Unique adaptations of the metabolic biochemistry of tunas and billfishes for life in the pelagic environment

Synopsis Virtually all characteristics of tunas and billfishes reflect their highly charged lifestyles as apex predators in the oceanic pelagic environment. The adaptations they possess for efficient and rapid swimming, efficient and rapid food processing, turnover of nutrients and storage and mobilization of internal fuel supplies, and for rapid recovery rates, are discussed. Overall, tunas and billfishes are designed for high performance, at both sustainable and burst swimming speeds, but there are several differences between tunas and billfishes. Tunas' aerobic metabolic capacities exceed those of ectothermic fishes, including billfishes and other scombrids, by virtue of their elevated red muscle temperatures, and because heart and white muscle aerobic capacities are significantly greater in tunas. The adaptations for high performance involve some costs, including the need for a constant high energy input to sustain high metabolic rates, high activity levels, and endothermy, Yet, tunas and billfishes have adopted successful lifestyles, as evidenced by their large numbers and biomass within the marine environment. Although our knowledge of these fishes has increased dramatically during the past 15 years, there are major gaps in our understanding of the metabolic biochemistry and physiology of these fishes, and these are highlighted so that additional research can be directed towards filling these gaps.Paper from the International Union of Biological Societies symposium The biology of tunas and billfishes: an examination of life on the knife edge, organized by Richard W. Brill and Kim N. Holland.     

Hindlimb muscle fiber types in two frogs (Rana catesbeiana and Litoria caerulea) with different locomotor behaviors: histochemical and enzymatic comparison

To test how differences in locomotor behaviors may be reflected in muscle fiber-type diversity within anurans, a comparison of hindlimb muscles between the powerful terrestrial hopper, Rana catesbeiana, and the tree frog, Litoria caerulea, was done. One postural muscle (tibialis posticus, TP) and one primary hopping muscle (plantaris longus, PL), were characterized to identify muscle fiber types using standard histochemical methods. In addition, spectophotometric analysis of activity levels of the oxidative enzyme citrate synthase (CS) and the glycolytic enzyme lactate dehydrogenase (LDH) were done in each muscle. In spite of presumed differences in behavior between the species, we found no significant differences in the proportions of the identified fiber types when the muscles were compared across species. In addition, there were no significant differences in the proportions of the different fiber types between the postural versus phasic muscles within species. Within Rana, the postural muscle (TP) had greater oxidative capacity (as measured by CS activity) than did the phasic muscle (PL). Both muscles had equivalent LDH activities. Within Litoria, PL and TP did not differ in either LDH or CS activities. Both PL and TP of Litoria had less LDH activity and greater CS activity than their homologs in Rana. Thus, in spite of the uniform populations of fiber types between muscles and species, the metabolic diversity based on enzyme activity is consistent with behavioral differences between the species. These results suggest that the range of functional diversity within fiber types may be very broad in anurans, and histochemical fiber typing alone is not a clear indicator of their metabolic or functional properties.     

Structural adaptations for ram ventilation: Gill fusions in scombrids and billfishes

For ram‐gill ventilators such as tunas and mackerels (family Scombridae) and billfishes (families Istiophoridae, Xiphiidae), fusions binding the gill lamellae and filaments prevent gill deformation by a fast and continuous ventilatory stream. This study examines the gills from 28 scombrid and seven billfish species in order to determine how factors such as body size, swimming speed, and the degree of dependence upon ram ventilation influence the site of occurrence and type of fusions. In the family Scombridae there is a progressive increase in the reliance on ram ventilation that correlates with the elaboration of gill fusions. This ranges from mackerels (tribe Scombrini), which only utilize ram ventilation at fast cruising speeds and lack gill fusions, to tunas (tribe Thunnini) of the genus Thunnus, which are obligate ram ventilators and have two distinct fusion types (one binding the gill lamellae and a second connecting the gill filaments). The billfishes appear to have independently evolved gill fusions that rival those of tunas in terms of structural complexity. Examination of a wide range of body sizes for some scombrids and billfishes shows that gill fusions begin to develop at lengths as small as 2.0 cm fork length. In addition to securing the spatial configuration of the gill sieve, gill fusions also appear to increase branchial resistance to slow the high‐speed current produced by ram ventilation to distribute flow evenly and optimally to the respiratory exchange surfaces. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Gill morphometrics in relation to gas transfer and ram ventilation in high‐energy demand teleosts: Scombrids and billfishes

This comparative study of the gill morphometrics in scombrids (tunas, bonitos, and mackerels) and billfishes (marlins, swordfish) examines features of gill design related to high rates of gas transfer and the high‐pressure branchial flow associated with fast, continuous swimming. Tunas have the largest relative gill surface areas of any fish group, and although the gill areas of non‐tuna scombrids and billfishes are smaller than those of tunas, they are also disproportionally larger than those of most other teleosts. The morphometric features contributing to the large gill surface areas of these high‐energy demand teleosts include: 1) a relative increase in the number and length of gill filaments that have, 2) a high lamellar frequency (i.e., the number of lamellae per length of filament), and 3) lamellae that are long and low in profile (height), which allows a greater number of filaments to be tightly packed into the branchial cavity. Augmentation of gill area through these morphometric changes represents a departure from the general mechanism of area enhancement utilized by most teleosts, which lengthen filaments and increase the size of the lamellae. The gill design of scombrids and billfishes reflects the combined requirements for ram ventilation and elevated energetic demands. The high lamellar frequencies and long lamellae increase branchial resistance to water flow which slows and streamlines the ram ventilatory stream. In general, scombrid and billfish gill surface areas correlate with metabolic requirements and this character may serve to predict the energetic demands of fish species for which direct measurement is not possible. The branching of the gill filaments documented for the swordfish in this study appears to increase its gill surface area above that of other billfishes and may allow it to penetrate oxygen‐poor waters at depth. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

Endothermy in fishes: a phylogenetic analysis of constraints,predispositions, and selection pressures

Synopsis Endothermy, the ability to raise body temperature by internal heat production, is unusual in teleost fishes and has only been documented within one suborder, the Scombroidei. Two separate modes of endothermy have evolved in the scombroidei; tunas warm their muscles, brain and viscera using heat exchangers in the circulation to these metabolically active tissues while billfishes and one primitive mackerel have a thermogenic organ situated beneath the brain. Both modes of endothermy emphasize common themes. Large body size coupled with heat exchangers are necessary to reduce convective and conductive heat exchange. A tissue with a high oxidative capacity is required for heat generation. Studies based upon morphology and mitochondrial DNA analyses indicate that endothermy has evolved independently at least three times within the scombroid lineage. Mapping of-morphological and physiological traits on a molecular phylogeny for scombroids provides evidence of selective pressures favoring evolution of diverse endothermic styles. The new results suggest anatomical constraints prevent most fish from using the tuna form of endothermy and indicate a possible linkage between endothermy and locomotory style (thunniform or sub-carangiform).Paper from the International Union of Biological Societies symposium The biology of tunas and billfishes: an examination of life on the knife edge, organized by Richard W. Brill and Kim N. Holland.     

Relationships between muscle mitochondrial DNA content, mitochondrial enzyme activity and oxidative capacity in man: alterations with disease

Muscle mitochondrial content is tightly regulated, and requires the expression of both nuclear and mitochondrial genes. In addition, muscle mitochondrial content is a major determinant of aerobic exercise capacity in healthy subjects. The current study was designed to test the hypothesis that in healthy humans, muscle mitochondrial DNA (mtDNA) content is correlated with citrate synthase activity (a representative nuclear-encoded mitochondrial enzyme) and aerobic exercise capacity as defined by whole-body peak oxygen consumption (VO2). Furthermore, it was postulated that these relationships might be altered with disease. Twelve healthy and five paraplegic subjects underwent exercise testing and vastus lateralis muscle biopsy sampling. An additional ten healthy subjects and eight patients with unilateral peripheral arterial disease (PAD) underwent exercise testing and gastrocnemius muscle biopsy sampling. Citrate synthase activity and mtDNA content were positively correlated in the vastus lateralis muscles from the healthy subjects. This relationship was similar in muscle from paraplegic subjects. mtDNA content was positively correlated with peak VO2 in the healthy subjects and in the paraplegic subjects in whom peak VO2 had been elicited by functional electrical stimulation of the muscle. In contrast, the PAD subjects demonstrated higher mtDNA contents than would have been predicted based on their claudication-limited peak VO2. Thus, in healthy humans there are strong relationships between muscle mtDNA content and both muscle citrate synthase activity and peak VO2. These relationships are consistent with coordinant nuclear DNA and mtDNA expression, and with mitochondrial content being a determinant of aerobic exercise capacity. The relationships seen in healthy humans are quantitatively similar in paraplegic patients, but not in patients with PAD, a disease which is associated with a metabolic myopathy. The relationships between mtDNA content, mitochondrial enzyme activities and exercise capacity provide insight into the physiologic and pathophysiologic regulation of muscle mitochondrial expression.     

Basic concepts relevant to heat transfer in fishes,and their use in measuring the physiological thermoregulatory abilities of tunas

Synopsis Aerobic heat production and heat loss via the gills are inexorably linked in all water breathing teleosts except tunas. These processes are decoupled in tunas by the presence of vascular counter-current heat exchangers, and sustained (i.e., steady state) muscle temperatures may exceed water temperature by 10° C or more in larger individuals. The presence of vascular counter-current heat exchangers is not clearly advantageous in all situations, however. Mathematical models predict that tunas could overheat during strenuous activity unless the efficacy of vascular heat exchangers can be reduced, and that they may be activity limited in warmer waters. Tunas may likewise be forced out of potentially usable habitats as they grow because they have to occupy cooler waters. Vascular counter-current heat exchangers also slow rates of heating and cooling. A reduced rate of muscle temperature decrease is clearly advantageous when diving into colder water to chase prey or avoid predators. A reduced rate of heat gain from the environment would be disadvantageous, however, when fish return to the warmer surface waters. When subjected to changes in ambient temperature, tunas cannot defend a specific body temperature and do not thermoregulate in the mammalian sense. Yet when appropriately analyzed, data taken under steady state and non-steady state conditions indicate that tunas are not strictly prisoners of their own thermoconserving mechanisms. They apparently can modify overall efficiency of their vascular counter-current heat exchangers and thus avoid overheating during bouts of strenuous activity, retard cooling after diving into colder water, and rapidly warm their muscles after voluntarily entering warmer water. The exact physiological mechanisms employed remain to be elucidated.Paper from the International Union of Biological Societies symposium The biology of tunas and billfishes: an examination of life on the knife edge, organized by Richard W. Brill and Kim N. Holland.     

Free imidazole compounds in white and dark muscles of migratory marine fish

1. Extractive nitrogenous components were analyzed in specimens of white and dark muscles of three tunas and the Pacific saury. 2. Tunas contained high concentrations of histidine, anserine and creatine in white muscle, and of taurine, anserine and creatine in dark muscle. 3. Imidazole compounds were determined in both muscles of 11 migratory marine fish. Histidine was found in great quantities in all species except swordfish, anserine was found in relatively large amounts in tunas and swordfish, but carnosine was only present in small amounts in yellowfin and skipjack tunas. 4. The dark muscle had a lower content of imidazole compounds than white muscle.     

Fiber-type differences in muscle mitochondrial profiles

Although striated muscles differ in mitochondrial content, the extent of fiber-type specific mitochondrial specializations is not well known. To address this issue, we compared mitochondrial structural and functional properties in red muscle (RM), white muscle (WM), and cardiac muscle of rainbow trout. Overall preservation of the basic relationships between oxidative phosphorylation complexes among fiber types was confirmed by kinetic analyses, immunoblotting of native holoproteins, and spectroscopic measurements of cytochrome content. Fiber-type differences in mitochondrial properties were apparent when parameters were expressed per milligram mitochondrial protein. However, the differences diminished when expressed relative to cytochrome oxidase (COX), possibly a more meaningful denominator than mitochondrial protein. Expressed relative to COX, there were no differences in oxidative phosphorylation enzyme activities, pyruvate-based respiratory rates, H2O2 production, or state 4 proton leak respiration. These data suggest most mitochondrial qualitative properties are conserved across fiber types. However, there remained modest differences ( approximately 50%) in stoichiometries of selected enzymes of the Krebs cycle, beta-oxidation, and antioxidant enzymes. There were clear differences in membrane fluidity (RM > cardiac, WM) and proton conductance (H+/min/mV/U COX: WM > RM > cardiac). The pronounced differences in mitochondrial content between fiber types could be attributed to a combination of differences in myonuclear domain and modest effects on the expression of nuclear- and mitochondrially encoded respiratory genes. Collectively, these studies suggest constitutive pathways that transcend fiber types are primarily responsible for determining most quantitative and qualitative properties of mitochondria.     

Metabolic enzyme activity patterns in muscle biopsy samples in different athletes

Citrate synthase (CS) and aldolase (ALD) activities and muscle fiber composition were compared in the muscles of high jumpers, sprinters, race walkers, middle distance runners and untrained men. Muscle biopsy samples were taken from vastus lateralis (VL) and gastrocnemius (G) in each group. Oxidative enzyme activity (CS, IU X g-1 ww) was highest (24.64 and 15.0 in G and VL, respectively) in endurance-trained top race walkers, followed in order by the middle distance runners (G: 17.28, VL: 12.29), untrained controls (G: 11.17, VL:8.10) and the high jumpers (G: 11.51, VL: 8.89). All athletes performing intense endurance exercise with the leg musculature displayed 30 to 60% higher CS activity and 20 to 40% higher ST% in G than in VL. Glycolytic enzyme activity (ALD approximately 28 IU X g-1 ww) was highest in both muscles in the sprinters, followed by the high jumpers (23 IU X g-1 ww). Novice runners had 30 to 50% lower ALD and CS activity than experienced sportsmen. The differences arise not only from age, but also from the periods of regular exercise and adaptation to training in elite sportsmen. It was concluded that the more intensive the sporting activity of a muscle, the higher its enzyme activity (as with oxidative or glycolytic metabolism). The correlations between fiber composition and enzyme activities differed in VL and G in the same sportsmen. Thus, the degree of adaptation due to training also differed.     

Temporal and spatial patterns of gene expression in skeletal muscles in response to swim training in adult zebrafish (Danio rerio)

In adult zebrafish, 4 weeks of exercise training is known to induce an increase in mitochondrial enzymes such as citrate synthase (CS) when determined in mixed (red and white) muscle. However, this remodeling is not accompanied by changes in PGC-1α mRNA, a potent inducer of mitochondrial biogenesis in mammals. To further understand this response, we examined absolute and relative changes in red muscle area by histochemistry after 4 weeks of swim training. We also examined fiber-type specific responses in the expression of metabolic genes and putative regulators in red and white muscle of adult zebrafish at 1 and 8 weeks of training and in recovery from a single bout of exercise. Total red muscle area was unaltered after 4 weeks of training. The mRNA expression of CS was unaffected in red muscle, while it was increased in white muscle after 1 week of training and remained elevated at 8 weeks of training, suggesting an increase in oxidative capacity of this fiber type. In contrast, PGC-1α mRNA was elevated in both muscles only after 1 week of training. In both muscles, an acute bout of exercise rapidly (within 0–2 h post-exercise) induced PGC-1α mRNA and a delayed (24 h) increase in CS mRNA post-exercise. These results suggest complex temporal and spatial adaptive molecular responses to exercise in the skeletal muscles of zebrafish.     

Patterning of fast and slow fibers within embryonic muscles is established independently of signals from the surrounding mesenchyme

During embryonic development, and before functional innervation, a highly stereotypic pattern of slow- and fast-contracting primary muscle fibers is established within individual muscles of the limbs, from distinct populations of myoblasts. A difference between the fiber-type pattern found within chicken and quail pectoral muscles was exploited to investigate the contributions of somite-derived myogenic precursors and lateral plate-derived mesenchymal stroma to the establishment of muscle fiber-type patterns. Chimeric chicken/quail embryos were constructed by reciprocal transplantation of somites or lateral plate mesoderm at stages prior to muscle formation. Muscle fibers derived from quail myogenic precursors that had migrated into chicken stroma showed a quail pattern of mixed fast- and slow-contracting muscle fibers. Conversely, chicken myogenic precursors that had migrated into quail stroma showed a chicken pattern of nearly exclusive fast muscle fiber formation. These results demonstrate in vivo an intrinsic commitment to fiber-type on the part of the myoblast, independent of extrinsic signals it receives from the mesenchymal stroma in which it differentiates.     

Selective advantages conferred by the high performance physiology of tunas,billfishes, and dolphin fish

Tunas are extensively distributed throughout world's oceans and grow and reproduce fast enough to support one of the world's largest commercial fisheries. Yet they are apex predators living in the energy depauperate pelagic environment. It is often presumed that tunas evolved their specialized anatomy, physiology, and biochemistry to be capable of (a) high maximum swimming speeds, (b) high sustained swimming speeds, and/or (c) very efficient swimming, all of which help account for their wide distribution and reproductive success. However, a growing body of data on the energetics and physiological abilities of tunas do not support these assumptions. The three things demonstratively “high performance” about tunas, and probably other pelagic species such as marlin (Makaira spp. and Tetrapturus spp.) and dolphin fish (Coryphaena spp.), are (a) rates of somatic and gonadal growth, (b) rates of digestion, (c) rates of recovery from exhaustive exercise (i.e., clearance of muscle lactate and the concomitant acid load). All of these are energy consuming processes requiring rates of oxygen and substrate delivery above those needed by the swimming muscles for sustained propulsion and for other routine metabolic activities. I hypothesize that the ability of high performance pelagic species (tunas, billfishes, and dolphin fish) to deliver oxygen and metabolic substrates to the tissues at high rates evolved to permit rapid somatic and gonadal growth, rapid digestion, and rapid recovery from exhaustive exercise (abilities central to success in the pelagic environment), not exceptionally high sustained swimming speeds.     

Phosphocreatine content of freeze-clamped muscle: influence of creatine kinase inhibition.

The study of cellular energetics is critically dependent on accurate measurement of high-energy phosphates. Muscle values of phosphocreatine (PCr) vary greatly between in vivo measurements (i.e., by nuclear magnetic resonance) and chemical measurements determined from muscles isolated and quick-frozen. The source of this difference has not been experimentally identified. A likely cause is activation of ATPases and phosphotransfer from PCr to ADP. Therefore, rat hindlimb skeletal muscle was perfused either with or without 2 mM iodoacetamide, a creatine kinase inhibitor, and muscle was freeze-clamped either at rest or after contraction. Creatine kinase inhibition resulted in approximately 6 micromol/g higher PCr and lower creatine in the freeze-clamped soleus, red gastrocnemius, and white gastrocnemius. This PCr content difference was reduced when the initial PCr content was decreased with prior contractions. Therefore, the amount of PCr artifact appears to scale with initial PCr content within a fiber-type section. This artifact directly affects the measurement and, thus, the calculations of muscle energetic parameters from studies using isolated and frozen muscle.     

Molecular Evolution of Cytochrome c Oxidase in High-Performance Fish (Teleostei: Scombroidei)

The 13 peptides encoded by vertebrate mitochondrial DNA (mtDNA) are essential subunits of oxidative phosphorylation (OXPHOS) enzymes. These genes normally experience purifying selection and also coevolve with nuclear-encoded subunits of OXPHOS complexes. However, the role of positive selection on mtDNA evolution is still unclear, as most examples of intergenomic coevolution appear to be the result of compensation by nuclear-encoded genes for mildly deleterious mtDNA mutations, and not simultaneous positive selection in both genomes. Organisms that have experienced strong selective pressures to increase aerobic capacity or adapt to changes in thermal environment may be better candidates in which to examine the impact of positively selected changes on mtDNA evolution. The tuna (suborder Scombroidei, family Scombridae) and billfish (suborder Scombroidei, families Xiphiidae and Istiophoridae) are highly aerobic fish with multiple specializations in muscle energetics, including a high mitochondrial content and regional endothermy. We examined the role of positively selected mtDNA substitutions in the production of these unique phenotypes. Focusing on a catalytic subunit of cytochrome c oxidase (COX II), we found that the rate ratio of nonsynonymous (d(N); amino acid changing)-to-synonymous (d(S); silent) substitutions was not increased in lineages leading to the tuna but was significantly increased in the lineage preceding the billfish. Furthermore, there are a number of individual positively selected sites that, when mapped onto the COX crystal structure, appear to interact with other COX subunits and may affect OXPHOS function and regulation in billfish.     

Human mitochondrial function during cardiac growth and development

Little information is presently available concerning mitochondrial respiratory and oxidative phosphorylation function in the normal human heart during growth and development. We investigated the levels of specific mitochondrial enzyme activities and content during cardiac growth and development from the early neonatal period (10-20 days) to adulthood (67 years). Biochemical analysis of enzyme specific activities and content and mitochondrial DNA (mtDNA) copy number was performed with left ventricular tissues derived from 30 control individuals. The levels of cytochrome c oxidase (COX) and complex V specific activity, mtDNA copy number and COX subunit II content remained unchanged in contrast to increased citrate synthase (CS) activity and content. The developmental increase in CS activity paralleled increasing CS polypeptide content, but was neither related to overall increases in mitochondrial number nor coordinately regulated with mitochondrial respiratory enzyme activities. Our findings of unchanged levels of cardiac mitochondrial respiratory enzyme activity during the progression from early childhood to older adult contrasts with the age-specific regulation found with CS, a Krebs cycle mitochondrial enzyme.     

Evolution and consequences of endothermy in fishes

Regional endothermy, the conservation of metabolic heat by vascular countercurrent heat exchangers to elevate the temperature of the slow-twitch locomotor muscle, eyes and brain, or viscera, has evolved independently among several fish lineages, including lamnid sharks, billfishes, and tunas. All are large, active, pelagic species with high energy demands that undertake long-distance migrations and move vertically within the water column, thereby encountering a range of water temperatures. After summarizing the occurrence of endothermy among fishes, the evidence for two hypothesized advantages of endothermy in fishes, thermal niche expansion and enhancement of aerobic swimming performance, is analyzed using phylogenetic comparisons between endothermic fishes and their ectothermic relatives. Thermal niche expansion is supported by mapping endothermic characters onto phylogenies and by combining information about the thermal niche of extant species, the fossil record, and paleoceanographic conditions during the time that endothermic fishes radiated. However, it is difficult to show that endothermy was required for niche expansion, and adaptations other than endothermy are necessary for repeated diving below the thermocline. Although the convergent evolution of the ability to elevate slow-twitch, oxidative locomotor muscle temperatures suggests a selective advantage for that trait, comparisons of tunas and their ectothermic sister species (mackerels and bonitos) provide no direct support of the hypothesis that endothermy results in increased aerobic swimming speeds, slow-oxidative muscle power, or energetic efficiency. Endothermy is associated with higher standard metabolic rates, which may result from high aerobic capacities required by these high-performance fishes to conduct many aerobic activities simultaneously. A high standard metabolic rate indicates that the benefits of endothermy may be offset by significant energetic costs.