Mechanisms of cardiac collapse at high temperature in a marine teleost (Girella nigrians).

Heat-induced mortality in ectotherms may be attributed to impaired cardiac performance, specifically a collapse in maximum heart rate (fHmax), although the physiological mechanisms driving this phenomenon are still unknown. Here, we tested two proposed factors which may restrict cardiac upper thermal limits: noxious venous blood conditions and oxygen limitation. We hypothesized elevated blood [K+] (hyperkalemia) and low oxygen (hypoxia) would reduce cardiac upper thermal limits in a marine teleost (Girella nigricans), while high oxygen (hyperoxia) would increase thermal limits. We also hypothesized higher acclimation temperatures would exacerbate the harmful effects of an oxygen limitation. Using the Arrhenius breakpoint temperature test, we measured fHmax in acutely warmed fish under control (saline injected) and hyperkalemic conditions (elevated plasma [K+]) while exposed to hyperoxia (200% air saturation), normoxia (100% air saturation), or hypoxia (20% air saturation). We also measured ventricle lactate content and venous blood oxygen partial pressure (PO2) to determine if there were universal thresholds in either metric driving cardiac collapse. Elevated [K+] was not significantly correlated with any cardiac thermal tolerance metric. Hypoxia significantly reduced cardiac upper thermal limits (Arrhenius breakpoint temperature [TAB], peak fHmax, temperature of peak heart rate [TPeak], and temperature at arrhythmia [TARR]). Hyperoxia did not alter cardiac thermal limits compared to normoxia. There was no evidence of a species-wide threshold in ventricular [lactate] or venous PO2. Here, we demonstrate that oxygen limits cardiac thermal tolerance only in instances of hypoxia, but that other physiological processes are responsible for causing temperature-induced heart failure when oxygen is not limited.

Diet changes thermal acclimation capacity, but not acclimation rate, in a marine ectotherm (Girella nigricans) during warming.

Global climate change is increasing thermal variability in coastal marine environments and the frequency, intensity and duration of marine heatwaves. At the same time, food availability and quality are being altered by anthropogenic environmental changes. Marine ectotherms often cope with changes in temperature through physiological acclimation, which can take several weeks and is a nutritionally demanding process. Here, we tested the hypothesis that different ecologically relevant diets (omnivorous, herbivorous, carnivorous) impact thermal acclimation rate and capacity, using a temperate omnivorous fish as a model (opaleye, Girella nigricans). We measured acute thermal performance curves for maximum heart rate because cardiac function has been observed to set upper thermal limits in ectotherms. Opaleye acclimated rapidly after raising water temperatures, but their thermal limits and acclimation rate were not affected by their diet. However, the fish's acclimation capacity for maximum heart rate was sensitive to diet, with fish in the herbivorous treatment displaying the smallest change in heart rate throughout acclimation. Mechanistically, ventricle fatty acid composition differed with diet treatment and was related to cardiac performance in ways consistent with homoviscous adaptation. Our results suggest that diet is an important, but often overlooked, determinant of thermal performance in ectotherms on environmentally relevant time scales.

Effect of thermal variation on the cardiac thermal limits of a eurythermal marine teleost (Girella nigricans).

Although most animals live in complex, thermally variable environments, the impact of this variability on specific physiological systems is still unresolved. The ectotherm heart is known to change in both structure and function to ensure appropriate oxygen delivery under different thermal regimes, but the plasticity of the upper thermal limits of the heart under stable or variable thermal acclimation conditions remains unknown. To investigate the role of thermal variability on cardiac acclimation potential, we acclimated a eurythermal fish, opaleye (Girella nigricans), to three static temperature treatments (13, 16, and 19 °C) as well as two oscillating treatments which cycled between maximum and minimum temperatures every 12 h (13-19 °C and 16-22 °C). These temperatures and daily thermal ranges were chosen to mimic the conditions observed in the rocky intertidal environments in Santa Barbara, CA, USA where the fish were collected. We hypothesized that increasing temperature would increase upper thermal limits of the heart, and that variable acclimations would result in broader acute thermal performance curves (TPCs) compared to static acclimations. We measured maximum heart rate during acute warming to determine cardiac thermal performance (i.e., the temperature corresponding to the onset of cardiac arrythmia, the temperature at maximum heart rate, absolute maximum heart rate, and the Arrhenius breakpoint temperature) and construct acute TPCs. Rising static acclimation temperatures increased upper thermal limits but had no impact on peak maximum heart rate. The warmest static temperature did, however, cause a narrowing of the acute TPC. Fish acclimated to variable conditions had the same upper thermal limits compared to fish acclimated to static conditions with the same mean temperature in all metrics of thermal performance. Further, there was no significant broadening of the acute TPC. This study suggests that cardiac plasticity is robust to thermal variation in this eurythermal fish.

A lack of red blood cell swelling in five elasmobranch fishes following air exposure and exhaustive exercise.

In teleost fishes, catecholamine-induced increases in the activity of cation exchangers compensate for decreases in hemoglobin oxygen affinity and maximum blood oxygen carrying capacity caused by decreases in plasma pH (i.e., metabolic acidosis). The resultant red blood cell (RBC) swelling has been documented in sandbar (Carcharhinus plumbeus) and epaulette (Hemiscyllium ocellatum) sharks following capture by rod-and-reel or after a 1.5 h exposure to anoxia (respectively), although the underlying mechanisms remain unknown. To determine if RBC swelling could be documented in other elasmobranch fishes, we collected blood samples from clearnose skate (Rostroraja eglanteria), blacktip reef shark (Carcharhinus melanopterus), and sicklefin lemon shark (Negaprion acutidens) subjected to exhaustive exercise or air exposure (or both) and measured hematocrit, hemoglobin concentration, RBC count, RBC volume, and mean corpuscular hemoglobin content. We did likewise with sandbar and epaulette sharks to further explore the mechanisms driving swelling when present. We could not document RBC swelling in any species; although hematocrit increased in all species (presumably due to RBC ejection from the spleen or fluid shifts out of the vascular compartment) except epaulette shark. Our results indicate RBC swelling and associated ion shifts in elasmobranch fishes is not inducible by exercise or hypoxia, thus implying this response maybe of lesser importance for maintaining oxygen delivery during acute acidosis than in teleost fishes.

The effects of elevated potassium, acidosis, reduced oxygen levels, and temperature on the functional properties of isolated myocardium from three elasmobranch fishes: clearnose skate (Rostroraja eglanteria), smooth dogfish (Mustelus canis), and sandbar shark (Carcharhinus plumbeus).

Elevated plasma potassium levels (hyperkalemia), reduced plasma pH (acidosis), reduced blood oxygen content, and elevated temperatures are associated with species-specific rates of at-vessel and post-release mortality in elasmobranch fishes. The mechanism linking these physiological disturbances to mortality remains undetermined however, and we hypothesize that the proximate cause is reduced myocardial function. We measured changes in the functional properties of isolated ventricular myocardial strips from clearnose skate (Rostroraja eglanteria), smooth dogfish (Mustelus canis), and sandbar shark (Carcharhinus plumbeus) when subjected to the following stressors (both in isolation and in combination): hyperkalemia (7.4 mM K+), acidosis (from 7.9 to 7.1), and reduced oxygen (to 31% O2 saturation) applied at temperatures 5 °C above and below holding temperatures. We selected these species based on phylogenetic distance, diverse routine activity levels, and their tolerance to capture and transport. Stressors had a few significant species-specific detrimental impacts on myocardial function (e.g., a 33-45% decrease in net force under acidosis + low O2). Net force production of myocardial strips from clearnose skate and smooth dogfish approximately doubled following exposure to isoproterenol, demonstrating that these species possess beta-adrenergic receptors and that their stimulation could provide a mechanism for preservation of cardiac function during stress. Our results suggest that disruption of physiological homeostasis associated with capture may fatally impair cardiac function in some elasmobranch species, although research with more severe stressors is needed.

The power struggle: assessing interacting global change stressors via experimental studies on sharks.

Ocean warming and acidification act concurrently on marine ectotherms with the potential for detrimental, synergistic effects; yet, effects of these stressors remain understudied in large predatory fishes, including sharks. We tested for behavioural and physiological responses of blacktip reef shark (Carcharhinus melanopterus) neonates to climate change relevant changes in temperature (28 and 31 °C) and carbon dioxide partial pressures (pCO2; 650 and 1050 µatm) using a fully factorial design. Behavioural assays (lateralisation, activity level) were conducted upon 7-13 days of acclimation, and physiological assays (hypoxia tolerance, oxygen uptake rates, acid-base and haematological status) were conducted upon 14-17 days of acclimation. Haematocrit was higher in sharks acclimated to 31 °C than to 28 °C. Significant treatment effects were also detected for blood lactate and minimum oxygen uptake rate; although, these observations were not supported by adequate statistical power. Inter-individual variability was considerable for all measured traits, except for haematocrit. Moving forward, studies on similarly 'hard-to-study' species may account for large inter-individual variability by increasing replication, testing larger, yet ecologically relevant, differences in temperature and pCO2, and reducing measurement error. Robust experimental studies on elasmobranchs are critical to meaningfully assess the threat of global change stressors in these data-deficient species.

Analysing tropical elasmobranch blood samples in the field: blood stability during storage and validation of the HemoCue® haemoglobin analyser.

Blood samples collected from wild-caught fishes can provide important information regarding the effects of capture (and thus post-release survival) as well as other stressors. Unfortunately, blood samples often cannot be analysed immediately upon sampling, and blood parameters (e.g. blood oxygen levels and acid-base parameters) are known to change with storage duration due to the metabolic activity of the red blood cells. We obtained blood samples from both untreated and stressed individuals of both blacktip reef shark (Carcharhinus melanopterus) and sicklefin lemon shark (Negaprion acutidens) to determine the effects of storage duration on blood pH, haematocrit and haemoglobin concentration ([Hb]). We found no significant effects after storage on ice for up to 180 minutes. Moreover, to validate the usability of a HemoCue haemoglobin analyser (a point-of-care device), we compared data from this device to [Hb] determined using the cyanomethaemoglobin method with blood samples from 10 individuals from each of the aforementioned species as well as epaulette shark (Hemiscyllium ocellatum). Values from the HemoCue consistently overestimated [Hb], and we therefore developed the necessary correction equations. The correction equations were not statistically different among the three elasmobranch species within the biologically relevant range but did differ from published corrections developed using blood from temperate teleost fishes. Although the HemoCue is useful in field situations, development of species-specific calibration equations may be necessary to ensure the reliability of inter-species comparisons of blood [Hb]. Together, these data should increase confidence in haematological stress indicators in elasmobranch fishes, measurements of which are critical for understanding the impact of anthropogenic stressors on these ecologically important species.

The impacts of warming and hypoxia on the performance of an obligate ram ventilator.

Climate change is causing the warming and deoxygenation of coastal habitats like Chesapeake Bay that serve as important nursery habitats for many marine fish species. As conditions continue to change, it is important to understand how these changes impact individual species' behavioral and metabolic performance. The sandbar shark (Carcharhinus plumbeus) is an obligate ram-ventilating apex predator whose juveniles use Chesapeake Bay as a nursery ground up to 10 years of age. The objective of this study was to measure juvenile sandbar shark metabolic and behavioral performance as a proxy for overall performance (i.e. fitness or success) when exposed to warm and hypoxic water. Juvenile sandbar sharks (79.5-113.5 cm total length) were collected from an estuary along the eastern shore of Virginia and returned to lab where they were fitted with an accelerometer, placed in a respirometer and exposed to varying temperatures and oxygen levels. Juvenile sandbar shark overall performance declined substantially at 32°C or when dissolved oxygen concentration was reduced below 3.5 mg l-1 (51% oxygen saturation between 24-32°C). As the extent of warm hypoxic water increases in Chesapeake Bay, we expect that the available sandbar shark nursery habitat will be reduced, which may negatively impact the population of sandbar sharks in the western Atlantic as well as the overall health of the ecosystem within Chesapeake Bay.

Combined Effects of Acute Temperature Change and Elevated pCO2 on the Metabolic Rates and Hypoxia Tolerances of Clearnose Skate (Rostaraja eglanteria), Summer Flounder (Paralichthys dentatus), and Thorny Skate (Amblyraja radiata).

Understanding how rising temperatures, ocean acidification, and hypoxia affect the performance of coastal fishes is essential to predicting species-specific responses to climate change. Although a population's habitat influences physiological performance, little work has explicitly examined the multi-stressor responses of species from habitats differing in natural variability. Here, clearnose skate (Rostaraja eglanteria) and summer flounder (Paralichthys dentatus) from mid-Atlantic estuaries, and thorny skate (Amblyraja radiata) from the Gulf of Maine, were acutely exposed to current and projected temperatures (20, 24, or 28 °C; 22 or 30 °C; and 9, 13, or 15 °C, respectively) and acidification conditions (pH 7.8 or 7.4). We tested metabolic rates and hypoxia tolerance using intermittent-flow respirometry. All three species exhibited increases in standard metabolic rate under an 8 °C temperature increase (Q10 of 1.71, 1.07, and 2.56, respectively), although this was most pronounced in the thorny skate. At the lowest test temperature and under the low pH treatment, all three species exhibited significant increases in standard metabolic rate (44-105%; p < 0.05) and decreases in hypoxia tolerance (60-84% increases in critical oxygen pressure; p < 0.05). This study demonstrates the interactive effects of increasing temperature and changing ocean carbonate chemistry are species-specific, the implications of which should be considered within the context of habitat.