Jeffrey G. Richards

Physiological, behavioral and biochemical adaptations of intertidal fishes to hypoxia

Summary Hypoxia survival in fish requires a well-coordinated response to either secure more O2 from the hypoxic environment or to limit the metabolic consequences of an O2 restriction at the mitochondria. Although there is a considerable amount of information available on the physiological, behavioral, biochemical and molecular responses of fish to hypoxia, very little research has attempted to determine the adaptive value of these responses. This article will review current attempts to use the phylogenetically corrected comparative method to define physiological and behavioral adaptations to hypoxia in intertidal fish and further identify putatively adaptive biochemical traits that should be investigated in the future. In a group of marine fishes known as sculpins, from the family Cottidae, variation in hypoxia tolerance, measured as a critical O2 tension (Pcrit), is primarily explained by variation in mass-specific gill surface area, red blood cell hemoglobin–O2 binding affinity, and to a lesser extent variation in routine O2 consumption rate (). The most hypoxia-tolerant sculpins consistently show aquatic surface respiration (ASR) and aerial emergence behavior during hypoxia exposure, but no phylogenetically independent relationship has been found between the thresholds for initiating these behaviors and Pcrit. At O2 levels below Pcrit, hypoxia survival requires a rapid reorganization of cellular metabolism to suppress ATP consumption to match the limited capacity for O2-independent ATP production. Thus, it is reasonable to speculate that the degree of metabolic rate suppression and the quantity of stored fermentable fuel is strongly selected for in hypoxia-tolerant fishes; however, these assertions have not been tested in a phylogenetic comparative model.

Molecular Evolution of Cytochrome c Oxidase Underlies High-Altitude Adaptation in the Bar-Headed Goose

Bar-headed geese (Anser indicus) fly at up to 9,000 m elevation during their migration over the Himalayas, sustaining high metabolic rates in the severe hypoxia at these altitudes. We investigated the evolution of cardiac energy metabolism and O(2) transport in this species to better understand the molecular and physiological mechanisms of high-altitude adaptation. Compared with low-altitude geese (pink-footed geese and barnacle geese), bar-headed geese had larger lungs and higher capillary densities in the left ventricle of the heart, both of which should improve O(2) diffusion during hypoxia. Although myoglobin abundance and the activities of many metabolic enzymes (carnitine palmitoyltransferase, citrate synthase, 3-hydroxyacyl-coA dehydrogenase, lactate dehydrogenase, and pyruvate kinase) showed only minor variation between species, bar-headed geese had a striking alteration in the kinetics of cytochrome c oxidase (COX), the heteromeric enzyme that catalyzes O(2) reduction in oxidative phosphorylation. This was reflected by a lower maximum catalytic activity and a higher affinity for reduced cytochrome c. There were small differences between species in messenger RNA and protein expression of COX subunits 3 and 4, but these were inconsistent with the divergence in enzyme kinetics. However, the COX3 gene of bar-headed geese contained a nonsynonymous substitution at a site that is otherwise conserved across vertebrates and resulted in a major functional change of amino acid class (Trp-116 → Arg). This mutation was predicted by structural modeling to alter the interaction between COX3 and COX1. Adaptations in mitochondrial enzyme kinetics and O(2) transport capacity may therefore contribute to the exceptional ability of bar-headed geese to fly high.

Do mitochondrial properties explain intraspecific variation in thermal tolerance

SUMMARY As global temperatures rise, there is a growing need to understand the physiological mechanisms that determine an organisms thermal niche. Here, we test the hypothesis that increases in mitochondrial capacity with cold acclimation and adaptation are associated with decreases in thermal tolerance using two subspecies of killifish (Fundulus heteroclitus) that differ in thermal niche. We assessed whole-organism metabolic rate, mitochondrial amount and mitochondrial function in killifish acclimated to several temperatures. Mitochondrial enzyme activities and mRNA levels were greater in fish from the northern subspecies, particularly in cold-acclimated fish, suggesting that the putatively cold-adapted northern subspecies has a greater capacity for increases in mitochondrial amount in response to cold acclimation. When tested at the fishs acclimation temperature, maximum ADP-stimulated (State III) rates of mitochondrial oxygen consumption in vitro were greater in cold-acclimated northern fish than in southern fish but did not differ between subspecies at higher acclimation temperatures. Whole-organism metabolic rate was greater in fish of the northern subspecies at all acclimation temperatures. Cold acclimation also changed the response of mitochondrial respiration to acute temperature challenge. Mitochondrial oxygen consumption was greater in cold-acclimated northern fish than in southern fish at low test temperatures, but the opposite was true at high test temperatures. These differences were reflected in whole-organism oxygen consumption. Our data indicate that the plasticity of mitochondrial function and amount differs between killifish subspecies, with the less high-temperature tolerant, and putatively cold adapted, northern subspecies having greater ability to increase mitochondrial capacity in the cold. However, there were few differences in mitochondrial properties between subspecies at warm acclimation temperatures, despite differences in both whole-organism oxygen consumption and thermal tolerance at these temperatures.

The effect of acclimation to hypoxia and sustained exercise on subsequent hypoxia tolerance and swimming performance in goldfish (Carassius auratus)

SUMMARY The objective of this study was to determine whether acclimation to hypoxia and sustained exercise would increase hypoxia tolerance (as indicated by a decrease in critical oxygen tension, Pcrit) and swimming performance in goldfish (Carassius auratus), and to investigate the relationship between changes in performance and gill remodelling and tissue metabolic capacity. Goldfish were acclimated to either hypoxia (48 h at 0.3 mg O2 l–1) or sustained exercise (48 h at 70% of critical swimming speed, Ucrit) and then Pcrit and Ucrit were determined in normoxia (10 mg O2 l–1) and hypoxia (1 mg O2 l–1) and compared with values from control fish. Acclimation to both hypoxia and sustained exercise improved hypoxia tolerance (Pcrit was reduced by 49% and 39%, respectively), which was associated with an increase in lamellar surface area (71% and 43%, respectively) and an increase in blood [Hb] (26% in both groups). Exercise acclimation also resulted in a decrease in routine (). Acclimation to both hypoxia and sustained exercise resulted in a significant increase in Ucrit in hypoxia (18% and 17%, respectively), which was associated with an increase in maximal O2 consumption rate at Ucrit (; 35% and 39%, respectively). While hypoxia acclimation resulted in an increase in Ucrit in normoxia, acclimation to sustained exercise did not improve subsequent swimming performance in normoxia. This lack of improvement was possibly due to depleted oxidizable substrates during exercise acclimation.

AMP-activated protein kinase activity during metabolic rate depression in the hypoxic goldfish, Carassius auratus.

SUMMARY Cell survival during hypoxia exposure requires a metabolic reorganization to decrease ATP demands to match the reduced capacity for ATP production. We investigated whether AMP-activated protein kinase (AMPK) activity responds to 12 h exposure to severe hypoxia (∼0.3 mg O2 l–1) in the anoxia-tolerant goldfish (Carassius auratus). Hypoxia exposure in goldfish was characterized by a strong activation of creatine phosphate hydrolysis and glycolysis in liver and muscle. AMPK activity increased by ∼5.5-fold in goldfish liver within 0.5 h hypoxia exposure and this increase in activity was temporally associated with an 11-fold increase in [AMPfree]/[ATP]. No changes in total AMPK protein amount were observed, suggesting that the changes in AMPK activity are due to post-translational phosphorylation of the protein. Hypoxia exposure had no effect on the expression of two identified AMPKα -subunit isoforms and caused an ∼50% decrease in the mRNA levels of AMPK β-subunit isoform. Changes in AMPK activity in the liver were associated with an increase in percentage phosphorylation of a well-characterized target of AMPK, eukaryotic elongation factor-2 (eEF2), and decreases in protein synthesis rates measured in liver cell-free extracts. No activation of AMPK was observed in muscle, brain, heart or gill during the 12 h hypoxia exposure suggesting a tissue-specific regulation of AMPK possibly related to a lack of change in cellular [AMPfree]/[ATP] as observed in muscle.

Evolution of muscle phenotype for extreme high altitude flight in the bar-headed goose

Bar-headed geese migrate over the Himalayas at up to 9000 m elevation, but it is unclear how they sustain the high metabolic rates needed for flight in the severe hypoxia at these altitudes. To better understand the basis for this physiological feat, we compared the flight muscle phenotype of bar-headed geese with that of low altitude birds (barnacle geese, pink-footed geese, greylag geese and mallard ducks). Bar-headed goose muscle had a higher proportion of oxidative fibres. This increased muscle aerobic capacity, because the mitochondrial volume densities of each fibre type were similar between species. However, bar-headed geese had more capillaries per muscle fibre than expected from this increase in aerobic capacity, as well as higher capillary densities and more homogeneous capillary spacing. Their mitochondria were also redistributed towards the subsarcolemma (cell membrane) and adjacent to capillaries. These alterations should improve O2 diffusion capacity from the blood and reduce intracellular O2 diffusion distances, respectively. The unique differences in bar-headed geese were much greater than the minor variation between low altitude species and existed without prior exercise or hypoxia exposure, and the correlation of these traits to flight altitude was independent of phylogeny. In contrast, isolated mitochondria had similar respiratory capacities, O2 kinetics and phosphorylation efficiencies across species. Bar-headed geese have therefore evolved for exercise in hypoxia by enhancing the O2 supply to flight muscle.

Swimming Performance and Energetics as a Function of Temperature in Killifish Fundulus heteroclitus

Populations of the common killifish Fundulus heteroclitus are found along a latitudinal temperature gradient in habitats with high thermal variability. The objectives of this study were to assess the effects of temperature and population of origin on killifish swimming performance (assessed as critical swimming speed, Ucrit). Acclimated fish from northern and southern killifish populations demonstrated a wide zone (from 7° to 33°C) over which Ucrit showed little change with temperature, with performance declining significantly only at lower temperatures. Although we observed significant differences in swimming performance between a northern and a southern population of killifish in one experiment, with northern fish having an ∼1.5‐fold‐greater Ucrit than southern fish across all acclimation temperatures, we were unable to replicate this finding in other populations or collection years, and performance was consistently high across all populations and at both low (7°C) and high (23°C) acclimation temperatures. The poor swimming performance of southern killifish from a single collection year was correlated with low muscle [glycogen] rather than with other indicators of fuel stores or body condition. Killifish acclimated to 18°C and acutely challenged at temperatures of 5°, 18°, 25°, or 34°C showed modest thermal sensitivity of Ucrit between 18° and 34°C, with performance declining substantially at 5°C. Thus, much of the zone of relative thermal insensitivity of swimming performance is intrinsic in this species rather than acquired as a result of acclimation. These data suggest that killifish are broadly tolerant of changing temperatures, whether acute or chronic, and demonstrate little evidence of local adaptation in endurance swimming performance in populations from different thermal habitats.

MIGRATION, MITOCHONDRIA, AND THE YELLOW-RUMPED WARBLER

Discordance between mitochondrial and nuclear DNA has been noted in many systems. Asymmetric introgression of mitochondria is a common cause of such discordances, although in most cases the drivers of introgression are unknown. In the yellow‐rumped warbler, evidence suggests that mtDNA from the eastern, myrtle warbler, has introgressed across much of the range of the western form, the Audubons warbler. Within the southwestern United States myrtle mtDNA comes into contact with another clade that occurs in the Mexican black‐fronted warbler. Both northern forms exhibit seasonal migration, whereas black‐fronted warblers are nonmigratory. We investigated the link between mitochondrial introgression, mitochondrial function, and migration using novel genetic, isotopic, biochemical, and phenotypic data obtained from populations in the transition zone. Isotopes suggest the zone is coincident with a shift in migration, with individuals in the south being resident and populations further north becoming increasingly more migratory. Mitochondrial respiration in flight muscles demonstrates that myrtle‐type individuals have a significantly greater acceptor control ratio of mitochondria, suggesting it may be more metabolically efficient. To our knowledge this is the first time this type of intraspecific variation in mitochondrial respiration has been measured in wild birds and we discuss how such mitochondrial adaptations may have facilitated introgression.

Effects of environmental hypoxia on cardiac energy metabolism and performance in tilapia

The ability of an animal to depress ATP turnover while maintaining metabolic energy balance is important for survival during hypoxia. In the present study, we investigated the responses of cardiac energy metabolism and performance in the hypoxia-tolerant tilapia (Oreochromis hybrid sp.) during exposure to environmental hypoxia. Exposure to graded hypoxia (> or =92% to 2.5% air saturation over 3.6 +/- 0.2 h) followed by exposure to 5% air saturation for 8 h caused a depression of whole animal oxygen consumption rate that was accompanied by parallel decreases in heart rate, cardiac output, and cardiac power output (CPO, analogous to ATP demand of the heart). These cardiac parameters remained depressed by 50-60% compared with normoxic values throughout the 8-h exposure. During a 24-h exposure to 5% air saturation, cardiac ATP concentration was unchanged compared with normoxia and anaerobic glycolysis contributed to ATP supply as evidenced by considerable accumulation of lactate in the heart and plasma. Reductions in the provision of aerobic substrates were apparent from a large and rapid (in <1 h) decrease in plasma nonesterified fatty acids concentration and a modest decrease in activity of pyruvate dehydrogenase. Depression of cardiac ATP demand via bradycardia and an associated decrease in CPO appears to be an integral component of hypoxia-induced metabolic rate depression in tilapia and likely contributes to hypoxic survival.

Chapter 11 Defining Hypoxia: An Integrative Synthesis of the Responses of Fish to Hypoxia

This chapter attempts to synthesize the responses of fish to hypoxia presented in this Fish Physiology volume. The previous chapters are built on by differentiating between environmental hypoxia and functional hypoxia, and by outlining the possible compensatory mechanisms that fish use to counteract these forms of hypoxia. Environmental hypoxia is most simply defined as the water PO 2 when physiological function is compromised, thus the definition of environmental hypoxia is dependent upon the physiological system under examination. Hypoxia-induced decrements in maximal oxygen consumption and thus reduced aerobic scope occur at higher water O 2 levels than changes in routine oxygen consumption, which when compromised, is quantified as the critical oxygen tension (P crit ). At water O 2 levels below P crit , duration of survival is dependent upon the capacity to reduce metabolic demands to match the limited supply of fermentable fuels. Functional hypoxia, on the other hand, occurs during situations where tissue O 2 demands exceed circulatory O 2 supply, which can be evident during exercise, temperature extremes, anemia, acidosis, and changes in gill structure, but the physiological strategies used to survive environmental hypoxia are not necessarily utilized to endure functional hypoxia.