant was discarded, resulting in clean symbiont pellets. After homogenising these pellets in 250 \u0026micro;L FASW, total sample volumes were determined. Next, two 15 \u0026micro;L subsamples were transferred to a Fuchs-Rosenthal counting chamber. With a BH2 microscope (Olympus, Japan), pictures were taken at 100x magnification using the software 2nd Look (version 2.0.4.0 64-bit, IO Industries, Canada) and symbionts were counted with ImageJ (version Java 1.8.0_172 64-bit). Counts were corrected for sample volume, normalised for settler surface area and expressed as cells per mm\u003csup\u003e2\u003c/sup\u003e. To measure chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence, a 200 \u0026micro;l sample obtained from each settler was pipetted into a flat-bottom 96-well plate (VWR, The Netherlands). The well plates were analysed for fluorescence on a CLARIOstar Plus plate reader (BMG Labtech, Germany). Sample excitation was done at 435\u0026thinsp;\u0026minus;\u0026thinsp;10 nm using the bottom optics, and emission measured at 678\u0026thinsp;\u0026minus;\u0026thinsp;20 nm with the dichroic filter set at 554 nm. For all readings, FASW was used in three separate wells to correct for background fluorescence. Readings were corrected for settler surface area.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data were collected in Microsoft Excel and analysed in SPSS Statistics 28.0.1.0 (IBM Corp., Armonk, USA). To quantify cumulative thermal stress experienced by settlers exposed to the heatwave, we used degree heating weeks (DHW, Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). DHW\u0026rsquo;s were calculated by summing values during the 48-day experiment that were greater than or equal to 1\u0026deg;C above the Maximum Monthly Mean (i.e. SST\u0026thinsp;\u0026ge;\u0026thinsp;MMM\u0026thinsp;+\u0026thinsp;1\u0026deg;C) of 28\u0026deg;C as determined for our collection site (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://coralreefwatch.noaa.gov/product/vs/timeseries/caribbean.php#abc_islands\u003c/span\u003e\u003cspan address=\"https://coralreefwatch.noaa.gov/product/vs/timeseries/caribbean.php#abc_islands\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) over the time period of 1985\u0026ndash;1990 plus 1993 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://coralreefwatch.noaa.gov/product/5km/methodology.php#dhw\u003c/span\u003e\u003cspan address=\"https://coralreefwatch.noaa.gov/product/5km/methodology.php#dhw\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Heron et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Data of collected end points was checked for normality with a Shapiro-Wilk test and homogeneity of variances with Levene\u0026rsquo;s test. Data for settler size, prey capture rate and areal symbiont density were log\u003csub\u003e10\u003c/sub\u003e transformed to meet the assumptions for parametric testing, after which analysis of variance (ANOVA) was used. Temperature and feeding regime were treated as between subjects factors, and time as a within subjects factor. As sphericity for settler size data was violated (Mauchly\u0026rsquo;s Test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), degrees of freedom were Greenhouse-Geisser corrected. For PSII yield, nonparametric signed rank tests were used to compare differences over time (Wilcoxon) and between temperature groups (Mann-Whitney U). Simple effects contrasts were used to follow up significant interactive terms, which were always Bonferroni-adjusted to compensate for family-wise errors. To this end, p-values were automatically multiplied by SPSS with the number of \u003cem\u003epost-hoc\u003c/em\u003e tests. P-values below α\u0026thinsp;=\u0026thinsp;0.05 were considered statistically significant, except for the Mann-Whitney and Wilcoxon tests, for which α was divided by the number of \u003cem\u003epost-hoc\u003c/em\u003e tests as automatic p-adjustment was unavailable. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;1 standard error of the mean (S.E.M.).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eGeneral observations\u003c/h2\u003e\u003cp\u003eSeawater temperatures in the control containers were highly stable over time (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The same was true for salinity, for both temperature regimes (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). After two weeks of acclimation, seawater temperature and cumulative temperature stress (expressed as degree heating weeks) of the heatwave treatments gradually increased as planned (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). An overview of all measured end points, which will discussed in detail below, can be found in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOverview of observed effects of temperature and feeding regime on \u003cem\u003eFavia fragum\u003c/em\u003e settlers. Arrows indicate direction of the effect (increase \u0026uarr;, decrease \u0026darr;).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemperature\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFeeding regime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAdditive/interactive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFigure\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEffective PSII yield\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026darr; 25% at 8.8 DHW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSettler size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026darr; growth stagnation between 4.1\u0026ndash;7.5 DHW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInteractive effect of feeding with time, \u0026uarr; 49% at 37 DPM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSurvival\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026darr; 0% at 14.4 DHW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrey capture rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026darr; 98%* at 9.4\u0026ndash;10.7 DHW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026uarr; 1371%*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInteractive, \u0026darr; heat wave (94\u0026ndash;98%**),\u003c/p\u003e\u003cp\u003eno feeding effect during heat wave\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSymbiont density\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026darr; 54%* at 9.4 DHW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInteractive, \u0026darr; heat wave (41\u0026ndash;64%**), no feeding effect during heat wave\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026darr; 53%* at 9.4 DHW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026uarr; 21%*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInteractive, \u0026darr; heat wave (46\u0026ndash;59%**), no feeding effect during heat wave\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e*See interactive effect, **Percentages refer to feeding regimes of 36 and 3600 Artemia nauplii L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. N/A: not applicable as the model used did not allow to test for an interactive effect (PSII yield) or because the data was treated as descriptive (survival).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePSII yield\u003c/h2\u003e\u003cp\u003eThe effective PSII yield of control settlers (~\u0026thinsp;28\u0026deg;C) was stable over time, with a slight decrease between 37 and 39 DPM (days post metamorphosis), regardless of feeding regime (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). PSII yield of the heatwave treatments declined between 35 and 37 DPM, and further between 37 and 39 DPM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). PSII yield of heatwave treatments was significantly lower compared to controls from 37 DPM onwards, corresponding to 31.8\u0026deg;C or 7.5 DHW (degree heating weeks), irrespective of feeding regime (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eGrowth\u003c/h2\u003e\u003cp\u003eSettler size gradually increased in all treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). An interactive effect of feeding regime and time on size was found (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). This was reflected by 49% larger corals provided the higher feeding regime of 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 37 DPM, regardless of temperature treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). In addition, an interactive effect of temperature regime and time was found (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), with growth of the heatwave treatments stagnating between 30 and 37 DPM, regardless of feeding regime (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). This growth cessation corresponded to 31.0-31.8\u0026deg;C or 4.1\u0026ndash;7.5 DHW. Growth of the control groups continued throughout the experiment, regardless of feeding regime (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Settler size of the heatwave treatments was 11% lower compared to controls at 37 DPM, corresponding to 31.8\u0026deg;C or 7.5 DHW, irrespective of feeding regime Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSurvival\u003c/h2\u003e\u003cp\u003eSettler survival of the controls ranged from 87 to 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Two settlers within the high feeding treatment died early in the experiment around 10 DPM, after which survival was stable for the duration of the experiment. During the heatwave, survival decreased rapidly from 100% to 0% between 39 to 48 DPM, at a similar rate between both feeding regimes, corresponding to 32.3\u0026ndash;32.4\u0026deg;C and 8.8\u0026ndash;14.4 DHW. Bleaching and tissue necrosis was observed for both heatwave groups in this time interval (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePrey capture rate\u003c/h2\u003e\u003cp\u003eFeeding regime and temperature affected prey capture rates of settlers over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Most importantly, the heatwave dramatically reduced prey capture: at 40\u0026ndash;42 DPM, feeding rates were reduced by 94 and 98% relative to controls at 36 and 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). In addition, no effect of feeding regime on prey capture rates was found during the heatwave (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). In contrast, elevating the prey density at control temperatures resulted in significantly higher prey capture rates, with a 1721 and 1633% increase at 5\u0026ndash;7 and 40\u0026ndash;42 DPM, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). A similar result was found for the heatwave groups at 5\u0026ndash;7 DPM before temperatures were elevated, with a 782% higher prey capture rate at 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Time (i.e. settler size) also had a strong effect; within the control temperature, prey capture rates at 40\u0026ndash;42 DPM were 411 and 387% higher as compared to those at 5\u0026ndash;7 DPM at 36 and 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), when the settlers still were 6\u0026ndash;9 times smaller. This was not the case within the heatwave treatments, of which prey capture rates during heat stress at 40\u0026ndash;42 DPM declined with 88 and 92% at 36 and 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSymbiont density and chlorophyll\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003efluorescence\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 40 DPM, corresponding to 32.4\u0026deg;C and 9.4 DHW, half the corals of each treatment (one from each 1.6L container where available) were sacrificed. Control settlers at 36 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e showed comparable areal symbiont densities as at the start of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). An interactive effect of feeding regime and temperature on symbiont densities was found (Table S5). This was reflected by a feeding effect for the control settlers only, with 40% higher symbiont densities at 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table S5). Feeding regime had no effect on the heatwave-exposed settlers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table S5). In addition, the heatwave resulted in a 41\u0026ndash;64% loss of symbiont densities at feeding regimes of 36 and 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table S5).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt 40 DPM, settlers at the control temperature had 194\u0026ndash;290% higher chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence compared to the start of the experiment at 36 and 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Beyond that, a pattern similar to symbiont densities was found, with an interactive effect of feeding regime and temperature on chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence (Table S6). This was reflected by a feeding effect for the control settlers only, with 33% higher chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence at 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Table S6). Feeding regime had no effect on chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence of heatwave-exposed settlers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Table S6). Chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence of the heatwave settlers was 46\u0026ndash;59% lower than that of the control settlers at feeding regimes of 36 and 3600 prey L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Table S6).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aim of this study was to investigate whether zooplankton provisioning would affect thermotolerance \u003cem\u003eof F. fragum\u003c/em\u003e settlers during a heatwave, and to link these observations to prey capture rates. Our expectation was that high prey availability would enhance settler survival and growth during a heatwave, as heterotrophic feeding would compensate for a loss of autotrophic input. However, we found that heavy plankton feeding did not mitigate the impact of a heatwave on \u003cem\u003eF. fragum\u003c/em\u003e settlers. Despite promoting growth rates under normal temperatures, feeding \u003cem\u003eArtemia salina\u003c/em\u003e nauplii at a high density did not enhance settler survival or growth during a heatwave, or delay a bleaching response. This is most likely due to the fact that settlers were unable to effectively feed during the heatwave, regardless of prey density, which will be discussed in detail below.\u003c/p\u003e\u003cp\u003eThe lack of enhanced settler thermotolerance despite heavy feeding became apparent from PSII yield, symbiont density and chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence. At 31.8\u0026deg;C (7.5 DHW), settlers exhibited reduced PSII yield, irrespective of feeding regime. This was followed by loss of symbionts and chlorophyll \u003cem\u003ea\u003c/em\u003e at 32.4\u0026deg;C (9.4 DHW), where feeding regime did not mitigate this response. Although the high feeding regime increased symbiont density and chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence at normal temperatures, in line with previous research (Houlbr\u0026egrave;que and Ferrier-Pag\u0026egrave;s \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Titlyanov et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), the heatwave reduced these end points by 64% and 59%, respectively, at the same feeding regime. This observed lack of enhanced thermotolerance was further reflected by settler survival, which rapidly declined between 32.3 and 32.4\u0026deg;C (8.8\u0026ndash;14.4 DHW), regardless of prey density. Finally, growth rates further substantiated the lack of any beneficial feeding effect under temperature stress. Although the high feeding regime enhanced settler growth rates at normal temperatures, in agreement with a previous study involving \u003cem\u003eF. fragum\u003c/em\u003e (Petersen et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), the heatwave resulted in stagnated growth between 31.0-31.8\u0026deg;C (4.1\u0026ndash;7.5 DHW) under both feeding regimes. Despite growing under non-stressful elevated temperatures for almost four weeks, potentially benefiting calcification rates (Clausen and Roth \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Lough and Barnes \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), settler size of the heatwave treatments generally was 27% lower compared to controls at 31.8\u0026deg;C (37 DPM or 7.5 DHW). The fact that growth already ceases between 31 and 31.8\u0026deg;C, just before PSII yield declines, suggests a direct negative temperature impact on the coral host itself, in addition to symbiosis dysfunction. Coral host heat stress responses include protein misfolding and increased DNA repair (R\u0026auml;decker et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; reviewed by Helgoe et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which may compromise growth rates. Another mechanism explaining growth stagnation is reduced photosynthate translocation by symbionts to the host coral under heat stress. Such reduced photosynthate transfer may be due to the incurred cost of cellular repair of heat-damaged DNA or D1 protein (Tremblay et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), or enhanced symbiont growth due to ammonium uptake from the catabolising host (R\u0026auml;decker et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe lack of a beneficial feeding effect on coral thermotolerance logically follows the observed impaired settler prey capture rates during the heatwave, which were reduced by 94 to 98% as compared to controls. For reef-building corals, it has been shown that they follow a type II saturating prey capture response under non-stressful conditions (Ferrier-Pages et al. 2003). This also held true for our settlers under control temperatures, with an ~\u0026thinsp;18-fold prey capture increase at a 100-fold prey density increase. This high prey capture ability was previously observed by Geertsma et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). During the heatwave, however, prey capture rates collapsed. This was likely due to decreased mucus synthesis and energy status as photosynthesis was impaired, reflected by low PSII yield. Corals may secrete up to 50% of their autotrophically acquired carbon as mucus (Davies et al. 1984; Muscatine et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Naumann et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e), which in turn is vital to effective prey capture and thus supporting heterotrophic input (Goldberg \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wijgerde et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). When photosynthesis is impaired under thermal stress, reduced mucus production will likely negatively affect prey capture. Indeed, a study using adult \u003cem\u003eStylophora pistillata\u003c/em\u003e colonies found that several days of thermal stress dramatically reduced the coral\u0026rsquo;s mucus layer (Fitt et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Related to this, Wijgerde et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) found that mucus-consuming epizoic flatworms (Naumann et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e) reduced tentacle adhesion and prey capture by \u003cem\u003eGalaxea fascicularis\u003c/em\u003e polyps. Unfortunately, our recorded videos did not provide sufficient detail to confidently show reduced prey adhesion to \u003cem\u003eF. fragum\u003c/em\u003e tentacles under temperature stress. This warrants future study using a microscopic setup which allows to visualise the feeding behaviour of these small polyps in higher detail. In addition, such a setup may show that heat-stressed polyps exhibit reduced tentacle activity, possibly due to reduced energy (ATP) availability. Our videos did suggest such reduced tentacle activity, but we were unable to quantify this due to lack of detail. Although reef-building corals can maintain normal ATP levels under thermal stress (Kochman et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), our prolonged heatwave may have exhausted this capacity by depleting energy reserves (Grottoli et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The coupling between auto- and heterotrophy is further substantiated by Love et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), who found that bleached corals exhibit reduced prey capture rates and lipid content. They suggested there may be an energetic cost to prey capture that normally is provided by the symbiont. Thus, when temperatures continued to increase above 31\u0026deg;C, both energy acquisition pathways became impaired, ultimately resulting in depletion of energy reserves, growth stagnation, tissue necrosis and mortality. This could be further substantiated by measuring lipid and fatty acid content (\u003cem\u003esensu\u003c/em\u003e Love et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which should be addressed in a future study.\u003c/p\u003e\u003cp\u003eOur results contrast with those reported by Connolly et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), who found that feeding increases survival rates of \u003cem\u003eAcropora intermedia\u003c/em\u003e during a heatwave and that it may aid in recovery. However, these authors used an acute, very short heatwave (\u0026lt;\u0026thinsp;1 DHW, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://coralreefwatch.noaa.gov\u003c/span\u003e\u003cspan address=\"https://coralreefwatch.noaa.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) rather than a more natural, prolonged one as in our study. A similar finding was reported based on a short-term, acute heatwave conducted by Ferrier-Pag\u0026egrave;s et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), who found that zooplankton supplementation prevented heat-induced PSII damage and bleaching of three coral species. They also found that one species, \u003cem\u003eStylophora pistillata\u003c/em\u003e, reduced its prey capture rates during thermal stress. Levas et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found that \u003cem\u003ePorites astreoides\u003c/em\u003e was able to increase its prey capture rate when in a bleached state during a mild heatwave (3\u0026ndash;4 DHW compared to controls), but not during a second round a year later, suggesting that cumulative thermal stress disrupts feeding abilities. Furthermore, Grottoli et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) found that bleached \u003cem\u003eMontipora capitata\u003c/em\u003e was able to meet its full metabolic demand through zooplankton capture, although they determined feeding rates after a two-week recovery period following experimental thermal bleaching. Finally, Borell and Bischof (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) found that feeding sustains PSII yield of \u003cem\u003eStylophora pistillata\u003c/em\u003e during thermal stress, although their heat wave lasted only 10 days with a high daily rise of 2\u0026ndash;3\u0026deg;C. In addition to the unnatural heat stress experiments outlined above, all these authors used adult colonies, which may behave differently from settlers. Rivera et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) arrived at a conclusion similar to ours, with a limited impact of feeding on thermotolerance of the temperate coral \u003cem\u003eOculina arbuscula\u003c/em\u003e. Although we cannot calculate DHW from their data, they applied a significant heatwave with a\u0026thinsp;~\u0026thinsp;9 degree Celsius increase over a period of two weeks. They conclude that, amongst others, coral energy status prior to heat stress and stress duration could limit the beneficial impact of increased prey availability. The energy status of our settlers is likely to have been favourable, as the parent colonies we used were healthy and had been fed year-round under optimal aquaculture conditions. Thermal stress duration and intensity, expressed as DHW, are likely to be the decisive factor when assessing the role of heterotrophy in coral thermotolerance, and may explain discrepancies between studies. Taken together, repeated or prolonged heat stress, such as in our study, may result in a point of no return after which corals are too weakened to effectively feed. A future study should determine prey capture dynamics during a prolonged heatwave in more detail, for both early settlers as well as adult corals. This would identify the temperature/DHW tipping point after which plankton feeding is disrupted to such an extent that it can no longer offset the incurred loss of autotrophic input. Although the cumulative temperature stress applied in our study (14.4 DHW) seems excessive, NOAA data over the 2015\u0026ndash;2025 period from our collection site show high values of up to 26 DHW (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://coralreefwatch.noaa.gov/product/vs/timeseries/caribbean.php#abc_islands\u003c/span\u003e\u003cspan address=\"https://coralreefwatch.noaa.gov/product/vs/timeseries/caribbean.php#abc_islands\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Mu\u0026ntilde;iz-Castillo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), owing to the 2023\u0026ndash;2024 ENSO event.\u003c/p\u003e\u003cp\u003eWhile this study provides valuable insight into the physiological response of early life-stage corals to temperature stress, it should be taken into account that our corals were fed solely with \u003cem\u003eArtemia salina\u003c/em\u003e nauplii, which, while a common aquaculture food source, do not fully represent the nutritional and size complexity of natural reef diets (Palardy et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Levas et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Future research should incorporate natural prey types, such as copepods, crab zoea or amphipods, which may alter coral response due to different nutritional composition. It also would be interesting to include molecular or transcriptomic analyses to help clarify the mechanisms underlying the observed responses. For example, carbohydrate, fatty acid and protein levels of settler tissue were likely negatively impacted (Rivera et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Love et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which could explain the observed cessation of prey capture during the heatwave.\u003c/p\u003e\u003cp\u003eWhile it is yet to be determined whether our results with \u003cem\u003eF. fragum\u003c/em\u003e larvae that vertically inherit symbionts can be generalised to aposymbiotic settlers, this study provides a valuable framework for understanding early-stage coral responses to environmental stress. Notably, the health performance of the control groups, evident by high PSII yield, robust growth and active prey capture, demonstrates the potential of these juvenile corals when provided with species-specific favourable conditions. Our results establish a critical growth baseline for future stress response studies using \u003cem\u003eF. fragum\u003c/em\u003e and provide new insight into the limited role of heterotrophy during heat stress of early life-stage corals. Zooplankton grazing, at least in the form of \u003cem\u003eArtemia\u003c/em\u003e nauplii, is not a decisive factor in settler survival during realistically prolonged heat stress such as experienced during ENSO temperature anomalies. Identifying temperature thresholds beyond which various coral species, during early or later life stages, can no longer temporarily rely on heterotrophic feeding for survival remains an important avenue of research. Our findings identify a clear coupling between symbiont physiological status and host performance in \u003cem\u003eF. fragum\u003c/em\u003e settlers. In addition, they provide an explanation for why recruitment of scleractinian corals on Caribbean reefs, which experience increasingly damaging ENSO events, is limited.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Joost Hamoen and the staff of Carus Aquatic Research Facility for their support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Wageningen University and Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll applicable international, national, and/or institutional guidelines for the care and use of organisms were followed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData collected and analyzed during this study are available as electronic supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTW designed and coordinated the study, performed data analyses, and wrote the manuscript. RCG, JP, DG and DP performed the experimental work, assisted with data analysis, and participated in manuscript review and editing. RO and AJM provided assistance with data analysis, and reviewed and edited a draft manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaker AC, Glynn PW, Riegl B (2008) Climate change and coral reef bleaching: an ecological assessment of long-term impacts, recovery trends and future outlook. Estuar Coast Shelf Sci 80: 435\u0026ndash;471\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBorell EM, Bischof K (2008) Feeding sustains photosynthetic quantum yield of a scleractinian coral during thermal stress. Oecologia 157: 593\u0026ndash;601\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBove CB, Mudge L, Bruno JF (2022) A century of warming on Caribbean reefs. 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Near-bottom depletion of zooplankton over coral reefs: I: diurnal dynamics and size distribution. Coral Reefs 24: 75\u0026ndash;85\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"coral-reefs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"core","sideBox":"Learn more about [Coral Reefs](http://link.springer.com/journal/338)","snPcode":"338","submissionUrl":"https://submission.nature.com/new-submission/338/3","title":"Coral Reefs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"coral bleaching, zooplankton, climate change, reef resilience, recruitment","lastPublishedDoi":"10.21203/rs.3.rs-7584376/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7584376/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecent reports suggest that reef-building corals can shift from autotrophic to heterotrophic feeding during episodic heatwaves, which may have mitigating effects on thermal stress. It is yet unclear to what extent this could occur during early life stages of corals, and how this affects survival. We exposed settlers (primary polyps) of the Caribbean Golf ball coral \u003cem\u003eFavia fragum\u003c/em\u003e to a lab-controlled heatwave lasting 48 days under two feeding regimes (36 versus 3600 \u003cem\u003eArtemia salina\u003c/em\u003e nauplii L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), starting from 28\u0026deg;C and peaking at 32.4\u0026deg;C with a daily increase of 0.19\u0026deg;C. Thermal stress, measured as declining effective photosystem II yield, became apparent at 31.8\u0026deg;C (or 7.5 Degree Heating Weeks, DHW). Growth of heat-exposed settlers ceased between 31.0-31.8\u0026deg;C (4.1\u0026ndash;7.5 DHW), regardless of feeding regime. All heat-exposed settlers had died at 14.4 DHW. Mortality was preceded by a 41\u0026ndash;64% loss of symbiont densities and a 46\u0026ndash;59% reduction of chlorophyll \u003cem\u003ea\u003c/em\u003e fluorescence at 32.4\u0026deg;C (9.4 DHW). No beneficial effect of feeding on thermotolerance was observed, likely because settlers were unable to feed on zooplankton during the heatwave: at 32.4\u0026deg;C (9.4\u0026ndash;10.7 DHW), prey capture was reduced by up to 98% as compared to controls. Our findings identify the coupling between symbiont and host performance under thermal stress in \u003cem\u003eF. fragum\u003c/em\u003e settlers, and help explain low recruitment on Caribbean reefs.\u003c/p\u003e","manuscriptTitle":"Zooplankton provisioning does not enhance thermotolerance and survival of Favia fragum settlers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 02:57:34","doi":"10.21203/rs.3.rs-7584376/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-03T22:20:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-25T00:52:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-04T06:03:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330472514138674019998113414465265796979","date":"2025-10-14T04:55:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120999280247631834535917679456361906700","date":"2025-10-07T22:45:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T21:50:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-27T02:42:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-19T14:24:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Coral Reefs","date":"2025-09-10T15:18:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"coral-reefs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"core","sideBox":"Learn more about [Coral Reefs](http://link.springer.com/journal/338)","snPcode":"338","submissionUrl":"https://submission.nature.com/new-submission/338/3","title":"Coral Reefs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"87225279-cc9e-4203-9a50-dd6506bd3a2a","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:07:39+00:00","versionOfRecord":{"articleIdentity":"rs-7584376","link":"https://doi.org/10.1007/s00338-026-02821-2","journal":{"identity":"coral-reefs","isVorOnly":false,"title":"Coral Reefs"},"publishedOn":"2026-02-05 15:59:42","publishedOnDateReadable":"February 5th, 2026"},"versionCreatedAt":"2025-09-12 02:57:34","video":"","vorDoi":"10.1007/s00338-026-02821-2","vorDoiUrl":"https://doi.org/10.1007/s00338-026-02821-2","workflowStages":[]},"version":"v1","identity":"rs-7584376","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7584376","identity":"rs-7584376","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}