Dynamic defence? Intertidal triplefin species show better maintenance of mitochondrial membrane potential than subtidal species at low oxygen pressures

Oxygen limitation experienced during hypoxic and/or anoxic conditions has a profound effect on all levels of biological organisation, and compromises organism physiology and metabolism (Galli and Richards, 2014). As O₂ delivery to cells diminishes, adenosine triphosphate (ATP) production through mitochondrial oxidative phosphorylation (OXPHOS) becomes disrupted (Devaux et al., 2018). Cellular energy demands to maintain ion balance and homeostasis may shift to anaerobic metabolism, which is ∼15-fold less efficient (Galli and Richards, 2014). When hypoxic exposure is prolonged, diminishing ATP production coupled with limited intracellular storage results in carbohydrate and ATP stores being quickly depleted, and creates a substrate-limited cap on survival (Bickler and Buck, 2007; Huss and Kelly, 2005; Richards, 2011; Richards et al., 2009). Further, ATP hydrolysis mediates proton release (H⁺) alongside metabolic waste product accumulation, which contributes to metabolic acidosis that can mediate irreversible cellular damage (Rehncrona, 1985; Rehncrona and Kågström, 1983; Siesjö, 1988).

Hypoxia-tolerant species, however, have evolved within a diverse array of environments, which routinely experience intermittent and/or chronic hypoxic episodes. These animals are useful to explore physiological adaptions that likely circumvent or efficiently buffer mitochondria from hypoxic damage. Adaptive modifications of the vertebrate O₂ transport cascade are well described in various hypoxia-tolerant groups, including intertidal sculpin species (Family: Cottidae) (Lau et al., 2017; Mandic et al., 2009), epaulette sharks (Hemiscyllium ocellatum) (Hickey et al., 2012; Routley et al., 2002; Speers-Roesch et al., 2012), high-altitude geese (Scott et al., 2009) and diving mammals (Allen and Vázquez-Medina, 2019). However, despite their central role in metabolism, the role of the mitochondria in hypoxia tolerance remains largely undefined.

The mitochondrial membrane potential (mtMP) is essential for developing the proton motive force, which drives ATP production (Zorova et al., 2018). During hypoxia, mtMP decreases below its endogenous steady-state level, which not only diminishes ATP production but also may induce reversal of the F₁F₀ATP-synthase, which then uses the energy released to pump H⁺ from the mitochondrial matrix to the intermembrane space in order to maintain physiological mtMP (Solaini et al., 2010). This creates an ATP sink and excessive consumption of ATP stores (Dudkina et al., 2006; Ikon and Ryan, 2017). A species' ability to maintain mtMP at homeostatic levels despite low O₂ conditions has been proposed as a possible hypoxia tolerance mechanism to sustain ATP production, drive Ca²⁺ and pyruvate uptake to stimulate the citric acid cycle (TCA cycle), as well as prevent the opening of the mitochondrial permeability transition pore (mPTP) and apoptotic downstream events (Dorn and Maack, 2013; Halestrap, 1975; Hochachka et al., 1996). This has been described in a number of hypoxia-tolerant species such as Trachemys scripta, Chrysemys picta and some species of aestivating frogs, which demonstrate maintenance of a stable polarised mtMP during anoxia and reoxygenation in response to the downregulation of F₁F₀-ATP synthase and decreases in H⁺ leak (Galli and Richards, 2014).

Mammalian brain function is particularly vulnerable to hypoxia and can be irreversibly impaired by even brief periods of low O₂ supply (Ravera et al., 2009; Siesjö, 1982). As a highly aerobic excitable tissue with sustained energetic demands, the resting brain is one of the greatest energy consumers among organs of resting vertebrates, and even within ectotherms the brain may use upwards of 20% of resting O₂ supplies (Obel et al., 2012; Vornanen and Paajanen, 2006). Moreover, brain cells remain active during low O₂, with obligatory maintenance of neuronal energy charge and H⁺ gradient across the mtMP to avoid deleterious increases in cytosolic Ca²⁺ influx and excitotoxic cell death (Pamenter et al., 2016). Action potentials and the consequent restoration of ion balance consume up to 80% of total ATP within active neurons, and these processes are O₂ dependent (Hochachka et al., 1996; Vornanen and Paajanen, 2006).

Intertidal marine ectotherms typically experience dramatic fluctuations in O₂ levels on both diurnal and tidal scales relative to subtidal species, and, as a result, show physiological adaptions to survive hypoxic stress (McArley et al., 2018; McArley et al., 2019). New Zealand triplefin fish (Family: Tripterygiidae) provide an excellent non-traditional model, as a large and closely related group of blennioid fishes, wherein species exhibit habitat partitioning by depth and exposure across the near-shore coastal environments (Hickey and Clements, 2003; Hickey et al., 2009). Three species were examined in the present study: Bellapiscis medius, Forsterygion lapillum and Forsterygion varium, which occupy niches within the high (0–2 m), mid (5–10 m) and subtidal (8–20 m) intertidal zones, respectively (Fig. 1) (McArley et al., 2018; Willis et al., 2021). Previous work on triplefin fish has shown clear differences in mitochondrial respiratory adaptations among species, with differing O₂ extraction capacities, critical oxygen partial pressures (P_crit) and metabolic scopes, likely mediated by habitat O₂ availability (Devaux et al., 2018; Hilton et al., 2010; McArley et al., 2019).

The aim of the present study was to investigate whether hypoxia-tolerant intertidal triplefin species evolved adaptive mechanisms to maintain mtMP and mitochondrial respiratory flux both in the transition into anoxia and to re-establish function following hypoxic exposure and reoxygenation. Oxygraph-2k respirometers were used to measure mitochondrial functional integrity through high-resolution respirometry, coupled with fluorometric measures of changes in mtMP in various ‘steady states’ as well as in the transition into and out of anoxia. These transition states provide the first measures of the rate of change of mtMP in hypoxic states relative to changes in O₂ flux, and permit comparison of classical measures of P₅₀ (the pressure at which haemoglobin is 50% saturated) and how mtMP changes with entry into anoxia.

Adult specimens of the three triplefin species (5–10 cm) were collected from different sites around the greater Auckland region (−36.08, 174.60). Rockpool-exclusive Bellapiscis medius (Günther 1861) and shallow subtidal Forsterygion lapillum Hardy 1989 were caught using hand nets from intertidal pools and nearshore subtidal sites (<5 m), and deep subtidal Forsterygion varium (Forster 1801) were caught using minnow traps by divers at 8–20 m depth. Fish were transported to the University of Auckland saltwater facilities, where they were held in 30 l aerated flow-through tanks (10–12 individuals per tank), with recirculating seawater at 18±1°C, 200 μm filtered, 35±1 ppt salinity. Fish were monitored daily and fed a standard mixture of shrimps and green-lipped mussels every 2 days, for a 2 week acclimation period prior to experimentation. All capture, housing and experimental procedures were performed under the approval of the University of Auckland Ethics Committee (AEC R002080).

Brain homogenates were selected over other methods of preparation (i.e. tissue permeabilisation or isolated mitochondria), as they (i) avoid shear stress and maintain mitochondrial integrity and (ii) minimise potential selection bias by retaining all mitochondria sub-populations in situ, while also (iii) conserving O₂ diffusion and potential cellular regulators of mitochondrial function (Larsen et al., 2014; Salin et al., 2016; Devaux et al., 2019). Moreover, triplefins are small fish with limited amounts of brain tissue and homogenisation requires only small tissue samples, which therefore minimises technical variation for greater reproducibility. This results in rapid processing that decreases degradation of the sample (Salin et al., 2016). However, as overall mitochondrial characteristics of sub-populations contained in brain homogenate were examined, differences in mitochondrial density were not assessed. In consequence, reported differences in mitochondrial respiration rates between species provide information on the overall mitochondrial capacity within a fixed volume of tissue, and do not examine differences between mitochondrial units, or adjustments within or among species' mitochondria.

Fish were euthanised by pithing of the spinal cord at the skull, followed by removal of the brain case and extraction of tissue. Tissue sample volumes were measured before being placed in a modified ice-cold marine teleost-specific respiration buffer (Fish MiR05; modified from Gnaiger, 2007); EGTA 0.5 mmol l⁻¹, lactobionic acid 60 mmol l⁻¹, taurine 20 mmol l⁻¹, KH₂PO₄ 10 mmol l⁻¹, Hepes 20 mmol l⁻¹, d-sucrose 160 mmol l⁻¹, BSA 1 g l⁻¹, pH 7.268, 18°C) at a 1:10 w/v dilution. Brain segments were gently homogenised in 1 ml cold Fish MiR05 via trituration through a modified 10 ml syringe with decreasing gauge needles (16–25 gauge).

Tissue dimensions were measured using a volume estimation device. The classic approach to quantifying tissue samples through mass poses problems for soft gelatinous brain segments. Most importantly, a large fraction of precious and limited brain sample from these small fish sticks to the blotting paper, and putatively antioxidants may leach from samples and impair function (Brahma et al., 2000; Kazmierska et al., 2012; Rice, 1999). Measurement of wet mass on a scale is also problematic, as there can be significant evaporation and variation in blotting that leads to miscalculation. A device was therefore designed to determine tissue mass through volume calculations (Fig. 2). This was simply constructed of two microscope slides separated from each other at a known distance via coverslips (0.15 mm) fixed at either end with clear-coat nail varnish. The base slide was also fixed to a black alloy heatsink and placed on ice to chill the sample and prevent tissue degradation. Tissue samples in assay buffer were placed in the centre of the base microscope slide and pressure placed on the top slide such that the sample spread to a maximal area between the slides (i.e. until it reached the thickness of the coverslips; 0.15 mm). An image of the tissue was then taken and analysed in ImageJ (Open source software package from NIH available at https://imagej.nih.gov/), wherein the area of the sample was determined and calibrated to premeasured markings on the slide (a white reference cross 2×2 mm) (Fig. 2). The total volume of each tissue sample was subsequently calculated as coverslip thickness multiplied by tissue area, assuming a brain density of 1.05 g cm⁻³ (Snoussi et al., 2015). This simple device, in addition to being highly portable, provides a simple accurate method for use in the field, and allowed retention of the majority of tissue without antioxidant leaching, while also preventing degradation through chilling of the tissue (see Supplementary Materials and Methods, Fig. S1).

Mitochondrial respiration assays were performed using Oroboros Oxygraph-2k respirometers (Oroboros Instruments, Innsbruck, Austria), which incorporated fluoro-spectrometers (Gnaiger, 2008). Chambers were calibrated to atmospheric O₂ pressure (20 kPa O₂ at 18°C) prior to experiments, and run down to ∼3 kPa O₂ using N₂ gas before being sealed, and inherent O₂ consumption of the tissue was allowed to deplete the chamber to near anoxia. Mass-specific mitochondrial respiration flux (J_O2) was calculated in real time using the negative time derivative of the O₂ pressure expressed in pmol O₂ s⁻¹ mg⁻¹ after instrumental background correction (Chowdhury et al., 2016). The assay temperature was chosen to represent an intermediate of the average seasonal habitat temperature experienced by the three species (∼18°C) in the greater Auckland area (McArley et al., 2018). A substrate–uncoupler–inhibitor titration (SUIT) assay was used to test components of the electron transport system (ETS) in a stepwise fashion, and their functional stability following anoxic exposure (Fig. 3).

Brain homogenate (∼10 mg wet mass equivalent; Fig. 2) was distributed equally between parallel respirometry chambers containing 2 ml O₂-saturated Fish MiR05 and left until a stable steady-state respiration was reached (routine state). The NADH₂-generating substrates pyruvate (10 mmol l⁻¹), malate (2.5 mmol l⁻¹) and glutamate (10 mmol l⁻¹) were added to induce a Leak respiration state supported by complex I (CI-Leak). The addition of ADP (5 mmol l⁻¹) initiated oxidative phosphorylation (CI-OXPHOS). Subsequent injection of succinate (10 mmol l⁻¹) stimulated complex II (CII) and allowed measurement of OXPHOS with combined electron inputs from CI and CII (CI+CII-OXPHOS). Anoxia was induced as described above and maintained for 30 min. Reoxygenation was achieved by opening the chamber until P_O2 had returned to normoxic levels (∼20 kPa). The stability of CI+CII-OXPHOS respiration following anoxic exposure (i.e. reoxygenation) was assessed after a secondary injection of ADP (5 mmol l⁻¹) to account for potential loss of substrates (CI+CII-OXPHOS_RO). Respiration attributed to proton leak (CI+CII-Leak_RO) was determined with the addition of the F₀F₁-ATP synthase inhibitor oligomycin (2.5 mmol l⁻¹). Mitochondria were uncoupled from CI+CII-OXPHOS with repeated titrations of protonophore carbonyl cyanide m-chlorophenyl hydrazine (CCCP; 0.5 μmol l⁻¹ titration steps) to determine maximum ETS_RO capacity without the limitation of the phosphorylation system. Net contribution of CI to CI+CII-OXPHOS_RO was determined with the inhibitor rotenone (CIII-OXPHOS_RO; 1 mmol l⁻¹). Finally, non-mitochondrial respiration was determined by the addition of the CIV inhibitor cyanide (2 mmol l⁻¹ KCN), which was subtracted from active states.

Tetramethylrhodamine methyl ester (TMRM) was used to estimate mtMP simultaneously with J_O2 measurements in homogenised brain tissue. TMRM is a cell-permeant cationic dye that undergoes fluorescence quenching upon accumulation in anionic sites within the mitochondrial matrix (Gerencser et al., 2012; Joshi and Bakowska, 2011). A near-linear correlation between the TMRM spectral shift and coupled mitochondria energised state permits estimates of mtMP (Nicholls and Ward, 2000). The O2K fluro-module was equipped with fluorometers with a TMRM filter set (excitation/emission 530±21 nm/592±22 nm). As per previous work, a final TMRM concentration of 2 μmol l⁻¹ was selected for this study, and the fluorescence signal was calibrated using a five-step titration protocol (0, 0.5, 1.0, 1.5, 2.0 μmol l⁻¹) into each O2K chamber prior to experimentation.

Mitochondrial ‘work’ (J_mt), or energy required to maintain mtMP during anoxia, was also estimated for each species, as per previous work by Devaux et al. (2018). Reference calculations are described in the Supplementary Materials and Methods.

Dynamic changes in mtMP during the transition into and out of anoxic were examined across species. The P_O2(kPa) at which the mtMP commenced depolarisation (i.e. the inflection point) with anoxic onset was estimated for each species, and the TMRM signal used as a proxy measure of mtMP. The point of inflection was taken as the point at which the TMRM signal depolarised by 2%, and the P_O2at this point was selected as the P_O2 of inflection. This was chosen instead of the apparent slight depolarisation during N₂ injections down to ∼3 kPa, because of possible differences in TMRM signal resulting from the speed of injection, which may cause changes in TMRM concentration with stopper movements (i.e. changes unrelated to mtMP depolarisation).

We further estimated the maximum rate at which mtMP depolarisation occurred with the onset of hypoxia and anoxia, and subsequent repolarisation with reoxygenation. This was determined as the maximum slope, reported as the change in TMRM concentration during the mtMP polarisation, which was converted into a change in mtMP (mV s⁻¹ mg⁻¹). The raw TMRM signal during anoxia was used as the baseline signal (Fig. 3, green), with the value of the maximum slope added to get a secondary point, and the Nernst equation was used to calculate this as a change in mV (Eqn 1), while also considering the mass and V_mt for each individual (Eqn 2).

Respiration and fluorometric measures were acquired in real time with DatLab software v.7.1 (OROBOROS Instruments), and exported to Microsoft Excel (Office v.16.16.20) for compilation and analysis. All statistical analyses were performed in Prism (v.9.4.1). Two-way repeated measures ANOVA were performed to test for differences among species among respiratory rates, mtMP and relative oxidation prior to and following anoxia. Pairwise Tukey post hoc tests were used to isolate differences amongst means. All values are presented as mean±s.e.m. (n=8), unless otherwise stated.

A SUIT protocol was applied to stimulate mitochondrial respiration and dynamic changes in mtMP in brain mitochondria for each species. While the stepwise addition of substrates and inhibitors influenced mitochondrial respiration, O₂ consumption remained relatively consistent among species at each state. However, during CI-Leak, F. varium exhibited higher respiration by 43% compared with that of B. medius (Fig. 4A; P<0.05). Steady-state mtMP was also examined as a measure of species’ mitochondrial structural integrity when exposed to progressive anoxia and subsequent reoxygenation. Both F. lapillum and B. medius showed higher mtMP during ETS_RO compared with F. varium (Fig. 4B; P<0.001 and P<0.05, respectively). CII-OXPHOS_RO also showed differences among species, with F. lapillum and B. medius maintaining mtMP 6.28% (Fig. 4B; P<0.0001) and 5.12% (Fig. 4B; P<0.005) higher than that of F. varium, respectively. Combined J_O2 and mtMP data allowed an estimate of the J_mt performed by mitochondria to maintain mtMP under various respiratory states. Calculation of J_mt allows direct assessment of mitochondrial efficiency and coupling by determining the amount of effort (i.e. respiration) attributed to sustaining mtMP at different states. While the J_mt required to maintain mtMP varied between respiratory states, there were no significant differences at steady state among species (Fig. 4C; P>0.05).

Respiratory control ratios (E−L) were calculated as either (CI-OXPHOS−CI-Leak)/CI-OXPHOS or (CI+CII-OXPHOS_RO−CI+CII-Leak_RO)/CI+CII-OXPHOS_RO, and represent the proportion of O₂ consumption typically coupled to ATP synthesis. High E−L values are indicative of tightly coupled mitochondria while low values represent dysfunctional mitochondria. Species demonstrated similar respiratory control ratios irrespective of either CI or CII substrates, or acclimation to normoxia compared with following anoxia exposure (Table 1; P>0.05). All species doubled their respiratory flux when ETS_RO was uncoupled from oxidative phosphorylation with CCCP (UCR_RO), with no differences in maximal flux rate (Table 1; P>0.05). While not statistically significant, CI contribution to overall OXPHOS trended higher in B. medius, and was 10.93% and 13.77% higher than both F. lapillum and F. varium, respectively. Comparison of OXPHOS prior to and following anoxic exposure demonstrated a 14% increase in F. lapillum respiratory flux, while flux decreased in both F. varium and B. medius by 8% and 9%, respectively (Table 1; P<0.05).

The P_O2 at which species’ mtMP commenced depolarisation in the transition into anoxia in part mirrors previously determined whole-animal P_crit values (Fig. 5; McArley et al., 2019). In F. varium, mtMP began to depolarise at P_O2 values that were 7- and 4.4-fold higher than in the hypoxia-tolerant B. medius and F. lapillum, respectively (Fig. 5; P<0.05). There was no difference in the P_O2 for the mtMP inflection between hypoxia-tolerant species (Fig. 5; P>0.05). The intertidal specialist B. medius had a lower P_crit than all other species (Fig. 5; P<0.001), while the intertidal generalist F. lapillum had a higher P_crit than the exclusively subtidal F. varium (Fig. 5; P<0.001).

Dynamic changes in species’ mtMP were examined at the onset of anoxia and upon reoxygenation. The maximum rate at which species' mtMP depolarised and repolarised was used as a measure of each species' ability to sustain mtMP, and therefore mitochondrial structural integrity and function during low O₂ conditions, and as a capacity to regenerate a mtMP upon the return of O₂. Forsterygion varium exhibited the fastest depolarisation rate, which was 50% higher than that of B. medius (Fig. 6; P<0.05). Forsterygion lapillum depolarisation rate was intermediate between that of the other two species (Fig. 6; P≥0.05). Repolarisation rates with reoxygenation were similar among species.

While tolerance to hypoxia has been shown to arise from a multitude of adaptive adjustments, the involvement of subcellular remodelling has remained largely unexplored. Mitochondria are responsible for producing over ∼90% of cellular ATP required by the brain to perform energy-demanding processes, such as maintenance of ion gradients and synaptic signalling (Pamenter et al., 2016). This central role in O₂ consumption and energy production results in mitochondria being the lynchpin in pathological dysfunction associated with low O₂ conditions (Galli and Richards, 2014). We demonstrate that while general measures of mitochondrial function (i.e. O₂ flux during ‘steady states’) do not differ among species, dynamic responses in brain mitochondria of intertidal triplefin species may reveal mechanisms underlying hypoxia tolerance. Our approach permitted us to track mitochondrial dynamics during the transition into and out of anoxia. The hypoxia-tolerant B. medius and F. lapillum maintained mtMP at significantly lower P_O2 values, and demonstrated a slower rate of mtMP depolarisation compared with the hypoxia-sensitive F. varium.

While absolute anoxia may not occur in rock pools (McArley et al., 2018), the P_O2 of active tissues is extremely low (P_O2<2 kPa). We therefore assessed the effect of 30 min of near anoxia in vitro, as this time generally triggers mitochondrial damage in other models (Devaux et al., 2023; Rouslin, 1983). Brain mitochondria of all triplefin species displayed similar respiration rates at each respiratory state, both before and after anoxia exposure (Fig. 4). While the data were not corrected for mitochondrial density, this implies that species exhibit a similar ability to produce total brain ATP both prior to and following anoxia exposure. This was consistent with coupling of oxidative phosphorylation, and the relative contributions of complex I, which did not differ among species (Table 1). All three species had relatively high reserve ETS_RO capacities (i.e. ETS>OXPHOS) and doubled their respiratory flux when oxidative phosphorylation was uncoupled (UC_RO) following anoxia and reoxygenation (Fig. 4, Table 1). This suggests that all species can accommodate some damage and loss of ETS function (i.e. reserve capacity), thereby preserving oxidative phosphorylation and ATP production rates (Devaux et al., 2019).

Exposure to anoxia and reoxygenation led to a decrease in CI+CII-OXPHOS flux in both hypoxia-tolerant B. medius and hypoxia-sensitive F. varium, while F. lapillum increased respiratory flux (Table 1). This response is possibly due to ETS damage resulting from reactive oxygen species (ROS) formed on reoxygenation, which damage mitochondrial membrane lipids and complexes (Kalogeris et al., 2011; Murphy, 2016; Sokolova, 2018). Elevated oxidative phosphorylation in F. lapillum following reoxygenation indicates improved ATP synthesis capacity. This may assist with restoring energy homeostasis following anoxic exposure and/or contribute to the increased O₂ consumption following recovery (i.e. O₂ debt) commonly found in hypoxia-tolerant invertebrates and fish (Lewis et al., 2007; Sokolov et al., 2021; Vismann and Hagerman, 2008).

From these data, and similar findings by Devaux et al. (2023), it appears that the response within the Forsterygion genus follows a trend that correlates with hypoxia tolerance. In contrast, the most hypoxia-tolerant and phylogenetically more distant species, B. medius, exhibited a response most similar to that of the more hypoxia-sensitive species, F. varium. Therefore, in the triplefin fish species examined, the respiratory responses to anoxia and reoxygenation do not appear to be associated with hypoxia tolerance, but instead seem to be species and/or genus specific.

The mtMP represents the partitioning of charge between the mitochondrial matrix and the mitochondrial intermembrane space, and is the primary driver of ATP production (Mitchell, 1961; Vasan et al., 2022). Mitochondrial membrane potential dissipates through H⁺ transfer into the matrix through negative flux (i.e. leak) induced by damage to the mtMP, and H⁺ flux through the F₀F₁-ATP synthase to produce ATP (Devaux et al., 2018; Mitchell, 1961). These act in opposition to H⁺ flux transferred by the ETS, which are used to sustain mtMP (i.e. J_mt). This study is one of the first to present changes in mtMP and the J_mt required to sustain it, which provides further information on the overall functioning and metabolic integrity of species' brain mitochondria when exposed to hypoxia (Vasan et al., 2022). Specifically, J_mt details an integrative measure of mitochondrial coupling efficiency and H⁺ leak compared with traditional respiratory control or ROS production ratios, as it directly assesses the amount of respiration attributed to maintaining mtMP under varying conditions and respiratory states.

During ETS_RO, the hypoxia-tolerant species F. lapillum and B. medius showed higher mtMP compared with that of the hypoxia-sensitive species F. varium, which is indicative of a more robust ETS and/or capacity to regenerate mtMP, despite exposure to anoxia and reoxygenation (Fig. 4B). This was also shown after inhibition of complex I. This has been observed in hypoxia-tolerant yeast, wherein expression of a hypoxia-specific CIV Vb isoform increases CIV turnover rate and therefore increases ETS efficiency (Kwast et al., 1999). Similarly, comparative analysis of mitochondrial CIV kinetics across species in intertidal sculpins demonstrated a significant relationship between hypoxia tolerance and CIV O₂ binding affinity (Lau et al., 2017). Modification of ETS complexes may allow greater capacities to import and oxidise mitochondrial substrates, increase electron transport capacity and/or enhance efficiency to consume O₂. Pyruvate requires H⁺ for symport into the mitochondrial matrix, and therefore maintenance of mtMP is required to maintain pyruvate influx and oxidation. In addition, mitochondria may exhibit greater plasticity to sustain reductive and oxidative damage without loss of function. This may explain the increased ETS function in hypoxia-tolerant triplefins to maintain a more polarised mtMP and prevent the excessive depolarisation with diminished proton flux apparent in the hypoxia-sensitive F. varium.

While steady-state measures of maximal and/or phosphorylating mtMP are informative of the overall functional integrity and stability of mitochondria, mtMP is likely highly conserved. Analysis of dynamic changes in mitochondrial function as they transition into and out of hypoxia and/or anoxia may provide more information on adaptive responses permitting continued function at lower O₂ conditions. These experiments permitted comparison of species’ sensitivity and tolerance to low O₂ fluctuations, where diminishing P_O2 is likely to impact mitochondrial function (Devaux et al., 2023; Di Santo et al., 2016; Rogers et al., 2016). While species’ O₂ consumption and measures of P_crit and loss of equilibrium are commonly tested, comparison of dynamic changes in mtMP during intermittent hypoxic transitions have remained poorly understood (Boutilier and St-Pierre, 2000; Di Santo et al., 2016; Rogers et al., 2016).

Comparison of the P_O2 at which mtMP depolarisation occurred revealed that B. medius and F. lapillum were able to maintain their normoxic mtMP to P_O2 values 7- and 4.4-fold lower, respectively, than that of F. varium (Fig. 5). This reflects greater mitochondrial stability and function to sustain ATP concentrations as O₂ declines. This pattern also mirrors that observed for whole-animal P_crit (Fig. 5) (McArley et al., 2019). 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 rockpools, thereby mitigating depletion of anaerobic fuel stores and toxic end products. Moreover, it should allow a greater proportion of energetic demand to be met through efficient mitochondrial ATP production (McArley et al., 2019). Notably, the P_O2 at mtMP inflection is lower than the P_crit for each species. This reflects the O₂ cascade where mitochondria function at a much lower P_O2 within cells (Brand et al., 2000). This is reflected in Devaux et al. (2023), wherein hypoxia-tolerant species also sustained higher mitochondrial respiratory capacity compared with the hypoxia-sensitive species as they approached anoxia, with electron transfer more efficiently directed to respiration rather than ROS production. These results show that while mtMP depolarisation is similar among species after 30 min, hypoxia-tolerant B. medius and F. lapillum are able to sustain a significant mtMP. This may be protective against ATP synthase reversal (and assumedly mitochondria function) at lower P_O2 values, and suggests that New Zealand hypoxia-tolerant species may benefit from more efficient mitochondria that may help to sustain repeated hypoxic episodes than those of the hypoxia-sensitive species F. varium.

Hypoxia-tolerant triplefin fish not only appear more able to maintain mtMP at lower P_O2 but also have slower mtMP depolarisation rates (Fig. 6). This may suggest adaptive responses of hypoxia-tolerant species’ mitochondria to maintain ATP synthesis capacity. Work by Devaux et al. (2018) indicates that intertidal hypoxia-tolerant triplefins may harness the extra mitochondria H⁺ which results from acidosis during hypoxic exposure, and utilise this to maintain the proton motive force (i.e. mtMP), thereby facilitating continued ATP production despite decreased respiration. Alternatively, mtMP may be retained by prevention or shortened opening time of the mPTP, which allows solutes up to 1500 Da to readily diffuse into and out of the mitochondria, enabling in part the release of apoptotic factors (Pamenter, 2014). Hypoxia-tolerant triplefins may display inhibition-like mechanisms of mPTP opening and maintain mtMP, which prevents neuronal apoptosis and necrosis following hypoxic damage (Pamenter, 2014).

Increased proton leak through adenine nucleotide translocase (ANT), which may reflect ADP–ATP exchange rate, is also displayed by hypoxia- and anoxia-tolerant species (Chinopoulos et al., 2014; Devaux et al., 2019). While counterintuitive, increased ANT leak may favour ADP–ATP exchange between mitochondria and the cytosol, subsequently restoring cytosolic ATP and mitochondrial ADP content (Klingenberg, 2008). Over-expression of ANT has been shown to protect mammalian cardiomyocytes when exposed to hypoxic (Winter et al., 2016) or oxidative stress (Klumpe et al., 2016). By sustaining OXPHOS, this should increase resistance to reductive stress and prolong cellular viability (Lemasters et al., 1997). Importantly, it should be noted that none of these mechanisms are mutually exclusive. Intertidal hypoxia-tolerant triplefin mitochondria are therefore not immune to hypoxic or anoxic insults, but are perhaps more efficient and better defended compared with hypoxia-sensitive subtidal species, and so can maintain function at lower P_O2.

The current study found few apparent differences in traditional ‘steady-state’ measures of mitochondrial function among triplefin species. However, relative to the subtidal hypoxia-sensitive F. varium, dynamic changes in mtMP for the hypoxia-tolerant species differed substantially as they entered anoxia. This should increase survival capacity for hypoxia-tolerant species during hypoxia exposure. In the transition into anoxia, brain mitochondria of hypoxia-tolerant B. medius and F. lapillum maintained mtMP to significantly lower P_O2, and had slower rates of depolarisation than the more sensitive species F. varium. These properties mirror the P_crit values of each species. Overall, these findings are congruent with predictions that hypoxia-tolerant species have mitochondria that resist hypoxic stress, a primary ecological requirement of life in intertidal pools.

The authors would like to thank Esther Stuck and Peter Schlegel for their invaluable help with animal husbandry. Our thanks to members of Applied Surgery and Metabolism Laboratory (ASML) for laboratory assistance, fish collection and the many discussions concerning hypoxic stress and triplefin physiology.