A novel protocol for rapid deployment of heart rate data storage tags in Atlantic bluefin tuna Thunnus thynnus reveals cardiac responses to temperature and feeding.

The Atlantic bluefin tuna (ABFT) is a highly prized species of large pelagic fish. Studies of their environmental physiology may improve understanding and management of their populations, but this is difficult for mature adults because of their large size. Biologging of heart rate holds promise in investigating physiological responses to environmental conditions in free-swimming fishes but it is very challenging to anesthetize large ABFT for invasive surgery to place a tag in the body cavity near to the heart. We describe a novel method for rapid deployment of a commercially available heart-rate tag on ABFT, using an atraumatic trocar to implant it in the musculature associated with the cleithrum. We performed three sequential experiments to show that the tagging method (1) is consistently repeatable and reliable, (2) can be used successfully on commercial fishing boats and does not seem to affect fish survival, and (3) is effective for long-term deployments. In experiment 3, a tag logged heart rate over 80 days on a 60-kg ABFT held in a farm cage. The logged data showed that heart rate was sensitive to prevailing seasonal temperature and feeding events. At low temperatures, there were clear responses to feeding but these all disappeared above a threshold temperature of 25.5°C. Overall, the results show that our method is simple, rapid, and repeatable, and can be used for long-term experiments to investigate physiological responses by large ABFT to environmental conditions.

Measuring oxygen uptake in fishes with bimodal respiration.

Respirometry is a robust method for measurement of oxygen uptake as a proxy for metabolic rate in fishes, and how species with bimodal respiration might meet their demands from water v. air has interested researchers for over a century. The challenges of measuring oxygen uptake from both water and air, preferably simultaneously, have been addressed in a variety of ways, which are briefly reviewed. These methods are not well-suited for the long-term measurements necessary to be certain of obtaining undisturbed patterns of respiratory partitioning, for example, to estimate traits such as standard metabolic rate. Such measurements require automated intermittent-closed respirometry that, for bimodal fishes, has only recently been developed. This paper describes two approaches in enough detail to be replicated by the interested researcher. These methods are for static respirometry. Measuring oxygen uptake by bimodal fishes during exercise poses specific challenges, which are described to aid the reader in designing experiments. The respiratory physiology and behaviour of air-breathing fishes is very complex and can easily be influenced by experimental conditions, and some general considerations are listed to facilitate the design of experiments. Air breathing is believed to have evolved in response to aquatic hypoxia and, probably, associated hypercapnia. The review ends by considering what realistic hypercapnia is, how hypercapnic tropical waters can become and how this might influence bimodal animals' gas exchange.

Swimming in air-breathing fishes.

Fishes with bimodal respiration differ in the extent of their reliance on air breathing to support aerobic metabolism, which is reflected in their lifestyles and ecologies. Many freshwater species undertake seasonal and reproductive migrations that presumably involve sustained aerobic exercise. In the six species studied to date, aerobic exercise in swim flumes stimulated air-breathing behaviour, and there is evidence that surfacing frequency and oxygen uptake from air show an exponential increase with increasing swimming speed. In some species, this was associated with an increase in the proportion of aerobic metabolism met by aerial respiration, while in others the proportion remained relatively constant. The ecological significance of anaerobic swimming activities, such as sprinting and fast-start manoeuvres during predator-prey interactions, has been little studied in air-breathing fishes. Some species practise air breathing during recovery itself, while others prefer to increase aquatic respiration, possibly to promote branchial ion exchange to restore acid-base balance, and to remain quiescent and avoid being visible to predators. Overall, the diversity of air-breathing fishes is reflected in their swimming physiology as well, and further research is needed to increase the understanding of the differences and the mechanisms through which air breathing is controlled and used during exercise.

Settleable atmospheric particulate matter affects the swimming performance and aerobic metabolic rate of Nile tilapia (Oreochromis niloticus).

Atmospheric particulate matter (APM) produced by the steel industry comprises a complex mixture of particles that includes a wide variety of metals and metallic nanoparticles. These particles settle out onto areas surrounding the industries. There is evidence that this 'settleable' APM (SePM) may cause air-to-water cross-contamination with significant effects on aquatic biota. Recent investigations have reported sublethal impacts on the gill structure and blood oxygen-carrying capacity of fishes, which raises the hypothesis that there will be consequences for gas exchange capacity and ability to support aerobic activities. Therefore, we investigated the effects of an environmentally relevant level of SePM contamination on swimming performance and associated aerobic metabolic rates in Nile tilapia, Oreochromis niloticus. Short-term exposure (96 h) to SePM reduced critical swimming speed, energetic efficiency of aerobic swimming, standard metabolic rate, maximum metabolic rate, and aerobic scope. The compromised swimming performance could have adverse ecological effects by limiting foraging ability, predator evasion, territorial protection, and migration. The impairments to aerobic capacity could also affect overall fish performance by influencing long-term energy balance and allocation to growth and reproduction. Thus, despite being sublethal, SePM contamination is considerably debilitating, and if its limiting effects are not compensated for in the longer term, this may reduce the survival and fitness of fish populations.

Settleable atmospheric particulate matter affects cardiorespiratory responses to hypoxia in Nile tilapia (Oreochromis niloticus).

Atmospheric particulate matter (APM) emitted by iron ore processing industries has a complex composition, including diverse metallic particles and nanoparticles. Settleable APM (SePM) causes air to water cross-contamination and has recently been demonstrated to have harmful sublethal impacts on fish, eliciting stress responses, affecting the immune system, and reducing blood oxygen-carrying capacity. These findings imply potential consequences for fish aerobic performance and energy allocation, particularly in their ability to tolerate respiratory challenges such as aquatic hypoxia. To assess that potential limitation, we analyzed metabolic, cardiorespiratory, and morphological alterations after exposing tilapia, Oreochromis niloticus, to an environmentally relevant concentration of SePM (96 h) and progressive hypoxia. The contamination initiated detectable gill damage, reducing respiratory efficiency, increasing ventilatory effort, and compromising fish capacity to deal with hypoxia. Even in normoxia, the resting respiratory frequency was elevated and limited respiratory adjustments during hypoxia. SePM increased O2crit from 26 to 34% of O2 (1.84 to 2.76 mg O2·L-1). Such ventilatory inefficacy implies higher ventilatory cost with relevant alterations in energy allocation. Progression in gill damage might be problematic and cause: infection, blood loss, ion imbalance, and limited cardiorespiratory performance. The contamination did not cause immediate lethality but may threaten fish populations due to limitations in physiological performance. This was the first investigation to evaluate the physiological responses of fish to hypoxia after SePM contamination. We suggest that the present level of environmental SePM deserves attention. The present results demonstrate the need for comprehensive studies on SePM effects in aquatic fauna.

Intraspecific variation in thermal tolerance differs between tropical and temperate fishes.

How ectothermic animals will cope with global warming is a critical determinant of the ecological impacts of climate change. There has been extensive study of upper thermal tolerance limits among fish species but how intraspecific variation in tolerance may be affected by habitat characteristics and evolutionary history has not been considered. Intraspecific variation is a primary determinant of species vulnerability to climate change, with implications for global patterns of impacts of ongoing warming. Using published critical thermal maximum (CTmax) data on 203 fish species, we found that intraspecific variation in upper thermal tolerance varies according to a species' latitude and evolutionary history. Overall, tropical species show a lower intraspecific variation in thermal tolerance than temperate species. Notably, freshwater tropical species have a lower variation in tolerance than freshwater temperate species, which implies increased vulnerability to impacts of thermal stress. The extent of variation in CTmax among fish species has a strong phylogenetic signal, which may indicate a constraint on evolvability to rising temperatures in tropical fishes. That is, in addition to living closer to their upper thermal limits, tropical species may have higher sensitivity and lower adaptability to global warming compared to temperate counterparts. This is evidence that freshwater tropical fish communities, worldwide, are especially vulnerable to ongoing climate change.

Individual variation and repeatability in aerobic and anaerobic swimming performance of European sea bass, Dicentrarchus labrax.

Studies of inter-individual variation in fish swimming performance may provide insight into how selection has influenced diversity in phenotypic traits. We investigated individual variation and short-term repeatability of individual swimming performance by wild European sea bass in a constant acceleration test (CAT). Fish were challenged with four consecutive CATs with 5 min rest between trials. We measured maximum anaerobic speed at exhaustion (U(CAT)), gait transition speed from steady aerobic to unsteady anaerobic swimming (U(gt)), routine metabolic rate (RMR), post-CAT maximum metabolic rate (MMR), aerobic scope and recovery time from the CATs. Fish achieved significantly higher speeds during the first CAT (U(CAT)=170 cm s(-1)), and had much more inter-individual variation in performance (coefficient of variation, CV=18.43%) than in the subsequent three tests (U(CAT)=134 cm s(-1); CV=7.3%), which were very repeatable among individuals. The individual variation in U(CAT) in the first trial could be accounted for almost exclusively by variation in anaerobic burst-and-coast performance beyond U(gt). The U(gt) itself varied substantially between individuals (CV=11.4%), but was significantly repeatable across all four trials. Individual RMR and MMR varied considerably, but the rank order of post-CAT MMR was highly repeatable. Recovery rate from the four CATs was highly variable and correlated positively with the first U(CAT) (longer recovery for higher speeds) but negatively with RMR and aerobic scope (shorter recovery for higher RMR and aerobic scope). This large variation in individual performance coupled with the strong correlations between some of the studied variables may reflect divergent selection favouring alternative strategies for foraging and avoiding predation.

Physiological mechanisms underlying a trade-off between growth rate and tolerance of feed deprivation in the European sea bass (Dicentrarchus labrax).

The specific growth rate (SGR) of a cohort of 2000 tagged juvenile European sea bass was measured in a common tank, during two sequential cycles comprising three-weeks feed deprivation followed by three-weeks ad libitum re-feeding. After correction for initial size at age as fork length, there was a direct correlation between negative SGR (rate of mass loss) during feed deprivation and positive SGR (rate of compensatory growth) during re-feeding (Spearman rank correlation R=0.388, P=0.000002). Following a period of rearing under standard culture conditions, individuals representing 'high growth' phenotypes (GP) and 'high tolerance of feed deprivation' phenotypes (DP) were selected from either end of the SGR spectrum. Static and swimming respirometry could not demonstrate lower routine or standard metabolic rate in DP to account for greater tolerance of feed deprivation. Increased rates of compensatory growth in GP were not linked to greater maximum metabolic rate, aerobic metabolic scope or maximum cardiac performance than DP. When fed a standard ration, however, GP completed the specific dynamic action (SDA) response significantly faster than DP. Therefore, higher growth rate in GP was linked to greater capacity to process food. There was no difference in SDA coefficient, an indicator of energetic efficiency. The results indicate that individual variation in growth rate in sea bass reflects, in part, a trade-off against tolerance of food deprivation. The two phenotypes represented the opposing ends of a spectrum. The GP aims to exploit available resources and grow as rapidly as possible but at a cost of physiological and/or behavioural attributes, which lead to increased energy dissipation when food is not available. An opposing strategy, exemplified by DP, is less 'boom and bust', with a lower physiological capacity to exploit resources but which is less costly to sustain during periods of food deprivation.

Effects of feeding and hypoxia on cardiac performance and gastrointestinal blood flow during critical speed swimming in the sea bass Dicentrarchus labrax.

Previous studies have shown that if European sea bass are exercised after feeding, they can achieve a significantly higher maximum metabolic rate (MMR) than when fasted. They can meet combined metabolic demands of digestion (specific dynamic action, SDA) and maximal aerobic exercise, with no decline in swimming performance. If, however, exposed to mild hypoxia (50% saturation), bass no longer achieve higher MMR after feeding but they swim as well fed as fasted, due to an apparent ability to defer the SDA response. This study explored patterns of cardiac output (Q(A)) and blood flow to the gastrointestinal tract (Q(GI)) associated with the higher MMR after feeding, and with the ability to prioritise swimming in hypoxia. Sea bass (mean mass approximately 325 g, forklength approximately 27 cm) were instrumented with flow probes to measure Q(A) and Q(GI) during an incremental critical swimming speed (U(crit)) protocol in a tunnel respirometer, to compare each animal either fasted or 6h after a meal of fish fillet equal to 3% body mass. Feeding raised oxygen uptake (M(O2)) prior to exercise, an SDA response associated with increased Q(A) (+30%) and Q(GI) (+100%) compared to fasted values. As expected, when exercised the fed bass maintained the SDA load throughout the protocol and achieved 14% higher MMR than when fasted, and the same U(crit) (approximately 100 cm s(-1)). Both fed and fasted bass showed pronounced increases in Q(A) and decreases in Q(GI) during exercise and the higher MMR of fed bass was not associated with higher maximum Q(A) relative to when fasted, or to any differences in Q(GI) at maximum Q(A). In hypoxia prior to exercise, metabolic and cardiac responses to feeding were similar compared to normoxia. Hypoxia caused an almost 60% reduction to MMR and 30% reduction to U(crit), but neither of these traits differed between fed or fasted bass. Despite hypoxic limitations to MMR and U(crit), maximum Q(A) and patterns of Q(GI) during exercise in fasted and fed bass were similar to normoxia. Estimating GI oxygen supply from Q(GI) indicated that the ability of bass to prioritise aerobic exercise over SDA when metabolically limited by hypoxia was linked to an ability to defer elements of the SDA response occurring outside the GI tract.

Reflex cardioventilatory responses to hypoxia in the flathead gray mullet (Mugil cephalus) and their behavioral modulation by perceived threat of predation and water turbidity.

In hypoxia, gray mullet surface to ventilate well-oxygenated water in contact with air, an adaptive response known as aquatic surface respiration (ASR). Reflex control of ASR and its behavioral modulation by perceived threat of aerial predation and turbid water were studied on mullet in a partly sheltered aquarium with free surface access. Injections of sodium cyanide (NaCN) into either the bloodstream (internal) or ventilatory water stream (external) revealed that ASR, hypoxic bradycardia, and branchial hyperventilation were stimulated by chemoreceptors sensitive to both systemic and water O2 levels. Sight of a model avian predator elicited bradycardia and hypoventilation, a fear response that inhibited reflex hyperventilation following external NaCN. The time lag to initiation of ASR following NaCN increased, but response intensity (number of events, time at the surface) was unchanged. Mullet, however, modified their behavior to surface under shelter or near the aquarium edges. Turbid water abolished the fear response and effects of the predator on gill ventilation and timing of ASR following external NaCN, presumably because of reduced visibility. However, in turbidity, mullet consistently performed ASR under shelter or near the aquarium edges. These adaptive modulations of ASR behavior would allow mullet to retain advantages of the chemoreflex when threatened by avian predators or when unable to perceive potential threats in turbidity.

Effects of plasma total ammonia content and pH on urea excretion in Nile tilapia.

Nile tilapia (Oreochromis niloticus) were infused with ammonium salts, acid, and base to investigate the effects of changes in arterial plasma total ammonia content (Tamm) and pH (pHa) on plasma urea-nitrogen (urea-N) levels and urea-N excretory fluxes (Jurea-N). The tilapia did not possess a functional hepatic ornithine urea-cycle (no significant carbamyl phosphate synthetase III activity). Infused substances were dissolved in a saline vehicle and injected twice (5 mL kg-1), the first infusion to "prime" the animal and promote a more marked response to the second infusion, given 2.5 h later. The results reported are those of the second infusion. Infusion of 200 mM NH4Cl increased Tamm, reduced pHa, and increased plasma urea-N and Jurea-N. Two hundred mM NH4HCO3 increased Tamm and arterial plasma total CO2 content (TaCO2), reduced pHa, and increased Jurea-N. Fifty mM HCl reduced pHa but had no effects on urea dynamics. Fifty mM NaOH increased pHa, plasma urea-N levels, and Jurea-N. Two hundred mM NaHCO3 increased pHa, TaCO2, plasma urea-N levels, and Jurea-N. Infusion of the saline vehicle was without effect. The results indicate that ammonia loading and plasma alkalosis both stimulate urea excretion in uricolytic fish. The responses to hyperammonemia or alkalosis were not modified when combined with elevated plasma bicarbonate levels.

Control of respiration in fish, amphibians and reptiles.

Fish and amphibians utilise a suction/force pump to ventilate gills or lungs, with the respiratory muscles innervated by cranial nerves, while reptiles have a thoracic, aspiratory pump innervated by spinal nerves. However, fish can recruit a hypobranchial pump for active jaw occlusion during hypoxia, using feeding muscles innervated by anterior spinal nerves. This same pump is used to ventilate the air-breathing organ in air-breathing fishes. Some reptiles retain a buccal force pump for use during hypoxia or exercise. All vertebrates have respiratory rhythm generators (RRG) located in the brainstem. In cyclostomes and possibly jawed fishes, this may comprise elements of the trigeminal nucleus, though in the latter group RRG neurons have been located in the reticular formation. In air-breathing fishes and amphibians, there may be separate RRG for gill and lung ventilation. There is some evidence for multiple RRG in reptiles. Both amphibians and reptiles show episodic breathing patterns that may be centrally generated, though they do respond to changes in oxygen supply. Fish and larval amphibians have chemoreceptors sensitive to oxygen partial pressure located on the gills. Hypoxia induces increased ventilation and a reflex bradycardia and may trigger aquatic surface respiration or air-breathing, though these latter activities also respond to behavioural cues. Adult amphibians and reptiles have peripheral chemoreceptors located on the carotid arteries and central chemoreceptors sensitive to blood carbon dioxide levels. Lung perfusion may be regulated by cardiac shunting and lung ventilation stimulates lung stretch receptors.