Thermally tolerant intertidal triplefin fish (Tripterygiidae) sustain ATP dynamics better than subtidal species under acute heat stress

Abstract

Temperature is a key factor that affects all levels of organization. Minute shifts away from thermal optima result in detrimental effects that impact growth, reproduction and survival. Metabolic rates of ectotherms are especially sensitive to temperature and for organisms exposed to high acute temperature changes, in particular intertidal species, energetic processes are often negatively impacted. Previous investigations exploring acute heat stress have implicated cardiac mitochondrial function in determining thermal tolerance. The brain, however, is by weight, one of the most metabolically active and arguably the most temperature sensitive organ. It is essentially aerobic and entirely reliant on oxidative phosphorylation to meet energetic demands, and as temperatures rise, mitochondria become less efficient at synthesising the amount of ATP required to meet the increasing demands. This leads to an energetic crisis. Here we used brain homogenate of three closely related triplefin fish species (Bellapiscis medius, Forsterygion lapillum, and Forsterygion varium) and measured respiration and ATP dynamics at three temperatures (15, 25 and 30 °C). We found that the intertidal B. medius and F. lapillum were able to maintain rates of ATP production above rates of ATP hydrolysis at high temperatures, compared to the subtidal F. varium, which showed no difference in rates at 30 °C. These results showed that brain mitochondria became less efficient at temperatures below their respective species thermal limits, and that energetic surplus of ATP synthesis over hydrolysis narrows. In subtidal species synthesis matches hydrolysis, leaving no scope to elevate ATP supply.

Figure 1

Triplefin species used in this study and their respective distributions. Bellapiscis medius is a high intertidal species that experiences a wide range of temperatures daily. Forsterygion lapillum is a shallow subtidal species that occurs at depths around 5–10 m and experiences a narrower temperature window than B. medius. Forsterygion varium is a deeper sub-tidal species found at depths of 8–20 m and experiences the narrowest temperature range of all three species. Temperatures in these rock pools vary with the tides and can peak in the summer months at ~ 30 °C (McArley et al., 2018; 2019). Triplefin images courtesy of Vivian Ward & Kendall Clements.

Figure 2

Representative trace of mitochondrial respiration assay from triplefin brain homogenates. Mitochondrial flux (pmol O₂s⁻¹ mg⁻¹) on the left axis and Mg²⁺ fluorescence (mA) on the right axis. Addition of tissue and titration of mitochondrial substrates and inhibitors at saturated concentrations are indicated by the arrows and follow the SUIT protocol outlined above. ADP Adenosine diphosphate, P Pyruvate, M Malate, G Glutamate, S Succinate, Oli Oligomycin, cAtr Carboxyatractyloside, CCCP Carbonyl cyanide m-chloro phenyl hydrazone, Ama Antimycin A. Different mitochondrial states are represented above the figure.

Figure 3

Respiratory flux in each respiration state of mitochondria at 15, 25, and 30 °C. Mean respiratory flux normalized to tissue wet weight (pmol O₂ s⁻¹ mg⁻¹) following the modified SUIT protocol for respiration and ATP determination. CI-OXP is initiated by the addition of PMG in the presence of ADP, CI&CII-OXP is initiated by the addition of S. CI&CII-Leak (denoted LEAK) is measured following the addition of the inhibitors Oligomycin (Oli) and carboxyatractyloside (cAtr) while rates of uncoupled respiration (denoted ETS) were measured after complete uncoupling with CCCP. (a) Mitochondrial flux during “CI-OXPHOS” for F. varium, F. lapillum and B. medius across the three experimental temperatures. (b) Mitochondrial flux during “CI&CII-OXPHOS” for F. varium, F. lapillum and B. medius across the three experimental temperatures. (c) Mitochondrial flux during “LEAK” for F. varium, F. lapillum and B. medius across the three experimental temperatures. (d) Mitochondrial flux during “ETS” for F. varium, F. lapillum and B. medius across the three experimental temperatures. Significant differences of p ≤ 0.05 between species within states are denoted by an asterisk (*), significant differences of p ≤ 0.01 are denoted (**), differences of p ≤ 0.001 are denoted (***) while differences of p ≤ 0.0001 are denoted (****).

Figure 4

Complex contributions (CI&CII) to OXPHOS and reserve respiratory capacity of mitochondria at 15, 25 and 30 °C. (a–c) Complex I (CI and complex II (CII) contribution to OXPHOS by temperature (a; 15 °C, b; 25 °C, c; 30 °C). (d) Complex II activity contribution to OXPHOS across temperature. (e) Respiratory reserve capacity calculated as ETS-CI&CII OXPHOS across temperature. Significant differences between species within temperature of p ≤ 0.05 are denoted by an asterisk (*), significant differences of p ≤ 0.01 are denoted (**) while differences of p ≤ 0.0001 are denoted (****).

Figure 5

Rates of ATP production and hydrolysis with Respiratory control ratios (RCR) and P/O ratios for all three species at the experimental temperatures (15, 25 and 30 °C). (a) ATP production and ATP hydrolysis rates. (b) Net ATP production rate. (c) RCR’s were calculated as (CI&CII-OXPHOS-LEAK)/CI&CII-OXPHOS and represent the proportion of oxygen consumption coupled to energy production. (d) P/O ratios calculated as the rate of overall ATP production divided by O₂ consumption during CI&CII-OXPHOS. Significant differences between species within temperature of p ≤ 0.05 are denoted by an asterisk (*), significant differences of p ≤ 0.01 are denoted (**) while differences of p ≤ 0.0001 are denoted (****). Significant differences within species across temperature are represented by letters (v/V = F.varium; l/L = F. Lapillum; m/M = B. medius). Capitalised letters represent differences from 15 °C while lowercase letters represent differences from 25 °C.