ABSTRACT Fish exposed to fluctuating oxygen concentrations often alter their metabolism and/or behaviour to survive. Hypoxia tolerance is typically associated with the ability to reduce energy demand by supressing metabolic processes such as protein synthesis. Arctic char is amongst the most sensitive salmonid to hypoxia, and typically engage in avoidance behaviour when faced with lack of oxygen. We hypothesized that a sensitive species will still have the ability (albeit reduced) to regulate molecular mechanisms during hypoxia. We investigated the tissue-specific response of protein metabolism during hypoxia. Little is known about protein degradation pathways during hypoxia in fish and we predict that protein degradation pathways are differentially regulated and play a role in the hypoxia response. We also studied the regulation of oxygen-responsive cellular signalling pathways [hypoxia inducible factor (HIF), unfolded protein response (UPR) and mTOR pathways] since most of what we know comes from studies on cancerous mammalian cell lines. Arctic char were exposed to cumulative graded hypoxia trials for 3 h at four air saturation levels (100%, 50%, 30% and 15%). The rate of protein synthesis was measured using a flooding dose technique, whereas protein degradation and signalling pathways were assessed by measuring transcripts and phosphorylation of target proteins. Protein synthesis decreased in all tissues measured (liver, muscle, gill, digestive system) except for the heart. Salmonid hearts have preferential access to oxygen through a well-developed coronary artery, therefore the heart is likely to be the last tissue to become hypoxic. Autophagy markers were upregulated in the liver, whereas protein degradation markers were downregulated in the heart during hypoxia. Further work is needed to determine the effects of a decrease in protein degradation on a hypoxic salmonid heart. Our study showed that protein metabolism in Arctic char is altered in a tissue-specific fashion during graded hypoxia, which is in accordance with the responses of the three major hypoxia-sensitive pathways (HIF, UPR and mTOR). The activation pattern of these pathways and the cellular processes that are under their control varies greatly among tissues, sometimes even going in the opposite direction. This study provides new insights on the effects of hypoxia on protein metabolism. Adjustment of these cellular processes is likely to contribute to shifting the fish phenotype into a more hypoxia-tolerant one, if more than one hypoxia event were to occur. Our results warrant studying these adjustments in fish exposed to long-term and diel cycling hypoxia.

ABSTRACT The present study investigated the potential role of hypoxia-inducible factor (HIF) in calcium homeostasis in developing zebrafish ( Danio rerio ). It was demonstrated that zebrafish raised in hypoxic water (30 mmHg; control, 155 mmHg P O 2 ) until 4 days post-fertilization exhibited a substantial reduction in whole-body Ca 2+ levels and Ca 2+ uptake. Ca 2+ uptake in hypoxia-treated fish did not return to pre-hypoxia (control) levels within 2 h of transfer back to normoxic water. Results from real-time PCR showed that hypoxia decreased the whole-body mRNA expression levels of the epithelial Ca 2+ channel ( ecac ), but not plasma membrane Ca 2+ -ATPase ( pmca2 ) or Na + /Ca 2+ -exchanger ( ncx1b ). Whole-mount in situ hybridization revealed that the number of ecac -expressing ionocytes was reduced in fish raised in hypoxic water. These findings suggested that hypoxic treatment suppressed the expression of ecac , thereby reducing Ca 2+ influx. To further evaluate the potential mechanisms for the effects of hypoxia on Ca 2+ regulation, a functional gene knockdown approach was employed to prevent the expression of HIF-1αb during hypoxic treatment. Consistent with a role for HIF-1αb in regulating Ca 2+ balance during hypoxia, the results demonstrated that the reduction of Ca 2+ uptake associated with hypoxic exposure was not observed in fish experiencing HIF-1αb knockdown. Additionally, the effects of hypoxia on reducing the number of ecac -expressing ionocytes was less pronounced in HIF-1αb-deficient fish. Overall, the current study revealed that hypoxic exposure inhibited Ca 2+ uptake in developing zebrafish, probably owing to HIF-1αb-mediated suppression of ecac expression.

SUMMARY Using implanted archival tags, we examined the effects of meal caloric value, food type (sardine or squid) and ambient temperature on the magnitude and duration of the heat increment of feeding in three captive juvenile Pacific bluefin tuna. The objective of our study was to develop a model that can be used to estimate energy intake in wild fish of similar body mass. Both the magnitude and duration of the heat increment of feeding (measured by visceral warming) showed a strong positive correlation with the caloric value of the ingested meal. Controlling for meal caloric value, the extent of visceral warming was significantly greater at lower ambient temperature. The extent of visceral warming was also significantly higher for squid meals compared with sardine meals. By using a hierarchical Bayesian model to analyze our data and treating individuals as random effects, we demonstrate how increases in visceral temperature can be used to estimate the energy intake of wild Pacific bluefin tuna of similar body mass to the individuals used in our study.

SUMMARY Specific dynamic action (SDA) is defined as the energy expended during ingestion, digestion, absorption and assimilation of a meal. This study presents the first data on the SDA response of individual tunas of any species. Juvenile Pacific bluefin tunas ( Thunnus orientalis ; body mass 9.7–11.0 kg; N =7) were individually fed known quantities of food consisting primarily of squid and sardine (meal energy range 1680–8749 kJ, ~4–13% of tuna body mass). Oxygen consumption rates ( ) were measured in a swim tunnel respirometer during the postprandial period at a swimming speed of 1 body length ( BL ) s −1 and a water temperature of 20°C. was markedly elevated above routine levels in all fish following meal consumption [routine metabolic rate (RMR)=174±9 mg kg −1 h −1 ]. The peak during the SDA process ranged from 250 to 440 mg kg −1 h −1 (1.5–2.3 times RMR) and was linearly related to meal energy content. The duration of the postprandial increment in ranged from 21 h to 33 h depending upon meal energy content. Consequently, the total energy used in SDA increased linearly with meal energy and ranged from 170 kJ to 688 kJ, such that the SDA process accounted for 9.2±0.7% of ingested energy across all experiments. These values suggest rapid and efficient food conversion in T . orientalis in comparison with most other fishes. Implanted archival temperature tags recorded the increment in visceral temperature ( T V ) in association with SDA. returned to routine levels at the end of the digestive period 2–3 h earlier than T V . The qualitative patterns in and T V during digestion were similar, strengthening the possibility that archival measurements of T V can provide new insight into the energetics and habitat utilization of free-swimming bluefin in the natural environment. Despite efficient food conversion, SDA is likely to represent a significant component of the daily energy budget of wild bluefin tunas due to a regular and high ingestion of forage. SUMMARY Specific dynamic action (SDA) is defined as the energy expended during ingestion, digestion, absorption and assimilation of a meal. This study presents the first data on the SDA response of individual tunas of any species. Juvenile Pacific bluefin tunas ( Thunnus orientalis ; body mass 9.7–11.0 kg; N =7) were individually fed known quantities of food consisting primarily of squid and sardine (meal energy range 1680–8749 kJ, ~4–13% of tuna body mass). Oxygen consumption rates ( ) were measured in a swim tunnel respirometer during the postprandial period at a swimming speed of 1 body length ( BL ) s −1 and a water temperature of 20°C. was markedly elevated above routine levels in all fish following meal consumption [routine metabolic rate (RMR)=174±9 mg kg −1 h −1 ]. The peak during the SDA process ranged from 250 to 440 mg kg −1 h −1 (1.5–2.3 times RMR) and was linearly related to meal energy content. The duration of the postprandial increment in ranged from 21 h to 33 h depending upon meal energy content. Consequently, the total energy used in SDA increased linearly with meal energy and ranged from 170 kJ to 688 kJ, such that the SDA process accounted for 9.2±0.7% of ingested energy across all experiments. These values suggest rapid and efficient food conversion in T . orientalis in comparison with most other fishes. Implanted archival temperature tags recorded the increment in visceral temperature ( T V ) in association with SDA. returned to routine levels at the end of the digestive period 2–3 h earlier than T V . The qualitative patterns in and T V during digestion were similar, strengthening the possibility that archival measurements of T V can provide new insight into the energetics and habitat utilization of free-swimming bluefin in the natural environment. Despite efficient food conversion, SDA is likely to represent a significant component of the daily energy budget of wild bluefin tunas due to a regular and high ingestion of forage. SUMMARY Specific dynamic action (SDA) is defined as the energy expended during ingestion, digestion, absorption and assimilation of a meal. This study presents the first data on the SDA response of individual tunas of any species. Juvenile Pacific bluefin tunas ( Thunnus orientalis ; body mass 9.7–11.0 kg; N =7) were individually fed known quantities of food consisting primarily of squid and sardine (meal energy range 1680–8749 kJ, ~4–13% of tuna body mass). Oxygen consumption rates ( ) were measured in a swim tunnel respirometer during the postprandial period at a swimming speed of 1 body length ( BL ) s −1 and a water temperature of 20°C. was markedly elevated above routine levels in all fish following meal consumption [routine metabolic rate (RMR)=174±9 mg kg −1 h −1 ]. The peak during the SDA process ranged from 250 to 440 mg kg −1 h −1 (1.5–2.3 times RMR) and was linearly related to meal energy content. The duration of the postprandial increment in ranged from 21 h to 33 h depending upon meal energy content. Consequently, the total energy used in SDA increased linearly with meal energy and ranged from 170 kJ to 688 kJ, such that the SDA process accounted for 9.2±0.7% of ingested energy across all experiments. These values suggest rapid and efficient food conversion in T . orientalis in comparison with most other fishes. Implanted archival temperature tags recorded the increment in visceral temperature ( T V ) in association with SDA. returned to routine levels at the end of the digestive period 2–3 h earlier than T V . The qualitative patterns in and T V during digestion were similar, strengthening the possibility that archival measurements of T V can provide new insight into the energetics and habitat utilization of free-swimming bluefin in the natural environment. Despite efficient food conversion, SDA is likely to represent a significant component of the daily energy budget of wild bluefin tunas due to a regular and high ingestion of forage. SUMMARY Specific dynamic action (SDA) is defined as the energy expended during ingestion, digestion, absorption and assimilation of a meal. This study presents the first data on the SDA response of individual tunas of any species. Juvenile Pacific bluefin tunas ( Thunnus orientalis ; body mass 9.7–11.0 kg; N =7) were individually fed known quantities of food consisting primarily of squid and sardine (meal energy range 1680–8749 kJ, ~4–13% of tuna body mass). Oxygen consumption rates ( ) were measured in a swim tunnel respirometer during the postprandial period at a swimming speed of 1 body length ( BL ) s −1 and a water temperature of 20°C. was markedly elevated above routine levels in all fish following meal consumption [routine metabolic rate (RMR)=174±9 mg kg −1 h −1 ]. The peak during the SDA process ranged from 250 to 440 mg kg −1 h −1 (1.5–2.3 times RMR) and was linearly related to meal energy content. The duration of the postprandial increment in ranged from 21 h to 33 h depending upon meal energy content. Consequently, the total energy used in SDA increased linearly with meal energy and ranged from 170 kJ to 688 kJ, such that the SDA process accounted for 9.2±0.7% of ingested energy across all experiments. These values suggest rapid and efficient food conversion in T . orientalis in comparison with most other fishes. Implanted archival temperature tags recorded the increment in visceral temperature ( T V ) in association with SDA. returned to routine levels at the end of the digestive period 2–3 h earlier than T V . The qualitative patterns in and T V during digestion were similar, strengthening the possibility that archival measurements of T V can provide new insight into the energetics and habitat utilization of free-swimming bluefin in the natural environment. Despite efficient food conversion, SDA is likely to represent a significant component of the daily energy budget of wild bluefin tunas due to a regular and high ingestion of forage. SUMMARY Specific dynamic action (SDA) is defined as the energy expended during ingestion, digestion, absorption and assimilation of a meal. This study presents the first data on the SDA response of individual tunas of any species. Juvenile Pacific bluefin tunas ( Thunnus orientalis ; body mass 9.7–11.0 kg; N =7) were individually fed known quantities of food consisting primarily of squid and sardine (meal energy range 1680–8749 kJ, ~4–13% of tuna body mass). Oxygen consumption rates ( ) were measured in a swim tunnel respirometer during the postprandial period at a swimming speed of 1 body length ( BL ) s −1 and a water temperature of 20°C. was markedly elevated above routine levels in all fish following meal consumption [routine metabolic rate (RMR)=174±9 mg kg −1 h −1 ]. The peak during the SDA process ranged from 250 to 440 mg kg −1 h −1 (1.5–2.3 times RMR) and was linearly related to meal energy content. The duration of the postprandial increment in ranged from 21 h to 33 h depending upon meal energy content. Consequently, the total energy used in SDA increased linearly with meal energy and ranged from 170 kJ to 688 kJ, such that the SDA process accounted for 9.2±0.7% of ingested energy across all experiments. These values suggest rapid and efficient food conversion in T . orientalis in comparison with most other fishes. Implanted archival temperature tags recorded the increment in visceral temperature ( T V ) in association with SDA. returned to routine levels at the end of the digestive period 2–3 h earlier than T V . The qualitative patterns in and T V during digestion were similar, strengthening the possibility that archival measurements of T V can provide new insight into the energetics and habitat utilization of free-swimming bluefin in the natural environment. Despite efficient food conversion, SDA is likely to represent a significant component of the daily energy budget of wild bluefin tunas due to a regular and high ingestion of forage. SUMMARY Specific dynamic action (SDA) is defined as the energy expended during ingestion, digestion, absorption and assimilation of a meal. This study presents the first data on the SDA response of individual tunas of any species. Juvenile Pacific bluefin tunas ( Thunnus orientalis ; body mass 9.7–11.0 kg; N =7) were individually fed known quantities of food consisting primarily of squid and sardine (meal energy range 1680–8749 kJ, ~4–13% of tuna body mass). Oxygen consumption rates ( ) were measured in a swim tunnel respirometer during the postprandial period at a swimming speed of 1 body length ( BL ) s −1 and a water temperature of 20°C. was markedly elevated above routine levels in all fish following meal consumption [routine metabolic rate (RMR)=174±9 mg kg −1 h −1 ]. The peak during the SDA process ranged from 250 to 440 mg kg −1 h −1 (1.5–2.3 times RMR) and was linearly related to meal energy content. The duration of the postprandial increment in ranged from 21 h to 33 h depending upon meal energy content. Consequently, the total energy used in SDA increased linearly with meal energy and ranged from 170 kJ to 688 kJ, such that the SDA process accounted for 9.2±0.7% of ingested energy across all experiments. These values suggest rapid and efficient food conversion in T . orientalis in comparison with most other fishes. Implanted archival temperature tags recorded the increment in visceral temperature ( T V ) in association with SDA. returned to routine levels at the end of the digestive period 2–3 h earlier than T V . The qualitative patterns in and T V during digestion were similar, strengthening the possibility that archival measurements of T V can provide new insight into the energetics and habitat utilization of free-swimming bluefin in the natural environment. Despite efficient food conversion, SDA is likely to represent a significant component of the daily energy budget of wild bluefin tunas due to a regular and high ingestion of forage.

SUMMARY Avian divers are confronted with a number of physiological challenges when foraging in cold water, especially at depth. Besides the obvious constraint imposed by the necessity to return to the surface for gas exchange, cold water temperatures and a reduction in body insulation due to the increase in pressure with dive depth will elevate the energetic costs of foraging in these endotherm divers. The complex effect that depth has on the diving energetics of aquatic birds has largely been ignored. To date, no study has assessed the impact of depth on diving energetics over a significant depth range, naturally encountered by the diver. We used open-circuit respirometry to study the energetic requirements of a foot-propelled pursuit diver, the double-crested cormorant ( Phalacrocorax auritus albociliatus ), when diving in a shallow (1 m) and deep (10 m) dive tank and when resting in air and water. We also investigated the modifying effects of air or water temperature and feeding status on the costs associated with diving and resting. Of all factors investigated, dive depth exercised the strongest influence on diving metabolic rate. Diving to 10 m depth increased metabolic rate on average by 22% when compared with shallow diving. Declining temperatures in air and water significantly elevated metabolic rate of cormorants resting in air and water as well as during diving. Feeding before resting in water or diving increased metabolic rate by 5–8% for at least 2 h. Cormorants maintained an elevated stomach temperature (>42°C) when resting in water and during diving, even at cold temperatures. The elevated dive costs during deep diving,when compared with shallow diving, are most likely a consequence of the increased thermoregulatory costs associated with a greater heat loss to the water at depth. Nevertheless, our study shows that dive costs in double-crested cormorants are similar to those of other foot-propelled avian divers.

SUMMARY Diving is believed to be very costly in cormorants (Phalacrocoracidae) when compared with other avian divers because of their poor insulation and less-efficient foot propulsion. It was therefore suggested that cormorants might employ a behavioural strategy to reduce daily energy expenditure by minimizing the amount of time spent in water. However, European shags( Phalacrocorax aristotelis ) have been observed to spend up to 7 h day -1 diving in water of around 5-6°C. To gain a better understanding of the energetic requirements in European shags, we measured their metabolic rates when resting in air/water and during shallow diving using respirometry. To investigate the effects of water temperature and feeding status on metabolic rate, birds dived at water temperatures ranging from 5 to 13°C in both post-absorptive and absorptive states. In parallel with respirometry, stomach temperature loggers were deployed to monitor body temperature. Basal metabolic rate ( BMR ) was almost identical to allometric predictions at 4.73 W kg -1 . Metabolic rate when resting on water, during diving and after feeding was significantly elevated when compared with the resting-in-air rate. During diving, the metabolic rate of post-absorptive shags increased to 22.66 W kg -1 , which corresponds to 4.8× BMR . Minimum cost of transport ( COT ) was calculated at 17.8 J kg -1 m -1 at a swim speed of 1.3 m s -1 . Feeding before diving elevated diving metabolic rate by 13%for up to 5 h. There was a significant relationship between diving metabolic rate and water temperature, where metabolic rate increased as water temperature declined. Thermal conductance when resting in air at 10-19°C was 2.05 W m -2 °C -1 and quadrupled during diving(7.88 W m -2 °C -1 ). Stomach temperature when resting in air during the day was 40.6°C and increased during activity. In dive trials lasting up to 50 min, stomach temperature fluctuated around a peak value of 42.0°C. Hence, there is no evidence that European shags might employ a strategy of regional hypothermia. The energetic costs during shallow diving in European shags are considerably lower than has previously been reported for great cormorants ( Phalacrocorax carbo ) and are comparable to other foot-propelled divers. The lower dive costs in shags might be the consequence of a more streamlined body shape reducing hydrodynamic costs as well as a greater insulative plumage air layer (estimated to be 2.71 mm), which reduces thermoregulatory costs. The latter might be of great importance for shags especially during winter when they spend extended periods foraging in cold water.