DOI: 10.5061/dryad.kh18932b5
title: Data for: Seasonal variability in resilience of a coral reef fish to marine heatwaves and hypoxia

description: 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 (Pcrit)) 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 Pcrit did not significantly vary
 between seasons, warming of +4°C during winter (i.e., 28.5°C)
 significantly increased Pcrit 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.
description: <strong>Study species and
 marine heatwave treatments</strong>
 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 <em>Acanthurus triostegus</em>,
 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.,
 &gt; 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<sub>winter</sub> = 11, n<sub>summer</sub> =
 8), a moderate MHW (i.e., 2°C
 above ambient temperature;
 n<sub>winter</sub> = 10, n<sub>summer</sub> =
 8), or a severe MHW (i.e., 4°C
 above ambient temperature;
 n<sub>winter</sub> = 7, n<sub>summer</sub> =
 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. <strong>Respirometry
 protocol</strong> Following each 7-day MHW treatment, rates of oxygen consumption
 (MO<sub>2</sub>) 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<sub>crit</sub>). 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 MO<sub>2</sub>
 measurements began within 30
 s. This process was then
 repeated until all respirometers in the system contained a fish.
 MO<sub>2</sub> 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<sub>crit</sub>. 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 MO<sub>2</sub> measurement was
 measured at each 5% increment for each fish, resulting in ~16 measurements per
 fish. <strong>Data analysis and
 statistics</strong> Mass-specific MO<sub>2</sub> (mg
 O<sub>2</sub> kg<sup>-1</sup>
 h<sup>-1</sup>) was automatically calculated using AquaResp
 software v3.04 by linear regression between oxygen concentration and time
 for each measurement cycle. Only MO<sub>2
 </sub>calculations with an R<sup>2</sup> ≥ 0.99 were kept
 for further analysis. To
 account for background respiration, bacterial MO<sub>2</sub> 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
 MO<sub>2</sub> 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<sub>O2</sub> was
 greater than 80%, resulting in an average
 197 MO<sub>2</sub> measurements per fish. Once
 determined, AS was calculated for each fish as the difference
 between MMR and SMR. The single highest
 MO<sub>2</sub> value measured during each respirometry trial was used as MMR
 for 13 individuals. Delays in maximal M<sub>O2</sub> 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<sub>O2</sub> 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<sub>crit</sub> was defined as the p<sub>O2</sub>
 below which
 MO<sub>2</sub> reduced significantly
 below SMR during declining ambient
 p<sub>O2</sub> and estimated following the guidelines of Reemeyer and
 Rees (2019). Based on visual analyses, the last four MO<sub>2</sub>
 calculations from the
 hypoxia experiments (i.e., MO<sub>2</sub> at and
 below P<sub>crit</sub>) were
 used to determine the relationship between MO<sub>2</sub>
 and p<sub>O2</sub> using a best-fit linear regression (Ultsch &
 Regan, 2019). The resulting linear
 function for each fish was then solved for the p<sub>O2</sub>
 where MO<sub>2</sub> equaled SMR using the following
 equation, <em>p<sub>O2</sub> = (m x
 MO<sub>2,std</sub>) + b
 +  ε </em> Where <em>m</em> equals the slope of
 the line, <em>b</em> equals the intercept, and
 <em>ε</em>
 equals the standard error. Average MMR, SMR, AS, and
 P<sub>crit</sub> was calculated for each MHW treatment group for both winter and summer
 cohorts.
description: Requires Microsoft Excel