Stress-hardening by environmental priming could increase the odds for corals to resist ocean warming. Natural environmental fluctuations, such as those observed on offshore reefs in the Andaman Sea, provide an ideal natural environment to study these effects. Here, internal waves (IW) generate short cold-water pulses that peak from January to June and are absent from August to November. Additionally, only western shores of islands are exposed to this stress-hardening stimulus of IWs, while eastern shores remain sheltered. Therefore, this study examined (1) whether exposed corals were more heat stress resistant than their sheltered conspecifics and (2) whether this trait would persist during the season of stimulus absence. We exemplify that thermal regimes featuring cold-temperature pulses successfully induced thermal stress-hardening in corals. Corals from the IW-sheltered shore responded strongly to heat stress irrespective of the season, while stress responses of IW-exposed corals were either undetectable (during stimulus presence) or very weak (during stimulus absence). However, this demonstrates the relevance of stimulus re-occurrence in maintaining heat resistance. Furthermore, priming stimuli do not need to exceed certain upper thermal thresholds to be effective and we argue that cooling pulses represent a safer stress-hardening regimen potentially implemented in conservation strategies since it avoids warming-stress accumulation.

Study sites and coral collection

Study sites were located at Racha Island in the Andaman Sea off the coast of Thailand, both at 15 m water depth (Figure 1 A-B). A reef on the western shore was chosen (7.595530°N, 98.354320°E, Figure 1 B) where internal wave (IW) forcing as a potential stress-hardening stimulus induced environmental variability through frequent upwelling of deep, cool, and nutrient rich water onto the shelf (Schmidt et al., 2016; Wall et al., 2012). A reef on the eastern shore, sheltered from the stimulus of IWs, was chosen to represent a low variability reef (7.598910°N, 98.373100°E, Figure 1 B). Temperature fluctuations were monitored in situ as a proxy for IW impact and environmental variability. Temperature loggers (HOBO Pendant Temperature/Light 8K Data Logger, Onset, USA) were deployed at the study sites one month before heat stress assays were performed. At each study site, visually healthy coral colonies of Pocillopora sp. and Porites sp. were permanently tagged to assess their thermal resistance levels during the two seasons (n = 8 to n = 18, Figure 1 C, Table S1). These two coral species are cosmopolitan reef-builders in Thailand and within the entire Indo-Pacific region (Brown & Phongsuwan, 2012; Jain et al., 2023; Schmidt et al., 2012). Coral fragments were collected at the end of April 2018, during the season of highest IW intensity, and at the end of October (Porites sp.) and November (Pocillopora sp.), during the seasonal absence of the IW stimulus. Two fragments (Porites sp.: ø ~ 6 cm; Pocillopora sp.: length ~ 5 cm) per colony were collected using a chisel and a hammer (Table S1).

Short-term heat stress assays

Collected fragments were instantly transported to the Phuket Marine Biological Center (Phuket, Thailand) where they were maintained in two 500 L flow-through tanks with a flow rate of 2.8 ± 1.31 L/min until the start of each heat stress assay. Another 500 L source tank constantly supplied both flow-through tanks with 5 μm-filtered seawater from the reef adjacent to the research center. Its temperature was held at constant 29.43 ± 0.32 °C using a temperature-controlling device including a chiller and a heater (Titanium Heater 100 W, Schego, Germany; Temperature Switch TS 125, HTRONIC, Germany; Aqua Medic Titan 1500 Chiller, Germany). LED lights (135 W, Hydra Fiftytwo HD LED, Aqua Illumination, USA) mimicked the average light conditions of the sampling sites (Text S1).

For each heat stress assay (Figure 1 D), two 40 L experimental tanks were set up inside each of the 500 L flow-through tanks that were used as temperature-controlling water baths (Table S2). The seawater of all four experimental tanks was supplied by daily, manual 50% water changes from the source tank. Each experimental tank was equipped with a temperature-controlling device, one heater, air supply, a small current pump and a temperature logger (Temperature Switch TS 125, HTRONIC, Germany; Titanium Heater 100 W, Schego, Germany; Koralia nano 900 L/h, Hydor, Italy; HOBO Pendant Temperature/Light 8K Data Logger, Onset, USA). Two coral fragments per coral colony were randomly distributed among the four tanks “34 °C” (n = 2) and “29 °C” (n = 2), resulting in one fragment per colony per treatment. The 34 °C-treatment was established over the course of one day by ramping temperatures from 29 °C to 34 °C for 4 h, holding at 34 °C for 5 h or 6 h (Pocillopora sp.) or for 6 h or 7 h (Porites sp.), and decreasing temperatures to 29 °C within 4 h. After the heat exposure, corals were maintained at ambient temperatures of 29 °C for 10 h until the next day. While Pocillopora sp. fragments were subjected to the short-term heat exposure once, resulting in a 24 h experiment, Porites sp. corals were exposed to the treatment over two consecutive days resulting in a duration of 72 h (Figure 1 D).

Coral stress response variables

We measured two variables that assessed the thermal stress response of each fragment before and after each heat stress assay (timepoints (1) and (2) in Figure 1 D). Tissue coloration, a proxy for microalgal symbiont cell density in coral tissues and therefore an indicator of holobiont health and coral bleaching severity, was assessed using a “bleaching score”. The coloration of each individual fragment was visually categorized on the scale from 1 (bleached, pale tissues) to 6 (healthy, dark tissues) using a coral bleaching chart (Siebeck et al., 2006). A minimum and maximum score was recorded per fragment and averaged. Photosynthetic efficiency of microalgal symbionts was determined by measuring effective quantum efficiency (yield Φ PSII = (Fm’ – F) / Fm’ = ΔF / Fm’, (Genty et al., 1989) of electron transport using a pulse amplitude-modulated fluorometer (Diving-PAM, Walz, Germany).

Statistical analyses

∆-values of each measured thermal stress response variable (end – start of each experimental part) were calculated to reflect their change over time. Based on these ∆-values, effect sizes were estimated using dabestR v0.2.3 6 (Ho et al., 2019). Effects of the high temperature treatment (“Heat” vs. “Ambient”) were compared between the sites of origin (“IW exposed shore” and “IW sheltered shore”) and between the seasons (“Season of IW stimulus presence” and “Season of IW stimulus absence”). Statistical significance was tested in R (R Core Team, 2013) using linear mixed effect models (nlme v4 3.1-148 and lme4 v1.1-23 package). Where applicable, coral colony genotype was used as a random factor.

References

Brown, B., & Phongsuwan, N. (2012). Delayed mortality in bleached massive corals on intertidal reef flats around Phuket, Andaman Sea, Thailand. Phuket Marine Biological Center Research Bulletin, 48(April 2010), 43–48.

Wall, M., Schmidt, G. M., Janjang, P., Khokiattiwong, S., & Richter, C. (2012). Differential Impact of Monsoon and Large Amplitude Internal Waves on Coral Reef Development in the Andaman Sea. PloS One, 7(11), e50207. https://doi.org/10.1371/journal.pone.0050207

Schmidt, G. M., Wall, M., Taylor, M., Jantzen, C., & Richter, C. (2016). Large-amplitude internal waves sustain coral health during thermal stress. Coral Reefs , 1–13. https://doi.org/10.1007/s00338-016-1450-z

Siebeck, U. E., Marshall, N. J., Klüter, A., & Hoegh-Guldberg, O. (2006). Monitoring coral bleaching using a colour reference card. Coral Reefs , 25(3), 453–460. https://doi.org/10.1007/s00338-006-0123-8

Ho, J., Tumkaya, T., Aryal, S., Choi, H., & Claridge-Chang, A. (2019). Moving beyond P values: data analysis with estimation graphics. Nature Methods, 16(7), 565–566. https://doi.org/10.1038/s41592-019-0470-3

Genty, B., Briantais, J.-M., & Baker, N. R. (1989). The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta (BBA) - General Subjects, 990(1), 87–92. https://doi.org/10.1016/S0304-4165(89)80016-9

Jain, T., Buapet, P., Ying, L., & Yucharoen, M. (2023). Differing Responses of Three Scleractinian Corals from Phuket Coast in the Andaman Sea to Experimental Warming and Hypoxia. Journal of Marine Science and Engineering, 11(2), 403. https://doi.org/10.3390/jmse11020403