Bridging disciplines to advance elasmobranch conservation: applications of physiological ecology.

A strength of physiological ecology is its incorporation of aspects of both species' ecology and physiology; this holistic approach is needed to address current and future anthropogenic stressors affecting elasmobranch fishes that range from overexploitation to the effects of climate change. For example, physiology is one of several key determinants of an organism's ecological niche (along with evolutionary constraints and ecological interactions). The fundamental role of physiology in niche determination led to the development of the field of physiological ecology. This approach considers physiological mechanisms in the context of the environment to understand mechanistic variations that beget ecological trends. Physiological ecology, as an integrative discipline, has recently experienced a resurgence with respect to conservation applications, largely in conjunction with technological advances that extended physiological work from the lab into the natural world. This is of critical importance for species such as elasmobranchs (sharks, skates and rays), which are an especially understudied and threatened group of vertebrates. In 2017, at the American Elasmobranch Society meeting in Austin, Texas, the symposium entitled `Applications of Physiological Ecology in Elasmobranch Research' provided a platform for researchers to showcase work in which ecological questions were examined through a physiological lens. Here, we highlight the research presented at this symposium, which emphasized the strength of linking physiological tools with ecological questions. We also demonstrate the applicability of using physiological ecology research as a method to approach conservation issues, and advocate for a more available framework whereby results are more easily accessible for their implementation into management practices.

Validation of the i-STAT system for the analysis of blood gases and acid-base status in juvenile sandbar shark (Carcharhinus plumbeus).

Accurate measurements of blood gases and acid-base status require an array of sophisticated laboratory equipment that is typically not available during field research; such is the case for many studies on the stress physiology, ecology and conservation of elasmobranch fish species. Consequently, researchers have adopted portable clinical analysers that were developed for the analysis of human blood characteristics, but often without thoroughly validating these systems for their use on fish. The aim of our study was to test the suitability of the i-STAT system, the most commonly used portable clinical analyser in studies on fish, for analysing blood gases and acid-base status in elasmobranchs, over a broad range of conditions and using the sandbar shark (Carcharhinus plumbeus) as a model organism. Our results indicate that the i-STAT system can generate useful measurements of whole blood pH, and the use of appropriate correction factors may increase the accuracy of results. The i-STAT system was, however, unable to generate reliable results for measurements of partial pressure of oxygen (PO2) and the derived parameter of haemoglobin O2 saturation. This is probably due to the effect of a closed-system temperature change on PO2 within the i-STAT cartridge and the fact that the temperature correction algorithms used by i-STAT assume a human temperature dependency of haemoglobin-O2 binding; in many ectotherms, this assumption will lead to equivocal i-STAT PO2 results. The in vivo partial pressure of CO2 (PCO2) in resting sandbar sharks is probably below the detection limit for PCO2 in the i-STAT system, and the measurement of higher PCO2 tensions was associated with a large measurement error. In agreement with previous work, our results indicate that the i-STAT system can generate useful data on whole blood pH in fishes, but not blood gases.

Metabolic and cardiorespiratory responses of summer flounder Paralichthys dentatus to hypoxia at two temperatures.

To quantify the tolerance of summer flounder Paralichthys dentatus to episodic hypoxia, resting metabolic rate, oxygen extraction, gill ventilation and heart rate were measured during acute progressive hypoxia at the fish's acclimation temperature (22° C) and after an acute temperature increase (to 30° C). Mean ±s.e. critical oxygen levels (i.e. the oxygen levels below which fish could not maintain aerobic metabolism) increased significantly from 27 ± 2% saturation (2·0 ± 0·1 mg O(2) l(-1)) at 22° C to 39 ± 2% saturation (2·4 ± 0·1 mg O(2) l(-1)) at 30° C. Gill ventilation and oxygen extraction changed immediately with the onset of hypoxia at both temperatures. The fractional increase in gill ventilation (from normoxia to the lowest oxygen level tested) was much larger at 22° C (6·4-fold) than at 30° C (2·7-fold). In contrast, the fractional decrease in oxygen extraction (from normoxia to the lowest oxygen levels tested) was similar at 22° C (1·7-fold) and 30° C (1·5-fold), and clearly smaller than the fractional changes in gill ventilation. In contrast to the almost immediate effects of hypoxia on respiration, bradycardia was not observed until 20 and 30% oxygen saturation at 22 and 30° C, respectively. Bradycardia was, therefore, not observed until below critical oxygen levels. The critical oxygen levels at both temperatures were near or immediately below the accepted 2·3 mg O(2) l(-1) hypoxia threshold for survival, but the increase in the critical oxygen level at 30° C suggests a lower tolerance to hypoxia after an acute increase in temperature.

Comparative metabolic rates of common western North Atlantic Ocean sciaenid fishes.

The resting metabolic rates (R(R)) of western North Atlantic Ocean sciaenids, such as Atlantic croaker Micropogonias undulatus, spot Leiostomus xanthurus and kingfishes Menticirrhus spp., as well as the active metabolic rates (R(A)) of M. undulatus and L. xanthurus were investigated to facilitate inter and intraspecific comparisons of their energetic ecology. The R(R) of M. undulatus and L. xanthurus were typical for fishes with similar lifestyles. The R(R) of Menticirrhus spp. were elevated relative to those of M. undulatus and L. xanthurus, but below those of high-energy-demand species such as tunas Thunnus spp. and dolphinfish Coryphaena hippurus. Repeated-measures non-linear mixed-effects models were applied to account for within-individual autocorrelation and corrected for non-constant variance typical of noisy R(A) data sets. Repeated-measures models incorporating autoregressive first-order [AR(1)] and autoregressive moving average (ARMA) covariances provided significantly superior fits, more precise parameter estimates (i.e. reduced s.e.) and y-intercept estimates that more closely approximated measured R(R) for M. undulatus and L. xanthurus than standard least-squares regression procedures.

The effect of stimulation frequency on the transmural ventricular monophasic action potential in yellowfin tuna Thunnus albacares.

Monophasic action potentials (MAPs) were recorded from the spongy and compact layers of the yellowfin tuna Thunnus albacares ventricle as stimulation frequency was increased. MAP duration decreased with increase in stimulation frequency in both the spongy and compact myocardial layers, but no significant difference in MAP duration was observed between the layers.

Responses of the red blood cells from two high-energy-demand teleosts, yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelamis), to catecholamines.

In fishes, catecholamines increase red blood cell intracellular pH through stimulation of a sodium/proton (Na+/H+) antiporter. This response can counteract potential reductions in blood O2 carrying capacity (due to Bohr and Root effects) when plasma pH and intracellular pH decrease during hypoxia, hypercapnia, or following exhaustive exercise. Tuna physiology and behavior dictate exceptionally high rates of O2 delivery to the tissues often under adverse conditions, but especially during recovery from exhaustive exercise when plasma pH may be reduced by as much as 0.4 pH units. We hypothesize that blood O2 transport during periods of metabolic acidosis could be especially critical in tunas and the response of rbc to catecholamines elevated to an extreme. We therefore investigated the in vitro response of red blood cells from yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelamis) to catecholamines. Tuna red blood cells had a typical response to catecholamines, indicated by a rapid decrease in plasma pH. Amiloride reduced the response, whereas 4,4'diisothiocyanatostilbene-2,2'-disulphonic acid enhanced both the decrease in plasma pH and the increase in intracellular pH. Changes in plasma [Na+], [Cl-], and [K+] were consistent with the hypothesis that tuna red blood cells have a Na+/H+ antiporter similar to that described for other teleost red blood cells. Red blood cells from both tuna species were more responsive to noradrenaline than adrenaline. At identical catecholamine concentrations, the decrease in plasma pH was greater in skipjack tuna blood, the more active of the two tuna species. Based on changes in plasma pH, the response of red blood cells to catecholamines from both tuna species was less than that of rainbow trout (Oncorhynchus mykiss) red blood cells, but greater than that of cod (Gadus morhua) red blood cells. Noradrenaline had no measurable influence on the O2 affinity of skipjack tuna blood and only slightly increased the O2 affinity of yellowfin tuna blood. Our results, therefore, do not support our original hypothesis. The catecholamine response of red blood cells from high-energy-demand teleosts (i.e., tunas) is not enhanced compared to other teleosts. There are data on changes in cardio-respiratory function in tunas caused by acute hypoxia and modest increases in activity, but there are no data on the changes in cardio-respiratory function in tunas accompanying the large increases in metabolic rate seen during recovery from exhaustive exercise. However, we conclude that during those instances where high rates of O2 delivery to the tissues are needed, tunas' ability to increase cardiac output, ventilation volume, blood O2 carrying capacity, and effective respiratory (i.e., gill) surface area are probably more important than are the responses of red blood cells to catecholamines. We also use our data to investigate the extent of the Haldane effect and its relationship to blood O2 and CO2 transport in yellowfin tuna. Yellowfin tuna blood shows a large Haldane effect; intracellular pH increases 0.20 units during oxygenation. The largest change in intracellular pH occurs between 40-100% O2 saturation, indicating that yellowfin tuna, like other teleosts, fully exploit the Haldane effect over the normal physiological range of blood O2 saturation.

Structural basis for oxygen delivery: muscle capillaries and manifolds in tuna red muscle.

We summarize our morphometric data on fiber vascularization and aerobic capacity in red muscle of tuna (Katsuwonus pelamis), compared to intensely aerobic flight muscles of hummingbird (Selasphorus rufus, BW 3-4 g) and bat (Eptesius fuscus, BW 15-16 g, Pipistrellus hesperus, BW 3-5 g). Three characteristic features of high flux paths for oxygen: (a) small fiber size, (b) dense capillary network and (c) high mitochondrial volume density were found in tuna, but they were not as pronounced as in hummingbird and bat flight muscles. A particular arrangement of capillary manifolds, also seen in flight muscle of birds but not in bats, was found in tuna, forming dense envelopes of capillary branches around portions of muscle fibers. However, all indexes of fiber capillarization were relatively low in tuna red muscle for its mitochondrial volume, compared with other intensely aerobic muscles. Capillary length per unit volume of mitochondria, and capillary surface per mitochondrial inner (and outer) membrane surface area, were about one half of those in hummingbird or bat flight muscles. Consistent differences exist in the size of the capillary network for the size of the mitochondrial compartment in highly aerobic red muscle of tuna compared with bird and mammal.

Physiological and behavioural thermoregulation in bigeye tuna (Thunnus obesus).

Tuna are unique among teleost fishes in being thermoconserving. Vascular counter-current heat exchangers maintain body temperatures above ambient water temperature, thereby improving locomotor muscle efficiency, especially at burst speeds and when pursuing prey below the thermocline. Because tuna also occasionally swim rapidly in warm surface waters, it has been hypothesized that tuna thermoregulate to accommodate changing activity levels or ambient temperatures. But previous field experiments have been unable to demonstrate definitively short-latency, mammalian-type physiological thermoregulation. Here we show using telemetered data that free-ranging bigeye tuna (Thunnus obesus) can rapidly alter whole-body thermal conductivity by two orders of magnitude. The heat exchangers are disengaged to allow rapid warming as the tuna ascend from cold water into warmer surface waters, and are reactivated to conserve heat when they return into the depths. Combining physiological and behavioural thermoregulation expands the foraging space of bigeye tuna into otherwise prohibitively cold, deep water.

Oxygen transport and cardiovascular responses in skipjack tuna (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) exposed to acute hypoxia.

Responses to acute hypoxia were measured in skipjack tuna (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) (approximately 1-3 kg body weight). Fish were prevented from making swimming movements by a spinal injection of lidocaine and were placed in front of a seawater delivery pipe to provide ram ventilation of the gills. Fish could set their own ventilation volumes by adjusting mouth gape. Heart rate, dorsal and ventral aortic blood pressures, and cardiac output were continuously monitored during normoxia (inhalant water (PO2 greater than 150 mmHg) and three levels of hypoxia (inhalant water PO2 approximately 130, 90, and 50 mmHg). Water and blood samples were taken for oxygen measurements in fluids afferent and efferent to the gills. From these data, various measures of the effectiveness of oxygen transfer, and branchial and systemic vascular resistance were calculated. Despite high ventilation volumes (4-7 l.min-1.kg-1), tunas extract approximately 50% of the oxygen from the inhalant water, in part because high cardiac outputs (115-132 ml.min-1.kg-1) result in ventilation/perfusion conductance ratios (0.75-1.1) close to the theoretically ideal value of 1.0. Therefore, tunas have oxygen transfer factors (ml O2.min-1.mmHg-1.kg-1) that are 10-50 times greater than those of other fishes. The efficiency of oxygen transfer from water in tunas (approximately 65%) matches that measured in teleosts with ventilation volumes an order of magnitude lower. The high oxygen transfer factors of tunas are made possible, in part, by a large gill surface area; however, this appears to carry a considerable osmoregulatory cost as the metabolic rate of gills may account for up 70% of the total metabolism in spinally blocked (i.e., non-swimming) fish. During hypoxia, skipjack and yellowfin tunas show a decrease in heart rate and increase in ventilation volume, as do other teleosts. However, in tunas hypoxic bradycardia is not accompanied by equivalent increases in stroke volume, and cardiac output falls as HR decreases. In both tuna species, oxygen consumption eventually must be maintained by drawing on substantial venous oxygen reserves. This occurs at a higher inhalant water PO2 (between 130 and 90 mmHg) in skipjack tuna than in yellowfin tuna (between 90 and 50 mmHg). The need to draw on venous oxygen reserves would make it difficult to meet the oxygen demand of increasing swimming speed, which is a common response to hypoxia in both species.(ABSTRACT TRUNCATED AT 400 WORDS)

CARDIO--a Lotus 1-2-3 based computer program for rapid calculation of cardiac output from dye or thermal dilution curves.

We have developed a menu-driven computer program (CARDIO), based on a Lotus 1-2-3 template and a series of macrocommands, that rapidly and semiautomatically calculates cardiac output from dye or thermal dilution curves. CARDIO works with any dye or thermal dilution recorder with an analog output, any analog to digital (A-to-D) conversion system, and any computer capable of running Lotus 1-2-3 version 2. No prior experience with Lotus 1-2-3 is needed to operate CARDIO, but experienced users can take full advantage of Lotus 1-2-3's graphics, data manipulation, and data retrieval capabilities.

Autocatalytic pathways to cell death: A new analysis of the tuna burn problem.

During capture and storage of tuna, a small but significant number of fish display a characteristic muscle degeneration termed tuna burn. Based on detailed amino acid analyses and on previous studies of metabolite changes during online swimming of tuna, a new model of the etiology of burnt muscle is developed. According to this model oxygen-lack to white muscle (developing initially during capture) leads to a metabolic collapse, to a drop in ATP concentration, to a consequent opening of ATP-dependent K(+) channels, with an efflux of K(+), and thus to a collapse of membrane potential. When the membrane potential falls far enough to open voltage-dependent Ca(++) channels, Ca(++) influx occurs leading to elevated Ca(++) concentrations in the cytosol. This process is augmented by simultaneous movement of Ca(++) from sarcoplasmic reticulum (SR) and from mitochondria into the cytosol. At high intracellular concentrations Ca(++) can be devastating. One of its more notable effects involves the activation of Ca(++)-dependent proteases, which preferentially target key components of the contractile machinery (troponins, tropomyosin, C-protein, M-protein, Z-discs, α-actinin) and thus cause disassembly of myofilaments prior to any significant hydrolysis of myosin or actin. This process is autocatalytic in the sense that Ca(++)-activated proteases may act upon SR, thus increasing Na(+) /Ca(++) exchange, and ultimately adding more Ca(++) to the cytosolic pool. According to this model, the difference between burnt and unburnt regions of the myotome is simply due to how far each region has moved along this self-destructive, autocatalytic pathway. The model is helpful in explaining previously perplexing data and in making useful (i.e. measurable) predictions for further studies of this important problem.

Oxygen sensitive afferent information arising from the first gill arch of yellowfin tuna.

Single nerve fiber discharge was recorded from O2 sensitive receptors in the first gill arch of the yellowfin tuna, Thunnus albacares, in vitro. These receptors were innervated by the vagus nerve and increased their discharge in response to decreasing perfusion rate, decreasing perfusion PO2 and, in most fibers, to decreasing external PO2. Fibers responding to environmental hypoxia exhibited an exponential increase in discharge to decreasing external PO2 with a sensitivity similar to that exhibited by cat carotid body chemoreceptors. Indirect evidence suggests that these receptors are located near the gill vasculature and are more sensitive to changes in arterial PO2 than water PO2. Their response characteristics and hypoxic sensitivity strongly implicate them as the afferent limb in the cardiac responses and perhaps also the ventilatory responses exhibited by tuna to environmental hypoxia.

Mammalian metabolite flux rates in a teleost: lactate and glucose turnover in tuna.

Lactate and glucose turnover rates were measured by bolus injection of [U-14C]lactate and [6-3H]glucose in cannulated lightly anesthetized skipjack tuna, Katsuwonus pelamis. Our goals were to find out whether the high rates of lactate clearance reported during recovery from burst swimming in tuna could be accounted for by high blood lactate fluxes; to extend the observed correlation between lactate turnover and lactate concentration in mammals to a nonmammalian system, and to assess the importance of lactate and glucose as metabolic fuels in tuna and to compare their flux rates with values reported for mammals. Measured lactate turnover rates ranged from 112 to 431 mumol X min-1 X kg-1 and were correlated with blood lactate concentration. Glucose turnover rate averaged 15.3 mumol X min-1 X kg-1. When correcting for body mass and temperature, skipjack tuna has at least as high or even higher lactate turnover rates than those recorded for mammals. Tuna glucose turnover rate is similar to that of mammals but much higher than levels found in other teleosts. Even the highest lactate turnover rate measured in tuna cannot fully account for the rate of blood lactate clearance observed during recovery, suggesting that some of the lactate produced in skeletal muscle must be metabolized in situ. After injection of [U-14C]lactate, less than 5% of the total blood activity was recovered in glucose, suggesting that the Cori cycle is not an important pathway of lactate metabolism in tuna.

Effects of temperature change on acid-base regulation in skipjack tuna (Katsuwonus pelamis) blood.

The effects of temperature change (in vitro) on acid-base balance of skipjack tuna blood were investigated. By examining the relationship between blood pH and temperature (in vitro) under conditions of constant CO2 tension (open system), it was observed that dpH/dT = -0.013 U/degrees C. This value falls well within the range of in vivo values reported for other ectothermic vertebrates, and is only slightly different than results obtained in vitro under conditions of constant CO2 content (closed system; dpH/dT = -0.0165 U/degrees C). It is concluded that changes in pH following temperature changes can be accounted for solely by the passive, in vitro behaviour of the chemical buffer system found in the blood, so that active regulatory mechanisms of pH adjustment need not be postulated for skipjack tuna.

On the stability of Innovar, a neuroleptic analgesic, for cardiovascular experiments.

The effects of 0.2 and 0.5 mL/kg Innovar (a neuroleptic analgesic) on cardiovascular functions and reflexes in rabbits were measured. We recorded the effects of Innovar on arterial pressure, heart rate, respiration rate, ventricular contractility, arterial oxygen tension, as well as the drug's effects on the bradycardia and vasoconstrictor response to cigarette smoke stimulation of the nose (the so-called "nasopharyngeal reflex"). In animals given 0.2 mL/kg Innovar, all steady state cardiovascular variables had returned to pre-Innovar levels in 45 min, as had the efficacy of the nasopharyngeal reflex. In animals given 0.5 mL/kg Innovar, all steady state cardiovascular variables, except PaO2, were slightly but significantly depressed for up to 135 min after injection. The nasopharyngeal reflex returned to normal within 90 min. Because of the calmative and analgesic effects of Innovar, and its only moderate effects on cardiovascular functions and reflexes, we feel it is a suitable analgesic agent for cardiovascular experiments where neither fully conscious nor surgically anesthetized animals can be used.