Climate change projections indicate more frequent and severe tropical marine heatwaves (MHWs) and accompanying hypoxia year-round. However, most studies have focused on peak summer peak conditions under the assumption that annual maximum temperatures will induce the greatest physiological consequences. This study challenges this idea by characterizing seasonal MHWs (i.e., mean, maximum, and cumulative intensities, durations, heating rates, and mean annual occurrence) and comparing metabolic traits (i.e., standard metabolic rate (SMR), Q10 of SMR, maximum metabolic rate (MMR), aerobic scope, and critical oxygen tension (P_crit)) of winter- and summer-acclimatized convict tang (Acanthurus triostegus) to the combined effects of MHWs and hypoxia. Fish were exposed to one of six MHW treatments with seasonally varying maximum intensities (winter: 24.5, 26.5, 28.5°C; summer: 28.5, 30.5, 32.5°C), representing past and future MHWs under IPCC projections (i.e., +0, +2, +4°C). Surprisingly, MHW characteristics did not significantly differ between seasons, yet SMR was more sensitive to winter MHWs (mean Q10 = 2.92) than summer MHWs (mean Q10 = 1.81), despite higher absolute summer temperatures. Concurrently, MMR increased similarly among winter +2°C and +4°C treatments (i.e., 26.5, 28.5°C) and all summer MHW treatments, suggesting a ceiling for maximal MMR increase. Aerobic scope did not significantly differ between seasons nor among MHW treatments. While mean P_crit did not significantly vary between seasons, warming of +4°C during winter (i.e., 28.5°C) significantly increased P_crit relative to the winter control group. Contrary to the idea of increased sensitivity to MHWs during the warmest time of year, our results reveal heightened sensitivity to the deleterious effects of winter MHWs, and that seasonal acclimatization to warmer summer conditions may bolster metabolic resilience to warming and hypoxia. Consequently, physiological sensitivity to MHWs and hypoxia may extend across larger parts of the year than previously expected, emphasizing the importance of evaluating climate change impacts during cooler seasons when essential fitness-related traits such as reproduction occur in many species.

Study species and marine heatwave treatments

A 26-year-long sea surface temperature dataset from 1994–2020 was collected at the National Oceanic and Atmospheric Adminstrationʻs Moku o Loʻe weather station (data retrieved online from tidesandcurrents.noaa.gov; Moku o Loʻe weather station; ID: 1612480; depth = 1 m; 21.433°N, 157.786°W) to characterize the local thermal environment to identify past marine heatwave (MHW) characteristics to simulate MHW conditions in the laboratory. To test seasonal acclimatization of thermal and hypoxia tolerance, experiments were conducted during the winter and summer for the winter and summer cohorts of Acanthurus triostegus, respectively. Collections were conducted at the end of the winter and summer of the year 2020, i.e., after ~3 month of seasonal acclimatization (winter collection temperature: 24.5 ± 0.5°C; summer collection temperature: 28.5 ± 0.5°C; N = 51). Fish were allowed to acclimate in the lab until they were consistently feeding before MHW treatments began (i.e., > 3 days of acclimation).

Individuals were exposed to simulated MHWs during the coldest (i.e., winter; January – March; water temperature 24.0 ± 0.1°C, mean ± S.E.M.) and warmest (i.e., summer; July – September; water temperature 27.4 ± 0.1°C, mean ± S.E.M.) months of the year. Fish were randomly assigned to one of three MHW treatments: ambient mean temperature (i.e., 0°C above ambient temperature; n_winter = 11, n_summer = 8), a moderate MHW (i.e., 2°C above ambient temperature; n_winter = 10, n_summer = 8), or a severe MHW (i.e., 4°C above ambient temperature; n_winter = 7, n_summer = 7). Water temperature was gradually increased by 1°C/day, and warming above the climatological mean lasted for 7 days total, resulting in 5 and 3 days at maximum intensity for the moderate and severe MHW treatments, respectively. These conditions closely matched the mean duration (days), maximum heating rate (°C/day), and maximum intensity (°C) for MHWs detected on the reefs within Kāne’ohe Bay, Hawaiʻi.

Respirometry protocol

Following each 7-day MHW treatment, rates of oxygen consumption (MO₂) were obtained at their respective maximum temperatures using automated intermittent-flow respirometry under the guidelines specified by Svendsen et al (2016). Equipment consisted of four cylindrical acrylic respirometers submerged in a dark 144 L experimental tank (length = 97 cm; width = 53 cm; height = 37 cm) containing fully aerated 35 ppt seawater. The working section of each respirometer had a volume of 600 mL (respirometer chamber dimensions: length = 15.5 cm; diameter = 7 cm). Water temperature was controlled using a 57 L sump (length = 19 cm; width = 10 cm; height = 30 cm) containing a Finnex 800W heater connected to an external temperature regulator (WILLHI WH1436). Water from the sump was pumped continuously into the experimental tank to maintain the desired temperature within ± 0.1°C. Each respirometer had a single small pump (DollaTek AD20P-1230E; 140L/h; 7V) continuously recirculating water in a loop. Oxygen levels were measured within each respirometer every second using fiber optic oxygen sensors and Pyro Oxygen Logger software v3.317 (Firesting O2, Pyro Science, Aachen, Germany) and automatically converted to mass-adjusted oxygen consumption values using AquaResp v3.04 software (www.aquaresp.com). The system ensured that a flush pump automatically replenished the water in the respirometers every ~7 min. These duty cycles differed slightly between seasons to ensure oxygen levels in the respirometers were maintained above 75% air saturation during the maximum metabolic rate (MMR) and standard metabolic rate (SMR) estimation (Steffensen 1989).

Fish were starved for 24 hours prior to experimentation to avoid measurement of specific dynamic action (Niimi & Beamish, 1974). Respirometry trials consisted of three measurements in the following order: (1) MMR, (2) SMR, and (3) the critical oxygen tension (P_crit). Before each respirometry trial, all equipment was disinfected with a bleach solution and thoroughly rinsed with freshwater. Additionally, background respiration was measured prior to placing the fish in the respirometers and again after fish were removed from the system to account for microbial respiration (see data analysis section below).

Maximum metabolic rate was obtained for each fish using a standard protocol of manually chasing fish until exhaustion (defined as when the experimenter could pick up the fish with no resistance), consisting of a 3-minute minimum chase period followed by 1 minute of air exposure. Individuals that could not sustain burst swimming for the entire 3-minute chase were continually chased to maintain prolonged swimming for the remainder of the period. Fish were placed directly into the respirometer following air exposure and MO₂ measurements began within 30 s. This process was then repeated until all respirometers in the system contained a fish. MO₂ measurements were then left to continue overnight in undisturbed fish (~16 hours) until the oxygen consumption rates of the fish had settled to a steady state minimum (reflective of SMR, see data analyses below). A 12:12 light:dark regime was used with lights set to turn on and off at 7AM HST and 7PM HST, respectively.

Once a steady state minimum had been reached, fish were exposed to incrementally declining oxygen concentrations to determine P_crit. A separate oxygen sensor (In-Situ, Aqua TROLL® 600 Multiparameter Sonde, Denver, CO, USA) was used to monitor oxygen levels in the sump while nitrogen gas was bubbled to reduce the oxygen level of the system without disturbing the fish in the experimental tank. Oxygen levels were reduced by increments of 5% air saturation beginning at 90-100% at a rate of ~0.7%/min. Hypoxia experiments stopped once a fish showed loss of equilibrium (LOE), demonstrated as the inability to maintain an upright position in the water column for 5 s, which usually occurred between 10–20% air saturation. A single MO₂ measurement was measured at each 5% increment for each fish, resulting in ~16 measurements per fish.

Data analysis and statistics

Mass-specific MO₂ (mg O₂ kg^-1 h^-1) was automatically calculated using AquaResp software v3.04 by linear regression between oxygen concentration and time for each measurement cycle. Only MO₂ calculations with an R² ≥ 0.99 were kept for further analysis. To account for background respiration, bacterial MO₂ measured at the beginning of experiments was subtracted from MMR. Linear regression over time using measures of bacterial respiration before and after each respirometry trial was used to subtract background respiration during the SMR and hypoxia experiments (Roche et al 2013).

SMR was initially evaluated using four techniques; the mean lowest normal distribution (MLND) was compared to the 10%, 15%, and 20% quantiles of MO₂ values to determine the most accurate estimate of SMR (sensu Chabot et al 2016). Per the recommendations of Chabot et al (2016), SMR was determined for each individual using all data from both the overnight and hypoxia experiments when ambient p_O2 was greater than 80%, resulting in an average 197 MO₂ measurements per fish. Once determined, AS was calculated for each fish as the difference between MMR and SMR. The single highest MO₂ value measured during each respirometry trial was used as MMR for 13 individuals. Delays in maximal M_O2 are thought to stem from recovery of oxygen debt and the accumulation of anaerobic metabolites incurred by the recruitment of large numbers of anaerobic white muscle and represent valid estimates of MMR (C. L. Milligan, 1996; C. Louise Milligan, Hooke, & Johnson, 2000; Norin & Clark, 2016; Pagnotta, Brooks, & Milligan, 1994; Peake & Farrell, 2006; Scarabello, Heigenhauser, & Wood, 1991, 1992). 

To demonstrate the relative sensitivity of SMR and MMR to seasonal MHWs, relative values were calculated by taking the quotient of M_O2 values of the MHW treatment groups (i.e., winter and summer +2°C and +4°C MHW treatments) and their respective seasonal control groups (i.e., winter and summer +0°C treatments). 

P_crit was defined as the p_O2 below which MO₂ reduced significantly below SMR during declining ambient p_O2 and estimated following the guidelines of Reemeyer and Rees (2019). Based on visual analyses, the last four MO₂ calculations from the hypoxia experiments (i.e., MO₂ at and below P_crit) were used to determine the relationship between MO₂ and p_O2 using a best-fit linear regression (Ultsch & Regan, 2019). The resulting linear function for each fish was then solved for the p_O2 where MO₂ equaled SMR using the following equation,

p_O2 = (m x MO_2,std) + b +  ε 

Where m equals the slope of the line, b equals the intercept, and ε equals the standard error. Average MMR, SMR, AS, and P_crit was calculated for each MHW treatment group for both winter and summer cohorts.

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