- Email: [email protected]
A3—Tribute to Graham Shelton (1930–2004)
Comparative Biochemistry and Physiology, Part A 143 (2006) S65 – S67 www.elsevier.com/locate/cbpa
Society for Experimental Biology Annual Main Meeting 2nd–7th April 2006, University of Kent at Canterbury, UK
A3–TRIBUTE TO GRAHAM SHELTON (1930–2004) Organised by D. Jones (University of British Columbia)
A3.1 Responses of fish to hypoxia
of FFA in the cytoplasm. It is interesting to note that the liver-type fatty acid-binding protein mRNA is down-regulated after 4 days of hypoxia, suggesting an increase in cytosolic FFA during hypoxia.
D. Randall, (City University of Hong Kong) Fish are subjected to hypoxia on a regular basis for both short and long periods. Initial reponses attempt to maintain oxygen supply in the face of decreased availability. Later responses are aimed at decreasing oxygen utilization and maintain energy supplies through aerobic metabolism. Feeding and reproduction are reduced and fish move to lower temperatures. A number of metabolic changes, many directed by hypoxia inducing factors (HIF), enable the fish to maintain function at a lower rate of energy expenditure.
A3.2 Mitochondrial uncoupling in fish T. Tsui, (McMaster University); C. Hung, (University of Guelph); J. Lai, R. Wu, D. Randall, (City University of Hong Kong) Hypoxia has been reported to cause increased oxidative damage in animals. Mitochondria is one of the main sources of reactive oxidative species (ROS). It is known that mitochondrial membrane potential is positively correlated to the rate of ROS production. Therefore, the regulatory mechanisms for mitochondrial membrane potential during hypoxia are of great interest. A mechanism of regulating mitochondrial membrane potential is via the mitochondrial uncoupling proteins (UCPs). UCPs act as a proton translocator to increase the mitochondrial basal proton leak and, thus, decrease the membrane potential. Using microarray for common carp, mRNA levels of UCP1 and UCP2 in the common carp liver were found to be up-regulated after 4 days of hypoxia. The effects of various inhibitors and activators of UCPs on the mitochondrial basal proton leak have been investigated. It was found that free fatty acids (FFA) increased the mitochondrial basal proton leak. The FFA-basal proton leak is thought to be partially mediated via UCPs. The FFA-induced proton leak was insensitive to GDP and ATP, which are inhibitor of mammalian UCPs. This might be an adaptation to prevent damage caused by sudden re-oxygenation. It was also found that superoxide did not cause induction of mitochondrial proton leak of common carp. It appears that the mitochondrial membrane potential of common carp is regulated merely by the level
doi:10.1016/j.cbpa.2006.01.038
A3.3 Varanid cardiac dynamics: ‘‘The balloon goes up now!’’ D. Jones, W. Milsom, (University of British Columbia); G. Shelton, (University of East Anglia (deceased)) We attempted to establish the degree to which physiologically separate pathways for pulmonary and systemic blood exist in the varanid heart (Varanus salvator) by independently manipulating flow rates in and out of the right and left side of the heart. We found no evidence that in systole contact between the muscular ridge and Bulbuslamelle broke down even under gross changes in pressure and flow. Unfortunately, it was more difficult to produce convincing evidence for separation of the blood streams entering the ventricular chamber by the atrio-ventricular valves during diastole. Deflation of a balloon occluding systemic venous return however resulted in restoration of pulmonary pressure and flow at least two heart beats before equivalent events in the systemic circuit, suggesting that blood from the vena cava selectively flows to the pulmonary artery and reaches the systemic system only after going through the lungs. The effects on cardiac function of separating the heart at different locations in diastole and systole will be discussed.
A3.4 Anoxic survival in Helix aspersa M. Braun, C. Schwiening, R. Boutilier, (University of Cambridge) Direct calorimetry was used to assess the metabolic responses to anoxia in the pulmonate snail, Helix aspersa. During anoxia, heat produced by the snails fell as low as 2% of normal and varied with oxygen levels during long (>72 hrs) and short (5 min) anoxic bouts. This oxyconformation occurred at PO2 values well in excess of the critical PO2 at which oxygen diffusion to the mitochondria is limited. Examinations of the brain and neurons show that these tissues also participate in the metabolic depression. Electrophysiological recordings were made of action potential firing rate and whole cell impedance
S66
Abstracts / Comparative Biochemistry and Physiology, Part A 143 (2006) S65 – S67
and it was found that the costs of ion pumping is limited by both decreasing action potential firing (spike arrest) and membrane leakiness (channel arrest). These processes allow maintenance of ionic gradients at a time when ion pumping has been slowed to conserve ATP. Calcium pumping, however, seems to be unaffected by the overall slowing of channel activity. The mitochondrial inhibitors myxothiazol and antimycin were used to examine whether the trigger for the anoxic response was mitochondrial inhibition or falling oxygen levels. During perfusion with myxothiazol, the impedance of the neurons decreased as ion channels opened when ATP levels fell. However, inhibition with antimycin resulted in responses very similar to that seen in anoxia. While the differential responses between anoxia and mitochondrial blockade suggest that snails possess an oxygen sensor, the differential responses between antimycin and myxothiazol suggest a location for this putative sensor in the bc-1 complex of the electron transport chain.
A3.5 A year in the life of barnacle geese: the relationship between body temperature, body composition and resting oxygen consumption
thought to behave in a way that reduces thermal stress and to be unable to tolerate large, abrupt changes in temperature. Consequently, it is believed that thermoclines of only a few degrees can constitute fairly impenetrable boundaries and that this may contribute to the segregation of cod populations in nature. Our studies of the movements and temperature experience of adult cod in the North Sea and further north using depth and temperature sensing electronic data storage tags indicate that the movements and distribution of individual animals are much less restricted by environmental temperature than laboratory studies suggest. Some individuals occupy shallow habitats in the southern North Sea where summer temperatures are consistently above 17 -C for several months, while others tagged at the same time move to deeper, cooler waters. Also, cod in the northeast Atlantic repeatedly experience abrupt temperature changes of up to 8 -C while moving through steep temperature gradients. We suggest that temperature may not be so critical in constraining the movements and distribution of adult cod and other environmental factors (e.g. prey distribution, suitable flow refuges etc.) may be equally, or even more, important.
A3.7 Physiological consequences of feeding in sharks
P. Butler, S. Portugal, J. Green, (University of Birmingham) Over a period spanning approximately 20 days and coinciding with the autumn migration, mean daily body temperature (Tb) of barnacle geese which breed on the island of Spitzbergen and overwinter in southern Scotland, fell by almost 4.5 -C. It was hypothesised that this controlled drop in body temperature (anapyrexia) was associated with a sparing of fat during migration and/or with the replacement of fat following migration. Preliminary observations on captive geese in Birmingham showed a similar, but smaller, reduction in Tb and changes in body mass which approximately coincided with the migratory periods of wild geese. The objective of the current study is to test the above hypothesis by recording and relating, throughout the year, body mass, body composition, body temperature and resting oxygen consumption of captive geese that have free access to food. While confirming the periods of change in body mass, by far the greater of the two is associated with moulting, which in wild birds precedes the autumn migration. There is a 21% decrease in body mass, but a 50% increase in absolute resting oxygen consumption. Current theories suggest that the loss in mass during moult is not the result of the moult process itself, but that it is an adaptation to regain flight faster with partially regrown feathers. The cause(s) of the large increase in oxygen consumption is unknown but could relate to any of the following: the cost of replacing the feathers, the catabolism of fat to accelerate mass loss, increased cost of thermoregulation.
A3.6 Cod in a sweat: from physiology to behaviour and back again J. Metcalfe, (CEFAS) Atlantic cod (Gadus morhua) are found in cooler waters over a wide area on the continental shelves of the northern Atlantic. Laboratory studies indicate that their thermal preferendum is in the range 11 – 14 -C, their metabolic scope is maximised in the temperature range 13 – 15 -C and that food-unlimited growth is optimal between about 6 – 14 -C. Although individuals can adapt to changes in temperatures, they are
C. Wood, M. Kajimura, (McMaster University); P. Walsh, (University of Miami); T. Mommsen, (University of Victoria) Sharks have a high N-demand because of their urea-N based osmoregulation, but in the wild they feed opportunistically and irregularly, sometimes consuming large meals (up to 10% body weight) in a single bout. We have examined the physiological consequences of feeding and starvation in the spiny dogfish Squalus acanthias using both natural (feeding frenzies) and experimental models (feeding via stomach intubation). Feeding (with teleost tissue) stimulates only a modest loss of N, mainly as ammonia-N and unknown-N; there is no change in urea-N excretion. N – retention is facilitated by an activation of the ornithine-urea cycle, mainly in skeletal muscle, and blood urea-N levels rise. The rectal gland is metabolically activated. Blood ketones and glucose fall after feeding and rise progressively during starvation. Despite a marked metabolic alkalosis (‘‘alkaline tide’’) in the systemic bloodstream indicative of stimulated acid-secretion, stomach pH (<2.0 in starved animals) rises to 3.0 – 4.0 after feeding and then slowly declines as digestion proceeds. The mass of the stomach and intestinal walls increase in sequence as the chyme proceeds down the tract. Despite complex ionic changes, chyme osmolality remains close to plasma levels. During the post-prandial period, both the volume and pH (6.5) of chyme in the intestine remains constant, while the bolus in the stomach is progressively reduced; absorption is complete by 5 days. During prolonged starvation (35 days), urea-N excretion is not reduced, but plasma urea-N levels and osmolality are well-maintained at the expense of body condition factor (NSERC Discovery, CRC program, NSF, Japan Fellowship).
A3.8 Matching supply and demand: integrating energetics from the genome to the organism J. Richards, (The University of British Columbia) The key biochemical strategy for hypoxia survival is the ability to maintain metabolic energy balance in the face of a reduced
Abstracts / Comparative Biochemistry and Physiology, Part A 143 (2006) S65 – S67
capacity for energy production. In hypoxia tolerant animals, the maintenance of energy balance is achieved through the activation of oxygen-independent pathways for ATP production and a controlled decrease in energy demand yielding whole body metabolic suppression. My laboratory studies the mechanisms and evolution of hypoxia tolerance in fish. During acclimation to moderate hypoxia, we hypothesize that changes in gene expression will act to optimize ATP production and limit ATP utilization. We chose pyruvate dehydrogenase kinase (PDK) as a candidate gene and examined its expression pattern in many species of intertidal sculpin and the common killifish (Fundulus heteroclitus), all hypoxia tolerant fish, during exposure to moderate hypoxia (0.5 to 0.8 mg O2/L). PDK inhibits pyruvate dehydrogenase (PDH) resulting in decreases in mitochondrial carbohydrate oxidation and lower oxygen demands. Two isoforms of PDK (PDK 2 and 2a) were identified in F. heteroclitus, each with a tissue specific distribution. Our data demonstrate a very rapid increase in PDK expression in F. heteroclitus in response to hypoxia, which coincides with decreases in PDH activity and initiation of metabolic suppression.
A3.9 Cardio-respiratory lessons from Graham Shelton ‘‘Be suspicious of straight lines’’ W. Burggren, (University of North Texas) Abstract not submitted.
S67
A3.10 Chronic hypoxia in zebrafish: an integrative approach to studying plasticity B. Bagatto, F. Moore, (University of Akron) Genetic and environmental variation are both known to influence development. Evolution of a developmental response that is optimized to the environment (adaptive plasticity) requires the existence of genetic variation for that developmental response. In complex traits composed of integrated sets of subsidiary traits, the adaptive process may be slowed by the existence of multiple possible integrated responses. This study tests for family (sibship) specific differences in plastic response to hypoxia in an integrated set of cardiovascular traits in zebrafish. Cardiac output, which is the integrated product of several subsidiary traits, varied highly significantly between families, and families differed significantly in the degree and direction of response to developmental oxygen level. The cardiac output response to oxygen environment was entirely family specific with no significant overall trend due to oxygen level. Constituent physiological variables that contribute to cardiac output all showed significant family specific response to hypoxia. Traits that were not directly related to cardiac output, such as arterial and venous diameter, and red blood cell velocities did not respond to hypoxia in a family specific manner. Zebrafish families vary in their plastic response to hypoxia. Genetic variation in plastic response to hypoxia may therefore provide the basic ingredient for adaptation to a variable environment. Considerable variation in the degree of familial response to hypoxia exists between different cardiovascular traits that may contribute to cardiac output. It is possible that the integration of several subsidiary traits into cardiac output allows the maintenance of genetic variance in cardiac response.
Society for Experimental Biology Annual Main Meeting 2nd–7th April 2006, University of Kent at Canterbury, UK
A3–TRIBUTE TO GRAHAM SHELTON (1930–2004) Organised by D. Jones (University of British Columbia)
A3.1 Responses of fish to hypoxia
of FFA in the cytoplasm. It is interesting to note that the liver-type fatty acid-binding protein mRNA is down-regulated after 4 days of hypoxia, suggesting an increase in cytosolic FFA during hypoxia.
D. Randall, (City University of Hong Kong) Fish are subjected to hypoxia on a regular basis for both short and long periods. Initial reponses attempt to maintain oxygen supply in the face of decreased availability. Later responses are aimed at decreasing oxygen utilization and maintain energy supplies through aerobic metabolism. Feeding and reproduction are reduced and fish move to lower temperatures. A number of metabolic changes, many directed by hypoxia inducing factors (HIF), enable the fish to maintain function at a lower rate of energy expenditure.
A3.2 Mitochondrial uncoupling in fish T. Tsui, (McMaster University); C. Hung, (University of Guelph); J. Lai, R. Wu, D. Randall, (City University of Hong Kong) Hypoxia has been reported to cause increased oxidative damage in animals. Mitochondria is one of the main sources of reactive oxidative species (ROS). It is known that mitochondrial membrane potential is positively correlated to the rate of ROS production. Therefore, the regulatory mechanisms for mitochondrial membrane potential during hypoxia are of great interest. A mechanism of regulating mitochondrial membrane potential is via the mitochondrial uncoupling proteins (UCPs). UCPs act as a proton translocator to increase the mitochondrial basal proton leak and, thus, decrease the membrane potential. Using microarray for common carp, mRNA levels of UCP1 and UCP2 in the common carp liver were found to be up-regulated after 4 days of hypoxia. The effects of various inhibitors and activators of UCPs on the mitochondrial basal proton leak have been investigated. It was found that free fatty acids (FFA) increased the mitochondrial basal proton leak. The FFA-basal proton leak is thought to be partially mediated via UCPs. The FFA-induced proton leak was insensitive to GDP and ATP, which are inhibitor of mammalian UCPs. This might be an adaptation to prevent damage caused by sudden re-oxygenation. It was also found that superoxide did not cause induction of mitochondrial proton leak of common carp. It appears that the mitochondrial membrane potential of common carp is regulated merely by the level
doi:10.1016/j.cbpa.2006.01.038
A3.3 Varanid cardiac dynamics: ‘‘The balloon goes up now!’’ D. Jones, W. Milsom, (University of British Columbia); G. Shelton, (University of East Anglia (deceased)) We attempted to establish the degree to which physiologically separate pathways for pulmonary and systemic blood exist in the varanid heart (Varanus salvator) by independently manipulating flow rates in and out of the right and left side of the heart. We found no evidence that in systole contact between the muscular ridge and Bulbuslamelle broke down even under gross changes in pressure and flow. Unfortunately, it was more difficult to produce convincing evidence for separation of the blood streams entering the ventricular chamber by the atrio-ventricular valves during diastole. Deflation of a balloon occluding systemic venous return however resulted in restoration of pulmonary pressure and flow at least two heart beats before equivalent events in the systemic circuit, suggesting that blood from the vena cava selectively flows to the pulmonary artery and reaches the systemic system only after going through the lungs. The effects on cardiac function of separating the heart at different locations in diastole and systole will be discussed.
A3.4 Anoxic survival in Helix aspersa M. Braun, C. Schwiening, R. Boutilier, (University of Cambridge) Direct calorimetry was used to assess the metabolic responses to anoxia in the pulmonate snail, Helix aspersa. During anoxia, heat produced by the snails fell as low as 2% of normal and varied with oxygen levels during long (>72 hrs) and short (5 min) anoxic bouts. This oxyconformation occurred at PO2 values well in excess of the critical PO2 at which oxygen diffusion to the mitochondria is limited. Examinations of the brain and neurons show that these tissues also participate in the metabolic depression. Electrophysiological recordings were made of action potential firing rate and whole cell impedance
S66
Abstracts / Comparative Biochemistry and Physiology, Part A 143 (2006) S65 – S67
and it was found that the costs of ion pumping is limited by both decreasing action potential firing (spike arrest) and membrane leakiness (channel arrest). These processes allow maintenance of ionic gradients at a time when ion pumping has been slowed to conserve ATP. Calcium pumping, however, seems to be unaffected by the overall slowing of channel activity. The mitochondrial inhibitors myxothiazol and antimycin were used to examine whether the trigger for the anoxic response was mitochondrial inhibition or falling oxygen levels. During perfusion with myxothiazol, the impedance of the neurons decreased as ion channels opened when ATP levels fell. However, inhibition with antimycin resulted in responses very similar to that seen in anoxia. While the differential responses between anoxia and mitochondrial blockade suggest that snails possess an oxygen sensor, the differential responses between antimycin and myxothiazol suggest a location for this putative sensor in the bc-1 complex of the electron transport chain.
A3.5 A year in the life of barnacle geese: the relationship between body temperature, body composition and resting oxygen consumption
thought to behave in a way that reduces thermal stress and to be unable to tolerate large, abrupt changes in temperature. Consequently, it is believed that thermoclines of only a few degrees can constitute fairly impenetrable boundaries and that this may contribute to the segregation of cod populations in nature. Our studies of the movements and temperature experience of adult cod in the North Sea and further north using depth and temperature sensing electronic data storage tags indicate that the movements and distribution of individual animals are much less restricted by environmental temperature than laboratory studies suggest. Some individuals occupy shallow habitats in the southern North Sea where summer temperatures are consistently above 17 -C for several months, while others tagged at the same time move to deeper, cooler waters. Also, cod in the northeast Atlantic repeatedly experience abrupt temperature changes of up to 8 -C while moving through steep temperature gradients. We suggest that temperature may not be so critical in constraining the movements and distribution of adult cod and other environmental factors (e.g. prey distribution, suitable flow refuges etc.) may be equally, or even more, important.
A3.7 Physiological consequences of feeding in sharks
P. Butler, S. Portugal, J. Green, (University of Birmingham) Over a period spanning approximately 20 days and coinciding with the autumn migration, mean daily body temperature (Tb) of barnacle geese which breed on the island of Spitzbergen and overwinter in southern Scotland, fell by almost 4.5 -C. It was hypothesised that this controlled drop in body temperature (anapyrexia) was associated with a sparing of fat during migration and/or with the replacement of fat following migration. Preliminary observations on captive geese in Birmingham showed a similar, but smaller, reduction in Tb and changes in body mass which approximately coincided with the migratory periods of wild geese. The objective of the current study is to test the above hypothesis by recording and relating, throughout the year, body mass, body composition, body temperature and resting oxygen consumption of captive geese that have free access to food. While confirming the periods of change in body mass, by far the greater of the two is associated with moulting, which in wild birds precedes the autumn migration. There is a 21% decrease in body mass, but a 50% increase in absolute resting oxygen consumption. Current theories suggest that the loss in mass during moult is not the result of the moult process itself, but that it is an adaptation to regain flight faster with partially regrown feathers. The cause(s) of the large increase in oxygen consumption is unknown but could relate to any of the following: the cost of replacing the feathers, the catabolism of fat to accelerate mass loss, increased cost of thermoregulation.
A3.6 Cod in a sweat: from physiology to behaviour and back again J. Metcalfe, (CEFAS) Atlantic cod (Gadus morhua) are found in cooler waters over a wide area on the continental shelves of the northern Atlantic. Laboratory studies indicate that their thermal preferendum is in the range 11 – 14 -C, their metabolic scope is maximised in the temperature range 13 – 15 -C and that food-unlimited growth is optimal between about 6 – 14 -C. Although individuals can adapt to changes in temperatures, they are
C. Wood, M. Kajimura, (McMaster University); P. Walsh, (University of Miami); T. Mommsen, (University of Victoria) Sharks have a high N-demand because of their urea-N based osmoregulation, but in the wild they feed opportunistically and irregularly, sometimes consuming large meals (up to 10% body weight) in a single bout. We have examined the physiological consequences of feeding and starvation in the spiny dogfish Squalus acanthias using both natural (feeding frenzies) and experimental models (feeding via stomach intubation). Feeding (with teleost tissue) stimulates only a modest loss of N, mainly as ammonia-N and unknown-N; there is no change in urea-N excretion. N – retention is facilitated by an activation of the ornithine-urea cycle, mainly in skeletal muscle, and blood urea-N levels rise. The rectal gland is metabolically activated. Blood ketones and glucose fall after feeding and rise progressively during starvation. Despite a marked metabolic alkalosis (‘‘alkaline tide’’) in the systemic bloodstream indicative of stimulated acid-secretion, stomach pH (<2.0 in starved animals) rises to 3.0 – 4.0 after feeding and then slowly declines as digestion proceeds. The mass of the stomach and intestinal walls increase in sequence as the chyme proceeds down the tract. Despite complex ionic changes, chyme osmolality remains close to plasma levels. During the post-prandial period, both the volume and pH (6.5) of chyme in the intestine remains constant, while the bolus in the stomach is progressively reduced; absorption is complete by 5 days. During prolonged starvation (35 days), urea-N excretion is not reduced, but plasma urea-N levels and osmolality are well-maintained at the expense of body condition factor (NSERC Discovery, CRC program, NSF, Japan Fellowship).
A3.8 Matching supply and demand: integrating energetics from the genome to the organism J. Richards, (The University of British Columbia) The key biochemical strategy for hypoxia survival is the ability to maintain metabolic energy balance in the face of a reduced
Abstracts / Comparative Biochemistry and Physiology, Part A 143 (2006) S65 – S67
capacity for energy production. In hypoxia tolerant animals, the maintenance of energy balance is achieved through the activation of oxygen-independent pathways for ATP production and a controlled decrease in energy demand yielding whole body metabolic suppression. My laboratory studies the mechanisms and evolution of hypoxia tolerance in fish. During acclimation to moderate hypoxia, we hypothesize that changes in gene expression will act to optimize ATP production and limit ATP utilization. We chose pyruvate dehydrogenase kinase (PDK) as a candidate gene and examined its expression pattern in many species of intertidal sculpin and the common killifish (Fundulus heteroclitus), all hypoxia tolerant fish, during exposure to moderate hypoxia (0.5 to 0.8 mg O2/L). PDK inhibits pyruvate dehydrogenase (PDH) resulting in decreases in mitochondrial carbohydrate oxidation and lower oxygen demands. Two isoforms of PDK (PDK 2 and 2a) were identified in F. heteroclitus, each with a tissue specific distribution. Our data demonstrate a very rapid increase in PDK expression in F. heteroclitus in response to hypoxia, which coincides with decreases in PDH activity and initiation of metabolic suppression.
A3.9 Cardio-respiratory lessons from Graham Shelton ‘‘Be suspicious of straight lines’’ W. Burggren, (University of North Texas) Abstract not submitted.
S67
A3.10 Chronic hypoxia in zebrafish: an integrative approach to studying plasticity B. Bagatto, F. Moore, (University of Akron) Genetic and environmental variation are both known to influence development. Evolution of a developmental response that is optimized to the environment (adaptive plasticity) requires the existence of genetic variation for that developmental response. In complex traits composed of integrated sets of subsidiary traits, the adaptive process may be slowed by the existence of multiple possible integrated responses. This study tests for family (sibship) specific differences in plastic response to hypoxia in an integrated set of cardiovascular traits in zebrafish. Cardiac output, which is the integrated product of several subsidiary traits, varied highly significantly between families, and families differed significantly in the degree and direction of response to developmental oxygen level. The cardiac output response to oxygen environment was entirely family specific with no significant overall trend due to oxygen level. Constituent physiological variables that contribute to cardiac output all showed significant family specific response to hypoxia. Traits that were not directly related to cardiac output, such as arterial and venous diameter, and red blood cell velocities did not respond to hypoxia in a family specific manner. Zebrafish families vary in their plastic response to hypoxia. Genetic variation in plastic response to hypoxia may therefore provide the basic ingredient for adaptation to a variable environment. Considerable variation in the degree of familial response to hypoxia exists between different cardiovascular traits that may contribute to cardiac output. It is possible that the integration of several subsidiary traits into cardiac output allows the maintenance of genetic variance in cardiac response.