Robert G. Boutilier

Ontogeny of Feeding and Respiration in Larval Atlantic Cod Gadus morhua (Teleostei, Gadiformes): II. Function

Cranial development in larval Atlantic cod Gadus morhua was studied throughout ontogeny using specimens treated by staining and clearing, scanning electron microscopy and histology. Newly hatched cod larvae have closed mouths, no operculii, five well‐developed branchial arches, and transversii ventralis muscles. During the endogenous feeding (yolk‐sac) stage, viscerocranial structures remain simple and nonarticulated. Six days after hatching at 5°C, articulation occurs between the quadrate/Meckels cartilage and the hyomandibula/cranium. Integration of skeletal elements results in a functional jaw that facilitates the transition from endogenous to exogenous feeding. During later ontogenetic stages, the opercular apparatus and levator‐operculi coupling develops, facilitating the transition of cutaneous to branchial respiration. Overall, feeding and respiratory needs are met by changes in form (including composition) and function during larval fish growth and are correlated with demands of energy acquisition essential to survival.

The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout : possible co-evolution with erythrocyte chloride/bicarbonate exchange

The subcellular distribution and kinetic properties of carbonic anhydrase were examined in red blood cells and gills of the lamprey, Petromyzon marinus, a primitive agnathan, and rainbow trout, Oncorhynchus mykiss, a modern teleost, in relation to the evolution of rapid Cl−/HCO3−exchange in the membrane of red blood cells. In the lamprey, which either lacks or has minimal red cell Cl−/HCO3−exchange, there has been no compensatory incorporation of carbonic anhydrase into the membrane fraction of either the red cell or the gill. Carbonic anhydrase activity in red cells is exclusively cytoplasmic, and the single isozyme displays kinetic properties typical of the type I, slow turnover, isozyme. In the red blood cells of the trout, however, which possess high amounts of the band-3 Cl−/HCO3−exchange protein, the single carbonic anhydrase isozyme appears to be kinetically similar to the type II, fast turnover, isozyme. It thus appears that the type I isozyme present in the red blood cells of primitive aquatic vertebrates was replaced in modern teleosts by the kinetically more efficient type II isozyme only after the incorporation and expression of a significant amount of the band-3 exchange protein in the membrane of the red cell.

Activity and metabolism of larval Atlantic cod (Gadus morhua) from Scotian Shelf and Newfoundland source populations

Patterns of activity and metabolism were investigated in larval Atlantic cod (Gadus morhua L.) between December 1991 and July 1992: (1) throughout larval development; (2) between two genetically discrete populations (Scotian Shelf and Newfoundland) and (3) as a function of two different culture temperatures. During the yolk-sac stage (0 to 5 d post-hatch), changes in swimming speed were not related to mass-specific metabolic rates; no portion of the mass-specific oxygen consumption could be explained by changes in activity. In the “mixed feeding” stage (6 to 14 d posthatch), there was a tendency for oxygen consumption to be related to changes in swimming speed. In the “exogenous feeding” stage (>14 d post-hatch), oxygen consumption significantly increased with swimming speed. These ontogenetic patterns of activity and metabolism were the same for larvae from the Scotian Shelf and Newfoundland populations. However, over the entire larval life and among ontogenetic stages, the metabolic cost of activity (Δmass-specific O2 consumption/Δswimming speed) of Scotian Shelf larvae was significantly higher than that of Newfoundland larvae. When cod larvae, that had developed at 5°C, were acutely exposed to 10°C, Scotian Shelf larvae had a higher intrinsic cost of activity than Newfoundland larvae, over the entire larval life. During the exogenous feeding stage, the mean metabolic cost of activity for Newfoundland larvae raised at 10°C and tested at 10°C was significantly higher and more variable than that of larvae raised at lower temperatures. However, the metabolic cost of activity of larvae raised and tested at 10°C was not significantly different between source populations. Together these findings suggest that differences in swimming energetics reflect changing energy requirements for activity among ontogenetic stages, and reflect adaptation to regional environments among genetically discrete populations.

Mechanisms of metabolic defense against hypoxia in hibernating frogs.

The cold submerged frog (Rana temporaria) serves as a useful model for many hibernating ectotherms that take refuge in hypoxic ponds and lakes until more favourable conditions of climate and food availability return. In all such animals, entry into a hypometabolic state effectively extends their survival time by lessening the impact of ATP demands on endogenous substrates. At the cellular level, metabolic depression may be brought about by decreasing energy-consuming processes and/or by increasing the efficiency of energy-producing pathways. Since the mitochondrion is the major contributor to the total energy production during aerobic metabolism and frog survival during winter depends on entry into a hypometabolic state, this review focuses on the respiratory properties of mitochondria that serve to increase the efficiency of energy production in hibernation. Energy conservation during overwintering also occurs through decreases in the ATP demand of the energy-consuming processes. For example, hibernating frogs decrease their ATP demands for Na(+)/K(+)-ATPase activity as part of a coordinated process of energy conservation wherein O(2)-limitation initiates a generalised suppression of ion channel densities and/or channel leak activities. The net result is that cell membrane permeabilities are reduced, thereby lowering the energetic costs of maintaining transmembrane ion gradients.

Gas exchange and heart rate in the harbour porpoise, Phocoena phocoena

Abstract The respiratory physiology, heart rates and metabolic rates of two captive juvenile male harbour porpoises (both 28 kg) were measured using a rapid-response respiratory gas analysis system in the laboratory. Breath-hold durations in the laboratory (12 ± 0.3 s, mean ± SEM) were shorter than field observations, although a few breath-holds of over 40 s were recorded. The mean percentage time spent submerged was 89 ± 0.4%. Relative to similarly-sized terrestrial mammals, the respiratory frequency was low (4.9 ± 0.19 breaths · min−1) but with high tidal volumes (1.1 ± 0.01 l), enabling a comparatively high minute rate of gas exchange. Oxygen consumption under these experimental conditions (247 ± 13.8 ml O2 · min−1) was 1.9-fold higher than predicted by standard scaling relations. These data together with an estimate of the total oxygen stores predicted an aerobic dive limit of 5.4 min. The peak end-tidal O2 values were related to the length of the previous breath-hold, demonstrating the increased oxygen uptake from the lung for the longer dives. Blood oxygen capacity was 23.5 ± 1.0 ml · 100 ml−1, and the oxygen affinity was high, enabling rapid oxygen loading during ventilation.

Metabolic suppression in anoxic frog muscle

Abstract Microcalorimetry is the only direct method for measuring moment-to-moment changes in whole-cell metabolism (as heat output) during anoxia. We have adapted this methodology, in conjunction with standard muscle isolation techniques, to monitor metabolic transitions in isolated frog (Rana temporaria) sartorius muscle during anoxia and recovery (reoxygenation). Anoxia (sustained 1 h, following 2 h progressive hypoxia) suppressed muscle heat output to 20% of the stable normoxic level. This effect was fully reversible upon reoxygenation. Metabolite profiles were consistent with other anoxia-tolerant vertebrates – most notably, adenosine triphosphate (ATP) content during anoxia and reoxygenation remained unchanged from normoxia (pre-anoxic control). In addition, the concentration of K+ ions ([K+]) in interstitial dialysates remained stable (2–3 mM) throughout anoxia and recovery. Interstitial [lactate−] increased slightly, in accord with anaerobiosis supporting suppressed metabolic rates during anoxia. The degree of anoxic suppression of metabolism observed is similar to other vertebrate models of anoxia tolerance. Furthermore, stable ATP concentrations and interstitial [K+] in the isolated tissue suggests that intrinsic mechanisms suppress metabolism in a manner that coordinates ATP supply and demand and avoids the severe ion imbalances that are characteristic of hypoxia-sensitive systems.

Effects of carbonic anhydrase inhibition on the acid base status in lamprey and trout

Inhibition of red cell carbonic anhydrase (CA) activity resulted in the rapid development of a respiratory acidosis (0.25 pH depression within 15 min post-injection) in the blood of trout. In the lamprey, however, the onset of the respiratory acidosis was delayed and its magnitude was less (0.18 pH depression at 6 h post-injection). Erythrocyte pH of both species decreased by about 0.12 units by 1 h after CA inhibition. These data, combined with the lack of rapid anion (Cl-/HCO3-) exchange in the red cells of agnathans but not in other lower vertebrates, support the hypotheses that (1) the majority of total CO2 in lamprey is transported within the erythrocyte, and (2) the limiting step in the evolution of a functioning Jacobs-Stewart cycle, and thus the evolution of the common mechanism of systemic CO2 transport in vertebrate blood, was the incorporation of the band-3 anion exchange protein into the membrane of the red cell.

Mitochondrial Proton Conductance, Standard Metabolic Rate and Metabolic Depression

Proton cycling across the mitochondrial inner membrane makes up a significant proportion (20–30%) of Standard Metabolic Rate (SMR) in rats. If proton cycling is equally important in other animals, those that metabolically depress to 25% or less of SMR have a problem: either their entire energy budget will be wasted by proton cycling, or they have to suppress the leak of protons across the mitochondrial membrane. Muscle mitochondria from metabolically depressed, hypoxic overwintering frogs (Rana temporaria) do have decreased proton leak rate. This is achieved not by decreasing the proton conductance of the membrane, but by lowering the protonmotive force (the driving force for the leak). Protonmotive force is lowered aerobically by restricting electron supply, and in anoxia by restricting mitochondrial ATPase activity. There is also a temperature component to the physiological depression of overwintering frogs. The proton conductance of frog muscle mitochondria decreases steeply with temperature. Frog hepatocytes also respond strongly to temperature, and decrease their proton cycling in parallel to other reactions, so preserving metabolic efficiency at different temperatures. Hepatopancreas cells from the land snail (Helix aspersa) provide a good new model system to study biochemical mechanisms of depression without the complications of temperature change. Cells from aestivating animals show a persistent metabolic depression to 30% of controls, partly through intrinsic effects and partly through the extrinsic effects of pH and pO2. In depressed cells, proton cycling decreases at least as much as cellular respiration rate. These results using frogs and snails show that mitochondrial proton cycling is strongly suppressed in metabolic depression, so that metabolic efficiency is maintained or even enhanced.

The protective effects of hypoxia-induced hypometabolism in the Nautilus

Abstract Specimens of Nautilus pompilius were trapped at depths of 225–300 m off the sunken barrier reef south-east of Port Moresby, Papua New Guinea. Animals transported to the Motupore Island laboratory were acclimated to normal habitat temperatures of 18 °C and then cannulated for arterial and venous blood sampling. When animals were forced to undergo a period of progressive hypoxia eventually to encounter ambient partial pressure of oxygen (PO2) levels of ∼10 mmHg (and corresponding arterial PO2s of ∼5 mmHg), they responded by lowering their aerobic metabolic rates to 5–10% of those seen in resting normoxic animals. Coincident with this profound metabolic suppression was an overall decrease in activity, with brief periods of jet propulsion punctuating long periods of rest. Below ambient PO2 levels of 30–40 mmHg, ventilatory movements became highly periodic and at the lowest PO2 levels encountered, ventilation occasionally ceased altogether. Cardiac output estimated by the Fick equation decreased during progressive hypoxia by as much as 75–80%, and in the deepest hypometabolic states heart rates slowed to one to two cycles of very low amplitude per minute. By the end of 500 min exposure to ambient PO2 levels of 10 mmHg or less, the anaerobic end products octopine and succinate had increased significantly in adductor muscle and heart, respectively. Increased concentrations of octopine in adductor muscle apparently contributed to a small intracellular acidosis and to the development of a combined respiratory and metabolic acidosis in the extracellular compartment. On the other hand, increases in succinate in heart muscle occurred in the absence of any change in cardiac pHi. Taken together, we estimate that these anaerobic end products would make up less than 2% of the energy deficit arising from the decrease in aerobic metabolism. Thus, metabolic suppression is combined with a massive downregulation of systemic O2 delivery to match metabolic supply to demand.

Constant set points for pH and PCO2 in cold-submerged skin-breathing frogs

The low temperatures encountered by overwintering frogs result in a large downregulation of metabolism and behaviour. However, little is known about acid-base regulation in the extreme cold, especially when frogs become exclusive skin-breathers during their winter submergence. Blood and muscle tissue acid-base parameters (pH, P(CO2), bicarbonate and lactic acid concentrations) were determined in submerged frogs exposed to a range of low temperatures (0.2-7 degrees C). At overwintering temperatures between T = 0.2 and 4 degrees C plasma pH and P(CO2) were maintained constant, whereas intracellular pH regulation resulted in larger pH-temperature slopes occurring in the presumably more active heart muscle (deltapH/deltaT = -0.0313) than in the gastrocnemius muscle (deltapH/deltaT = -0.00799). Although blood pH was not significantly affected by submergence between 0.2 and 4 degrees C (pH = 8.220-8.253), it declined in the 7 degrees C frogs (pH = 8.086), a decrease not linked to the recruitment of anaerobiosis. Plasma P(CO2) and pH in the cold appear to be regulated at constant levels, implying that cutaneous CO2 conductance in submerged frogs is adjusted within the range of overwintering temperatures. This is likely geared toward facilitating the uptake of oxygen under conditions of greater metabolic demand, however there remains the possibility that acid-base balance itself is maintained at a constant set point at the frogs natural overwintering temperatures.