Superior hypoxia tolerance of intertidal triplefin fish is associated with low critical oxygen tension and high phosphorylating capacity in brain mitochondria

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Devaux, Tristan J. McArley, Neill Herbert, Anthony J.R. Hickey This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4722244/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract At the terminus of the O 2 cascade, mitochondria play an important role in O 2 utilisation and energy conservation, with adaptive modifications occasionally shared among hypoxia-tolerant species. Here, we sought to determine whether mitochondrial adaptations in brain tissue explain the hypoxia tolerance of New Zealand triplefin fishes ( Tripterygiidae ). We compared two intertidal species ( Bellapiscis medius and Forsterygion lapillum ), both likely adapted to hypoxia-reoxygenation exposures, and two subtidal species ( F. varium and F. malcomi ), which inhabit normoxic waters. To assess hypoxia tolerance, we determined loss of equilibrium (LOE) during hypoxia exposure and measured the critical O 2 tension (P crit ). Intertidal species displayed superior hypoxia tolerance as assessed by LOE and also had lower P crit (LOE versus P crit R 2 = 0.92). High-resolution respirometry was used to measure mitochondrial respiration in homogenate and permeabilised fragments of brain. While a weak relationship was apparent between mitochondrial O 2 binding affinity (mP 50 ) and hypoxia tolerance, maximum phosphorylating O 2 flux (OxPhos) and O 2 catalytic rates were strongly correlated with hypoxia tolerance. Although cytochrome- c -oxidase activity was highest in the most hypoxia-tolerant species B. medius , it was only weakly correlated with hypoxia tolerance across species. Notably, the high OxPhos capacity of intertidal species was not associated with higher whole animal resting O 2 consumption, suggesting intertidal species maintain high capacity for ATP production without incurring increased basal energetic costs. While somewhat paradoxical, the low P crit /high OxPhos strategy of intertidal species may provide flexibility in the dynamic intertidal environment where short, severe periods of hypoxia are interspersed with high energy demand environmental conditions (e.g. acute warming). respirometry Pcrit physiology cytochrome C oxidase loss of equilibrium oxidative phosphorylation Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Adaptations to hypoxia come in many forms and occur at all levels of biological organisation, from behavioural adjustments to transcriptomic alterations (A.L. Val, 1998). Ultimately, to benefit hypoxia tolerance, these adaptations must contribute to the ability of an organism to maintain energy balance under conditions where ATP production via efficient oxidative phosphorylation is compromised (Richards, 2011). At the terminus of the O 2 cascade, mitochondria produce ATP via pathways that oxidise carbon sources and reduce O 2 to water (Koch and Britton, 2008). Multiple adaptations of mitochondrial structure and function have been identified in hypoxia-tolerant organisms (reviewed in Pamenter, 2014); however, these vary across hypoxia and anoxia tolerant species (Bickler and Buck, 2007; Galli and Richards, 2014; Gorr et al., 2010). In particular, mitochondrial adaptations in the brain, which is often highly sensitive to O 2 depravation (Cervós-Navarro and Diemer, 1991), appear to play a key role in hypoxia tolerance (Del Rio and Montaner, 2021; Larson et al., 2014; Nilsson and Lutz, 2004). For instance, to conserve ATP, brain mitochondria of the anoxia-tolerant turtle ( Trachemys scripta ) appear to remodel, partially uncouple and decrease F 0 -F 1 -ATPase activity during anoxia (Galli et al., 2013; Pamenter et al., 2016). Additionally, in the epaulette shark ( Hemiscyllium ocellatum ), the electron transport chain in brain mitochondria remodels in anoxia with a decrease in complex II mediated O 2 flux, and this may protect against oxidative stress during reperfusion (Devaux et al., 2019b). Brain mitochondria of intertidal sculpin species ( Cottidae family) display greater O 2 affinities, which is partially explained by CCO isoforms having greater O 2 binding properties (Lau et al., 2017). It is anticipated that both higher mitochondrial O 2 binding affinity and higher mitochondrial O 2 consumption would facilitate improved O 2 extraction (Gnaiger et al., 1998) from the blood to brain tissue, thereby allowing intertidal fish to better utilize available O 2 during periodic hypoxia in rock pools. The New-Zealand triplefin fish (Family Tripterygiidae ) group consists of 26 endemic species of which most occupy stable, normoxic subtidal habitats. Three species, however, are known to inhabit intertidal rock pools, which can become severely hypoxic during night-time low tides (Hickey and Clements, 2005; Hilton, 2010; Hilton et al., 2008; McArley et al., 2018). While not linked to hypoxia, there is some evidence of selective pressure on mitochondrial genes within rock pool species relative to subtidal species (Hickey et al., 2009). Moreover, heart (Hilton et al., 2010) and brain (Willis et al., 2021) mitochondria of the exclusively intertidal rock pool species Bellapiscis medius had greater respiratory efficiencies and stabilities than two strictly subtidal triplefin species when exposed to elevated temperatures. Relative to the two subtidal triplefins, brain mitochondria of B. medius also showed adaptations that enhance ATP production under acidifying conditions, such as those occurring in hypoxic brain (Devaux et al., 2019a). This suggests that mitochondrial function may vary among triplefin species challenged with hypoxia; thus, the aim of this study was to determine whether adaptations in mitochondrial function contribute to variation in hypoxia tolerance among intertidal and subtidal triplefins. The present study includes four triplefin species, which occupy a range of habitats varying in terms of the likelihood of environmental hypoxia exposure. Bellapiscis medius is an intertidal specialist, which exclusively inhabits rockpools that can routinely decrease to less than 20% air saturated O 2 during night-time low tides (Hilton et al., 2008; McArley et al., 2019). Fosterygion lapillum occupies lower intertidal rock pools and shallow subtidal habitats, and F. varium and F. malcomi are exclusively subtidal species occupying rocky reef habitats to depths of ~ 35 m (Hilton et al., 2008; McArley et al., 2019). We have previously shown that the intertidal species B. medius and F. lapillum have a lower critical O 2 tension (P crit ) than their subtidal counterparts F. varium and F. malcolmi (McArley et al., 2019). This suggests intertidal triplefins have a superior ability to meet O 2 demand associated with standard metabolic rate (SMR; an estimate of basal metabolic rate) in hypoxia and indicates they are also likely to be more hypoxia tolerant. In the present investigation, time to loss of equilibrium (LOE) during severe hypoxia exposure was measured alongside P crit to establish differences in absolute hypoxia tolerance among the intertidal and subtidal species. Firstly, we established differences in whole animal hypoxia tolerance among intertidal and subtidal species at 20 o C. We predicted that relative to subtidal species ( F. varium and F. malcolmi ), intertidal species ( B. medius and F. lapillum ) would have lower P crit , as has been previously established at 18 o C (McArley et al., 2019), and also be more hypoxia tolerant as assessed by time to LOE. We then measured O 2 consumption in vitro in brain homogenate and permeabilised brain and assessed the mitochondrial affinity to O 2 (mP 50 ) and O 2 catalytic efficiencies (K cat,app ) using high resolution respirometry at the same temperature. We chose brain as this excitable tissue is acutely sensitive to low O 2 (Cervós-Navarro and Diemer, 1991). Brain homogenate preparations were used as these contain endogenous substrates and allows for a greater O 2 diffusion, likely more representative mitochondrial environment in situ (Gnaiger, 2020; Gnaiger et al., 2000), and to some extent, in vivo . However, O 2 and substrate diffusion barriers may remain in homogenate preparations, and tissue permeabilisation may be required to addresscal maximal mitochondrial respiration. Therefore, measuring O 2 consumption using both preparations provides complementary information regarding state-dependent mitochondrial respiration, including in the context of declining O 2 (20.5 kPa to anoxia). In addition, given that others reported that CCO could be a determinant of hypoxia tolerance in other intertidal fish species (Lau et al., 2017), we measured CCO activity and derived inhibition-curves using sodium-azide to determine whether control from CCO differed among species. Thus, we predicted that the mitochondria and CCO of hypoxia-tolerant intertidal triplefins would display a greater affinity to O 2 relative to subtidal species. 2. Material and Methods 2.1. Experimental animals and housing The animals used in this study were adult specimens collected from sites on the Northeast coast of the Auckland region. The rock pool specialist B. medius ( BM ) was caught from high intertidal pools (> 1m) using hand nets, while the occasionally intertidal and shallow subtidal species F. lapillum (FL) was caught using minnow traps from nearshore subtidal sites (< 1m). The deeper dwelling exclusively subtidal specimens F. varium ( FV ) and F. malcomi ( FM ) were caught with hand nets on Scuba dives at a depth of 10-15m. The experiments carried out in this study were performed at two research facilities. The fish used in the LOE trials and mitochondrial assays were housed in a recirculated seawater facility at the University of Auckland’s School of Biological Sciences. These fish were held in 30 L tanks provided with a constant flow of recirculated seawater (20 ± 1°C, air saturated, 200 µm filtered, 35 ppt salinity). The fish used for whole animal respirometry were housed at the Leigh Marine Laboratory in 30 L flow-through seawater tanks (20 ± 0.5°C, air saturated, 200 µm filtered, 35 ppt salinity). All fish were acclimated to laboratory conditions for at least 2 weeks prior to the start of experiments and were fed ad libitum on a mixture of shrimp, mussel and fish. Mass and length data for the fish used in each part of the study are found in Table 1 . Food was withheld for a period of 48 h prior to the start of experiments. All capture, housing and experimental procedures were performed under the approval of the University of Auckland Ethics Committee (Approval 001551). Table 1 Anatomical features of the four New Zealand triplefin fish species used in each part of the present study. Data presented as mean ± s.e.m, with sample sizes indicated in brackets in the left-hand column. Brian mass to body mass ratio was determined in 8 individuals of each species used in the mitochondrial function trials. Statistical differences among species were tested using one-way ANOVA. Significance was set at P < 0.05, and significant post-hoc differences between species are shown by uncommon superscript letters Study (n) Body length (mm) Body weight (g) Brain (g.g − 1 ) B. medius P crit (10) 64.80 ± 1.37 a 2.71 ± 0.11 a P LOE (9) 62.33 ± 3.01 a 3.01 ± 0.37 ab Mito. (8) 54.36 ± 4.81 a 2.54 ± 0.29 a 0.73 ± 0.04 a Total (27) 60.50 ± 2.94 a 2.75 ± 0.17 a F. lapillum P crit (10) 62.90 ± 2.47 a 2.13 ± 0.08 a P LOE (9) 67.89 ± 1.72 ab 2.32 ± 0.10 b Mito. (8) 68.57 ± 1.41 b 2.32 ± 0.07 a 0.72 ± 0.02 a Total (27) 66.45 ± 1.41 a 2.26 ± 0.06 a F. varium P crit (10) 80.90 ± 2.04 b 4.63 ± 0.32 bc P LOE (9) 64.11 ± 2.00 a 3.94 ± 0.18 a Mito. (8) 65.00 ± 2.80 bc 4.01 ± 0.21 b 0.44 ± 0.01 b Total (27) 70.00 ± 2.43 a 4.19 ± 0.19 b F. malcomi P crit (10) 75.20 ± 2.90 b 4.37 ± 0.59 c P LOE (9) 83.00 ± 3.54 b 7.12 ± 0.32 c Mito. (8) 80.64 ± 2.83 c 6.84 ± 0.25 c 0.34 ± 0.01 c Total (27) 79.61 ± 2.08 b 6.11 ± 0.38 c 2.2. Whole animal respirometry and determination of critical oxygen tension The P crit of each species (N = 10; see Table 1 for mass and length) was determined at 20°C using automated intermittent stop-flow respirometry (Steffensen, 1989) to measure mass-specific O 2 consumption ( Ṁ O 2 ; mg O 2 g -1 h -1 ). The design of the respirometers, general respirometry methods and procedure for Ṁ O 2 calculation are described in detail in McArley et al., (2018). P crit was defined as the O 2 tension where Ṁ O 2 under a progressive hypoxia exposure could no longer be maintained above standard metabolic rate (SMR; Ṁ O 2 in a rested, unfed animal) (Claireaux and Chabot, 2016). The protocol for P crit determination began with an overnight period (~ 16 h) of respirometry where Ṁ O 2 was assessed repeatedly over 7–8 min cycles in undisturbed fish under normoxia. SMR was defined as the mean of the lowest 10% of Ṁ O 2 measurements made during the overnight period (Khan et al., 2014; Mandic et al., 2009; McArley et al., 2017; Norin et al., 2014), which likely corresponded to periods when fish were completely inactive as these species are benthic and tend to perch in a stationary position on the bottom of the respirometers. Ṁ O 2 measurements were then made at decreasing O 2 tensions (~ 15.3, 11.6, 7.4, 6.3, 5.2, 4.2, 3.3, 2.3 and 1.6 kPa), with the required water O 2 levels achieved by bubbling N 2 into the seawater reservoir supplying respirometers. Three 7–8 min Ṁ O 2 measurements were made at 15.3, 11.6, 7.4, 6.3, 5.2 and 4.2 kPa, and one 7–8 min measurement at 3.3, 2.3 and 1.6 kPa. The entire progressive decline in O 2 tension was completed in ~ 3 h, and the time of exposure to each O 2 tension was the same for each species. To estimate P crit , SMR and routine Ṁ O 2 during progressive hypoxia were first mass corrected (see below) and then plotted against water PO 2 . A linear regression (forced through zero) was then established on Ṁ O 2 values that fell below SMR, and P crit was calculated by dividing SMR by the slope of this regression line (i.e. the point where routine Ṁ O 2 under progressive hypoxia could no longer be maintained above SMR; see Fig. 1 A) (as per method of Behrens and Steffensen, 2007; Cook et al., 2013; Cumming and Herbert, 2016; Schurmann and Steffensen, 1997). To account for body mass differences between species (Table 1 ), Ṁ O 2 values were standardised to the mean body mass of all fish (3.5 g) from whole animal respirometry and P crit assessments. Body mass correction of Ṁ O 2 values was carried out using the standard formula outlined in Schurmann and Steffensen (1997) and a mass scaling exponent of 0.8 Clarke and Johnston (1999). 2.3. Loss of equilibrium Across three 40L tanks, three individuals of each species were placed into each tank (a total of 12 fish per tank) and left to recover overnight (~ 16 h) in fully aerated (normoxic) seawater (see Table 1 for mass and length). Seawater PO 2 in each tank was monitored using NeoFox-GT sensors (Ocean Optics © , Inc), and a clear plastic film was placed over the water surface to prevent the possibility of aerial surface respiration. After overnight recovery, hypoxia exposure was induced by bubbling N 2 gas into the tanks holding the fish. Bubbling N 2 continued until the tank PO 2 reached the target water O 2 level of ~ 1.4 kPa, which was achieved in approximately 30 min. Once the target water O 2 level was reached, N 2 bubbling was adjusted manually to maintain a constant PO 2 of ~ 1.4 kPa (Fig. 1 .B). LOE was deemed to occur when a fish could no longer maintain an upright body position for a period of 10 s. In FV and FM and some FL , this occurred spontaneously, usually following a short burst of exercise, and occurred either before or soon after the target water PO 2 was reached. In BM and the most hypoxia tolerant FL , however, individuals remained largely stationary on the tank bottom throughout the duration of the hypoxia exposure. To determine LOE in these individuals, the fish were periodically challenged by turning them over with a small stick and observing whether they could right themselves to an upright position within 10 s. To take into account the water O 2 level of LOE and the length of time which hypoxia was survived, a composite measure of hypoxia tolerance was determined by the area above the curve of water O 2 level versus time of hypoxia exposure. An O 2 level of 21 kPa was set as the ceiling of the curve, and the analysis was performed using numpy.trapz module with default dx = 1 in Python 3.7. We refereed to this composite measure as total LOE, and it was expressed in units of kPa O2 .min. 2.1. Tissue and mitochondrial respirometry Fish were euthanized by section of the spinal cord at the skull and the intact brain was dissected and then weighed ensuring any excess blood was removed. In experiments using homogenates, intact brains was triturated gently by suction through a 10 ml syringe with decreasing gauge needles (16–25 gauge) prior to being introduced to the respirometry chamber, which occurred within 30 s of the initial brain dissection. In experiments using permeabilised samples, intact brains were immediately placed in ice-cold biopsy buffer containing (in mM hereon, unless stated) 2.77 CaK 2 EGTA, K 2 7.23 EGTA, 5.77 Na 2 ATP, 6.56 MgCl 2 .6H 2 O, 20 taurine, 15 Na 2 -phosphocreatine, 20 imidazole, 0.5 DTT, 50 KMES, 50 sucrose, pH 7.22 at 20°C (Gnaiger et al., 2000). Cellular permeabilisation was undertaken by the addition of 50 µg.ml -1 of freshly prepared saponin to plastic cell culture plates held on ice. The brains were then gently agitated in the culture plates for 30 min on ice, after which the permeabilised tissue was removed and washed three times for 10 min in ice-cold respiration medium (containing 0.5 EGTA, 3 MgCl 2 .6H 2 O, 60 K-lactobionate, 20 taurine, 10 KH 2 PO 4 , 2.5 HEPES, 30 MES, 160 sucrose, 1 g.l -1 BSA, pH 7.22 at 20°C) prior to its addition to respirometry chambers. Fish were weighed and measured (see Table 1 for mass and length) after dissection and the proportion of brain mass relative to body mass was calculated. Respiration was measured using Oroboros™ O2k high resolution respirometers (Innsbruck, Austria). The O 2 electrode was calibrated from 0-20.46 kPa PO 2 (0-100% air saturation, 262 µM dissolved O 2 equivalent at 20ºC and 101 kPa) prior respirometry assays. Brains (around ~ 5 mg homogenate or permeabilised) were introduced in the respirometry chambers containing 2 ml respiration medium calibrated prior experiment at 100% O 2 . After signal stabilisation and the measurement of the routine state in brain homogenates, saturating mitochondrial substrates (pyruvate, malate, glutamate and succinate) and ADP were added to maximise oxidative phosphorylation (OxPhos PMGS ). The medium was then re-aerated fully and brains were left to deplete O 2 and held in anoxia for ~ 5 min, after which re-oxygenation was performed. In chambers containing permeabilised brain, oligomycin (5 µM) was added to measure respiration attributed to proton leak (Leak), followed by carbonyl cyanide m-chlorophenyl hydrazone (0.5 µM titration steps until signal stabilisation) to measure the maximum O 2 consumption capacity of the electron transport system (ETS) when uncoupled from OxPhos. In all assays, potassium-cyanide (1 mM) was added to measure non-mitochondrial O 2 consumption, which was then subtracted from raw respiration data. Substrate-Uncoupler-Inhibitor-Titration protocols are detailed in the supplementary file (Table S.1). 2.2. Determination of mitochondrial affinity to O 2 Respirometry data was recorded with DatLab (v7.1) software with the minimum smoothing to maximise resolution, especially at low PO 2 . Correction for time response of the electrode was also accounted for (Gnaiger, 2008). Above 2.05 kPa, an exponential moving average of 20 seconds (i.e. 10 recordings) was calculated in Excel to lower the signal to noise ration. The mitochondrial affinity to O 2 (mP 50 ) was then determined as the PO 2 at which the respiration rate is half of the maximum OxPhos rate. We also made an estimate of efficiency, which typically relates to kinetic parameters of purified enzymes. Given that mitochondrial respiration is a composite or pathways we make a proxy for the classical k cat /K M and present a measure of efficiency in the context JO 2Max /mP 50 . 2.3. CCO capacity and catalytic efficiency The CCO capacity was assessed in two independent set of assays using two different methods (Table S.2). In permeabilised brain induced in OxPhos (saturated pyruvate, malate, glutamate, succinate and ADP), sodium azide was titrated to gradually inhibit CCO, until full CCO inhibition (final concentration of 12 mM). Inhibition dose response curves (Hill curves) were fitted with the least-squares method using GraphPad © Prism. Using another set of assay, permeabilised brain was induced in uncoupled state (ETS), and electron feeding to CCO was inhibited by the addition of 2.5µM antimycin A. Maximum CCO oxidation rates were then assessed by excess electron feeding with N , N , N ’, N ’-tetramethyl-p-phenylenediamine (TMPD, 0.5 mM) and additional ascorbate (2 mM) to measure TMPD auto-oxidation. Background chemical auto-oxidation was measured following additional potassium-cyanide (2 mM) and subtracted to CCO consumption rates. Net CCO rates were then normalised by the ETS capacity determined in the uncoupled state described in paragraph 2.2. 2.4. Statistical analysis Statistical analyses were performed with GraphPad © Prism 7, with significance set at P < 0.05. One-way ANOVA or two-way ANOVA followed by Turkey’s post-hoc tests were used to test for interactions and differences among and between species and/or parameters. For both mitochondrial respiration from 20.5 kPa to anoxia (Fig. 2 .B) and CCO inhibition (Fig. 3 .A) data, curves were fitted for each species to a three-parameters dose-response curve (hill curve) using the least-squares method. Resulting fitted curves were then compared using the extra-sum of squares F-test to test for shared parameters, including mP 50 (Fig. 2 .C), IC50 and Hill slope (respectively Fig. 3 .C and D). In Fig. 4 , linear regression using least squares method was used to test for correlation between parameters of the mitochondrial function to LOE determined in 2.3. 3. Results 3.1. Hypoxia tolerance of triplefin fish species SMR under normoxia was similar among species, except for FV having a slightly higher resting Ṁ O 2 than BM (P < 0.05; Fig. 1 .A). The intertidal specialist BM had a lower P crit than all other species (P < 0.05; Fig. 1 .A), and the occasional intertidal occupant FL had a lower P crit than the exclusively subtidal species FV and FM (P < 0.05; Fig. 1 .A). There was no difference in P crit between the exclusively subtidal species FV and FM . The two subtidal species could not match the lowest PO 2 achieved by the two rock-pool species and lost equilibrium at ~ 2.8 ± 0.2 and 1.8 ± 0.1 kPa for FM and FV , respectively (Fig. 1 . B ). While some FL reached LOE around 2 kPa, the most tolerant FL maintained equilibrium for ~ 1.25h at ~ 1.4 kPa (Fig. 1 .B). BM tolerated severe hypoxia (~ 1.4 kPa) for an average time of 1.5h before LOE. Both the O 2 tension at LOE (Fig. 1 . C ) and the time duration to LOE (Fig. 1 .D) showed BM and FL to have superior hypoxia tolerance than their subtidal counterparts. Indeed, when total LOE was expressed as the area above the curve from 0 min to time of LOE during hypoxia exposure, this revealed that the exclusively intertidal BM was more hypoxia tolerant than all other species and that the occasional intertidal occupant FL tended to be more hypoxia tolerant than the exclusively subtidal species (P < 0.05; Fig. 1 .E). A strong linear correlation between P crit and total LOE was verified among species (P < 0.01 ,R 2 = 0.87, Fig. 1 .D). 3.2. Oxygen kinetics of brain tissues Overall, O 2 flux was higher in the brain of BM relative to the subtidal species FV and FM , regardless of preparation or mitochondrial state (P < 0.01; Fig. 2 .A). With the brain homogenate of all species, routine respiration was ~ 31% lower than OxPhos respiration (P < 0.01). In permeabilised brain without ADP, leak respiration rates were less than a third of OxPhos rates (P < 0.01) and only FV and FM showed an increase in O 2 flux with uncoupling (P < 0.05). OxPhos was however ~ 20% lower in brain homogenates (P < 0.05). To span the O 2 levels the brain likely encounters in vivo , we assessed O 2 consumption kinetics in vitro with brain homogenate and permeabilised brain in the OxPhos state (i.e. without substrate limitation). In brain homogenates, respiration was the highest in BM (P < 0.001), which retained almost two times higher O 2 flux at PO 2 2.05 kPa (P < 0.05), while at PO 2 0.001;). In all species, with the exception of FL , respiration rates were ~ 30% lower in permeabilised tissues than in homogenised tissues (P < 0.0001). No difference in mP 50 was found between the fish species with no correlation between maximum OxPhos respiration (Fig. 2 .C). However, in brain homogenates the JO 2max /mP 50 was highest in the rock-pool BM relative to the intermediate and subtidal species (P < 0.01). This relationship was less apparent for permeabilised brain (Fig. 2 .D). 3.3. Cytochrome C oxidase Dose response CCO inhibition by sodium azide was different among species (F 6,375 =7.16; P < 0.001; Fig. 3 .A). Although, IC 50 (Fig. 3 .B) and Hill slopes (Fig. 3 .C) extracted from the fitted curves were not significantly different. While in the Forsterygion genus, hypoxia-tolerant FL had the lowest CCO activity (P < 0.001; Fig. 3 .D), it was the highest in the most hypoxia-tolerant BM (P < 0.05), with 10% more activity than the least hypoxia-tolerant FM. 3.4. Correlation between mitochondrial function and hypoxia tolerance At the subcellular level, mitochondrial O 2 affinity (mP 50 ) had a weak correlation with species hypoxia tolerance, here represented with total LOE (R 2 = 0.51; Fig. 4 .A). Paradoxically, higher mitochondrial O 2 consumption rates in OxPhos state (Fig. 4 .B) and greater mitochondrial O 2 catalytic rates (Fig. 4 .C) correlated strongly with hypoxia tolerance (R 2 = 0.99 and 0.95, respectively). At a lower biological level, however, activity rates of CCO (the enzyme at the end of the O 2 cascade) did not correlate linearly with total LOE (R 2 = 0.12). 4. Discussion 4.1. Intertidal triplefins have superior hypoxia tolerance and lower P crit than their subtidal counterparts Here, we show intertidal triplefin species survive severe hypoxia for longer than their subtidal counterparts, which likely reflects the fact that the rock pools inhabited by these species can become severely hypoxic during night-time low tides (McArley et al., 2019). We and others have previously demonstrated intertidal triplefin species have a lower P crit than subtidal triplefin species and have speculated this should promote tolerance to hypoxia (Hilton et al., 2010; McArley et al., 2019). In the current study, the association between lower P crit in intertidal species and improved hypoxia tolerance was confirmed by a strong inverse correlation between total LOE and P crit among the four species examined. Time to LOE and P crit were also correlated among intertidal and subtidal sculpin species (Mandic et al., 2013), which together with the current findings suggests that a relatively low P crit is important to the hypoxia survival strategy of intertidal fishes not undergoing metabolic depression. A low P crit may benefit hypoxia tolerance in intertidal fish by allowing them to delay the onset of anaerobic metabolism during progressive hypoxia in rock pools, thereby mitigating depletion of anaerobic fuel stores and accumulation of toxic end products of anaerobic ATP production. Moreover, in the absence of metabolic rate depression, which is not yet known to occur in triplefins and we did not observe here, a low P crit should allow a greater proportion of energetic demand to be met through efficient mitochondrial ATP production as hypoxia worsens. Indeed, the hypoxia tolerant BM maintained an Ṁ O 2 equivalent to 51% of SMR at a PO 2 of ~ 1.5 kPa, whereas the least hypoxia tolerant FM had an Ṁ O 2 equivalent to only 27% of SMR at the same PO 2 . The ability of BM to extract more O 2 from severely hypoxic water is explained, at least partly, by the possession of gills with a high diffusive capacity for O 2 . Indeed, gill secondary lamellae are thin and numerous in this species (McArley et al., 2019). Furthermore, relative to FM , BM has a higher blood O 2 carrying capacity as indicated by higher haemoglobin concentration (McArley et al., 2019). We also believe the typical behaviour of BM observed during LOE determination, where this species remains relatively inactive and stationary on the tank bottom, is likely to permit maintenance of a metabolic rate close to SMR at water PO 2 below P crit . This behaviour contrasts starkly with that of the exclusively subtidal species, which become active, likely in an attempt to behaviourally avoid hypoxia, as water PO 2 descends below P crit and LOE is approached. Of course, high activity levels mean that metabolic rate will increase substantially beyond that associated with SMR and maintaining energy balance will become even more challenging. For subtidal species, hypoxia is escapable in nature and a behavioural avoidance strategy would be advantageous. However, hypoxia is inescapable in rock pools and also within the LOE trials we performed, so the quiescent low energy demand behaviour of BM is likely to convey a greater chance of surviving periods of severe hypoxia that develop in rock pools prior to replenishment of water O 2 levels on the incoming tide. Quiescent behaviour in severe hypoxia has also been observed in other hypoxia tolerant intertidal fishes (Yoshiyama et al., 1995). Lastly, we have previously demonstrated intertidal triplefins have higher white muscle and hepatic glycogen stores than subtidal species, which could also contribute to superior hypoxia tolerance through fuelling anaerobic respiration (McArley et al., 2019). Large tissue glycogen stores and higher glycolytic enzyme activities in brain have also been demonstrated in intertidal sculpins (Mandic et al., 2013). 4.2. Oxygen utilization is greater in brain mitochondria of intertidal species Brain mitochondria of hypoxia-tolerant and hypoxia sensitive triplefin species had similar affinities to O 2 , with mP 50 a little above 0.10 kPa. In comparison, the least hypoxia-tolerant sculpin displayed brain tissue mP 50 of around 0.08 kPa, although this was measured 2ºC below our experimental temperature (i.e. 18ºC vs 20ºC) (Lau et al., 2017) and higher temperatures increase binding affinities (Blair et al., 1986). Note that within sculpin species a strong correlation was observed between isolated mitochondria mP 50 determined at 18ºC and P crit measured at 12ºC (Lau et al., 2017). In triplefin species, no such correlation was verified when measuring both parameter at 20ºC using brain homogenates or permeabilised tissue. Intracellularly, recent measurements of mitochondrial PO 2 in vivo indicate levels of around 2.6-5 kPa (Mik, 2013; Mik et al., 2008), levels at which brain homogenate mitochondria operates here at ~ 70% of OxPhos for all fish species. Routine respiration rates, mediated by endogenous substrates and closely representative of in vivo conditions, were also ~ 70% that of OxPhos, yet Routine respiration was measured under air saturated O 2 . This indicates that above 2 kPa, substrates, but not O 2, limit mitochondrial respiration in brain homogenates. Interestingly, subtidal species lost equilibrium around 2 kPa, which indicates that below such level, Routine respiration may be affected by O 2 levels, altering brain metabolism which subsequently compromises fish spatial awareness and reflexes upon acute exposure. Such PO 2 is > 25x higher than mP 50 measured here in vitro in both subtidal and intertidal fish, which indicates that all species studied here have marginal PO 2 scope before reaching 50% of maximal mitochondrial respiration capacities. In both homogenate and permeabilised brain, basal O 2 consumption (Routine and Leak, respectively) and OxPhos were consistently greater in the intertidal hypoxia-tolerant species. As indicated by the greater JO 2max /mP 50 (a proxy for k cat /K m ), intertidal species also have greater efficiency to bind and turnover O 2 . This at first appears paradoxical, given that O 2 is limiting in hypoxia and that the mP 50 of mitochondria are similar among species. However, elevation of O 2 consumption can increase O 2 gradients (Gnaiger et al., 1998), thereby enhancing O 2 flow to mitochondria to better support ATP synthesis in hypoxia sensitive species. However, the elevated O 2 consumption observed in brain tissue was not associated with a higher whole animal Ṁ O 2 in intertidal species. Thus, if the high basal O 2 consumption and OxPhos observed in brain is common in other tissues of intertidal species, it does not come at the cost of a higher resting metabolic demand, which would likely impair hypoxia tolerance through increasing basal energetic demands. We have also observed that intertidal triplefins can achieve a higher maximum O 2 consumption rate than their subtidal counterparts (McArley et al., 2019), and this matches the high OxPhos observed in brain tissues of intertidal species here. Rather than high metabolic capacity being an adaptation for hypoxia tolerance per se, it might be that intertidal species retain the potential for high ATP production via OxPhos (Devaux et al., 2019a), and this would meet the energy requirements of other high demand environmental conditions that occur in rock pools. For example, high aerobic capacity would be beneficial for meeting the energetic requirements of wave surge and for avoiding temperature-induced tissue O 2 limitation (functional hypoxia) during acute warming events during daytime low tides. Moreover, thermal ramping events in rock pools tend to coincide with hyperoxia (O 2 supersaturation) (McArley et al., 2019; Richards, 2011), where the high OxPhos capacity of intertidal species could potentially act as a sink for excess O 2 at the tissues and mitigate oxidative stress. This could also occur upon replenishment of rock pool water by the incoming tide, where a high OxPhos capacity could potentially mitigate oxidative stress associated with rapid hypoxia re-oxygenation of tissues (Starkov, 2008). 4.3. Cytochrome c oxidase is not associated with hypoxia tolerance of triplefin fish A greater mitochondrial O 2 utilisation may reside from multiple adjustments. This includes a greater capacity to import and oxidise mitochondrial substrates, greater electron transport capacities of the ETS or a more tuned cytochrome c oxidase – the enzyme at the end of the O 2 cascade. CCO subunit 4 has two paralogues in most vertebrates, including fish (Porplycia et al., 2017), with CCO4-2 expression being elevated in response to hypoxia (Fukuda et al., 2007), decreasing O 2 affinity and consumption (Pajuelo Reguera et al., 2020). Notably, CCO inhibition curves were similar across species (Fig. 3 . A ), with similar apparent Km and Hill slopes, suggesting that triplefin species likely share similar forms of CCO with conserved O 2 binding properties. In addition, no clear correlation between CCO and hypoxia tolerance was apparent. This again differs from sculpin species that showed a strong correlation between mP 50 and CCO-O2 affinity (Lau et al., 2017). Maximum CCO activity was similar to maximum OxPhos rates in FL , suggesting that unlike the other species, CCO reserve capacity is limited. This indicates O 2 consumption and therefore ATP synthesis may be affected in FL if CCO activity were downregulated. As expected, however, the CCO capacity was higher than ETS capacity, and this suggests that CCO is not limiting and that regulation of O 2 consumption in hypoxia-tolerant triplefin fish likely occurs at a higher biological level. Conclusion Here, we show that intertidal triplefin fish are more hypoxia tolerant than their subtidal counterparts. Indeed, the exclusively intertidal BM survived severe hypoxia (~ 1.4 kPa) for up to 2 h, whereas the exclusively subtidal FM could only tolerate brief periods of exposure to water O 2 levels between ~ 2-3.5 kPa. Brain mitochondria in hypoxia-tolerant species displayed higher respiration rates than brain of hypoxia sensitive triplefins, even at PO 2 where mitochondria function in vivo . High OxPhos capacity, while potentially aiding hypoxia tolerance through steepening O 2 diffusion gradients between blood and tissues, may also provide reserve aerobic capacity to cope with other high energy demand environmental conditions that occur in the intertidal zone (e.g. acute warming). Although CCO, the enzyme at the end of the O 2 cascade, was not correlated with hypoxia tolerance, it does not appear to limit O 2 consumption in phosphorylating mitochondria. Despite similar mitochondrial affinity to O 2 in brain tissue between hypoxia-tolerant and hypoxia sensitive triplefins, a greater capacity of intertidal species to access O 2 in hypoxic environments was indicated by a lower P crit at the whole animal level. Furthermore, intertidal species, despite the high OxPhos capacity identified in brain tissue, did not have increased resting Ṁ O 2 . This suggests that when exposed to severe hypoxia, intertidal species are able to meet a greater proportion of their energetic demand through efficient mitochondrial ATP production, which would likely aid hypoxia tolerance through, at least in part, mitigating unsustainable levels of anaerobic respiration. A low P crit strategy, particularly when combined with quiescent behaviour, may be best suited to the nature of hypoxic events in rock pools inhabited by tripflefins, where severe hypoxia is of short duration and there is always some O 2 available. Abbreviations CCO Cytochrome C oxidase ETS Mitochondrial electron transport system LOE Loss of equilibrium Ṁ O 2 Mass-specific O 2 consumption mP 50 Oxygen tension at which mitochondrial respiration is half of its maximum OxPhos Oxidative respiration P crit Critical oxygen tension PO 2 Partial O 2 pressure Declarations Competing interests The authors declare no competing interest. Funding This work was fully supported by the Royal Society of New Zealand Marsden fund (14-UOA-210: How to avoid brain damage during oxygen deprivation? Intertidal fish provide a unique test model). Author Contribution All authors designed the research; T.M. performed the whole animal respirometry and J.B.L. performed mitochondrial experiments; J.B.L., T.M., N.H. and A.J.R.H. analysed the data and critically discussed the results; J.B.L. and T.M. wrote the paper and all authors approved submission. Acknowledgement The authors would like to thank Jaime Willis, Alicia Hellens, Peter Schlegel, Craig Norrie and Esther Stuck for their help with the animal collection. Data Availability Data supporting the results presented in this article are available at the University of Auckland repository (upon acceptance of this present manuscript). References A.L. Val, M. N. P. S., V. M. F. Almeida-Val. (1998). Hypoxia adaptation in fish of the Amazon: a never-ending task. South African journal of zoology. Suid-Afrikaanse tydskrif vir dierkunde. 33 , 107–114. Behrens, J. W. and Steffensen, J. F. (2007). The effect of hypoxia on behavioural and physiological aspects of lesser sandeel, Ammodytes tobianus (Linnaeus, 1785). Marine Biology 150 , 1365–1377. Bickler, P. E. and Buck, L. T. (2007). Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability. Annu Rev Physiol 69 , 145 − 70. Blair, D. F., Ellis, W. R., Wang, H., Gray, H. B. and Chan, S. I. (1986). Spectroelectrochemical study of cytochrome c oxidase: pH and temperature dependences of the cytochrome potentials. Characterization of site-site interactions. Journal of Biological Chemistry 261 , 11524–11537. Cervós-Navarro, J. and Diemer, N. H. (1991). Selective vulnerability in brain hypoxia. Critical reviews in neurobiology 6 , 149–182. Claireaux, G. and Chabot, D. (2016). Responses by fishes to environmental hypoxia: integration through Fry's concept of aerobic metabolic scope. J Fish Biol 88 , 232 − 51. Clarke, A. and Johnston, N. M. (1999). Scaling of metabolic rate with body mass and temperature in teleost fish. Journal of Animal Ecology 68 , 893–905. Cook, D. G., Iftikar, F. I., Baker, D. W., Hickey, A. J. and Herbert, N. A. (2013). Low-O(2) acclimation shifts the hypoxia avoidance behaviour of snapper (Pagrus auratus) with only subtle changes in aerobic and anaerobic function. J Exp Biol 216 , 369 − 78. Cumming, H. and Herbert, N. A. (2016). Gill structural change in response to turbidity has no effect on the oxygen uptake of a juvenile sparid fish. Conserv Physiol 4 , cow033. Del Rio, C. and Montaner, J. (2021). Hypoxia Tolerant Species: The Wisdom of Nature Translated into Targets for Stroke Therapy. Int J Mol Sci 22 . Devaux, J. B. L., Hedges, C. P., Birch, N., Herbert, N., Renshaw, G. M. C. and Hickey, A. J. R. (2019a). Acidosis Maintains the Function of Brain Mitochondria in Hypoxia-Tolerant Triplefin Fish: A Strategy to Survive Acute Hypoxic Exposure? Front Physiol 9 . Devaux, J. B. L., Hickey, A. J. R. and Renshaw, G. M. C. (2019b). Mitochondrial plasticity in the cerebellum of two anoxia-tolerant sharks: contrasting responses to anoxia/reoxygenation. J Exp Biol , jeb.191353. Fukuda, R., Zhang, H., Kim, J. W., Shimoda, L., Dang, C. V. and Semenza, G. L. (2007). HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell 129 , 111 − 22. Galli, G. L., Lau, G. Y. and Richards, J. G. (2013). Beating oxygen: chronic anoxia exposure reduces mitochondrial F1FO-ATPase activity in turtle (Trachemys scripta) heart. J Exp Biol 216 , 3283-93. Galli, G. L. and Richards, J. G. (2014). Mitochondria from anoxia-tolerant animals reveal common strategies to survive without oxygen. J Comp Physiol B 184 , 285–302. Gnaiger, E. (2008). Polarographic Oxygen Sensors, the Oxygraph, and High-Resolution Respirometry to Assess Mitochondrial Function. In Drug-Induced Mitochondrial Dysfunction . Gnaiger, E. (2020). Mitochondrial physiology. Bioenergetics Communications . Gnaiger, E., Kuznetsov, A. V., Schneeberger, S., Seiler, R., Brandacher, G., Steurer, W. and Margreiter, R. (2000). Mitochondria in the Cold. In Life in the Cold: Eleventh International Hibernation Symposium , eds. G. Heldmaier and M. Klingenspor), pp. 431–442. Berlin, Heidelberg: Springer Berlin Heidelberg. Gnaiger, E., Lassnig, B., Kuznetsov, A., Rieger, G. and Margreiter, R. (1998). Mitochondrial oxygen affinity, respiratory flux control and excess capacity of cytochrome c oxidase. J Exp Biol 201 , 1129-39. Gorr, T. A., Wichmann, D., Hu, J., Hermes-Lima, M., Welker, A. F., Terwilliger, N., Wren, J. F., Viney, M., Morris, S., Nilsson, G. E. et al. (2010). Hypoxia tolerance in animals: biology and application. Physiol Biochem Zool 83 , 733 − 52. Hickey, A. J. and Clements, K. D. (2005). Genome size evolution in New Zealand triplefin fishes. J Hered 96 , 356 − 62. Hickey, A. J., Lavery, S. D., Hannan, D. A., Baker, C. S. and Clements, K. D. (2009). New Zealand triplefin fishes (family Tripterygiidae): contrasting population structure and mtDNA diversity within a marine species flock. Mol Ecol 18 , 680 − 96. Hilton, Z. (2010). Physiological adaptation in the radiation of New Zealand triplefin fishes (Family Tripterygiidae). In School of Biological Sciences , vol. PhD: University of Auckland. Hilton, Z., Clements, K. D. and Hickey, A. J. (2010). Temperature sensitivity of cardiac mitochondria in intertidal and subtidal triplefin fishes. J Comp Physiol B 180 , 979 − 90. Hilton, Z., Wellenreuther, M. and Clements, K. D. (2008). Physiology underpins habitat partitioning in a sympatric sister-species pair of intertidal fishes. Functional Ecology 22 , 1108–1117. Khan, J. R., Pether, S., Bruce, M., Walker, S. P. and Herbert, N. A. (2014). Optimum temperatures for growth and feed conversion in cultured hapuku (Polyprion oxygeneios) — Is there a link to aerobic metabolic scope and final temperature preference? Aquaculture 430 , 107–113. Koch, L. G. and Britton, S. L. (2008). Aerobic metabolism underlies complexity and capacity. The Journal of Physiology 586 , 83–95. Larson, J., Drew, K. L., Folkow, L. P., Milton, S. L. and Park, T. J. (2014). No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates. J Exp Biol 217 , 1024-39. Lau, G. Y., Mandic, M. and Richards, J. G. (2017). Evolution of Cytochrome c Oxidase in Hypoxia Tolerant Sculpins (Cottidae, Actinopterygii). Mol Biol Evol 34 , 2153–2162. Mandic, M., Speers-Roesch, B. and Richards, J. G. (2013). Hypoxia tolerance in sculpins is associated with high anaerobic enzyme activity in brain but not in liver or muscle. Physiol Biochem Zool 86 , 92–105. Mandic, M., Todgham, A. E. and Richards, J. G. (2009). Mechanisms and evolution of hypoxia tolerance in fish. Proc Biol Sci 276 , 735 − 44. McArley, T. J., Hickey, A. J. R. and Herbert, N. A. (2017). Chronic warm exposure impairs growth performance and reduces thermal safety margins in the common triplefin fish (Forsterygion lapillum). J Exp Biol 220 , 3527–3535. McArley, T. J., Hickey, A. J. R. and Herbert, N. A. (2018). Hyperoxia increases maximum oxygen consumption and aerobic scope of intertidal fish facing acutely high temperatures. J Exp Biol . McArley, T. J., Hickey, A. J. R., Wallace, L., Kunzmann, A. and Herbert, N. A. (2019). Intertidal triplefin fishes have a lower critical oxygen tension (Pcrit), higher maximal aerobic capacity, and higher tissue glycogen stores than their subtidal counterparts. J Comp Physiol B 189 , 399–411. Mik, E. G. (2013). Special article: measuring mitochondrial oxygen tension: from basic principles to application in humans. Anesth Analg 117 , 834 − 46. Mik, E. G., Johannes, T., Zuurbier, C. J., Heinen, A., Houben-Weerts, J. H., Balestra, G. M., Stap, J., Beek, J. F. and Ince, C. (2008). In vivo mitochondrial oxygen tension measured by a delayed fluorescence lifetime technique. Biophys J 95 , 3977-90. Nilsson, G. E. and Lutz, P. L. (2004). Anoxia tolerant brains. J Cereb Blood Flow Metab 24 , 475 − 86. Norin, T., Malte, H. and Clark, T. D. (2014). Aerobic scope does not predict the performance of a tropical eurythermal fish at elevated temperatures. J Exp Biol 217 , 244 − 51. Pajuelo Reguera, D., Cunatova, K., Vrbacky, M., Pecinova, A., Houstek, J., Mracek, T. and Pecina, P. (2020). Cytochrome c Oxidase Subunit 4 Isoform Exchange Results in Modulation of Oxygen Affinity. Cells 9 . Pamenter, M. E. (2014). Mitochondria: a multimodal hub of hypoxia tolerance. Canadian Journal of Zoology 92 , 569–589. Pamenter, M. E., Gomez, C. R., Richards, J. G. and Milsom, W. K. (2016). Mitochondrial responses to prolonged anoxia in brain of red-eared slider turtles. Biol Lett 12 , 20150797. Porplycia, D., Lau, G. Y., McDonald, J., Chen, Z., Richards, J. G. and Moyes, C. D. (2017). Subfunctionalization of COX4 paralogs in fish. Am J Physiol Regul Integr Comp Physiol 312 , R671-R680. Richards, J. G. (2011). Physiological, behavioral and biochemical adaptations of intertidal fishes to hypoxia. J Exp Biol 214 , 191-9. Schurmann, H. and Steffensen, J. F. (1997). Effects of temperature, hypoxia and activity on the metabolism of juvenile Atlantic cod. J Fish Biol 50 , 1166–1180. Starkov, A. A. (2008). The role of mitochondria in reactive oxygen species metabolism and signaling. Ann N Y Acad Sci 1147 , 37–52. Steffensen, J. F. (1989). Some errors in respirometry of aquatic breathers: How to avoid and correct for them. Fish Physiol Biochem 6 , 49–59. Willis, J. R., Hickey, A. J. and Devaux, J. B. (2021). Thermally tolerant intertidal triplefin fish (Tripterygiidae) sustain ATP dynamics better than subtidal species under acute heat stress. Scientific Reports 11 , 1–10. Yoshiyama, R. M., Valpey, C. J., Schalk, L. L., Oswald, N. M., Vaness, K. K., Lauritzen, D. and Limm, M. (1995). Differential propensities for aerial emergence in intertidal sculpins (Teleostei; Cottidae). Journal of Experimental Marine Biology and Ecology 191 , 195–207. Additional Declarations No competing interests reported. Supplementary Files HTTriplefinsSupFigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4722244","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":375641704,"identity":"3a6abbb6-3752-42e4-9e70-aab47a4ab94a","order_by":0,"name":"Jules B.L. Devaux","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYBACxgYGAzCDH8wpAJIHiNUi2QDiGBChBQggWgwOEKuFuYF542OemsOJm4+fMfw4w8Auj+8A88MPjDvq8DiMrdiY59hhY7MzOcaSGwySiyUPsBlLMJ5hw6OFx0xyBtthObMDOWaMDwyYEzccYDBjYGzjwafF/OeMf4d5jPvfgLTUA7WwfwNqkcBrC8PHtsNyBhJAWzYYHAZq4QHZYoBbSzNbscTHvnRjiRvPiiVnGBxPnHmYp1gisS0BpxbD9uaNHxK+WSf29ydv/NhTUZ3Yd7x944ePbbhDzLAZQ4gZiHHbwcAgj0duFIyCUTAKRgEEAABNnlLlp+PYogAAAABJRU5ErkJggg==","orcid":"","institution":"The University of Auckland","correspondingAuthor":true,"prefix":"","firstName":"Jules","middleName":"B.L.","lastName":"Devaux","suffix":""},{"id":375641705,"identity":"4ff4cbbf-5c6d-4506-ad00-5224c2d81234","order_by":1,"name":"Tristan J. McArley","email":"","orcid":"","institution":"The University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Tristan","middleName":"J.","lastName":"McArley","suffix":""},{"id":375641706,"identity":"d63d913f-a414-4478-bc19-ab6e7b2496b2","order_by":2,"name":"Neill Herbert","email":"","orcid":"","institution":"The University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Neill","middleName":"","lastName":"Herbert","suffix":""},{"id":375641707,"identity":"f8bcdd1d-1c72-4482-8b4e-06fffeb85f27","order_by":3,"name":"Anthony J.R. Hickey","email":"","orcid":"","institution":"The University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"J.R.","lastName":"H","suffix":"J.R."}],"badges":[],"createdAt":"2024-07-11 06:42:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4722244/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4722244/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70467900,"identity":"18a17b79-c9f9-43ae-9577-619fb2d6f746","added_by":"auto","created_at":"2024-12-03 12:50:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":36868,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCritical O\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u003cstrong\u003etension (P\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u003cstrong\u003ecrit\u003c/strong\u003e\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u003cstrong\u003e) and tolerance to hypoxia among intertidal and subtidal triplefin fish species.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Bellapiscis medius (BM): exclusively intertidal rock pools; Forsterygiion lapillum (FL): intertidal rock pools and shallow subtidal; F. varium (FV): exclusively subtidal; F. malcomi (FM): exclusively subtidal. Data points show means ± s.e.m. N=10 individuals for each species in panel A and N=9 individuals for each species in panel C-E.\u0026nbsp; (A) P\u003c/em\u003e\u003csub\u003e\u003cem\u003ecrit \u003c/em\u003e\u003c/sub\u003e\u003cem\u003e(vertical-coloured lines) was defined as the O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2 \u003c/em\u003e\u003c/sub\u003e\u003cem\u003epartial pressure (PO\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e) at which mass-specific O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e consumption rate (measured by intermittent stop-flow respirometry) during stepwise hypoxia began to decline progressively below standard metabolic rate (SMR). (B) Profile showing the point of loss of equilibrium (LOE) for individuals of each species during a hypoxia exposure where PO\u003c/em\u003e\u003csub\u003e\u003cem\u003e2 \u003c/em\u003e\u003c/sub\u003e\u003cem\u003ewas reduced from normoxia to \u0026lt;1.5 kPa in ~30 min. (C) Partial pressure of O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2 \u003c/em\u003e\u003c/sub\u003e\u003cem\u003eat LOE. (D) Time to LOE. (E) To account for the temporal aspect of hypoxia tolerance, total LOE was determined as the area above the curve of water O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2 \u003c/em\u003e\u003c/sub\u003e\u003cem\u003elevel versus time. (F) The relationship between P\u003c/em\u003e\u003csub\u003e\u003cem\u003ecrit\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e and total LOE among species (dashed lines represent 95% confidence intervals). Differences in P\u003c/em\u003e\u003csub\u003e\u003cem\u003ecrit, \u003c/em\u003e\u003c/sub\u003e\u003cem\u003eSMR and LOE among species were tested with one-way ANOVA. Significance was set at P \u0026lt; 0.05, and significant post-hoc differences between species are shown by uncommon letters.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4722244/v1/a11e1a9261e589a94b3dde95.png"},{"id":70467499,"identity":"5f8c925b-4161-4aab-9629-473386314961","added_by":"auto","created_at":"2024-12-03 12:42:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxygen kinetics at the cellular level.\u003c/strong\u003e (A) Mitochondrial respiration was measured in brain homogenates (left) with no added substrates (i.e. only endogenous substrates present; “Routine”) and in the presence of saturated mitochondrial substrates pyruvate, malate and succinate (OxPhos\u003csub\u003ePMS\u003c/sub\u003e). Similarly, in permeabilised brain fragments (right), basal respiration attributed to proton leak (Leak\u003csub\u003eOmy\u003c/sub\u003e, measured in the presence of the ATP\u003csub\u003eF0-F1\u003c/sub\u003e inhibitor oligomycin), OxPhos\u003csub\u003ePMS\u003c/sub\u003e and maximum respiration when uncoupled from OxPhos\u003csub\u003ePMS\u003c/sub\u003e with CCCP. (B) In air saturated medium and saturated respiratory substrates, oxygen consumption was measured in brain homogenate (left) and permeabilised brain (right), let to deplete O\u003csub\u003e2\u003c/sub\u003e until anoxia. The dashed line at 2.05 kPa (10% air saturation) corresponds to the level below which the O\u003csub\u003e2\u003c/sub\u003e tension is likely encountered intracellularly. (C) In both homogenate and permeabilised brain, all species displayed similar mitochondrial apparent affinity to O\u003csub\u003e2\u003c/sub\u003e (mP\u003csub\u003e50\u003c/sub\u003e). Data expressed in kPa and extracted from the respiration curves in (B). (D) Brain homogenates of the most hypoxia tolerant species has a higher O\u003csub\u003e2\u003c/sub\u003e turnover rate (JO\u003csub\u003e2Max\u003c/sub\u003e/mP\u003csub\u003e50\u003c/sub\u003e) than the two subtidal species.\u0026nbsp; Species indicated as “BM”: B. medius, “FL”: F. lapillum, “FV”: F. varium and “FM”: F. malcomi. Data are mean of 8 and 7 (respectively homogenate and permeabilised) ± s.e.m. Statistical difference in black between species and in blue between states chosen at P \u0026lt; 0.05, 0.01 and 0.001 represented as *, ** and *** respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4722244/v1/8a3ee6ab541924e503b4fe0e.png"},{"id":70467500,"identity":"28649123-7963-490e-91a7-b7ed11bf0cb7","added_by":"auto","created_at":"2024-12-03 12:42:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCytochrome c oxidase properties in permeabilised brains of triplefin fishes.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e (A) Graded inhibition of cytochrome c oxidase was performed with the titration of sodium azide in situ. (B) Half inhibition (IC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e) and (C) cooperativity (Hill slope) extracted from (A), with no apparent difference among fish species. (D) The maximum activity of the enzyme in situ was assessed with TMPD-ascorbate with correction for auto-oxidation and expressed in pmolO\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e.s\u003c/em\u003e\u003csup\u003e\u003cem\u003e-1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e.mg\u003c/em\u003e\u003csup\u003e\u003cem\u003e-1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e. (E) Affinity of an enzyme to its substrate, i.e. O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e, is not independent of the enzyme concentration. Therefore, CCO activity was normalised by the mitochondrial electron transport system capacity (ETS). Intertidal fish species are “BM”: B. medius and “FL”: F. lapillum, and subtidal species are “FV”: F. varium and “FM”: F. varium. Data presented as mean of 6 individuals ± s.e.m. Statistical difference between species chosen at P \u0026lt; 0.05, 0.01 and 0.001 represented as *, ** and *** respectively.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4722244/v1/148a78c9ce1cbdbe2bbb0c7d.png"},{"id":70467502,"identity":"833592ca-ec1d-475b-a9ff-cc385d5b21f7","added_by":"auto","created_at":"2024-12-03 12:42:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":31703,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe paradox\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e:\u0026nbsp; Mitochondrial affinity to O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e does not correlate with hypoxia-tolerance but higher O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e extraction and consumption does. (A) mitochondrial affinity to O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e (mP50), (B) mitochondrial O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e consumption (OxPhos respiration), (C) O\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e turnover rate and (D) maximal cytochrome c oxidase (CCO) activity correlation with loss of equilibrium (LOE) in the triplefin fish species (from most to least hypoxia-tolerant) “BM”: B. medius, “FL”: F. lapillum, “FV”: F. varium and “FM”: F. malcomi. Data extracted from previous figures using parameters from brain homogenates (A, B \u0026amp; C). Linear regression was performed using the least squares method and displayed as goodness of fit (full line) with confidence bands (dotted lines) chosen at 95%.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4722244/v1/7d87614deb5419e3cdce2210.png"},{"id":72487333,"identity":"67ea1312-5703-4637-85af-1c212b8dcb1b","added_by":"auto","created_at":"2024-12-27 19:31:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1524662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4722244/v1/053f49b4-55a1-48d1-99ab-56605023956b.pdf"},{"id":70467501,"identity":"0b8a1311-7ddd-459d-ad3f-91065ba7c839","added_by":"auto","created_at":"2024-12-03 12:42:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":62064,"visible":true,"origin":"","legend":"","description":"","filename":"HTTriplefinsSupFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-4722244/v1/5d36a051562d45f54cb65101.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Superior hypoxia tolerance of intertidal triplefin fish is associated with low critical oxygen tension and high phosphorylating capacity in brain mitochondria","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAdaptations to hypoxia come in many forms and occur at all levels of biological organisation, from behavioural adjustments to transcriptomic alterations (A.L. Val, 1998). Ultimately, to benefit hypoxia tolerance, these adaptations must contribute to the ability of an organism to maintain energy balance under conditions where ATP production via efficient oxidative phosphorylation is compromised (Richards, 2011). At the terminus of the O\u003csub\u003e2\u003c/sub\u003e cascade, mitochondria produce ATP via pathways that oxidise carbon sources and reduce O\u003csub\u003e2\u003c/sub\u003e to water (Koch and Britton, 2008). Multiple adaptations of mitochondrial structure and function have been identified in hypoxia-tolerant organisms (reviewed in Pamenter, 2014); however, these vary across hypoxia and anoxia tolerant species (Bickler and Buck, 2007; Galli and Richards, 2014; Gorr et al., 2010). In particular, mitochondrial adaptations in the brain, which is often highly sensitive to O\u003csub\u003e2\u003c/sub\u003e depravation (Cerv\u0026oacute;s-Navarro and Diemer, 1991), appear to play a key role in hypoxia tolerance (Del Rio and Montaner, 2021; Larson et al., 2014; Nilsson and Lutz, 2004). For instance, to conserve ATP, brain mitochondria of the anoxia-tolerant turtle (\u003cem\u003eTrachemys scripta\u003c/em\u003e) appear to remodel, partially uncouple and decrease F\u003csub\u003e0\u003c/sub\u003e-F\u003csub\u003e1\u003c/sub\u003e-ATPase activity during anoxia (Galli et al., 2013; Pamenter et al., 2016). Additionally, in the epaulette shark (\u003cem\u003eHemiscyllium ocellatum\u003c/em\u003e), the electron transport chain in brain mitochondria remodels in anoxia with a decrease in complex II mediated O\u003csub\u003e2\u003c/sub\u003e flux, and this may protect against oxidative stress during reperfusion (Devaux et al., 2019b). Brain mitochondria of intertidal sculpin species (\u003cem\u003eCottidae\u003c/em\u003e family) display greater O\u003csub\u003e2\u003c/sub\u003e affinities, which is partially explained by CCO isoforms having greater O\u003csub\u003e2\u003c/sub\u003e binding properties (Lau et al., 2017). It is anticipated that both higher mitochondrial O\u003csub\u003e2\u003c/sub\u003e binding affinity and higher mitochondrial O\u003csub\u003e2\u003c/sub\u003e consumption would facilitate improved O\u003csub\u003e2\u003c/sub\u003e extraction (Gnaiger et al., 1998) from the blood to brain tissue, thereby allowing intertidal fish to better utilize available O\u003csub\u003e2\u003c/sub\u003e during periodic hypoxia in rock pools.\u003c/p\u003e \u003cp\u003eThe New-Zealand triplefin fish (Family \u003cem\u003eTripterygiidae\u003c/em\u003e) group consists of 26 endemic species of which most occupy stable, normoxic subtidal habitats. Three species, however, are known to inhabit intertidal rock pools, which can become severely hypoxic during night-time low tides (Hickey and Clements, 2005; Hilton, 2010; Hilton et al., 2008; McArley et al., 2018). While not linked to hypoxia, there is some evidence of selective pressure on mitochondrial genes within rock pool species relative to subtidal species (Hickey et al., 2009). Moreover, heart (Hilton et al., 2010) and brain (Willis et al., 2021) mitochondria of the exclusively intertidal rock pool species \u003cem\u003eBellapiscis medius\u003c/em\u003e had greater respiratory efficiencies and stabilities than two strictly subtidal triplefin species when exposed to elevated temperatures. Relative to the two subtidal triplefins, brain mitochondria of \u003cem\u003eB. medius\u003c/em\u003e also showed adaptations that enhance ATP production under acidifying conditions, such as those occurring in hypoxic brain (Devaux et al., 2019a). This suggests that mitochondrial function may vary among triplefin species challenged with hypoxia; thus, the aim of this study was to determine whether adaptations in mitochondrial function contribute to variation in hypoxia tolerance among intertidal and subtidal triplefins.\u003c/p\u003e \u003cp\u003eThe present study includes four triplefin species, which occupy a range of habitats varying in terms of the likelihood of environmental hypoxia exposure. \u003cem\u003eBellapiscis medius\u003c/em\u003e is an intertidal specialist, which exclusively inhabits rockpools that can routinely decrease to less than 20% air saturated O\u003csub\u003e2\u003c/sub\u003e during night-time low tides (Hilton et al., 2008; McArley et al., 2019). \u003cem\u003eFosterygion lapillum\u003c/em\u003e occupies lower intertidal rock pools and shallow subtidal habitats, and \u003cem\u003eF. varium\u003c/em\u003e and \u003cem\u003eF. malcomi\u003c/em\u003e are exclusively subtidal species occupying rocky reef habitats to depths of ~\u0026thinsp;35 m (Hilton et al., 2008; McArley et al., 2019). We have previously shown that the intertidal species \u003cem\u003eB. medius\u003c/em\u003e and \u003cem\u003eF. lapillum\u003c/em\u003e have a lower critical O\u003csub\u003e2\u003c/sub\u003e tension (P\u003csub\u003ecrit\u003c/sub\u003e) than their subtidal counterparts \u003cem\u003eF. varium\u003c/em\u003e and \u003cem\u003eF. malcolmi\u003c/em\u003e (McArley et al., 2019). This suggests intertidal triplefins have a superior ability to meet O\u003csub\u003e2\u003c/sub\u003e demand associated with standard metabolic rate (SMR; an estimate of basal metabolic rate) in hypoxia and indicates they are also likely to be more hypoxia tolerant. In the present investigation, time to loss of equilibrium (LOE) during severe hypoxia exposure was measured alongside P\u003csub\u003ecrit\u003c/sub\u003e to establish differences in absolute hypoxia tolerance among the intertidal and subtidal species.\u003c/p\u003e \u003cp\u003eFirstly, we established differences in whole animal hypoxia tolerance among intertidal and subtidal species at 20\u003csup\u003eo\u003c/sup\u003eC. We predicted that relative to subtidal species (\u003cem\u003eF. varium\u003c/em\u003e and \u003cem\u003eF. malcolmi\u003c/em\u003e), intertidal species (\u003cem\u003eB. medius\u003c/em\u003e and \u003cem\u003eF. lapillum\u003c/em\u003e) would have lower P\u003csub\u003ecrit\u003c/sub\u003e, as has been previously established at 18\u003csup\u003eo\u003c/sup\u003eC (McArley et al., 2019), and also be more hypoxia tolerant as assessed by time to LOE. We then measured O\u003csub\u003e2\u003c/sub\u003e consumption \u003cem\u003ein vitro\u003c/em\u003e in brain homogenate and permeabilised brain and assessed the mitochondrial affinity to O\u003csub\u003e2\u003c/sub\u003e (mP\u003csub\u003e50\u003c/sub\u003e) and O\u003csub\u003e2\u003c/sub\u003e catalytic efficiencies (K\u003csub\u003ecat,app\u003c/sub\u003e) using high resolution respirometry at the same temperature. We chose brain as this excitable tissue is acutely sensitive to low O\u003csub\u003e2\u003c/sub\u003e (Cerv\u0026oacute;s-Navarro and Diemer, 1991). Brain homogenate preparations were used as these contain endogenous substrates and allows for a greater O\u003csub\u003e2\u003c/sub\u003e diffusion, likely more representative mitochondrial environment \u003cem\u003ein situ\u003c/em\u003e (Gnaiger, 2020; Gnaiger et al., 2000), and to some extent, \u003cem\u003ein vivo\u003c/em\u003e. However, O\u003csub\u003e2\u003c/sub\u003e and substrate diffusion barriers may remain in homogenate preparations, and tissue permeabilisation may be required to addresscal maximal mitochondrial respiration. Therefore, measuring O\u003csub\u003e2\u003c/sub\u003e consumption using both preparations provides complementary information regarding state-dependent mitochondrial respiration, including in the context of declining O\u003csub\u003e2\u003c/sub\u003e (20.5 kPa to anoxia). In addition, given that others reported that CCO could be a determinant of hypoxia tolerance in other intertidal fish species (Lau et al., 2017), we measured CCO activity and derived inhibition-curves using sodium-azide to determine whether control from CCO differed among species. Thus, we predicted that the mitochondria and CCO of hypoxia-tolerant intertidal triplefins would display a greater affinity to O\u003csub\u003e2\u003c/sub\u003e relative to subtidal species.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Experimental animals and housing\u003c/h2\u003e \u003cp\u003eThe animals used in this study were adult specimens collected from sites on the Northeast coast of the Auckland region. The rock pool specialist \u003cem\u003eB. medius\u003c/em\u003e (\u003cem\u003eBM\u003c/em\u003e) was caught from high intertidal pools (\u0026gt;\u0026thinsp;1m) using hand nets, while the occasionally intertidal and shallow subtidal species \u003cem\u003eF. lapillum (FL)\u003c/em\u003e was caught using minnow traps from nearshore subtidal sites (\u0026lt;\u0026thinsp;1m). The deeper dwelling exclusively subtidal specimens \u003cem\u003eF. varium\u003c/em\u003e (\u003cem\u003eFV\u003c/em\u003e) and \u003cem\u003eF. malcomi\u003c/em\u003e (\u003cem\u003eFM\u003c/em\u003e) were caught with hand nets on Scuba dives at a depth of 10-15m. The experiments carried out in this study were performed at two research facilities. The fish used in the LOE trials and mitochondrial assays were housed in a recirculated seawater facility at the University of Auckland\u0026rsquo;s School of Biological Sciences. These fish were held in 30 L tanks provided with a constant flow of recirculated seawater (20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, air saturated, 200 \u0026micro;m filtered, 35 ppt salinity). The fish used for whole animal respirometry were housed at the Leigh Marine Laboratory in 30 L flow-through seawater tanks (20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, air saturated, 200 \u0026micro;m filtered, 35 ppt salinity). All fish were acclimated to laboratory conditions for at least 2 weeks prior to the start of experiments and were fed \u003cem\u003ead libitum\u003c/em\u003e on a mixture of shrimp, mussel and fish. Mass and length data for the fish used in each part of the study are found in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Food was withheld for a period of 48 h prior to the start of experiments. All capture, housing and experimental procedures were performed under the approval of the University of Auckland Ethics Committee (Approval 001551).\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\u003e\u003cb\u003eAnatomical features of the four New Zealand triplefin fish species used in each part of the present study.\u003c/b\u003e Data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.e.m, with sample sizes indicated in brackets in the left-hand column. Brian mass to body mass ratio was determined in 8 individuals of each species used in the mitochondrial function trials. Statistical differences among species were tested using one-way ANOVA. Significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and significant post-hoc differences between species are shown by uncommon superscript letters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStudy (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eBody length (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eBody weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eBrain (g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. medius\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003csub\u003ecrit\u003c/sub\u003e (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003csub\u003eLOE\u003c/sub\u003e (9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;3.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMito. (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e54.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal (27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eF. lapillum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003csub\u003ecrit\u003c/sub\u003e (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003csub\u003eLOE\u003c/sub\u003e (9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e67.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.72\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMito. (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal (27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e66.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eF. varium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003csub\u003ecrit\u003c/sub\u003e (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e80.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003csub\u003eLOE\u003c/sub\u003e (9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMito. (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e65.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.80\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal (27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e70.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eF. malcomi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003csub\u003ecrit\u003c/sub\u003e (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.90\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.59\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003csub\u003eLOE\u003c/sub\u003e (9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e83.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;3.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMito. (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e80.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.83\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal (27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e79.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Whole animal respirometry and determination of critical oxygen tension\u003c/h2\u003e \u003cp\u003eThe P\u003csub\u003ecrit\u003c/sub\u003e of each species (N\u0026thinsp;=\u0026thinsp;10; see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for mass and length) was determined at 20\u0026deg;C using automated intermittent stop-flow respirometry (Steffensen, 1989) to measure mass-specific O\u003csub\u003e2\u003c/sub\u003e consumption (\u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e; mg O\u003csub\u003e2\u003c/sub\u003e g \u003csup\u003e-1\u003c/sup\u003e h \u003csup\u003e-1\u003c/sup\u003e). The design of the respirometers, general respirometry methods and procedure for \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e calculation are described in detail in McArley et al., (2018). P\u003csub\u003ecrit\u003c/sub\u003e was defined as the O\u003csub\u003e2\u003c/sub\u003e tension where \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e under a progressive hypoxia exposure could no longer be maintained above standard metabolic rate (SMR; \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e in a rested, unfed animal) (Claireaux and Chabot, 2016). The protocol for P\u003csub\u003ecrit\u003c/sub\u003e determination began with an overnight period (~\u0026thinsp;16 h) of respirometry where \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e was assessed repeatedly over 7\u0026ndash;8 min cycles in undisturbed fish under normoxia. SMR was defined as the mean of the lowest 10% of \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e measurements made during the overnight period (Khan et al., 2014; Mandic et al., 2009; McArley et al., 2017; Norin et al., 2014), which likely corresponded to periods when fish were completely inactive as these species are benthic and tend to perch in a stationary position on the bottom of the respirometers. \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e measurements were then made at decreasing O\u003csub\u003e2\u003c/sub\u003e tensions (~\u0026thinsp;15.3, 11.6, 7.4, 6.3, 5.2, 4.2, 3.3, 2.3 and 1.6 kPa), with the required water O\u003csub\u003e2\u003c/sub\u003e levels achieved by bubbling N\u003csub\u003e2\u003c/sub\u003e into the seawater reservoir supplying respirometers. Three 7\u0026ndash;8 min \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e measurements were made at 15.3, 11.6, 7.4, 6.3, 5.2 and 4.2 kPa, and one 7\u0026ndash;8 min measurement at 3.3, 2.3 and 1.6 kPa. The entire progressive decline in O\u003csub\u003e2\u003c/sub\u003e tension was completed in ~\u0026thinsp;3 h, and the time of exposure to each O\u003csub\u003e2\u003c/sub\u003e tension was the same for each species. To estimate P\u003csub\u003ecrit\u003c/sub\u003e, SMR and routine \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e during progressive hypoxia were first mass corrected (see below) and then plotted against water PO\u003csub\u003e2\u003c/sub\u003e. A linear regression (forced through zero) was then established on \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e values that fell below SMR, and P\u003csub\u003ecrit\u003c/sub\u003e was calculated by dividing SMR by the slope of this regression line (i.e. the point where routine \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e under progressive hypoxia could no longer be maintained above SMR; see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) (as per method of Behrens and Steffensen, 2007; Cook et al., 2013; Cumming and Herbert, 2016; Schurmann and Steffensen, 1997).\u003c/p\u003e \u003cp\u003eTo account for body mass differences between species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e values were standardised to the mean body mass of all fish (3.5 g) from whole animal respirometry and P\u003csub\u003ecrit\u003c/sub\u003e assessments. Body mass correction of \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e values was carried out using the standard formula outlined in Schurmann and Steffensen (1997) and a mass scaling exponent of 0.8 Clarke and Johnston (1999).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Loss of equilibrium\u003c/h2\u003e \u003cp\u003eAcross three 40L tanks, three individuals of each species were placed into each tank (a total of 12 fish per tank) and left to recover overnight (~\u0026thinsp;16 h) in fully aerated (normoxic) seawater (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for mass and length). Seawater PO\u003csub\u003e2\u003c/sub\u003e in each tank was monitored using NeoFox-GT sensors (Ocean Optics\u003csup\u003e\u0026copy;\u003c/sup\u003e, Inc), and a clear plastic film was placed over the water surface to prevent the possibility of aerial surface respiration. After overnight recovery, hypoxia exposure was induced by bubbling N\u003csub\u003e2\u003c/sub\u003e gas into the tanks holding the fish. Bubbling N\u003csub\u003e2\u003c/sub\u003e continued until the tank PO\u003csub\u003e2\u003c/sub\u003e reached the target water O\u003csub\u003e2\u003c/sub\u003e level of ~\u0026thinsp;1.4 kPa, which was achieved in approximately 30 min. Once the target water O\u003csub\u003e2\u003c/sub\u003e level was reached, N\u003csub\u003e2\u003c/sub\u003e bubbling was adjusted manually to maintain a constant PO\u003csub\u003e2\u003c/sub\u003e of ~\u0026thinsp;1.4 kPa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B). LOE was deemed to occur when a fish could no longer maintain an upright body position for a period of 10 s. In \u003cem\u003eFV\u003c/em\u003e and \u003cem\u003eFM\u003c/em\u003e and some \u003cem\u003eFL\u003c/em\u003e, this occurred spontaneously, usually following a short burst of exercise, and occurred either before or soon after the target water PO\u003csub\u003e2\u003c/sub\u003e was reached. In \u003cem\u003eBM\u003c/em\u003e and the most hypoxia tolerant \u003cem\u003eFL\u003c/em\u003e, however, individuals remained largely stationary on the tank bottom throughout the duration of the hypoxia exposure. To determine LOE in these individuals, the fish were periodically challenged by turning them over with a small stick and observing whether they could right themselves to an upright position within 10 s. To take into account the water O\u003csub\u003e2\u003c/sub\u003e level of LOE and the length of time which hypoxia was survived, a composite measure of hypoxia tolerance was determined by the area above the curve of water O\u003csub\u003e2\u003c/sub\u003e level versus time of hypoxia exposure. An O\u003csub\u003e2\u003c/sub\u003e level of 21 kPa was set as the ceiling of the curve, and the analysis was performed using numpy.trapz module with default dx\u0026thinsp;=\u0026thinsp;1 in Python 3.7. We refereed to this composite measure as total LOE, and it was expressed in units of kPa\u003csub\u003eO2\u003c/sub\u003e.min.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.1. Tissue and mitochondrial respirometry\u003c/h3\u003e\n\u003cp\u003eFish were euthanized by section of the spinal cord at the skull and the intact brain was dissected and then weighed ensuring any excess blood was removed. In experiments using homogenates, intact brains was triturated gently by suction through a 10 ml syringe with decreasing gauge needles (16\u0026ndash;25 gauge) prior to being introduced to the respirometry chamber, which occurred within 30 s of the initial brain dissection. In experiments using permeabilised samples, intact brains were immediately placed in ice-cold biopsy buffer containing (in mM hereon, unless stated) 2.77 CaK\u003csub\u003e2\u003c/sub\u003e EGTA, K\u003csub\u003e2\u003c/sub\u003e 7.23 EGTA, 5.77 Na\u003csub\u003e2\u003c/sub\u003e ATP, 6.56 MgCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO, 20 taurine, 15 Na\u003csub\u003e2\u003c/sub\u003e-phosphocreatine, 20 imidazole, 0.5 DTT, 50 KMES, 50 sucrose, pH 7.22 at 20\u0026deg;C (Gnaiger et al., 2000). Cellular permeabilisation was undertaken by the addition of 50 \u0026micro;g.ml\u003csup\u003e-1\u003c/sup\u003e of freshly prepared saponin to plastic cell culture plates held on ice. The brains were then gently agitated in the culture plates for 30 min on ice, after which the permeabilised tissue was removed and washed three times for 10 min in ice-cold respiration medium (containing 0.5 EGTA, 3 MgCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO, 60 K-lactobionate, 20 taurine, 10 KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.5 HEPES, 30 MES, 160 sucrose, 1 g.l\u003csup\u003e-1\u003c/sup\u003e BSA, pH 7.22 at 20\u0026deg;C) prior to its addition to respirometry chambers. Fish were weighed and measured (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for mass and length) after dissection and the proportion of brain mass relative to body mass was calculated.\u003c/p\u003e \u003cp\u003eRespiration was measured using Oroboros\u0026trade; O2k high resolution respirometers (Innsbruck, Austria). The O\u003csub\u003e2\u003c/sub\u003e electrode was calibrated from 0-20.46 kPa PO\u003csub\u003e2\u003c/sub\u003e (0-100% air saturation, 262 \u0026micro;M dissolved O\u003csub\u003e2\u003c/sub\u003e equivalent at 20\u0026ordm;C and 101 kPa) prior respirometry assays. Brains (around ~\u0026thinsp;5 mg homogenate or permeabilised) were introduced in the respirometry chambers containing 2 ml respiration medium calibrated prior experiment at 100% O\u003csub\u003e2\u003c/sub\u003e. After signal stabilisation and the measurement of the routine state in brain homogenates, saturating mitochondrial substrates (pyruvate, malate, glutamate and succinate) and ADP were added to maximise oxidative phosphorylation (OxPhos\u003csub\u003ePMGS\u003c/sub\u003e). The medium was then re-aerated fully and brains were left to deplete O\u003csub\u003e2\u003c/sub\u003e and held in anoxia for ~\u0026thinsp;5 min, after which re-oxygenation was performed. In chambers containing permeabilised brain, oligomycin (5 \u0026micro;M) was added to measure respiration attributed to proton leak (Leak), followed by carbonyl cyanide m-chlorophenyl hydrazone (0.5 \u0026micro;M titration steps until signal stabilisation) to measure the maximum O\u003csub\u003e2\u003c/sub\u003e consumption capacity of the electron transport system (ETS) when uncoupled from OxPhos. In all assays, potassium-cyanide (1 mM) was added to measure non-mitochondrial O\u003csub\u003e2\u003c/sub\u003e consumption, which was then subtracted from raw respiration data. Substrate-Uncoupler-Inhibitor-Titration protocols are detailed in the supplementary file (Table S.1).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Determination of mitochondrial affinity to O\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eRespirometry data was recorded with DatLab (v7.1) software with the minimum smoothing to maximise resolution, especially at low PO\u003csub\u003e2\u003c/sub\u003e. Correction for time response of the electrode was also accounted for (Gnaiger, 2008). Above 2.05 kPa, an exponential moving average of 20 seconds (i.e. 10 recordings) was calculated in Excel to lower the signal to noise ration. The mitochondrial affinity to O\u003csub\u003e2\u003c/sub\u003e (mP\u003csub\u003e50\u003c/sub\u003e) was then determined as the PO\u003csub\u003e2\u003c/sub\u003e at which the respiration rate is half of the maximum OxPhos rate. We also made an estimate of efficiency, which typically relates to kinetic parameters of purified enzymes. Given that mitochondrial respiration is a composite or pathways we make a proxy for the classical k\u003csub\u003ecat\u003c/sub\u003e/K\u003csub\u003eM\u003c/sub\u003e and present a measure of efficiency in the context JO\u003csub\u003e2Max\u003c/sub\u003e/mP\u003csub\u003e50\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3. CCO capacity and catalytic efficiency\u003c/h2\u003e \u003cp\u003eThe CCO capacity was assessed in two independent set of assays using two different methods (Table S.2). In permeabilised brain induced in OxPhos (saturated pyruvate, malate, glutamate, succinate and ADP), sodium azide was titrated to gradually inhibit CCO, until full CCO inhibition (final concentration of 12 mM). Inhibition dose response curves (Hill curves) were fitted with the least-squares method using GraphPad\u003csup\u003e\u0026copy;\u003c/sup\u003e Prism. Using another set of assay, permeabilised brain was induced in uncoupled state (ETS), and electron feeding to CCO was inhibited by the addition of 2.5\u0026micro;M antimycin A. Maximum CCO oxidation rates were then assessed by excess electron feeding with \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e\u0026rsquo;,\u003cem\u003eN\u003c/em\u003e\u0026rsquo;-tetramethyl-p-phenylenediamine (TMPD, 0.5 mM) and additional ascorbate (2 mM) to measure TMPD auto-oxidation. Background chemical auto-oxidation was measured following additional potassium-cyanide (2 mM) and subtracted to CCO consumption rates. Net CCO rates were then normalised by the ETS capacity determined in the uncoupled state described in paragraph 2.2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed with GraphPad\u003csup\u003e\u0026copy;\u003c/sup\u003e Prism 7, with significance set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. One-way ANOVA or two-way ANOVA followed by Turkey\u0026rsquo;s \u003cem\u003epost-hoc\u003c/em\u003e tests were used to test for interactions and differences among and between species and/or parameters. For both mitochondrial respiration from 20.5 kPa to anoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.B) and CCO inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A) data, curves were fitted for each species to a three-parameters dose-response curve (hill curve) using the least-squares method. Resulting fitted curves were then compared using the extra-sum of squares F-test to test for shared parameters, including mP\u003csub\u003e50\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.C), IC50 and Hill slope (respectively Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.C and D). In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, linear regression using least squares method was used to test for correlation between parameters of the mitochondrial function to LOE determined in 2.3.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Hypoxia tolerance of triplefin fish species\u003c/h2\u003e\n \u003cp\u003eSMR under normoxia was similar among species, except for \u003cem\u003eFV\u003c/em\u003e having a slightly higher resting \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e than \u003cem\u003eBM\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.A). The intertidal specialist \u003cem\u003eBM\u003c/em\u003e had a lower P\u003csub\u003ecrit\u003c/sub\u003e than all other species (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.A), and the occasional intertidal occupant \u003cem\u003eFL\u003c/em\u003e had a lower P\u003csub\u003ecrit\u003c/sub\u003e than the exclusively subtidal species \u003cem\u003eFV\u003c/em\u003e and \u003cem\u003eFM\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.A). There was no difference in P\u003csub\u003ecrit\u003c/sub\u003e between the exclusively subtidal species \u003cem\u003eFV\u003c/em\u003e and \u003cem\u003eFM\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eThe two subtidal species could not match the lowest PO\u003csub\u003e2\u003c/sub\u003e achieved by the two rock-pool species and lost equilibrium at ~\u0026thinsp;2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 and 1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 kPa for \u003cem\u003eFM\u003c/em\u003e and \u003cem\u003eFV\u003c/em\u003e, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cstrong\u003eB\u003c/strong\u003e). While some \u003cem\u003eFL\u003c/em\u003e reached LOE around 2 kPa, the most tolerant \u003cem\u003eFL\u003c/em\u003e maintained equilibrium for ~\u0026thinsp;1.25h at ~\u0026thinsp;1.4 kPa (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.B). \u003cem\u003eBM\u003c/em\u003e tolerated severe hypoxia (~\u0026thinsp;1.4 kPa) for an average time of 1.5h before LOE. Both the O\u003csub\u003e2\u003c/sub\u003e tension at LOE (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cstrong\u003eC\u003c/strong\u003e) and the time duration to LOE (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.D) showed \u003cem\u003eBM\u003c/em\u003e and \u003cem\u003eFL\u003c/em\u003e to have superior hypoxia tolerance than their subtidal counterparts. Indeed, when total LOE was expressed as the area above the curve from 0 min to time of LOE during hypoxia exposure, this revealed that the exclusively intertidal \u003cem\u003eBM\u003c/em\u003e was more hypoxia tolerant than all other species and that the occasional intertidal occupant \u003cem\u003eFL\u003c/em\u003e tended to be more hypoxia tolerant than the exclusively subtidal species (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.E). A strong linear correlation between P\u003csub\u003ecrit\u003c/sub\u003e and total LOE was verified among species (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 ,R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.87, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Oxygen kinetics of brain tissues\u003c/h2\u003e\n \u003cp\u003eOverall, O\u003csub\u003e2\u003c/sub\u003e flux was higher in the brain of \u003cem\u003eBM\u003c/em\u003e relative to the subtidal species \u003cem\u003eFV\u003c/em\u003e and \u003cem\u003eFM\u003c/em\u003e, regardless of preparation or mitochondrial state (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.A). With the brain homogenate of all species, routine respiration was ~\u0026thinsp;31% lower than OxPhos respiration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In permeabilised brain without ADP, leak respiration rates were less than a third of OxPhos rates (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and only \u003cem\u003eFV\u003c/em\u003e and \u003cem\u003eFM\u003c/em\u003e showed an increase in O\u003csub\u003e2\u003c/sub\u003e flux with uncoupling (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). OxPhos was however\u0026thinsp;~\u0026thinsp;20% lower in brain homogenates (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eTo span the O\u003csub\u003e2\u003c/sub\u003e levels the brain likely encounters \u003cem\u003ein vivo\u003c/em\u003e, we assessed O\u003csub\u003e2\u003c/sub\u003e consumption kinetics \u003cem\u003ein vitro\u003c/em\u003e with brain homogenate and permeabilised brain in the OxPhos state (i.e. without substrate limitation). In brain homogenates, respiration was the highest in \u003cem\u003eBM\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), which retained almost two times higher O\u003csub\u003e2\u003c/sub\u003e flux at PO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;2.05 kPa (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.B). Respiration rates in \u003cem\u003eFL\u003c/em\u003e was significantly higher than in \u003cem\u003eFV\u003c/em\u003e and \u003cem\u003eFM\u003c/em\u003e at PO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;2.05 kPa (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while at PO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;2.05 kPa, it was similar to the two subtidal species. Similarly, in permeabilised brain, respiration was more than 75% higher for the two intertidal species (P\u0026thinsp;\u0026gt;\u0026thinsp;0.001;). In all species, with the exception of \u003cem\u003eFL\u003c/em\u003e, respiration rates were ~\u0026thinsp;30% lower in permeabilised tissues than in homogenised tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\n \u003cp\u003eNo difference in mP\u003csub\u003e50\u003c/sub\u003e was found between the fish species with no correlation between maximum OxPhos respiration (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.C). However, in brain homogenates the JO\u003csub\u003e2max\u003c/sub\u003e/mP\u003csub\u003e50\u003c/sub\u003e was highest in the rock-pool \u003cem\u003eBM\u003c/em\u003e relative to the intermediate and subtidal species (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This relationship was less apparent for permeabilised brain (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Cytochrome C oxidase\u003c/h2\u003e\n \u003cp\u003eDose response CCO inhibition by sodium azide was different among species (F\u003csub\u003e6,375\u003c/sub\u003e=7.16; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.A). Although, IC\u003csub\u003e50\u003c/sub\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.B) and Hill slopes (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.C) extracted from the fitted curves were not significantly different. While in the \u003cem\u003eForsterygion\u003c/em\u003e genus, hypoxia-tolerant \u003cem\u003eFL\u003c/em\u003e had the lowest CCO activity (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.D), it was the highest in the most hypoxia-tolerant \u003cem\u003eBM\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with 10% more activity than the least hypoxia-tolerant FM.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Correlation between mitochondrial function and hypoxia tolerance\u003c/h2\u003e\n \u003cp\u003eAt the subcellular level, mitochondrial O\u003csub\u003e2\u003c/sub\u003e affinity (mP\u003csub\u003e50\u003c/sub\u003e) had a weak correlation with species hypoxia tolerance, here represented with total LOE (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.51; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.A). Paradoxically, higher mitochondrial O\u003csub\u003e2\u003c/sub\u003e consumption rates in OxPhos state (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.B) and greater mitochondrial O\u003csub\u003e2\u003c/sub\u003e catalytic rates (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.C) correlated strongly with hypoxia tolerance (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.99 and 0.95, respectively). At a lower biological level, however, activity rates of CCO (the enzyme at the end of the O\u003csub\u003e2\u003c/sub\u003e cascade) did not correlate linearly with total LOE (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.12).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1. Intertidal triplefins have superior hypoxia tolerance and lower P\u003csub\u003ecrit\u003c/sub\u003e than their subtidal counterparts\u003c/h2\u003e\n \u003cp\u003eHere, we show intertidal triplefin species survive severe hypoxia for longer than their subtidal counterparts, which likely reflects the fact that the rock pools inhabited by these species can become severely hypoxic during night-time low tides (McArley et al., 2019). We and others have previously demonstrated intertidal triplefin species have a lower P\u003csub\u003ecrit\u003c/sub\u003e than subtidal triplefin species and have speculated this should promote tolerance to hypoxia (Hilton et al., 2010; McArley et al., 2019). In the current study, the association between lower P\u003csub\u003ecrit\u003c/sub\u003e in intertidal species and improved hypoxia tolerance was confirmed by a strong inverse correlation between total LOE and P\u003csub\u003ecrit\u003c/sub\u003e among the four species examined. Time to LOE and P\u003csub\u003ecrit\u003c/sub\u003e were also correlated among intertidal and subtidal sculpin species (Mandic et al., 2013), which together with the current findings suggests that a relatively low P\u003csub\u003ecrit\u003c/sub\u003e is important to the hypoxia survival strategy of intertidal fishes not undergoing metabolic depression. A low P\u003csub\u003ecrit\u003c/sub\u003e may benefit hypoxia tolerance in intertidal fish by allowing them to delay the onset of anaerobic metabolism during progressive hypoxia in rock pools, thereby mitigating depletion of anaerobic fuel stores and accumulation of toxic end products of anaerobic ATP production. Moreover, in the absence of metabolic rate depression, which is not yet known to occur in triplefins and we did not observe here, a low P\u003csub\u003ecrit\u003c/sub\u003e should allow a greater proportion of energetic demand to be met through efficient mitochondrial ATP production as hypoxia worsens. Indeed, the hypoxia tolerant \u003cem\u003eBM\u003c/em\u003e maintained an \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e equivalent to 51% of SMR at a PO\u003csub\u003e2\u003c/sub\u003e of ~\u0026thinsp;1.5 kPa, whereas the least hypoxia tolerant \u003cem\u003eFM\u003c/em\u003e had an \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e equivalent to only 27% of SMR at the same PO\u003csub\u003e2\u003c/sub\u003e. The ability of \u003cem\u003eBM\u003c/em\u003e to extract more O\u003csub\u003e2\u003c/sub\u003e from severely hypoxic water is explained, at least partly, by the possession of gills with a high diffusive capacity for O\u003csub\u003e2\u003c/sub\u003e. Indeed, gill secondary lamellae are thin and numerous in this species (McArley et al., 2019). Furthermore, relative to \u003cem\u003eFM\u003c/em\u003e, \u003cem\u003eBM\u003c/em\u003e has a higher blood O\u003csub\u003e2\u003c/sub\u003e carrying capacity as indicated by higher haemoglobin concentration (McArley et al., 2019). We also believe the typical behaviour of \u003cem\u003eBM\u003c/em\u003e observed during LOE determination, where this species remains relatively inactive and stationary on the tank bottom, is likely to permit maintenance of a metabolic rate close to SMR at water PO\u003csub\u003e2\u003c/sub\u003e below P\u003csub\u003ecrit\u003c/sub\u003e. This behaviour contrasts starkly with that of the exclusively subtidal species, which become active, likely in an attempt to behaviourally avoid hypoxia, as water PO\u003csub\u003e2\u003c/sub\u003e descends below P\u003csub\u003ecrit\u003c/sub\u003e and LOE is approached. Of course, high activity levels mean that metabolic rate will increase substantially beyond that associated with SMR and maintaining energy balance will become even more challenging. For subtidal species, hypoxia is escapable in nature and a behavioural avoidance strategy would be advantageous. However, hypoxia is inescapable in rock pools and also within the LOE trials we performed, so the quiescent low energy demand behaviour of \u003cem\u003eBM\u003c/em\u003e is likely to convey a greater chance of surviving periods of severe hypoxia that develop in rock pools prior to replenishment of water O\u003csub\u003e2\u003c/sub\u003e levels on the incoming tide. Quiescent behaviour in severe hypoxia has also been observed in other hypoxia tolerant intertidal fishes (Yoshiyama et al., 1995). Lastly, we have previously demonstrated intertidal triplefins have higher white muscle and hepatic glycogen stores than subtidal species, which could also contribute to superior hypoxia tolerance through fuelling anaerobic respiration (McArley et al., 2019). Large tissue glycogen stores and higher glycolytic enzyme activities in brain have also been demonstrated in intertidal sculpins (Mandic et al., 2013).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e4.2. Oxygen utilization is greater in brain mitochondria of intertidal species\u003c/h2\u003e\n \u003cp\u003eBrain mitochondria of hypoxia-tolerant and hypoxia sensitive triplefin species had similar affinities to O\u003csub\u003e2\u003c/sub\u003e, with mP\u003csub\u003e50\u003c/sub\u003e a little above 0.10 kPa. In comparison, the least hypoxia-tolerant sculpin displayed brain tissue mP\u003csub\u003e50\u003c/sub\u003e of around 0.08 kPa, although this was measured 2\u0026ordm;C below our experimental temperature (i.e. 18\u0026ordm;C vs 20\u0026ordm;C) (Lau et al., 2017) and higher temperatures increase binding affinities (Blair et al., 1986). Note that within sculpin species a strong correlation was observed between isolated mitochondria mP\u003csub\u003e50\u003c/sub\u003e determined at 18\u0026ordm;C and P\u003csub\u003ecrit\u003c/sub\u003e measured at 12\u0026ordm;C (Lau et al., 2017). In triplefin species, no such correlation was verified when measuring both parameter at 20\u0026ordm;C using brain homogenates or permeabilised tissue. Intracellularly, recent measurements of mitochondrial PO\u003csub\u003e2\u003c/sub\u003e \u003cem\u003ein vivo\u003c/em\u003e indicate levels of around 2.6-5 kPa (Mik, 2013; Mik et al., 2008), levels at which brain homogenate mitochondria operates here at ~\u0026thinsp;70% of OxPhos for all fish species. Routine respiration rates, mediated by endogenous substrates and closely representative of \u003cem\u003ein vivo\u003c/em\u003e conditions, were also ~\u0026thinsp;70% that of OxPhos, yet Routine respiration was measured under air saturated O\u003csub\u003e2\u003c/sub\u003e. This indicates that above 2 kPa, substrates, but not O\u003csub\u003e2,\u003c/sub\u003e limit mitochondrial respiration in brain homogenates. Interestingly, subtidal species lost equilibrium around 2 kPa, which indicates that below such level, Routine respiration may be affected by O\u003csub\u003e2\u003c/sub\u003e levels, altering brain metabolism which subsequently compromises fish spatial awareness and reflexes upon acute exposure. Such PO\u003csub\u003e2\u003c/sub\u003e is \u0026gt;\u0026thinsp;25x higher than mP\u003csub\u003e50\u003c/sub\u003e measured here \u003cem\u003ein vitro\u003c/em\u003e in both subtidal and intertidal fish, which indicates that all species studied here have marginal PO\u003csub\u003e2\u003c/sub\u003e scope before reaching 50% of maximal mitochondrial respiration capacities.\u003c/p\u003e\n \u003cp\u003eIn both homogenate and permeabilised brain, basal O\u003csub\u003e2\u003c/sub\u003e consumption (Routine and Leak, respectively) and OxPhos were consistently greater in the intertidal hypoxia-tolerant species. As indicated by the greater JO\u003csub\u003e2max\u003c/sub\u003e/mP\u003csub\u003e50\u003c/sub\u003e (a proxy for k\u003csub\u003ecat\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e), intertidal species also have greater efficiency to bind and turnover O\u003csub\u003e2\u003c/sub\u003e. This at first appears paradoxical, given that O\u003csub\u003e2\u003c/sub\u003e is limiting in hypoxia and that the mP\u003csub\u003e50\u003c/sub\u003e of mitochondria are similar among species. However, elevation of O\u003csub\u003e2\u003c/sub\u003e consumption can increase O\u003csub\u003e2\u003c/sub\u003e gradients (Gnaiger et al., 1998), thereby enhancing O\u003csub\u003e2\u003c/sub\u003e flow to mitochondria to better support ATP synthesis in hypoxia sensitive species. However, the elevated O\u003csub\u003e2\u003c/sub\u003e consumption observed in brain tissue was not associated with a higher whole animal \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e in intertidal species. Thus, if the high basal O\u003csub\u003e2\u003c/sub\u003e consumption and OxPhos observed in brain is common in other tissues of intertidal species, it does not come at the cost of a higher resting metabolic demand, which would likely impair hypoxia tolerance through increasing basal energetic demands. We have also observed that intertidal triplefins can achieve a higher maximum O\u003csub\u003e2\u003c/sub\u003e consumption rate than their subtidal counterparts (McArley et al., 2019), and this matches the high OxPhos observed in brain tissues of intertidal species here. Rather than high metabolic capacity being an adaptation for hypoxia tolerance per se, it might be that intertidal species retain the potential for high ATP production via OxPhos (Devaux et al., 2019a), and this would meet the energy requirements of other high demand environmental conditions that occur in rock pools. For example, high aerobic capacity would be beneficial for meeting the energetic requirements of wave surge and for avoiding temperature-induced tissue O\u003csub\u003e2\u003c/sub\u003e limitation (functional hypoxia) during acute warming events during daytime low tides. Moreover, thermal ramping events in rock pools tend to coincide with hyperoxia (O\u003csub\u003e2\u003c/sub\u003e supersaturation) (McArley et al., 2019; Richards, 2011), where the high OxPhos capacity of intertidal species could potentially act as a sink for excess O\u003csub\u003e2\u003c/sub\u003e at the tissues and mitigate oxidative stress. This could also occur upon replenishment of rock pool water by the incoming tide, where a high OxPhos capacity could potentially mitigate oxidative stress associated with rapid hypoxia re-oxygenation of tissues (Starkov, 2008).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e4.3. Cytochrome c oxidase is not associated with hypoxia tolerance of triplefin fish\u003c/h2\u003e\n \u003cp\u003eA greater mitochondrial O\u003csub\u003e2\u003c/sub\u003e utilisation may reside from multiple adjustments. This includes a greater capacity to import and oxidise mitochondrial substrates, greater electron transport capacities of the ETS or a more tuned cytochrome \u003cem\u003ec\u003c/em\u003e oxidase \u0026ndash; the enzyme at the end of the O\u003csub\u003e2\u003c/sub\u003e cascade. CCO subunit 4 has two paralogues in most vertebrates, including fish (Porplycia et al., 2017), with CCO4-2 expression being elevated in response to hypoxia (Fukuda et al., 2007), decreasing O\u003csub\u003e2\u003c/sub\u003e affinity and consumption (Pajuelo Reguera et al., 2020). Notably, CCO inhibition curves were similar across species (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003cstrong\u003eA\u003c/strong\u003e), with similar apparent Km and Hill slopes, suggesting that triplefin species likely share similar forms of CCO with conserved O\u003csub\u003e2\u003c/sub\u003e binding properties. In addition, no clear correlation between CCO and hypoxia tolerance was apparent. This again differs from sculpin species that showed a strong correlation between mP\u003csub\u003e50\u003c/sub\u003e and CCO-O2 affinity (Lau et al., 2017). Maximum CCO activity was similar to maximum OxPhos rates in \u003cem\u003eFL\u003c/em\u003e, suggesting that unlike the other species, CCO reserve capacity is limited. This indicates O\u003csub\u003e2\u003c/sub\u003e consumption and therefore ATP synthesis may be affected in \u003cem\u003eFL\u003c/em\u003e if CCO activity were downregulated. As expected, however, the CCO capacity was higher than ETS capacity, and this suggests that CCO is not limiting and that regulation of O\u003csub\u003e2\u003c/sub\u003e consumption in hypoxia-tolerant triplefin fish likely occurs at a higher biological level.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eHere, we show that intertidal triplefin fish are more hypoxia tolerant than their subtidal counterparts. Indeed, the exclusively intertidal \u003cem\u003eBM\u003c/em\u003e survived severe hypoxia (~\u0026thinsp;1.4 kPa) for up to 2 h, whereas the exclusively subtidal \u003cem\u003eFM\u003c/em\u003e could only tolerate brief periods of exposure to water O\u003csub\u003e2\u003c/sub\u003e levels between ~\u0026thinsp;2-3.5 kPa. Brain mitochondria in hypoxia-tolerant species displayed higher respiration rates than brain of hypoxia sensitive triplefins, even at PO\u003csub\u003e2\u003c/sub\u003e where mitochondria function \u003cem\u003ein vivo\u003c/em\u003e. High OxPhos capacity, while potentially aiding hypoxia tolerance through steepening O\u003csub\u003e2\u003c/sub\u003e diffusion gradients between blood and tissues, may also provide reserve aerobic capacity to cope with other high energy demand environmental conditions that occur in the intertidal zone (e.g. acute warming). Although CCO, the enzyme at the end of the O\u003csub\u003e2\u003c/sub\u003e cascade, was not correlated with hypoxia tolerance, it does not appear to limit O\u003csub\u003e2\u003c/sub\u003e consumption in phosphorylating mitochondria. Despite similar mitochondrial affinity to O\u003csub\u003e2\u003c/sub\u003e in brain tissue between hypoxia-tolerant and hypoxia sensitive triplefins, a greater capacity of intertidal species to access O\u003csub\u003e2\u003c/sub\u003e in hypoxic environments was indicated by a lower P\u003csub\u003ecrit\u003c/sub\u003e at the whole animal level. Furthermore, intertidal species, despite the high OxPhos capacity identified in brain tissue, did not have increased resting \u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e. This suggests that when exposed to severe hypoxia, intertidal species are able to meet a greater proportion of their energetic demand through efficient mitochondrial ATP production, which would likely aid hypoxia tolerance through, at least in part, mitigating unsustainable levels of anaerobic respiration. A low P\u003csub\u003ecrit\u003c/sub\u003e strategy, particularly when combined with quiescent behaviour, may be best suited to the nature of hypoxic events in rock pools inhabited by tripflefins, where severe hypoxia is of short duration and there is always some O\u003csub\u003e2\u003c/sub\u003e available.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCytochrome C oxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eETS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial electron transport system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLOE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLoss of equilibrium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eṀ\u003c/em\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMass-specific O\u003csub\u003e2\u003c/sub\u003e consumption\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emP\u003csub\u003e50\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxygen tension at which mitochondrial respiration is half of its maximum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOxPhos\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxidative respiration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003csub\u003ecrit\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCritical oxygen tension\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePartial O\u003csub\u003e2\u003c/sub\u003e pressure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was fully supported by the Royal Society of New Zealand Marsden fund (14-UOA-210: How to avoid brain damage during oxygen deprivation? Intertidal fish provide a unique test model).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors designed the research; T.M. performed the whole animal respirometry and J.B.L. performed mitochondrial experiments; J.B.L., T.M., N.H. and A.J.R.H. analysed the data and critically discussed the results; J.B.L. and T.M. wrote the paper and all authors approved submission.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Jaime Willis, Alicia Hellens, Peter Schlegel, Craig Norrie and Esther Stuck for their help with the animal collection.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData supporting the results presented in this article are available at the University of Auckland repository (upon acceptance of this present manuscript).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e\u003cb\u003eA.L. Val, M. N. P. S., V. M. F. Almeida-Val.\u003c/b\u003e (1998). Hypoxia adaptation in fish of the Amazon: a never-ending task. \u003cem\u003eSouth African journal of zoology. Suid-Afrikaanse tydskrif vir dierkunde.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 107\u0026ndash;114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eBehrens, J. W. and Steffensen, J. F.\u003c/b\u003e (2007). The effect of hypoxia on behavioural and physiological aspects of lesser sandeel, Ammodytes tobianus (Linnaeus, 1785). \u003cem\u003eMarine Biology\u003c/em\u003e \u003cb\u003e150\u003c/b\u003e, 1365\u0026ndash;1377.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eBickler, P. E. and Buck, L. T.\u003c/b\u003e (2007). Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability. \u003cem\u003eAnnu Rev Physiol\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e, 145\u0026thinsp;\u0026minus;\u0026thinsp;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eBlair, D. F., Ellis, W. R., Wang, H., Gray, H. B. and Chan, S. I.\u003c/b\u003e (1986). Spectroelectrochemical study of cytochrome c oxidase: pH and temperature dependences of the cytochrome potentials. Characterization of site-site interactions. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cb\u003e261\u003c/b\u003e, 11524\u0026ndash;11537.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eCerv\u0026oacute;s-Navarro, J. and Diemer, N. H.\u003c/b\u003e (1991). Selective vulnerability in brain hypoxia. \u003cem\u003eCritical reviews in neurobiology\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 149\u0026ndash;182.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eClaireaux, G. and Chabot, D.\u003c/b\u003e (2016). Responses by fishes to environmental hypoxia: integration through Fry's concept of aerobic metabolic scope. \u003cem\u003eJ Fish Biol\u003c/em\u003e \u003cb\u003e88\u003c/b\u003e, 232\u0026thinsp;\u0026minus;\u0026thinsp;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eClarke, A. and Johnston, N. M.\u003c/b\u003e (1999). Scaling of metabolic rate with body mass and temperature in teleost fish. \u003cem\u003eJournal of Animal Ecology\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e, 893\u0026ndash;905.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eCook, D. G., Iftikar, F. I., Baker, D. W., Hickey, A. J. and Herbert, N. A.\u003c/b\u003e (2013). Low-O(2) acclimation shifts the hypoxia avoidance behaviour of snapper (Pagrus auratus) with only subtle changes in aerobic and anaerobic function. \u003cem\u003eJ Exp Biol\u003c/em\u003e \u003cb\u003e216\u003c/b\u003e, 369\u0026thinsp;\u0026minus;\u0026thinsp;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eCumming, H. and Herbert, N. A.\u003c/b\u003e (2016). Gill structural change in response to turbidity has no effect on the oxygen uptake of a juvenile sparid fish. \u003cem\u003eConserv Physiol\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, cow033.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eDel Rio, C. and Montaner, J.\u003c/b\u003e (2021). Hypoxia Tolerant Species: The Wisdom of Nature Translated into Targets for Stroke Therapy. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eDevaux, J. B. L., Hedges, C. P., Birch, N., Herbert, N., Renshaw, G. M. C. and Hickey, A. J. R.\u003c/b\u003e (2019a). Acidosis Maintains the Function of Brain Mitochondria in Hypoxia-Tolerant Triplefin Fish: A Strategy to Survive Acute Hypoxic Exposure? \u003cem\u003eFront Physiol\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eDevaux, J. B. L., Hickey, A. J. R. and Renshaw, G. M. C.\u003c/b\u003e (2019b). Mitochondrial plasticity in the cerebellum of two anoxia-tolerant sharks: contrasting responses to anoxia/reoxygenation. \u003cem\u003eJ Exp Biol\u003c/em\u003e, jeb.191353.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eFukuda, R., Zhang, H., Kim, J. W., Shimoda, L., Dang, C. V. and Semenza, G. L.\u003c/b\u003e (2007). HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e129\u003c/b\u003e, 111\u0026thinsp;\u0026minus;\u0026thinsp;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eGalli, G. L., Lau, G. Y. and Richards, J. G.\u003c/b\u003e (2013). Beating oxygen: chronic anoxia exposure reduces mitochondrial F1FO-ATPase activity in turtle (Trachemys scripta) heart. \u003cem\u003eJ Exp Biol\u003c/em\u003e \u003cb\u003e216\u003c/b\u003e, 3283-93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eGalli, G. L. and Richards, J. G.\u003c/b\u003e (2014). Mitochondria from anoxia-tolerant animals reveal common strategies to survive without oxygen. \u003cem\u003eJ Comp Physiol B\u003c/em\u003e \u003cb\u003e184\u003c/b\u003e, 285\u0026ndash;302.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eGnaiger, E.\u003c/b\u003e (2008). Polarographic Oxygen Sensors, the Oxygraph, and High-Resolution Respirometry to Assess Mitochondrial Function. In \u003cem\u003eDrug-Induced Mitochondrial Dysfunction\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eGnaiger, E.\u003c/b\u003e (2020). Mitochondrial physiology. \u003cem\u003eBioenergetics Communications\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eGnaiger, E., Kuznetsov, A. V., Schneeberger, S., Seiler, R., Brandacher, G., Steurer, W. and Margreiter, R.\u003c/b\u003e (2000). Mitochondria in the Cold. In \u003cem\u003eLife in the Cold: Eleventh International Hibernation Symposium\u003c/em\u003e, eds. G. Heldmaier and M. Klingenspor), pp. 431\u0026ndash;442. Berlin, Heidelberg: Springer Berlin Heidelberg.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eGnaiger, E., Lassnig, B., Kuznetsov, A., Rieger, G. and Margreiter, R.\u003c/b\u003e (1998). Mitochondrial oxygen affinity, respiratory flux control and excess capacity of cytochrome c oxidase. \u003cem\u003eJ Exp Biol\u003c/em\u003e \u003cb\u003e201\u003c/b\u003e, 1129-39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eGorr, T. A., Wichmann, D., Hu, J., Hermes-Lima, M., Welker, A. F., Terwilliger, N., Wren, J. F., Viney, M., Morris, S., Nilsson, G. E. et al.\u003c/b\u003e (2010). Hypoxia tolerance in animals: biology and application. \u003cem\u003ePhysiol Biochem Zool\u003c/em\u003e \u003cb\u003e83\u003c/b\u003e, 733\u0026thinsp;\u0026minus;\u0026thinsp;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eHickey, A. J. and Clements, K. D.\u003c/b\u003e (2005). Genome size evolution in New Zealand triplefin fishes. \u003cem\u003eJ Hered\u003c/em\u003e \u003cb\u003e96\u003c/b\u003e, 356\u0026thinsp;\u0026minus;\u0026thinsp;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eHickey, A. J., Lavery, S. D., Hannan, D. A., Baker, C. S. and Clements, K. D.\u003c/b\u003e (2009). New Zealand triplefin fishes (family Tripterygiidae): contrasting population structure and mtDNA diversity within a marine species flock. \u003cem\u003eMol Ecol\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 680\u0026thinsp;\u0026minus;\u0026thinsp;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eHilton, Z.\u003c/b\u003e (2010). Physiological adaptation in the radiation of New Zealand triplefin fishes (Family Tripterygiidae). In \u003cem\u003eSchool of Biological Sciences\u003c/em\u003e, vol. PhD: University of Auckland.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eHilton, Z., Clements, K. D. and Hickey, A. J.\u003c/b\u003e (2010). Temperature sensitivity of cardiac mitochondria in intertidal and subtidal triplefin fishes. \u003cem\u003eJ Comp Physiol B\u003c/em\u003e \u003cb\u003e180\u003c/b\u003e, 979\u0026thinsp;\u0026minus;\u0026thinsp;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eHilton, Z., Wellenreuther, M. and Clements, K. D.\u003c/b\u003e (2008). Physiology underpins habitat partitioning in a sympatric sister-species pair of intertidal fishes. \u003cem\u003eFunctional Ecology\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 1108\u0026ndash;1117.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eKhan, J. R., Pether, S., Bruce, M., Walker, S. P. and Herbert, N. A.\u003c/b\u003e (2014). Optimum temperatures for growth and feed conversion in cultured hapuku (Polyprion oxygeneios) \u0026mdash; Is there a link to aerobic metabolic scope and final temperature preference? \u003cem\u003eAquaculture\u003c/em\u003e \u003cb\u003e430\u003c/b\u003e, 107\u0026ndash;113.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eKoch, L. G. and Britton, S. L.\u003c/b\u003e (2008). Aerobic metabolism underlies complexity and capacity. \u003cem\u003eThe Journal of Physiology\u003c/em\u003e \u003cb\u003e586\u003c/b\u003e, 83\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eLarson, J., Drew, K. L., Folkow, L. P., Milton, S. L. and Park, T. J.\u003c/b\u003e (2014). No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates. \u003cem\u003eJ Exp Biol\u003c/em\u003e \u003cb\u003e217\u003c/b\u003e, 1024-39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eLau, G. Y., Mandic, M. and Richards, J. G.\u003c/b\u003e (2017). Evolution of Cytochrome c Oxidase in Hypoxia Tolerant Sculpins (Cottidae, Actinopterygii). \u003cem\u003eMol Biol Evol\u003c/em\u003e \u003cb\u003e34\u003c/b\u003e, 2153\u0026ndash;2162.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eMandic, M., Speers-Roesch, B. and Richards, J. G.\u003c/b\u003e (2013). Hypoxia tolerance in sculpins is associated with high anaerobic enzyme activity in brain but not in liver or muscle. \u003cem\u003ePhysiol Biochem Zool\u003c/em\u003e \u003cb\u003e86\u003c/b\u003e, 92\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eMandic, M., Todgham, A. E. and Richards, J. G.\u003c/b\u003e (2009). Mechanisms and evolution of hypoxia tolerance in fish. \u003cem\u003eProc Biol Sci\u003c/em\u003e \u003cb\u003e276\u003c/b\u003e, 735\u0026thinsp;\u0026minus;\u0026thinsp;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eMcArley, T. J., Hickey, A. J. R. and Herbert, N. A.\u003c/b\u003e (2017). Chronic warm exposure impairs growth performance and reduces thermal safety margins in the common triplefin fish (Forsterygion lapillum). \u003cem\u003eJ Exp Biol\u003c/em\u003e \u003cb\u003e220\u003c/b\u003e, 3527\u0026ndash;3535.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eMcArley, T. J., Hickey, A. J. R. and Herbert, N. A.\u003c/b\u003e (2018). Hyperoxia increases maximum oxygen consumption and aerobic scope of intertidal fish facing acutely high temperatures. \u003cem\u003eJ Exp Biol\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eMcArley, T. J., Hickey, A. J. R., Wallace, L., Kunzmann, A. and Herbert, N. A.\u003c/b\u003e (2019). Intertidal triplefin fishes have a lower critical oxygen tension (Pcrit), higher maximal aerobic capacity, and higher tissue glycogen stores than their subtidal counterparts. \u003cem\u003eJ Comp Physiol B\u003c/em\u003e \u003cb\u003e189\u003c/b\u003e, 399\u0026ndash;411.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eMik, E. G.\u003c/b\u003e (2013). Special article: measuring mitochondrial oxygen tension: from basic principles to application in humans. \u003cem\u003eAnesth Analg\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e, 834\u0026thinsp;\u0026minus;\u0026thinsp;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eMik, E. G., Johannes, T., Zuurbier, C. J., Heinen, A., Houben-Weerts, J. H., Balestra, G. M., Stap, J., Beek, J. F. and Ince, C.\u003c/b\u003e (2008). In vivo mitochondrial oxygen tension measured by a delayed fluorescence lifetime technique. \u003cem\u003eBiophys J\u003c/em\u003e \u003cb\u003e95\u003c/b\u003e, 3977-90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eNilsson, G. E. and Lutz, P. L.\u003c/b\u003e (2004). Anoxia tolerant brains. \u003cem\u003eJ Cereb Blood Flow Metab\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 475\u0026thinsp;\u0026minus;\u0026thinsp;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eNorin, T., Malte, H. and Clark, T. D.\u003c/b\u003e (2014). Aerobic scope does not predict the performance of a tropical eurythermal fish at elevated temperatures. \u003cem\u003eJ Exp Biol\u003c/em\u003e \u003cb\u003e217\u003c/b\u003e, 244\u0026thinsp;\u0026minus;\u0026thinsp;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003ePajuelo Reguera, D., Cunatova, K., Vrbacky, M., Pecinova, A., Houstek, J., Mracek, T. and Pecina, P.\u003c/b\u003e (2020). Cytochrome c Oxidase Subunit 4 Isoform Exchange Results in Modulation of Oxygen Affinity. \u003cem\u003eCells\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003ePamenter, M. E.\u003c/b\u003e (2014). Mitochondria: a multimodal hub of hypoxia tolerance. \u003cem\u003eCanadian Journal of Zoology\u003c/em\u003e \u003cb\u003e92\u003c/b\u003e, 569\u0026ndash;589.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003ePamenter, M. E., Gomez, C. R., Richards, J. G. and Milsom, W. K.\u003c/b\u003e (2016). Mitochondrial responses to prolonged anoxia in brain of red-eared slider turtles. \u003cem\u003eBiol Lett\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 20150797.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003ePorplycia, D., Lau, G. Y., McDonald, J., Chen, Z., Richards, J. G. and Moyes, C. D.\u003c/b\u003e (2017). Subfunctionalization of COX4 paralogs in fish. \u003cem\u003eAm J Physiol Regul Integr Comp Physiol\u003c/em\u003e \u003cb\u003e312\u003c/b\u003e, R671-R680.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eRichards, J. G.\u003c/b\u003e (2011). Physiological, behavioral and biochemical adaptations of intertidal fishes to hypoxia. \u003cem\u003eJ Exp Biol\u003c/em\u003e \u003cb\u003e214\u003c/b\u003e, 191-9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eSchurmann, H. and Steffensen, J. F.\u003c/b\u003e (1997). Effects of temperature, hypoxia and activity on the metabolism of juvenile Atlantic cod. \u003cem\u003eJ Fish Biol\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 1166\u0026ndash;1180.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eStarkov, A. A.\u003c/b\u003e (2008). The role of mitochondria in reactive oxygen species metabolism and signaling. \u003cem\u003eAnn N Y Acad Sci\u003c/em\u003e \u003cb\u003e1147\u003c/b\u003e, 37\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eSteffensen, J. F.\u003c/b\u003e (1989). Some errors in respirometry of aquatic breathers: How to avoid and correct for them. \u003cem\u003eFish Physiol Biochem\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 49\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eWillis, J. R., Hickey, A. J. and Devaux, J. B.\u003c/b\u003e (2021). Thermally tolerant intertidal triplefin fish (Tripterygiidae) sustain ATP dynamics better than subtidal species under acute heat stress. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eYoshiyama, R. M., Valpey, C. J., Schalk, L. L., Oswald, N. M., Vaness, K. K., Lauritzen, D. and Limm, M.\u003c/b\u003e (1995). Differential propensities for aerial emergence in intertidal sculpins (Teleostei; Cottidae). \u003cem\u003eJournal of Experimental Marine Biology and Ecology\u003c/em\u003e \u003cb\u003e191\u003c/b\u003e, 195\u0026ndash;207.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"respirometry, Pcrit, physiology, cytochrome C oxidase, loss of equilibrium, oxidative phosphorylation","lastPublishedDoi":"10.21203/rs.3.rs-4722244/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4722244/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAt the terminus of the O\u003csub\u003e2\u003c/sub\u003e cascade, mitochondria play an important role in O\u003csub\u003e2\u003c/sub\u003e utilisation and energy conservation, with adaptive modifications occasionally shared among hypoxia-tolerant species. Here, we sought to determine whether mitochondrial adaptations in brain tissue explain the hypoxia tolerance of New Zealand triplefin fishes (\u003cem\u003eTripterygiidae\u003c/em\u003e). We compared two intertidal species (\u003cem\u003eBellapiscis medius\u003c/em\u003e and \u003cem\u003eForsterygion lapillum\u003c/em\u003e), both likely adapted to hypoxia-reoxygenation exposures, and two subtidal species (\u003cem\u003eF. varium\u003c/em\u003e and \u003cem\u003eF. malcomi\u003c/em\u003e), which inhabit normoxic waters. To assess hypoxia tolerance, we determined loss of equilibrium (LOE) during hypoxia exposure and measured the critical O\u003csub\u003e2\u003c/sub\u003e tension (P\u003csub\u003ecrit\u003c/sub\u003e). Intertidal species displayed superior hypoxia tolerance as assessed by LOE and also had lower P\u003csub\u003ecrit\u003c/sub\u003e (LOE versus P\u003csub\u003ecrit\u003c/sub\u003e R\u003csup\u003e2\u003c/sup\u003e = 0.92). High-resolution respirometry was used to measure mitochondrial respiration in homogenate and permeabilised fragments of brain. While a weak relationship was apparent between mitochondrial O\u003csub\u003e2\u003c/sub\u003e binding affinity (mP\u003csub\u003e50\u003c/sub\u003e) and hypoxia tolerance, maximum phosphorylating O\u003csub\u003e2\u003c/sub\u003e flux (OxPhos) and O\u003csub\u003e2\u003c/sub\u003e catalytic rates were strongly correlated with hypoxia tolerance. Although cytochrome-\u003cem\u003ec\u003c/em\u003e-oxidase activity was highest in the most hypoxia-tolerant species \u003cem\u003eB. medius\u003c/em\u003e, it was only weakly correlated with hypoxia tolerance across species. Notably, the high OxPhos capacity of intertidal species was not associated with higher whole animal resting O\u003csub\u003e2\u003c/sub\u003e consumption, suggesting intertidal species maintain high capacity for ATP production without incurring increased basal energetic costs. While somewhat paradoxical, the low P\u003csub\u003ecrit\u003c/sub\u003e/high OxPhos strategy of intertidal species may provide flexibility in the dynamic intertidal environment where short, severe periods of hypoxia are interspersed with high energy demand environmental conditions (e.g. acute warming).\u003c/p\u003e","manuscriptTitle":"Superior hypoxia tolerance of intertidal triplefin fish is associated with low critical oxygen tension and high phosphorylating capacity in brain mitochondria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-03 12:42:46","doi":"10.21203/rs.3.rs-4722244/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1866b6a4-b5cd-4053-864d-e9730983c763","owner":[],"postedDate":"December 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-27T19:23:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-03 12:42:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4722244","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4722244","identity":"rs-4722244","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}