ABSTRACT The nests of embryonic turtles naturally experience elevated CO 2 (hypercarbia), which leads to increased blood P CO 2 and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pH e )]. Some fishes preferentially regulate tissue pH [intracellular pH (pH i )] against changes in pH e ; this has been proposed to be associated with exceptional CO 2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO 2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pH i , conferring tolerance to severe acute acid–base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles ( Chelydra serpentina ) reared in normoxia then exposed to hypercarbia (13 kPa P CO 2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pH e but not pH i , at all developmental ages. At 70% of incubation, pH e was depressed by 0.324 pH units while pH i of brain, white muscle and lung increased; heart, liver and kidney pH i remained unchanged. At 90% of incubation, pH e was depressed by 0.352 pH units but heart pH i increased with no change in pH i of other tissues. Yearlings exhibited a pH e reduction of 0.235 pH units but had no changes in pH i of any tissues. The results indicate common snapping turtles preferentially regulate pH i during development, but the degree of response is reduced throughout development. This is the first time preferential pH i regulation has been identified in an amniote. These findings may provide insight into the evolution of acid–base homeostasis during development of amniotes, and vertebrates in general.

ABSTRACT Broadcast spawning marine invertebrates are susceptible to environmental stressors such as climate change, as their reproduction depends on the successful meeting and fertilization of gametes in the water column. Under near-future scenarios of ocean acidification, the swimming behaviour of marine invertebrate sperm is altered. We tested whether this was due to changes in sperm mitochondrial activity by investigating the effects of ocean acidification on sperm metabolism and swimming behaviour in the sea urchin Centrostephanus rodgersii . We used a fluorescent molecular probe (JC-1) and flow cytometry to visualize mitochondrial activity (measured as change in mitochondrial membrane potential, MMP). Sperm MMP was significantly reduced in ΔpH −0.3 (35% reduction) and ΔpH −0.5 (48% reduction) treatments, whereas sperm swimming behaviour was less sensitive with only slight changes (up to 11% decrease) observed overall. There was significant inter-individual variability in responses of sperm swimming behaviour and MMP to acidified seawater. We suggest it is likely that sperm exposed to these changes in pH are close to their tipping point in terms of physiological tolerance to acidity. Importantly, substantial inter-individual variation in responses of sperm swimming to ocean acidification may increase the scope for selection of resilient phenotypes, which, if heritable, could provide a basis for adaptation to future ocean acidification.

SUMMARY Trigeminally innervated, mechanically sensitive chemoreceptors (M) were previously identified in rainbow trout, Oncorhynchus mykiss , but it is not known whether these receptors are responsive only to noxious, chemical irritants or have a general chemosensory function. This study aimed to characterise the stimulus–response properties of these receptors in comparison with polymodal nociceptors (P). Both P and M gave similar response profiles to acetic acid concentrations. The electrophysiological properties were similar between the two different afferent types. To determine whether the receptors have a nociceptive function, a range of chemical stimulants was applied to these receptors, including non-noxious stimuli such as ammonium chloride, bile, sodium bicarbonate and alarm pheromone, and potentially noxious chemical irritants such as acetic acid, carbon dioxide, low pH, citric acid, citric acid phosphate buffer and sodium chloride. Only irritant stimuli evoked a response, confirming their nociceptive function. All receptor afferents tested responded to carbon dioxide (CO 2 ) in the form of mineral water or soda water. The majority responded to 1% acetic acid, 2% citric acid, citric acid phosphate buffer (pH 3) and 5.0 mol l –1 NaCl. CO 2 receptors have been characterised in the orobranchial cavity and gill arches in fish; however, this is the first time that external CO 2 receptors have been identified on the head of a fish. Because the fish skin is in constant contact with the aqueous environment, contaminants with a low pH or hypercapnia may stimulate the nociceptive system in fish.

SUMMARY The critical O 2 tension of whole-animal O 2 consumption rate ( ), or P crit , is the water P O2 ( P w O 2 ) at which an animal transitions from an oxyregulator to an oxyconformer. Although P crit is a popular measure of hypoxia tolerance in fishes because it reflects the capacity for O 2 uptake from the environment at low P w O 2 , little is known about the interrelationships between P crit and blood O 2 transport characteristics and increased use of anaerobic metabolism during hypoxia exposure in fishes, especially elasmobranchs. We addressed this knowledge gap using progressive hypoxia exposures of two elasmobranch species with differing hypoxia tolerance. The P crit of the hypoxia-tolerant epaulette shark ( Hemiscyllium ocellatum , 5.10±0.37 kPa) was significantly lower than that of the comparatively hypoxia-sensitive shovelnose ray ( Aptychotrema rostrata , 7.23±0.40 kPa). Plasma [lactate] was elevated above normoxic values at around P crit in epaulette sharks, but increased relative to normoxic values at P w O 2 below P crit in shovelnose rays, providing equivocal support for the hypothesis that P crit is associated with increased anaerobic metabolism. The , arterial P O2 and arterial blood O 2 content ( C a O 2 ) were similar between the two species under normoxia and decreased in both species with progressive hypoxia, but as P w O 2 declined, epaulette sharks had a consistently higher and C a O 2 than shovelnose rays, probably due to their significantly greater in vivo haemoglobin (Hb)–O 2 binding affinity ( in vivo Hb–O 2 P 50 =4.27±0.57 kPa for epaulette sharks vs 6.35±0.34 kPa for shovelnose rays). However, at P w O 2 values representing the same percentage of each species' P crit (up to ∼175% of P crit ), Hb–O 2 saturation and C a O 2 were similar between species. These data support the hypothesis that Hb–O 2 P 50 is an important determinant of P crit and suggest that P crit can predict Hb–O 2 saturation and C a O 2 during hypoxia exposure, with a lower P crit being associated with greater O 2 supply at a given P w O 2 and consequently better hypoxia tolerance. Thus, P crit is a valuable predictor of environmental hypoxia tolerance and hypoxia exposures standardized at a given percentage of P crit will yield comparable levels of arterial hypoxaemia, facilitating cross-species comparisons of responses to hypoxia. SUMMARY The critical O 2 tension of whole-animal O 2 consumption rate ( ), or P crit , is the water P O2 ( P w O 2 ) at which an animal transitions from an oxyregulator to an oxyconformer. Although P crit is a popular measure of hypoxia tolerance in fishes because it reflects the capacity for O 2 uptake from the environment at low P w O 2 , little is known about the interrelationships between P crit and blood O 2 transport characteristics and increased use of anaerobic metabolism during hypoxia exposure in fishes, especially elasmobranchs. We addressed this knowledge gap using progressive hypoxia exposures of two elasmobranch species with differing hypoxia tolerance. The P crit of the hypoxia-tolerant epaulette shark ( Hemiscyllium ocellatum , 5.10±0.37 kPa) was significantly lower than that of the comparatively hypoxia-sensitive shovelnose ray ( Aptychotrema rostrata , 7.23±0.40 kPa). Plasma [lactate] was elevated above normoxic values at around P crit in epaulette sharks, but increased relative to normoxic values at P w O 2 below P crit in shovelnose rays, providing equivocal support for the hypothesis that P crit is associated with increased anaerobic metabolism. The , arterial P O2 and arterial blood O 2 content ( C a O 2 ) were similar between the two species under normoxia and decreased in both species with progressive hypoxia, but as P w O 2 declined, epaulette sharks had a consistently higher and C a O 2 than shovelnose rays, probably due to their significantly greater in vivo haemoglobin (Hb)–O 2 binding affinity ( in vivo Hb–O 2 P 50 =4.27±0.57 kPa for epaulette sharks vs 6.35±0.34 kPa for shovelnose rays). However, at P w O 2 values representing the same percentage of each species' P crit (up to ∼175% of P crit ), Hb–O 2 saturation and C a O 2 were similar between species. These data support the hypothesis that Hb–O 2 P 50 is an important determinant of P crit and suggest that P crit can predict Hb–O 2 saturation and C a O 2 during hypoxia exposure, with a lower P crit being associated with greater O 2 supply at a given P w O 2 and consequently better hypoxia tolerance. Thus, P crit is a valuable predictor of environmental hypoxia tolerance and hypoxia exposures standardized at a given percentage of P crit will yield comparable levels of arterial hypoxaemia, facilitating cross-species comparisons of responses to hypoxia. SUMMARY The critical O 2 tension of whole-animal O 2 consumption rate ( ), or P crit , is the water P O2 ( P w O 2 ) at which an animal transitions from an oxyregulator to an oxyconformer. Although P crit is a popular measure of hypoxia tolerance in fishes because it reflects the capacity for O 2 uptake from the environment at low P w O 2 , little is known about the interrelationships between P crit and blood O 2 transport characteristics and increased use of anaerobic metabolism during hypoxia exposure in fishes, especially elasmobranchs. We addressed this knowledge gap using progressive hypoxia exposures of two elasmobranch species with differing hypoxia tolerance. The P crit of the hypoxia-tolerant epaulette shark ( Hemiscyllium ocellatum , 5.10±0.37 kPa) was significantly lower than that of the comparatively hypoxia-sensitive shovelnose ray ( Aptychotrema rostrata , 7.23±0.40 kPa). Plasma [lactate] was elevated above normoxic values at around P crit in epaulette sharks, but increased relative to normoxic values at P w O 2 below P crit in shovelnose rays, providing equivocal support for the hypothesis that P crit is associated with increased anaerobic metabolism. The , arterial P O2 and arterial blood O 2 content ( C a O 2 ) were similar between the two species under normoxia and decreased in both species with progressive hypoxia, but as P w O 2 declined, epaulette sharks had a consistently higher and C a O 2 than shovelnose rays, probably due to their significantly greater in vivo haemoglobin (Hb)–O 2 binding affinity ( in vivo Hb–O 2 P 50 =4.27±0.57 kPa for epaulette sharks vs 6.35±0.34 kPa for shovelnose rays). However, at P w O 2 values representing the same percentage of each species' P crit (up to ∼175% of P crit ), Hb–O 2 saturation and C a O 2 were similar between species. These data support the hypothesis that Hb–O 2 P 50 is an important determinant of P crit and suggest that P crit can predict Hb–O 2 saturation and C a O 2 during hypoxia exposure, with a lower P crit being associated with greater O 2 supply at a given P w O 2 and consequently better hypoxia tolerance. Thus, P crit is a valuable predictor of environmental hypoxia tolerance and hypoxia exposures standardized at a given percentage of P crit will yield comparable levels of arterial hypoxaemia, facilitating cross-species comparisons of responses to hypoxia.

SUMMARY Experiments were carried out to test the hypothesis that ventilatory and cardiovascular responses to hypercarbia (elevated water P CO2 ) in the tambaqui Colossoma macropomum are stimulated by externally oriented receptors that are sensitive to water CO 2 tension as opposed to water pH. Cardiorespiratory responses to acute hypercarbia were evaluated in both the absence and presence of internal hypercarbia (elevated blood P CO2 ), achieved by treating fish with the carbonic anhydrase inhibitor acetazolamide. Exposure to acute hypercarbia (15 min at each level, final water CO 2 tensions of 7.2,15.5 and 26.3 mmHg) elicited significant increases in ventilation frequency(at 26.3 mmHg, a 42% increase over the normocarbic value) and amplitude(128%), together with a fall in heart rate (35%) and an increase in cardiac stroke volume (62%). Rapid washout of CO 2 from the water reversed these effects, and the timing of the changes in cardiorespiratory variables corresponded more closely to the fall in water P CO2 ( P w CO2 ) than to that in blood P CO2 ( P a CO2 ). Similar responses to acute hypercarbia (15 min,final P w CO2 of 13.6 mmHg) were observed in acetazolamide-treated (30 mg kg -1 ) tambaqui. Acetazolamide treatment itself, however, increased P a CO2 (from 4.81±0.58 to 13.83±0.91 mmHg, mean ± s . e . m .; N =8) in the absence of significant change in ventilation, heart rate or cardiac stroke volume. The lack of response to changes in blood P CO2 and/or pH were confirmed by comparing responses to the bolus injection of hypercarbic saline(5% or 10% CO 2 ; 2 ml kg -1 ) into the caudal vein with those to the injection of CO 2 -enriched water (1%, 3%, 5% or 10%CO 2 ; 50 ml kg -1 ) into the buccal cavity. Whereas injections of hypercarbic saline were ineffective in eliciting cardiorespiratory responses, changes in ventilation and cardiovascular parameters accompanied injection of CO 2 -laden water into the mouth. Similar injections of CO 2 -free water acidified to the corresponding pH of the hypercarbic water (pH 6.3, 5.6, 5.3 or 4.9, respectively) generally did not stimulate cardiorespiratory responses. These results are in agreement with the hypothesis that in tambaqui, externally oriented chemoreceptors that are predominantly activated by increases in water P CO2 ,rather than by accompanying decreases in water pH, are linked to the initiation of cardiorespiratory responses to hypercarbia.

SUMMARY Scenarios of rising CO 2 concentration in surface waters due to atmospheric accumulation of anthropogenic CO 2 , or in the deep sea due to anticipated industrial dumping of CO 2 , suggest that hypercapnia (elevated partial pressure of CO 2 ) will become a general stress factor in aquatic environments, with largely unknown effects on species survival and well being, especially in cold and deep waters. For an analysis of CO 2 effects at the cellular level, isolated hepatocytes were prepared from two representatives of the Antarctic fish fauna, Pachycara brachycephalum and Lepidonotothen kempi . Correlated changes in energy and protein metabolism were investigated by determining the rates of oxygen consumption at various levels of P CO 2 , of intra- and extracellular pH, and after inhibition of protein synthesis by cycloheximide. A decrease in extracellular pH (pHe) from control levels (pHe 7.90) to pHe 6.50 caused a reduction in aerobic metabolic rate of 34-37% under both normocapnic and hypercapnic conditions. Concomitantly, protein biosynthesis was inhibited by about 80%under conditions of severe acidosis in hepatocytes from both species. A parallel drop in intracellular pH probably mediates this effect. In conclusion, the present data indicate that elevated P CO 2 may limit the functional integrity of the liver due to a pronounced depression in protein anabolism. This process may contribute to the limits of whole-animal tolerance to raised CO 2 levels.

In normoxic water at 25°C, the reedfish ( Erpetoichthys calabaricus ) can breathe in both air and water, although water is the primary source of oxygen. This pattern of oxygen partitioning is altered by a number of factors. Increases in metabolic costs (e.g. locomotor activity) and decreases in aquatic oxygen concentration are met by increases in aerial oxygen uptake. Reedfish acclimated to 33°C have increased lung breathing compared with fish acclimated to 25°C. Also, activity stimulates lung breathing more at 33°C than at 25°C. Unexpectedly, weight-specific oxygen uptake rates of reedfish at these two temperatures were not significantly different. Increased lung-breathing compensates for changes in locomotor activity, dissolved oxygen and temperature, allowing reedfish to meet their overall oxygen requirements. Exposure of reedfish to six different combinations of oxygen and carbon dioxide indicated that lung ventilation frequencies were more labile than gill ventilation frequencies. The maximum change in lung ventilation rates was 849% above controls whereas the largest change in gill ventilation frequency was 27%. Exposure of reedfish to hyperoxia significantly depressed both gill and lung ventilation. Hypercapnic gases stimulated gill ventilation at both low (0.5%) and high (5%) concentrations; however, lung ventilation was only stimulated at the higher carbon dioxide concentration. The observed depression of gill ventilation frequencies in reedfish exposed to combinations of hypercapnic and hypoxic gases indicates that the inhibitory effect of low oxygen on gill ventilation dominates the stimulatory effect of hypercapnia. Overall, these data suggest that oxygen exerts a stronger influence in the control of respiration than carbon dioxide in this air- and water-breathing fish. Reedfish survived out of water for 6 and 8h at 25°C without obvious ill effects. Oxygen consumption rates of these fish were significantly less than those of similar sized reedfish measured in water at either 25 or 33°C. The respiratory physiology of the reedfish frees it from aquatic oxygen constraints and allows it to tolerate at least short-term terrestrial exposures.