Effect of acute exposure to settleable atmospheric particulate matter emitted by the steel industry on hematology and innate immunity of fat snook (Centropomus parallelus).

The steel industry is a significant worldwide source of atmospheric particulate matter (PM). Part of PM may settle (SePM) and deposit metal/metalloid and metallic nanoparticles in aquatic ecosystems. However, such an air-to-water cross-contamination is not observed by most monitoring agencies. The region of Vitoria City is the main location of iron processing for exports in Brazil, and it has rivers, estuaries, and coastal areas affected by SePM. We have evaluated the effects of SePM on a local representative fish species, the fat snook, Centropomus parallelus. After acclimation, 48 fishes (61.67 ± 27.83 g) were individually exposed for 96 h to diverse levels of SePM (0.0, 0.01, 0.1 and 1 g/L-1). The presence of metals in the blood and several blood biomarkers were analyzed to evaluate the impact of SePM on stress signaling, blood oxygen transport capacity, and innate immune activity. Metal bioaccumulation was measured from blood in two separately analyzed compartments: intracellular (erythrocytes plus white blood cells) and extracellular (plasma). The major metals present at all contamination levels in both compartments were Fe and Zn, followed by Al and Cu, plus traces of 'Emerging metals': Ba, Ce, La, Rb, Se, Sr, and Ti. Emerging metals refer to those that have recently been identified in water as contaminants, encompassing rare earth elements and critical technology elements, as documented in previous studies (See REEs and TCEs in Cobelo-García et al., 2015; Batley et al., 2022). Multivariate analysis revealed that SePM had strong, dose-dependent correlations with all biomarker groups and indicated that blood oxygen-carrying capacity had the highest contamination responsiveness. Metal contamination also increased cortisol and blood glucose levels, attesting to increased stress signaling, and had a negative effect on innate immune activity. Knowledge of the risks related to SePM contamination remains rudimentary. However, the fact that there was metal bioaccumulation, causing impairment of fundamental physiological and cellular processes in this ecologically relevant fish species, consumed by the local human population, highlights the pressing need for further monitoring and eventual control of SePM contamination.

Tolerance of an acute warming challenge declines with body mass in Nile tilapia: evidence of a link to capacity for oxygen uptake.

It has been proposed that larger individuals within fish species may be more sensitive to global warming, as a result of limitations in their capacity to provide oxygen for aerobic metabolic activities. This could affect size distributions of populations in a warmer world but evidence is lacking. In Nile tilapia Oreochromis niloticus (n=18, mass range 21-313 g), capacity to provide oxygen for aerobic activities (aerobic scope) was independent of mass at an acclimation temperature of 26°C. Tolerance of acute warming, however, declined significantly with mass when evaluated as the critical temperature for fatigue from aerobic swimming (CTSmax). The CTSmax protocol challenges a fish to meet the oxygen demands of constant aerobic exercise while their demands for basal metabolism are accelerated by incremental warming, culminating in fatigue. CTSmax elicited pronounced increases in oxygen uptake in the tilapia but the maximum rates achieved prior to fatigue declined very significantly with mass. Mass-related variation in CTSmax and maximum oxygen uptake rates were positively correlated, which may indicate a causal relationship. When fish populations are faced with acute thermal stress, larger individuals may become constrained in their ability to perform aerobic activities at lower temperatures than smaller conspecifics. This could affect survival and fitness of larger fish in a future world with more frequent and extreme heatwaves, with consequences for population productivity.

Using aerobic exercise to evaluate sub-lethal tolerance of acute warming in fishes.

We investigated whether fatigue from sustained aerobic swimming provides a sub-lethal endpoint to define tolerance of acute warming in fishes, as an alternative to loss of equilibrium (LOE) during a critical thermal maximum (CTmax) protocol. Two species were studied, Nile tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus). Each fish underwent an incremental swim test to determine gait transition speed (UGT), where it first engaged the unsteady anaerobic swimming mode that preceded fatigue. After suitable recovery, each fish was exercised at 85% of their own UGT and warmed 1°C every 30 min, to identify the temperature at which they fatigued, denoted as CTswim Fish were also submitted to a standard CTmax, warming at the same rate as CTswim, under static conditions until LOE. All individuals fatigued in CTswim, at a mean temperature approximately 2°C lower than their CTmax Therefore, if exposed to acute warming in the wild, the ability to perform aerobic metabolic work would be constrained at temperatures significantly below those that directly threatened survival. The collapse in performance at CTswim was preceded by a gait transition qualitatively indistinguishable from that during the incremental swim test. This suggests that fatigue in CTswim was linked to an inability to meet the tissue oxygen demands of exercise plus warming. This is consistent with the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis, regarding the mechanism underlying tolerance of warming in fishes. Overall, fatigue at CTswim provides an ecologically relevant sub-lethal threshold that is more sensitive to extreme events than LOE at CTmax.

Interactive effects of mercury exposure and hypoxia on ECG patterns in two Neotropical freshwater fish species: Matrinxã, Brycon amazonicus and traíra, Hoplias malabaricus.

Hypoxia and mercury contamination often co-occur in tropical freshwater ecosystems, but the interactive effects of these two stressors on fish populations are poorly known. The effects of mercury (Hg) on recorded changes in the detailed form of the electrocardiogram (ECG) during exposure to progressive hypoxia were investigated in two Neotropical freshwater fish species, matrinxã, Brycon amazonicus and traíra, Hoplias malabaricus. Matrinxã were exposed to a sublethal concentration of 0.1 mg L-1 of HgCl2 in water for 96 h. Traíra were exposed to dietary doses of Hg by being fed over a period of 30 days with juvenile matrinxãs previously exposed to HgCl2, resulting in a dose of 0.45 mg of total Hg per fish, each 96 h. Both species showed a bradycardia in progressive hypoxia. Hg exposure impaired cardiac electrical excitability, leading to first-degree atrioventricular block, plus profound extension of the ventricular action potential (AP) plateau. Moreover, there was the development of cardiac arrhythmias and anomalies such as occasional absence of QRS complexes, extra systoles, negative Q-, R- and S-waves (QRS complex), and T wave inversion, especially in hypoxia below O2 partial pressures (PO2) of 5.3 kPa. Sub-chronic dietary Hg exposure induced intense bradycardia in normoxia in traira, plus lengthening of ventricular AP duration coupled with prolonged QRS intervals. This indicates slower ventricular AP conduction during ventricular depolarization. Overall, the data indicate that both acute waterborne and sub-chronic dietary exposure (trophic level transfer), at sublethal concentrations of mercury, cause damage in electrical stability and rhythm of the heartbeat, leading to myocardial dysfunction, which is further intensified during hypoxia. These changes could lead to impaired cardiac output, with consequences for swimming ability, foraging capacity, and hence growth and/or reproductive performance.

Heart rate variability in the tegu lizard, Salvator merianae, its neuroanatomical basis and role in the assessment of recovery from experimental manipulation.

Using long-term, remote recordings of heart rate (fH) on fully recovered, undisturbed lizards, we identified several components of heart rate variability (HRV) associated with respiratory sinus arrhythmia (RSA): 1.) A peak in the spectral representation of HRV at the frequency range of ventilation. 2.) These cardiorespiratory interactions were shown to be dependent on the parasympathetic arm of the autonomic nervous system. 3.) Vagal preganglionic neurons are located in discrete groups located in the dorsal motor nucleus of the vagus and also, in a ventro-lateral group, homologous to the nucleus ambiguus of mammals. 4.) Myelinated nerve fibers in the cardiac vagus enabling rapid communication between the central nervous system and the heart. Furthermore, the study of the progressive recovery of fH in tegu following anesthesia and instrumentation revealed that 'resting' levels of mean fH and reestablishment of HRV occurred over different time courses. Accordingly, we suggest that, when an experiment is designed to study a physiological variable reliant on autonomic modulation at its normal, resting level, then postsurgical reestablishment of HRV should be considered as the index of full recovery, rather than mean fH.

Respiratory sinus arrhythmia is a major component of heart rate variability in undisturbed, remotely monitored rattlesnakes, Crotalus durissus.

ECG recordings were obtained using an implanted telemetry device from the South American rattlesnake, Crotalus durissus, held under stable conditions without restraining cables or interaction with researchers. Mean heart rate (fH) recovered rapidly (<24 h) from anaesthesia and operative procedures. This preceded a more gradual development of heart rate variability (HRV), with instantaneous fH increasing during each lung ventilation cycle. Atropine injection increased mean fH and abolished HRV. Complete autonomic blockade revealed a cholinergic tonus on the heart of 55% and an adrenergic tonus of 37%. Power spectral analysis of HRV identified a peak at the same frequency as ventilation. This correlation was sustained after temperature changes and it was more evident, marked by a more prominent power spectrum peak, when ventilation is less episodic. This HRV component is homologous to that observed in mammals, termed respiratory sinus arrhythmia (RSA). Evidence for instantaneous control of fH indicated rapid conduction of activity in the cardiac efferent nervous supply, as supported by the description of myelinated fibres in the cardiac vagus. Establishment of HRV 10 days after surgical intervention seems a reliable indicator of the re-establishment of control of integrative functions by the autonomic nervous system. We suggest that this criterion could be applied to other animals exposed to natural or imposed trauma, thus improving protocols involving animal handling, including veterinarian procedures.

Convective oxygen transport during development in embryos of the snapping turtle Chelydra serpentina.

This study investigated the maturation of convective oxygen transport in embryos of the snapping turtle (Chelydra serpentina). Measurements included: mass, oxygen consumption (V̇O2 ), heart rate, blood oxygen content and affinity and blood flow distribution at 50%, 70% and 90% of the incubation period. Body mass increased exponentially, paralleled by increased cardiac mass and metabolic rate. Heart rate was constant from 50% to 70% incubation but was significantly reduced at 90% incubation. Hematocrit and hemoglobin concentration were constant at the three points of development studied but arteriovenous difference doubled from 50% to 90% incubation. Oxygen affinity was lower for the early 50% incubation group (stage 19) compared with all other age groups. Blood flow was directed predominantly to the embryo but was highest to the chorioallantoic membrane (CAM) at 70% incubation and was directed away from the yolk as it was depleted at 90% incubation. The findings indicate that the plateau or reduction in egg V̇O2  characteristic of the late incubation period of turtle embryos may be related to an overall reduction in mass-specific V̇O2  that is correlated with decreasing relative heart mass and plateaued CAM blood flow. Importantly, if the blood properties remain unchanged prior to hatching, as they did during the incubation period studied in the current investigation, this could account for the pattern of V̇O2 previously reported for embryonic snapping turtles prior to hatching.

Cardiorespiratory interactions previously identified as mammalian are present in the primitive lungfish.

The present study has revealed that the lungfish has both structural and functional features of its system for physiological control of heart rate, previously considered solely mammalian, that together generate variability (HRV). Ultrastructural and electrophysiological investigation revealed that the nerves connecting the brain to the heart are myelinated, conferring rapid conduction velocities, comparable to mammalian fibers that generate instantaneous changes in heart rate at the onset of each air breath. These respiration-related changes in beat-to-beat cardiac intervals were detected by complex analysis of HRV and shown to maximize oxygen uptake per breath, a causal relationship never conclusively demonstrated in mammals. Cardiac vagal preganglionic neurons, responsible for controlling heart rate via the parasympathetic vagus nerve, were shown to have multiple locations, chiefly within the dorsal vagal motor nucleus that may enable interactive control of the circulatory and respiratory systems, similar to that described for tetrapods. The present illustration of an apparently highly evolved control system for HRV in a fish with a proven ancient lineage, based on paleontological, morphological, and recent genetic evidence, questions much of the anthropocentric thinking implied by some mammalian physiologists and encouraged by many psychobiologists. It is possible that some characteristics of mammalian respiratory sinus arrhythmia, for which functional roles have been sought, are evolutionary relics that had their physiological role defined in ancient representatives of the vertebrates with undivided circulatory systems.

Vagal tone regulates cardiac shunts during activity and at low temperatures in the South American rattlesnake, Crotalus durissus.

The undivided ventricle of non-crocodilian reptiles allows for intracardiac admixture of oxygen-poor and oxygen-rich blood returning via the atria from the systemic circuit and the lungs. The distribution of blood flow between the systemic and pulmonary circuits may vary, based on differences between systemic and pulmonary vascular conductances. The South American rattlesnake, Crotalus durissus, has a single pulmonary artery, innervated by the left vagus. Activity in this nerve controls pulmonary conductance so that left vagotomy abolishes this control. Experimental left vagotomy to abolish cardiac shunting had no effect on long-term survival and failed to identify a functional role in determining metabolic rate, growth or resistance to food deprivation. Accordingly, the present investigation sought to evaluate the extent to which cardiac shunt patterns are actively controlled during changes in body temperature and activity levels. We compared hemodynamic parameters between intact and left-vagotomized rattlesnakes held at different temperatures and subjected to enforced physical activity. Increased temperature and enforced activity raised heart rate, cardiac output, pulmonary and systemic blood flow in both groups, but net cardiac shunt was reversed in the vagotomized group at lower temperatures. We conclude that vagal control of pulmonary conductance is an active mechanism regulating cardiac shunts in C. durissus.

The progressive onset of cholinergic and adrenergic control of heart rate during development in the green iguana, Iguana iguana.

The autonomic control of heart rate was studied throughout development in embryos of the green iguana, Iguana iguana by applying receptor agonists and antagonists of the parasympathetic and sympathetic systems. Acetylcholine (Ach) slowed or stopped the heart and atropine antagonized the response to Ach indicating the presence of muscarinic cholinoceptors on the heart of early embryos. However, atropine injections had no impact on heart rate until immediately before hatching, when it increased heart rate by 15%. This cholinergic tonus increased to 34% in hatchlings and dropped to 24% in adult iguanas. Although epinephrine was without effect, injection of propranolol slowed the heart throughout development, indicating the presence of ß-adrenergic receptors on the heart of early embryos, possibly stimulated by high levels of circulating catecholamines. The calculated excitatory tonus varied between 33% and 68% until immediately before hatching when it fell to 25% and 29%, a level retained in hatchlings and adults. Hypoxia caused a bradycardia in early embryos that was unaffected by injection of atropine indicating that hypoxia has a direct effect upon the heart. In later embryos and hatchlings hypoxia caused a tachycardia that was unaffected by injection of atropine. Subsequent injection of propranolol reduced heart rate both uncovering a hypoxic bradycardia in late embryos and abolishing tachycardia in hatchlings. Hypercapnia was without effect on heart rate in late stage embryos and in hatchlings.

An appraisal of the use of an infrared digital monitoring system for long-term measurement of heart rate in reptilian embryos.

Measurement of heart rate (fH) in embryonic reptiles has previously imposed some degree of invasive treatment on the developing embryo. Recently a non-invasive technique of fH detection from intact eggs was developed for commercial avian breeders and has since been used in biological research. This device uses infrared light, enabling it to detect heartbeats in very early embryos. However, infrared light is a source of heat and extended enclosure of an egg in the device is likely to affect temperature with consequent effects on physiological processes, including fH. We studied the effect of use of the monitor on the temperature of eggs and on fH in two species of reptiles, the snapping turtle (Chelydra serpentina) and the green iguana (Iguana iguana). Egg temperature increased from a room temperature of 27-28 °C, by 26% in turtles and 14% in iguanas over 1h of enclosure, resulting in an increase in fH of 76-81% in turtles and 35-50% iguanas. These effects on fH can either be avoided by brief enclosure of each egg in the monitor or measured and accounted for during the design of long-term experiments.

The phylogeny and ontogeny of autonomic control of the heart and cardiorespiratory interactions in vertebrates.

Heart rate in vertebrates is controlled by activity in the autonomic nervous system. In spontaneously active or experimentally prepared animals, inhibitory parasympathetic control is predominant and is responsible for instantaneous changes in heart rate, such as occur at the first air breath following a period of apnoea in discontinuous breathers like inactive reptiles or species that surface to air breathe after a period of submersion. Parasympathetic control, exerted via fast-conducting, myelinated efferent fibres in the vagus nerve, is also responsible for beat-to-beat changes in heart rate such as the high frequency components observed in spectral analysis of heart rate variability. These include respiratory modulation of the heartbeat that can generate cardiorespiratory synchrony in fish and respiratory sinus arrhythmia in mammals. Both may increase the effectiveness of respiratory gas exchange. Although the central interactions generating respiratory modulation of the heartbeat seem to be highly conserved through vertebrate phylogeny, they are different in kind and location, and in most species are as yet little understood. The heart in vertebrate embryos possesses both muscarinic cholinergic and ß-adrenergic receptors very early in development. Adrenergic control by circulating catecholamines seems important throughout development. However, innervation of the cardiac receptors is delayed and first evidence of a functional cholinergic tonus on the heart, exerted via the vagus nerve, is often seen shortly before or immediately after hatching or birth, suggesting that it may be coordinated with the onset of central respiratory rhythmicity and subsequent breathing.

Ablation of the ability to control the right-to-left cardiac shunt does not affect oxygen uptake, specific dynamic action or growth in the rattlesnake Crotalus durissus.

The morphologically undivided ventricle of the heart in non-crocodilian reptiles permits the mixing of oxygen-rich blood returning from the lungs and oxygen-poor blood from the systemic circulation. A possible functional significance for this intra-cardiac shunt has been debated for almost a century. Unilateral left vagotomy rendered the single effective pulmonary artery of the South American rattlesnake, Crotalus durissus, unable to adjust the magnitude of blood flow to the lung. The higher constant perfusion of the lung circulation and the incapability of adjusting the right-left shunt in left-denervated snakes persisted over time, providing a unique model for investigation of the long-term consequences of cardiac shunting in a squamate. Oxygen uptake recorded at rest and during spontaneous and forced activity was not affected by removing control of the cardiac shunt. Furthermore, metabolic rate and energetic balance during the post-prandial metabolic increment, plus the food conversion efficiency and growth rate, were all similarly unaffected. These results show that control of cardiac shunting is not associated with a clear functional advantage in adjusting metabolic rate, effectiveness of digestion or growth rates.

The contribution of air breathing to aerobic scope and exercise performance in the banded knifefish Gymnotus carapo L.

The contribution of air breathing to aerobic metabolic scope and exercise performance was investigated in a teleost with bimodal respiration, the banded knifefish, submitted to a critical swimming speed (U(crit)) protocol at 30°C. Seven individuals (mean ± s.e.m. mass 89±7 g, total length 230±4 mm) achieved a U(crit) of 2.1±1 body lengths (BL) s(-1) and an active metabolic rate (AMR) of 350±21 mg kg(-1) h(-1), with 38±6% derived from air breathing. All of the knifefish exhibited a significant increase in air-breathing frequency (f(AB)) with swimming speed. If denied access to air in normoxia, these individuals achieved a U(crit) of 2.0±0.2 BL s(-1) and an AMR of 368±24 mg kg(-1) h(-1) by gill ventilation alone. In normoxia, therefore, the contribution of air breathing to scope and exercise was entirely facultative. In aquatic hypoxia (P(O(2))=4 kPa) with access to normoxic air, the knifefish achieved a U(crit) of 2.0±0.1 BL s(-1) and an AMR of 338±29 mg kg(-1) h(-1), similar to aquatic normoxia, but with 55±5% of AMR derived from air breathing. Indeed, f(AB) was higher than in normoxia at all swimming speeds, with a profound exponential increase during exercise. If the knifefish were denied access to air in hypoxia, U(crit) declined to 1.2±0.1 BL s(-1) and AMR declined to 199±29 mg kg(-1) h(-1). Therefore, air breathing allowed the knifefish to avoid limitations to aerobic scope and exercise performance in aquatic hypoxia.

Evidence for a respiratory component, similar to mammalian respiratory sinus arrhythmia, in the heart rate variability signal from the rattlesnake, Crotalus durissus terrificus.

Autonomic control of heart rate variability and the central location of vagal preganglionic neurones (VPN) were examined in the rattlesnake (Crotalus durissus terrificus), in order to determine whether respiratory sinus arrhythmia (RSA) occurred in a similar manner to that described for mammals. Resting ECG signals were recorded in undisturbed snakes using miniature datalogging devices, and the presence of oscillations in heart rate (fh) was assessed by power spectral analysis (PSA). This mathematical technique provides a graphical output that enables the estimation of cardiac autonomic control by measuring periodic changes in the heart beat interval. At fh above 19 min(-1) spectra were mainly characterised by low frequency components, reflecting mainly adrenergic tonus on the heart. By contrast, at fh below 19 min(-1) spectra typically contained high frequency components, demonstrated to be cholinergic in origin. Snakes with a fh >19 min(-1) may therefore have insufficient cholinergic tonus and/or too high an adrenergic tonus acting upon the heart for respiratory sinus arrhythmia (RSA) to develop. A parallel study monitored fh simultaneously with the intraperitoneal pressures associated with lung inflation. Snakes with a fh<19 min(-1) exhibited a high frequency (HF) peak in the power spectrum, which correlated with ventilation rate (fv). Adrenergic blockade by propranolol infusion increased the variability of the ventilation cycle, and the oscillatory component of the fh spectrum broadened accordingly. Infusion of atropine to effect cholinergic blockade abolished this HF component, confirming a role for vagal control of the heart in matching fh and fv in the rattlesnake. A neuroanatomical study of the brainstem revealed two locations for vagal preganglionic neurones (VPN). This is consistent with the suggestion that generation of ventilatory components in the heart rate variability (HRV) signal are dependent on spatially distinct loci for cardiac VPN. Therefore, this study has demonstrated the presence of RSA in the HRV signal and a dual location for VPN in the rattlesnake. We suggest there to be a causal relationship between these two observations.

The role of nitric oxide in regulation of the cardiovascular system in reptiles.

The roles that nitric oxide (NO) plays in the cardiovascular system of reptiles are reviewed, with particular emphasis on its effects on central vascular blood flows in the systemic and pulmonary circulations. New data is presented that describes the effects on hemodynamic variables in varanid lizards of exogenously administered NO via the nitric oxide donor sodium nitroprusside (SNP) and inhibition of nitric oxide synthase (NOS) by l-nitroarginine methyl ester (l-NAME). Furthermore, preliminary data on the effects of SNP on hemodynamic variables in the tegu lizard are presented. The findings are compared with previously published data from our laboratory on three other species of reptiles: pythons (), rattlesnakes () and turtles (). These five species of reptiles possess different combinations of division of the heart and structural complexity of the lungs. Comparison of their responses to NO donors and NOS inhibitors may reveal whether the potential contribution of NO to vascular tone correlates with pulmonary complexity and/or with blood pressure. All existing studies on reptiles have clearly established a potential role for NO in regulating vascular tone in the systemic circulation and NO may be important for maintaining basal systemic vascular tone in varanid lizards, pythons and turtles, through a continuous release of NO. In contrast, the pulmonary circulation is less responsive to NO donors or NOS inhibitors, and it was only in pythons and varanid lizards that the lungs responded to SNP. Both species have a functionally separated heart, so it is possible that NO may exert a larger role in species with low pulmonary blood pressures, irrespective of lung complexity.

The role of nitric oxide in the regulation of the systemic and pulmonary vasculature of the rattlesnake, Crotalus durissus terrificus.

The functional role of nitric oxide (NO) was investigated in the systemic and pulmonary circulations of the South American rattlesnake, Crotalus durissus terrificus. Bolus, intra-arterial injections of the NO donor, sodium nitroprusside (SNP) caused a significant systemic vasodilatation resulting in a reduction in systemic resistance (Rsys). This response was accompanied by a significant decrease in systemic pressure and a rise in systemic blood flow. Pulmonary resistance (Rpul) remained constant while pulmonary pressure (Ppul) and pulmonary blood flow (Qpul) decreased. Injection of L-Arginine (L-Arg) produced a similar response to SNP in the systemic circulation, inducing an immediate systemic vasodilatation, while Rpul was unaffected. Blockade of NO synthesis via the nitric oxide synthase inhibitor, L-NAME, did not affect haemodynamic variables in the systemic circulation, indicating a small contribution of NO to the basal regulation of systemic vascular resistance. Similarly, Rpul and Qpul remained unchanged, although there was a significant rise in Ppul. Via injection of SNP, this study clearly demonstrates that NO causes a systemic vasodilatation in the rattlesnake, indicating that NO may contribute in the regulation of systemic vascular resistance. In contrast, the pulmonary vasculature seems far less responsive to NO.