Respiratory acidosis and O2 supply capacity do not affect the acute temperature tolerance of rainbow trout (Oncorhynchus mykiss).

The mechanisms that determine the temperature tolerances of fish are poorly understood, creating barriers to disentangle how additional environmental challenges-such as CO2-induced aquatic acidification and fluctuating oxygen availability-may exacerbate vulnerability to a warming climate and extreme heat events. Here, we explored whether two acute exposures (~0.5 hours or ~72 hours) to increased CO2 impact acute temperature tolerance limits in a freshwater fish, rainbow trout (Oncorhynchus mykiss). We separated the potential effects of acute high CO2 exposure on critical thermal maximum (CTmax), caused via either respiratory acidosis (reduced internal pH) or O2 supply capacity (aerobic scope), by exposing rainbow trout to ~1 kPa CO2 (~1% or 10 000 µatm) in combination with normoxia or hyperoxia (~21 or 42 kPa O2, respectively). In normoxia, acute exposure to high CO2 caused a large acidosis in trout (blood pH decreased by 0.43 units), while a combination of hyperoxia and ~1 kPa CO2 increased the aerobic scope of trout by 28%. Despite large changes in blood pH and aerobic scope between treatments, we observed no impacts on the CTmax of trout. Our results suggest that the mechanisms that determine the maximum temperature tolerance of trout are independent of blood acid-base balance or the capacity to deliver O2 to tissues.

Fish feeds supplemented with calcium-based buffering minerals decrease stomach acidity, increase the blood alkaline tide and cost more to digest.

Predatory fish in the wild consume whole prey including hard skeletal parts like shell and bone. Shell and bone are made up of the buffering minerals calcium carbonate (CaCO3) and calcium phosphate (Ca3(PO4)2). These minerals resist changes in pH, meaning they could have physiological consequences for gastric acidity, digestion and metabolism in fish. Using isocaloric diets supplemented with either CaCO3, Ca3(PO4)2 or CaCl2 as non-buffering control, we investigated the impacts of dietary buffering on the energetic cost of digestion (i.e. specific dynamic action or SDA), gastric pH, the postprandial blood alkalosis (the "alkaline tide") and growth in juvenile rainbow trout (Oncorhynchus mykiss). Increases in dietary buffering were significantly associated with increased stomach chyme pH, postprandial blood HCO3-, net base excretion, the total SDA and peak SDA but did not influence growth efficiency in a 21 day trial. This result shows that aspects of a meal that have no nutritional value can influence the physiological and energetic costs associated with digestion in fish, but that a reduction in the SDA will not always lead to improvements in growth efficiency. We discuss the broader implications of these findings for the gastrointestinal physiology of fishes, trade-offs in prey choice in the wild, anthropogenic warming and feed formulation in aquaculture.

Characterizing the hypoxic performance of a fish using a new metric: PAAS-50.

The hypoxic constraint on peak oxygen uptake (MO2,peak) was characterized in rainbow trout over a range of ambient oxygen tensions with different testing protocols and statistical models. The best-fit model was selected using both statistical criteria (R2 and AIC) and the model's prediction of three anchor points for hypoxic performance: critical PO2 (Pcrit), maximum MO2 and a new metric, the minimum PO2 that supports 50% of absolute aerobic scope (PAAS-50). The best-fitting model was curvilinear using five strategically selected PO2 values. This model predicted PAAS-50 as 70 mmHg (coefficient of variation, CV=9%) for rainbow trout. Thus, while a five-point hypoxic performance curve can characterize the limiting effects of hypoxia in fish, as envisaged by Fry over 75 years ago, PAAS-50 is a promising metric to compare hypoxic constraints on performance in a standardized manner both within and across fish species.

Rapid blood acid-base regulation by European sea bass (Dicentrarchus labrax) in response to sudden exposure to high environmental CO2.

Fish in coastal ecosystems can be exposed to acute variations in CO2 of between 0.2 and 1 kPa CO2 (2000-10,000 µatm). Coping with this environmental challenge will depend on the ability to rapidly compensate for the internal acid-base disturbance caused by sudden exposure to high environmental CO2 (blood and tissue acidosis); however, studies about the speed of acid-base regulatory responses in marine fish are scarce. We observed that upon sudden exposure to ∼1 kPa CO2, European sea bass (Dicentrarchus labrax) completely regulate erythrocyte intracellular pH within ∼40 min, thus restoring haemoglobin-O2 affinity to pre-exposure levels. Moreover, blood pH returned to normal levels within ∼2 h, which is one of the fastest acid-base recoveries documented in any fish. This was achieved via a large upregulation of net acid excretion and accumulation of HCO3- in blood, which increased from ∼4 to ∼22 mmol l-1. While the abundance and intracellular localisation of gill Na+/K+-ATPase (NKA) and Na+/H+ exchanger 3 (NHE3) remained unchanged, the apical surface area of acid-excreting gill ionocytes doubled. This constitutes a novel mechanism for rapidly increasing acid excretion during sudden blood acidosis. Rapid acid-base regulation was completely prevented when the same high CO2 exposure occurred in seawater with experimentally reduced HCO3- and pH, probably because reduced environmental pH inhibited gill H+ excretion via NHE3. The rapid and robust acid-base regulatory responses identified will enable European sea bass to maintain physiological performance during large and sudden CO2 fluctuations that naturally occur in coastal environments.

Rising CO2 enhances hypoxia tolerance in a marine fish.

Global environmental change is increasing hypoxia in aquatic ecosystems. During hypoxic events, bacterial respiration causes an increase in carbon dioxide (CO2) while oxygen (O2) declines. This is rarely accounted for when assessing hypoxia tolerances of aquatic organisms. We investigated the impact of environmentally realistic increases in CO2 on responses to hypoxia in European sea bass (Dicentrarchus labrax). We conducted a critical oxygen (O2crit) test, a common measure of hypoxia tolerance, using two treatments in which O2 levels were reduced with constant ambient CO2 levels (~530 µatm), or with reciprocal increases in CO2 (rising to ~2,500 µatm). We also assessed blood acid-base chemistry and haemoglobin-O2 binding affinity of sea bass in hypoxic conditions with ambient (~650 µatm) or raised CO2 (~1770 µatm) levels. Sea bass exhibited greater hypoxia tolerance (~20% reduced O2crit), associated with increased haemoglobin-O2 affinity (~32% fall in P50) of red blood cells, when exposed to reciprocal changes in O2 and CO2. This indicates that rising CO2 which accompanies environmental hypoxia facilitates increased O2 uptake by the blood in low O2 conditions, enhancing hypoxia tolerance. We recommend that when impacts of hypoxia on aquatic organisms are assessed, due consideration is given to associated environmental increases in CO2.