Use of complex physiological traits as ecotoxicological biomarkers in tropical freshwater fishes.

We review the use of complex physiological traits, of tolerance and performance, as biomarkers of the toxicological effects of contaminants in subtropical and tropical freshwater fishes. Such traits are growing in relevance due to climate change, as exposure to contaminants may influence the capacity of fishes to tolerate and perform in an increasingly stressful environment. We review the evidence that the critical oxygen level, a measure of hypoxia tolerance, provides a valuable biomarker of impacts of diverse classes of contaminants. When coupled with measures of cardiorespiratory variables, it can provide insight into mechanisms of toxicity. The critical thermal maximum, a simple measure of tolerance of acute warming, also provides a valuable biomarker despite a lack of understanding of its mechanistic basis. Its relative ease of application renders it useful in the rapid evaluation of multiple species, and in understanding how the severity of contaminant impacts depends upon prevailing environmental temperature. The critical swimming speed is a measure of exercise performance that is widely used as a biomarker in temperate species but very few studies have been performed on subtropical or tropical fishes. Overall, the review serves to highlight a critical lack of knowledge for subtropical and tropical freshwater fishes. There is a real need to expand the knowledge base and to use physiological biomarkers in support of decision making to manage tropical freshwater fish populations and their habitats, which sustain rich biodiversity but are under relentless anthropogenic pressure.

Effects of change in temperature on the cardiac contractility of broad-snouted caiman (Caiman latirostris) during digestion.

In many reptiles, digestion has been associated with the selection of higher body temperatures, the so-called post-prandial thermophilic response. This study aimed to investigate the excitation-contraction (E-C) coupling in postprandial broad-snouted caimans (Caiman latirostris) in response to acute warming within a preferred body temperature range of crocodiles. Isometric preparations subjected to a temperature transition from 25°C to 30°C were used to investigate myocardial contractility of postprandial caimans, that is, 48 h after the animals ingested a rodent meal corresponding to 15% of body mass. The caiman heart exhibits a negative force-frequency relationship that is independent of the temperature. At 25°C, cardiac muscle was able to maintain a constant force up to 36 bpm, above which it decreased significantly, reaching minimum values at the highest frequency of 84 bpm. Moreover, E-C coupling is predominantly dependent on transsarcolemmal Ca2+ transport denoted by the lack of significant ryanodine effects on force generation. On the contrary, ventricular strips at 30°C were able to sustain the cardiac contractility at higher pacing frequencies (from 12 to 144 bpm) due to an important role of Na+ /Ca2+ exchanger in Ca2+ cycling, as indicated by the decay of the post-rest contraction, and a significant contribution of the sarcoplasmic reticulum above 72 bpm. Our results demonstrated that the myocardium of postprandial caimans exhibits a significant degree of thermal plasticity of E-C coupling during acute warming. Therefore, myocardial contractility can be maximized when postprandial broad-snouted caimans select higher body temperatures (preferred temperature zone) following feeding.

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.

Impact of waterborne and trophic mercury exposures on cardiac function of two ecologically distinct Neotropical freshwater fish Brycon amazonicus and Hoplias malabaricus.

Metal pollutants have been considered one of the main factors underlying the depletion of biodiversity in natural populations unbalancing aquatic environments. The aim of this study was to evaluate the effects of exposure to inorganic Hg on myocardial contractility and the electrocardiogram (ECG) of two ecologically distinct Neotropical fish species, namely: matrinxã (Brycon amazonicus) and trahira (Hoplias malabaricus). Matrinxãs were exposed to a sublethal concentration of 0.1mgL-1 of Hg in water for 96h. Trahiras were exposed to dietary Hg doses (0.45mg of Hg, each 4days, for 30days) using juvenile B. amazonicus as the prey vehicle. Hg exposures decreased myocardial isometric twitch force development, harmed contraction/relaxation dynamics and cardiac pumping capacity (CPC), and reduced the relative contribution of the calcium stored in the sarcoplasmic reticulum (SR) to excitation contraction (EC) coupling in both fish species. Analysis of the ECG revealed that Hg impaired electrical conduction across the heart, inducing first degree atrioventricular block and lengthening the plateau phase of action potential duration. In trahira trophic doses of Hg induced a marked bradycardia, increasing the duration of the ventricular action potential and delaying atrial and ventricular depolarization. These findings indicate that both acute and long-term Hg exposure, by different routes is cardiotoxic to matrinxã and trahira. Hg potently impaired intracellular calcium kinetics in the cardiomyocytes, myocardium contractility, and electrical conduction across the heart, all of which can be implicated in decreased cardiac output and putative heart failure.

Impact of an environmental relevant concentration of 17α-ethinylestradiol on the cardiac function of bullfrog tadpoles.

This study evaluated if a concentration of 17α-ethinylestradiol (EE2 - 10 ng L(-1) for 96 h) normally found in Brazilian surface waters exerts any impact on cardiac function of bullfrog tadpoles (25 Gosner stage), Lithobates catesbeianus. During exposure, the animals' activity level (AL -% of active individuals) was monitored twice a day. Then, the in loco heart rate (f(H) - bpm) was determined, as well as the relative ventricular mass (RVM - % of body mass). Afterwards, cardiac ventricles were mounted for isometric force recordings (CS - mN mm(-2)), and determination of the cardiac pumping capacity (CPC - mN mm(-2) min(-1)). EE2 did not affect tadpoles' AL, although it resulted in a tachycardia in animals exposed to EE2 (f(H) = 66 bpm) when compared to controls (f(H) = 52 bpm), suggesting that EE2 acts directly on the cardiac muscle of tadpoles, rather than being a result of an increased cardiac demand due to a higher activity level (i.e., avoidance response). Additionally, EE2 exerted a positive inotropic response, which resulted in a higher CPC, which occurred independently of an increase in the number of myofibrils of EE2-exposed animals, since RVM remained similar between experimental groups. Thus, the increase on cardiac demand induced by the exposure to EE2 elevates considerably the animal energy expenditure, diverting a large amount of energy that tadpoles could use for their growth and development. These alterations can make amphibians more susceptible to predators and reduce the likelihood to reach reproductive stage.

Branchial O(2) chemoreceptors in Nile tilapia Oreochromis niloticus: Control of cardiorespiratory function in response to hypoxia.

This study examined the distribution and orientation of gill O(2) chemoreceptors in Oreochromis niloticus and their role in cardiorespiratory responses to graded hypoxia. Intact fish, and a group with the first gill arch excised (operated), were submitted to graded hypoxia and their cardiorespiratory responses (oxygen uptake - V˙O(2) , breathing frequency - fR, ventilatory stroke volume - VT, gill ventilation - V˙G, O(2) extraction from the ventilatory current - EO(2) , and heart rate - fH) were compared. Their responses to bolus injections of NaCN into the bloodstream (internal) or ventilatory water stream (external) were also determined. The V˙O(2) of operated fish was significantly lower at the deepest levels of hypoxia. Neither reflex bradycardia nor ventilatory responses were completely abolished by bilateral excision of the first gill arch. EO(2) of the operated group was consistently lower than the intact group. The responses to internal and external NaCN included transient decreases in fH and increases in fR and Vamp (ventilation amplitude). These cardiorespiratory responses were attenuated but not abolished in the operated group, indicating that chemoreceptors are not restricted to the first gill arch, and are sensitive to oxygen levels in both blood and water.

The relationship between O2 chemoreceptors, cardio-respiratory reflex and hypoxia tolerance in the neotropical fish Hoplias lacerdae.

The localization, distribution and orientation of O(2) chemoreceptors associated with the control of cardio-respiratory responses were investigated in the neotropical, Hoplias lacerdae. Selective denervation of the cranial nerves (IX and X) was combined with chemical stimulation (NaCN) to characterize the gill O(2) chemoreceptors, and the fish were then exposed to gradual hypoxia to examine the extent of each cardio-respiratory response. Changes in heart rate (f(H)) and ventilation amplitude (V(amp)) were allied with chemoreceptors distributed on both internal and external surfaces of all gill arches, while ventilation rate (f) was allied to the O(2) chemoreceptors located only in the internal surface of the first gill arch. H. lacerdae exposed to gradual hypoxia produced a marked bradycardia (45%) and 50% increase in V(amp), but only a relatively small change in f (32%). Thus, the low f(R) response yet high V(amp) were in accord with the characterization of the O(2) chemoreceptors. Comparing these results from H. lacerdae with hypoxia-tolerant species revealed a relationship existent between general oxygenation of the individual species environment, its cardio-respiratory response to hypoxia and the characterization of O(2) chemoreceptors.

Oxidative stress biomarkers and heart function in bullfrog tadpoles exposed to Roundup Original.

Oxidative stress biomarkers, in vivo heart rate (f (H)), and contraction dynamics of ventricle strips of bullfrog (Lithobates catesbeiana) tadpoles were evaluated after 48 h of exposure to a sub-lethal concentration (1 ppm) of the herbicide Roundup Original (glyphosate 41%). The activities of the antioxidant enzymes superoxide dismutase and catalase were increased in the liver and decreased in muscle, while oxidative damage to lipids increased above control values in both tissues, showing that the generation of reactive oxygen species and oxidative stress are involved in the toxicity induced by Roundup. Additionally, tadpoles' hyperactivity was associated with tachycardia in vivo, probably due to a stress-induced adrenergic stimulation. Ventricle strips of Roundup-exposed tadpoles (R-group) presented a faster relaxation and also a higher cardiac pumping capacity at the in vivo contraction frequency, indicating that bullfrog tadpoles were able to perform cardiac mechanistic adjustments to face Roundup-exposure. However, the lower maximal in vitro contraction frequency of the R-group could limit its in vivo cardiac performance, when the adrenergic-stimulation is present. The association between the high energetic cost to counteract the harmful effects of this herbicide and the induction of oxidative stress suggest that low and realistic concentrations of Roundup can have an impact on tadpoles' performance and success, jeopardizing their survival and/or population establishment.

Importance of the sarcoplasmic reticulum and adrenergic stimulation on the cardiac contractility of the neotropical teleost Synbranchus marmoratus under different thermal conditions.

Experiments were carried out to investigate the heart rate of Synbranchus marmoratus after changing the temperature of the water contained in the experimental chamber of the acclimated fish (from 25 to 35 degrees C and from 25 to 15 degrees C). Then, an isometric cardiac muscle preparation was used to test the relative importance of Ca(2+) released from the sarcoplasmic reticulum and Ca(2+) influx across the sarcolemma for the cardiac performance under different thermal conditions: 25 degrees C (acclimation temperature), 15 and 35 degrees C. Adrenaline and ryanodine were used to modulate the Ca(2+) flux through the sarcolemma and the sarcoplasmic reticulum, respectively. Ryanodine reduced the peak tension by approximately 47% at 25 degrees C, and by 53% at 35 degrees C; however, it had no effect at 15 degrees C. A high adrenaline concentration was able to ameliorate the negative effects of ryanodine. Despite increasing the peak tension, adrenaline increased the times necessary for contraction and relaxation. We conclude that the sarcoplasmic reticulum is active in contributing Ca(2+) to the development of tension at physiological contraction frequencies. The adrenaline-stimulated Ca(2+) influx is able to increase the peak tension, even after addition of ryanodine, at physiologically relevant temperatures and pacing frequencies.

The role of branchial and orobranchial O2 chemoreceptors in the control of aquatic surface respiration in the neotropical fish tambaqui (Colossoma macropomum): progressive responses to prolonged hypoxia.

The present study examined the role of branchial and orobranchial O(2) chemoreceptors in the cardiorespiratory responses, aquatic surface respiration (ASR), and the development of inferior lip swelling in tambaqui during prolonged (6 h) exposure to hypoxia. Intact fish (control) and three groups of denervated fish (bilateral denervation of cranial nerves IX+X (to the gills), of cranial nerves V+VII (to the orobranchial cavity) or of cranial nerves V alone), were exposed to severe hypoxia (Pw(O)2=10 mmHg) for 360 min. Respiratory frequency (fr) and heart rate (fh) were recorded simultaneously with ASR. Intact (control) fish increased fr, ventilation amplitude (V(AMP)) and developed hypoxic bradycardia in the first 60 min of hypoxia. The bradycardia, however, abated progressively and had returned to normoxic levels by the last hour of exposure to hypoxia. The changes in respiratory frequency and the hypoxic bradycardia were eliminated by denervation of cranial nerves IX and X but were not affected by denervation of cranial nerves V or V+VII. The V(AMP) was not abolished by the various denervation protocols. The fh in fish with denervation of cranial nerves V or V+VII, however, did not recover to control values as in intact fish. After 360 min of exposure to hypoxia only the intact and IX+X denervated fish performed ASR. Denervation of cranial nerve V abolished the ASR behavior. However, all (control and denervated (IX+X, V and V+VII) fish developed inferior lip swelling. These results indicate that ASR is triggered by O(2) chemoreceptors innervated by cranial nerve V but that other mechanisms, such as a direct effect of hypoxia on the lip tissue, trigger lip swelling.

Cardiorespiratory reflexes and aquatic surface respiration in the neotropical fish tambaqui (Colossoma macropomum): acute responses to hypercarbia.

We examined the cardiorespiratory responses to 6 h of acute hypercarbia (1, 2.5, and 5% CO(2)) in intact and gill-denervated (bilateral denervation of branchial branches of cranial nerves IX and X) tambaqui, Colossoma macropomum. Intact fish exposed to 1 and 2.5% CO(2) increased respiratory frequency ( f(R)) and ventilation amplitude ( V(AMP)) slowly over a 1- to 3-h period. Denervated fish did not show this response, suggesting that tambaqui possess receptors in the gills that will produce excitatory responses to low levels of hypercarbia (1 and 2.5% CO(2)) if the exposure is prolonged. The cardiac response to stimulation of these receptors with this level of CO(2) was a tachycardia and not a bradycardia. During exposure to 5% CO(2), intact fish increased f(R) and V(AMP), and showed a pronounced bradycardia after 1 h. After 2 h, the heart rate ( f(H)) started to increase, but returned to control values after 6 h. In denervated fish, the increase in f(R) was abolished. The slow increase in V(AMP) and the bradycardia were not abolished, suggesting that these changes arose from extra-branchial receptors. Neither intact nor denervated fish developed the swelling of the lower lip or performed aquatic surface respiration, even after 6 h, suggesting that these are unique responses to hypoxia and not hypercarbia.