Changes in blood parameters and metabolism in bullfrog tadpoles, Lithobates catesbeianus, (Shaw, 1802) after exposure to the Sorocaba River (São Paulo, Brazil) water.

This study evaluated the genetic damage, oxidative stress, neurotoxicity, and energy metabolism in bullfrog tadpoles (Lithobates catesbeianus) exposed to water from two sites of the Sorocaba River, Ibiúna (PI), and Itupararanga reservoir (PIR), in summer and winter. After 96-h exposure, the erythrocyte number decreased in PI and increase in PIR in summer. Bullfrogs show oxidative unbalance (liver, kidney, and muscle), with alterations in the nitric oxide synthase and glucose 6-phosphate dehydrogenase. Cholinesterase increased in the brain in PI and PIR in the summer and decreased in PI in the winter. It also increased in the muscle in both PI and PIR in the winter. Tadpoles show alterations in the activity of the metabolic enzymes (liver, kidney, and muscle), such as phosphofructokinase, pyruvatokinase, malate dehydrogenase, and lactate dehydrogenase; and in the amount of glucose and triglycerides metabolites. Exposure to the Sorocaba River reflected a stressful situation for L. catesbeianus as the changes caused to their metabolism associated with oxidative stress and neurotoxicity may have effects on the development of tadpoles.

Bombardier beetles repel invasive bullfrogs.

Invasive non-native predators negatively affect native species; however, some native species can survive the predation pressures of invasive species by using pre-existing antipredator strategies or evolving defenses against invasive predators. The American bullfrog Lithobates catesbeianus (Anura: Ranidae) has been intentionally introduced to many countries and regions, and has impacted native animals through direct predation. Bombardier beetles (Coleoptera: Carabidae: Brachininae: Brachinini) discharge chemicals at a temperature of approximately 100 °C from the tip of the abdomen when they are attacked by predators. This "bombing" can successfully repel predators. However, adults of a native bombardier beetle Pheropsophus (Stenaptinus) occipitalis jessoensis have been reportedly found in the gut contents of the introduced bullfrog L. catesbeianus in Japan. These records suggest that the invasive bullfrog L. catesbeianus attacks the native bombardier beetle P. occipitalis jessoensis under field conditions in Japan; however, the effectiveness of the bombing defense against invasive bullfrogs is unclear. To test the effectiveness of the bombing defense against bullfrogs, we investigated the behavioral responses of L. catesbeianus juveniles to P. occipitalis jessoensis adults under laboratory conditions. Contrary to previous gut content results, almost all the bullfrogs (96.3%) rejected bombardier beetles before swallowing them; 88.9% rejected the beetles after being bombed, and 7.4% stopped attacking the beetles before being bombed. Only 3.7% successfully swallowed and digested the beetle. All of the beetles collected from non-bullfrog-invaded sites could deter bullfrogs, suggesting that the pre-existing defenses of bombardier beetles played an essential role in repelling bullfrogs. When treated beetles that were unable to discharge hot chemicals were provided, 77.8% of bullfrogs successfully swallowed and digested the treated beetles. These results indicate that bombing is important for the successful defense of P. occipitalis jessoensis against invasive bullfrogs. Although invasive bullfrogs have reportedly impacted native insect species, P. occipitalis jessoensis has an existing defense mechanism strong enough to repel the invasive predators.

Efferent Activity Controls Hair Cell Response to Mechanical Overstimulation.

The efferent pathway strengthens the auditory system for optimal performance by fine-tuning the response and protecting the inner ear from noise-induced damage. Although it has been well documented that efference helps defend against hair cell and synaptic extinction, the mechanisms of its otoprotective role have still not been established. Specifically, the effect of efference on an individual hair cell's recovery from mechanical overstimulation has not been demonstrated. In the current work, we explored the impact of efferent stimulation on this recovery using in vitro preparations of hair cells situated in the sacculi of American bullfrogs (Rana catesbeiana). In the absence of efferent stimulus, exposure of a hair bundle to high-amplitude mechanical deflection detuned it from its oscillatory regime, with the extent of detuning dependent on the applied signal. Efferent actuation concomitant with the hair bundle's relaxation from a high-amplitude deflection notably changed the recovery profile and often entirely eliminated the transition to quiescence. Our findings indicate that the efferent system acts as a control mechanism that determines the dynamic regime in which the hair cell is poised.

Presence of invasive American bullfrogs may reduce infectious disease in a native frog species.

Amphibians breeding in aquatic environments may encounter a myriad of threats during their life cycle. One species known to prey on native amphibians in aquatic habitats is the invasive North American bullfrog Lithobates catesbeianus, which, besides being a voracious predator and competitor, often acts as a pathogen carrier and disease superspreader because it tolerates high infection loads of the frog-killing fungus Batrachochytrium dendrobatidis (Bd). Here, we hypothesized that the presence of the bullfrogs in microcosms should either (1) decrease Bd disease severity in native frog species by discouraging them from using the aquatic environment, or (2) increase the mortality of the native species. We tested these 2 mutually exclusive hypotheses by co-housing the snouted treefrog Scinax x-signatus (native to our study area) with L. catesbeianus in the laboratory, exposing them to Bd, and using qPCR analysis to quantify the resulting Bd infection loads in the native frogs. Our experiment had the following replicated treatments: (1) native-only treatment (3 individuals of S. x-signatus), (2) native-predominant treatment (2 S. x-signatus + 1 L. catesbeianus), and (3) exotic-predominant treatment (1 S. x-signatus + 2 L. catesbeianus). We found that Bd infection loads in the native S. x-signatus were highest in the native-only treatment, and lowest in the exotic-predominant treatment, indicating that bullfrogs may discourage native frogs from occupying the aquatic habitat, thus reducing encounter rates between native frogs and the waterborne pathogen. This effect could be driven by the bullfrogs' predatory behavior and their high philopatry to aquatic habitats. Our results highlight that predation risk adds to the complexity of host-species interactions in Bd epidemiology.

Effects of corticosterone treatment and wound healing on reproductive traits of American bullfrogs.

During reproductive season, calling anuran males display high testosterone (T) and episodically high corticosterone (CORT) plasma levels, which are positively associated with higher calling rates and immunocompetence. However, exposure to constant stress stimuli can result in chronically elevated CORT levels, possibly leading to inhibition of reproductive and immune activity. Reproduction and immune responses are energetically expensive, so when an animal is immunologically challenged, a tradeoff might be expressed, with CORT potentially mediating it. Our aim was to test how episodic and chronic CORT treatments, alongside wound healing, would affect reproduction in American bullfrog males (Lithobates catesbeianus). Forty animals were divided in four groups: Episodic CORT (daily transdermic application of CORT), placebo (daily transdermic application of sesame oil), chronic CORT (subcutaneous CORT silastic implants), and sham control (subcutaneous empty silastic implants). One week after treatments began, animals were punctured in the leg with a biopsy needle and the wound was photographed after 45 days to determine wound healing status (WS). Blood samples were collected throughout the experiment to measure CORT and T plasma levels. After animal euthanasia, testes were dissected, fixed, and analyzed histologically to determine spermatogenic activity (germinative cyst [GmC] morphometrics). As expected, the episodic CORT treatment had no effect on T plasma levels or spermatogenic activity. On the other hand, chronic CORT treatment reduced GmC morphometric traits, indicating suppression of reproduction, although T levels were not altered. In addition, animals from sham control and chronic CORT treatments with higher T levels presented higher WS, which indicates an immune-enhancing T effect.

Metamorphic acceleration following the exposure to lithium and selenium on American bullfrog tadpoles (Lithobates catesbeianus).

To regulate the presence of contaminants in Brazilian water, the Brazilian Environmental Council (CONAMA) promulgates regulations regarding the concentrations of given compounds that are supposed to be safe for aquatic life. Considering these regulations, this study tested the effects of considered safe levels of lithium (2.5 mgL-1) and selenium (0.01 mgL-1), isolated and mixed, on the American bullfrog (Lithobates catesbeianus) tadpoles. The evaluation was done through the use of biomarkers of larval development as total wet weight (TWW), snout-vent-length (SVL), hind-limb-length (HLL), activity level (AL), histologic evaluation of the thyroid gland and the mortality rate. The tadpoles were allocated into four groups (n = 20 each): a control group (CT); a group exposed to lithium (LI), a group exposed to selenium (SE), and a group exposed to both lithium and selenium (SELI). The whole assay was carried out over 21 days, with two rounds of data collection (on 7th and 21st day) to evaluate the responses over time. A statistical reduction in the AL was observed in the tadpoles from the LI and SELI groups after 7 days of exposure, the same pattern was observed after 21 days. Histological analyses of the thyroid gland showed signs of up-regulation (i.e. statistic reduction in number and area of the follicles, as well a significant reduction in the area of the gland) in all exposed groups, which represents an endocrine response as an adaptative strategy to deal with polluted aquatic environment. The stress triggered by the polluted medium is discussed.

Neuromodulation or energy failure? Metabolic limitations silence network output in the hypoxic amphibian brainstem.

Hypoxia tolerance in the vertebrate brain often involves chemical modulators that arrest neuronal activity to conserve energy. However, in intact networks, it can be difficult to determine whether hypoxia triggers modulators to stop activity in a protective manner or whether activity stops because rates of ATP synthesis are insufficient to support network function. Here, we assessed the extent to which neuromodulation or metabolic limitations arrest activity in the respiratory network of bullfrogs-a circuit that survives moderate periods of oxygen deprivation, presumably, by activating an inhibitory noradrenergic pathway. We confirmed that hypoxia and norepinephrine (NE) reduce network output, consistent with the view that hypoxia may cause the release of NE to inhibit activity. However, these responses differed qualitatively; hypoxia, but not NE, elicited a large motor burst and silenced the network. The stereotyped response to hypoxia persisted in the presence of both NE and an adrenergic receptor blocker that eliminates sensitivity to NE, indicating that noradrenergic signaling does not cause the arrest. Pharmacological inhibition of glycolysis and mitochondrial respiration recapitulated all features of hypoxia on network activity, implying that reduced ATP synthesis underlies the effects of hypoxia. Finally, activating modulatory mechanisms that dampen neuronal excitability when ATP levels fall, KATP channels and AMP-dependent protein kinase, did not resemble the hypoxic response. These results suggest that energy failure-rather than inhibitory modulation-silences the respiratory network during hypoxia and emphasize the need to account for metabolic limitations before concluding that modulators arrest activity as an adaptation for energy conservation in the nervous system.

Thermal sensitivity of Bullfrog's immune response kept at different temperatures.

Climate change and emerging infectious diseases are often described as the main factors associated with the worldwide amphibian population decline. In this context, rising temperatures due to global warming might act as a chronic stressor for many amphibians, leading to immunosuppression. This study aimed to characterize the thermal sensitivity of the Bullfrog's (Lithobates catesbeianus) immune response and the effect of acclimation at different temperatures on it. Plasma bacterial killing ability (BKA) and phagocytosis activity of blood leukocytes were measured at different incubation temperatures (5-40°C) in individuals kept at 28°C and 34°C. First, all individuals were held under 28°C and sampled on the 16th day. Subsequently, one group was kept at 28°, and the other one was transferred to 34°C. Both groups were sampled at 83 and 106 days of maintenance. Plasma corticosterone (CORT) and testosterone (T) were assessed to evidence thermal stress and possible endocrine correlates of immune changes over time. The incubation temperature affected BKA both on animals kept at 28°C and 34°C, with maximum values at lower temperatures (5-20°C). Phagocytosis activity was constant over the range of assay temperatures. Immune and endocrine variables decreased over time in both thermal regimes, but frogs maintained at 34°C showed lower T and immunosuppression, evidencing stress response. Therefore, exposure to high temperatures might decrease immune function in bullfrogs due to chronic stress response and by exposition to temperatures of lower performance according to the thermal sensitivity curve, which might increase vulnerability to diseases in this anuran species.

Effects of dehydration on thermoregulatory behavior and thermal tolerance limits of Rana catesbeiana ().

Predicting the effects of high environmental temperatures and drought on populations requires understanding how these conditions will influence the thermoregulatory behavior and thermal tolerance of organisms. Ectotherms show proportional (fine-tuned) and all-or-none (abrupt) responses to avoid overheating. Scattered evidence suggests that dehydration alters these behavioral responses and thermal tolerance, but these effects have not been evaluated in an integrative manner. We examined the effects of hydration level on the behavioral thermoregulation and behavioral and physiological thermal limits of the "bullfrog" (Rana catesbeiana), a well-studied and important invasive species. To examine the effects of dehydration on proportional responses, we compared the Preferred Body Temperatures (PBT) of frogs with restricted and unrestricted access to water. To assess the effect of dehydration on all-or-none responses, we measured and compared the Voluntary Thermal Maximum (VTMax) at different hydration levels (100%, 90%, 80% of body weight at complete hydration). Finally, to understand the effect of dehydration on physiological thermal tolerance, we measured the Critical Thermal Maximum (CTMax) of frogs at matched hydration levels. PBT, VTMax, and CTMax all decreased in response to higher dehydration levels. However, bullfrogs changed their PBT more than their VTMax or CTMax in response to dehydration. Moreover, some severely dehydrated individuals did not exhibit a VTMax response. We discuss the implications of our results in the context of plasticity of thermoregulatory responses and thermal limits, and its potential application to mechanistic modeling.

Assessment of antioxidant system, cholinesterase activity and histopathology in Lithobates catesbeianus tadpoles exposed to water from an urban stream.

Anthropogenic activities promote changes in community structure and decrease the species abundance of amphibians. The aim of this study was to assess potential alterations in the antioxidant system and cholinesterase activity, histopathological and oxidative damage in Lithobates catesbeianus tadpoles exposed to water from the Cascavel River, in Southern Brazil. Water samples (140 L each) were collected from the headwater, urban and rural areas of the river. Tadpoles were acclimated for seven days. After acclimatization tadpoles were reared in water from the river, except for the control aquarium. After seven days, a portion of the liver was removed and prepared for cholinesterase (ChE), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) analysis; another part of the tissue was prepared for histological examination. An elevation of CAT activity was observed for water from both urban and rural environments. A decrease in LPO reaction was detected, mainly among the tadpoles exposed to water from the rural area. These alternations can cause delay the metamorphosis and lead to metabolic dysfunction, interfering in survival capacity and diminishing, not only individual fitness, but that of the whole population.

Is there a common drive for buccal movements associated with buccal and lung 'breath' in Lithobates catesbeianus?

In amphibians, there is some evidence that (1) anatomically separate brainstem respiratory oscillators are involved in rhythm generation, one for the buccal rhythm and another for the lung rhythm and (2) they become functionally coupled during metamorphosis. The present analysis, performed on neurograms recorded using brainstem preparations from Lithobates catesbeianus, aims to investigate the temporal organisation of lung and buccal burst types. Continuous Wavelet Transfom applied to the separated buccal and lung signals of a neurogram revealed that both buccal and lung frequency profiles exhibited the same low frequency peak around 1 Hz. This suggests that a common 'clock' organises both rhythms within an animal. A cross-correlation analysis applied to the buccal and lung burst signals revealed their similar intrinsic oscillation features, occurring at approximately 25 Hz. These observations suggest that a coupling between the lung and buccal oscillators emerges at metamorphosis. This coupling may be related to inter-connectivity between the two oscillators, and to a putative common drive.

Estimation of Forces on Actin Filaments in Living Muscle from X-ray Diffraction Patterns and Mechanical Data.

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.

Passive muscle tension increases in proportion to intramuscular fluid volume.

During extended bouts of exercise, muscle can increase in volume by as much as 20% as vascular fluid moves into the tissue. Recent findings suggest that the fluid content of muscle can influence the mechanics of force production; however, the extent to which natural volume fluctuations should be expected to influence muscle mechanics in vivo remains unclear. Here, using osmotic perturbations of bullfrog muscle, we explored the impacts of physiologically relevant volume fluctuations on a fundamental property of muscle: passive force production. We found that passive force and fluid volume were correlated over a 20% increase in muscle volume, with small changes in volume having significant effects on force (e.g. a 5% volume increase results in a >10% passive force increase). A simple physical model of muscle morphology reproduces these effects. These findings suggest that physiologically relevant fluid fluxes could alter passive muscle mechanics in vivo and affect organismal performance.

Excitation and electroporation by MHz bursts of nanosecond stimuli.

Intense nanosecond pulsed electric field (nsPEF) is a novel modality for cell activation and nanoelectroporation. Applications of nsPEF in research and therapy are hindered by a high electric field requirement, typically from 1 to over 50 kV/cm to elicit any bioeffects. We show how this requirement can be overcome by engaging temporal summation when pulses are compressed into high-rate bursts (up to several MHz). This approach was tested for excitation of ventricular cardiomyocytes and peripheral nerve fibers; for membrane electroporation of cardiomyocytes, CHO, and HEK cells; and for killing EL-4 cells. MHz compression of nsPEF bursts (100-1000 pulses) enables excitation at only 0.01-0.15 kV/cm and electroporation already at 0.4-0.6 kV/cm. Clear separation of excitation and electroporation thresholds allows for multiple excitation cycles without membrane disruption. The efficiency of nsPEF bursts increases with the duty cycle (by increasing either pulse duration or repetition rate) and with increasing the total time "on" (by increasing either pulse duration or number). For some endpoints, the efficiency of nsPEF bursts matches a single "long" pulse whose amplitude and duration equal the time-average amplitude and duration of the bursts. For other endpoints this rule is not valid, presumably because of nsPEF-specific bioeffects and/or possible modification of targets already during the burst. MHz compression of nsPEF bursts is a universal and efficient way to lower excitation thresholds and facilitate electroporation.

Pyrethrum extract encapsulated in nanoparticles: Toxicity studies based on genotoxic and hematological effects in bullfrog tadpoles.

The environment receives about 2.7 kg.ha-1 annually of pesticides, used in crop production. Pesticides may have a negative impact on environmental biodiversity and potentially induce physiological effects on non-target species. Advances in technology and nanocarrier systems for agrochemicals led to new alternatives to minimize these impacts, such as nanopesticides, considered more efficient, safe and sustainable. However, it is important to evaluate the risk potential, action and toxicity of nanopesticides in aquatic and terrestrial organisms. This study aims to evaluate genotoxic and hematological biomarkers in bullfrog tadpoles (Lithobates catesbeianus) submitted to acute exposure (48 h) to pyrethrum extract (PYR) and solid lipid nanoparticles loaded with PYR. Results showed increased number of leukocytes during acute exposure, specifically eosinophils in nanoparticle-exposed groups, and basophil in PYR-exposed group. Hematological analysis showed that PYR encapsulated in nanoparticles significantly increased the erythrocyte number compared to the other exposed groups. Data from the comet assay indicated an increase in frequency of the classes that correspond to more severe DNA damages in exposed groups, being that the PYR-exposed group showed a high frequency of class-4 DNA damage. Moreover, erythrocyte nuclear abnormalities were triggered by short-time exposure in all treatments, which showed effects significantly higher than the control group. These results showed genotoxic responses in tadpoles, which could trigger cell death pathways. Concluding, these analyses are important for applications in assessment of contaminated aquatic environments and their biomonitoring, which will evaluate the potential toxicity of xenobiotics, for example, the nanoparticles and pyrethrum extract in frog species. However, further studies are needed to better understand the effects of nanopesticides and botanical insecticides on non-target organisms, in order to contribute to regulatory aspects of future uses for these systems.

Buccal rhythmogenesis and CO2 sensitivity in Lithobates catesbeianus tadpole brainstems across metamorphosis.

Bullfrog tadpoles ventilate both the buccal cavity and lung. In isolated brainstems, the midbrain/pons influences CO2 responsiveness and timing of lung ventilatory bursting, depending on larval development. However, little is known about midbrain/pons influences on buccal burst patterns. As such, we investigated how removal of this region affects buccal burst shape and CO2 responsiveness across development. We measured facial nerve activity in brainstems isolated from tadpoles during early and late developmental stages, under normal and elevated levels of CO2. Brainstems were either left intact or transected by removing the midbrain/pons. In late stage preparations, buccal burst pattern differed between intact and reduced preparations, and bursts were responsive to elevated CO2 in these reduced preparations. These results suggest the midbrain/pons affects tadpole buccal burst pattern and CO2 responsiveness, perhaps similar to its influences on lung ventilation.

Respiratory motoneuron properties during the transition from gill to lung breathing in the American bullfrog.

Amphibian respiratory development involves a dramatic metamorphic transition from gill to lung breathing and coordination of distinct motor outputs. To determine whether the emergence of adult respiratory motor patterns was associated with similarly dramatic changes in motoneuron (MN) properties, we characterized the intrinsic electrical properties of American bullfrog trigeminal MNs innervating respiratory muscles comprising the buccal pump. In premetamorphic tadpoles (TK stages IX-XVIII) and adult frogs, morphometric analyses and whole cell recordings were performed in trigeminal MNs identified by fluorescent retrograde labeling. Based on the amplitude of the depolarizing sag induced by hyperpolarizing voltage steps, two MN subtypes (I and II) were identified in tadpoles and adults. Compared with type II MNs, type I MNs had larger sag amplitudes (suggesting a larger hyperpolarization-activated inward current), greater input resistance, lower rheobase, hyperpolarized action potential threshold, steeper frequency-current relationships, and fast firing rates and received fewer excitatory postsynaptic currents. Postmetamorphosis, type I MNs exhibited similar sag, enhanced postinhibitory rebound, and increased action potential amplitude with a smaller-magnitude fast afterhyperpolarization. Compared with tadpoles, type II MNs from frogs received higher-frequency, larger-amplitude excitatory postsynaptic currents. Input resistance decreased and rheobase increased postmetamorphosis in all MNs, concurrent with increased soma area and hyperpolarized action potential threshold. We suggest that type I MNs are likely recruited in response to smaller, buccal-related synaptic inputs as well as larger lung-related inputs, whereas type II MNs are likely recruited in response to stronger synaptic inputs associated with larger buccal breaths, lung breaths, or nonrespiratory behaviors involving powerful muscle contractions.

Differential expression of RNA exosome subunits in the amphibian Lithobates catesbeianus during reproductive and non-reproductive periods.

OBJECTIVE: The RNA exosome is an evolutionarily conserved 3'-5' exoribonucleolytic protein complex involved in processing and degradation of different classes of nuclear and cytoplasmic RNAs, and, therefore, important for the posttranscriptional control of gene expression. Despite the extensive in vivo functional studies and the structural data on the RNA exosome, few studies have been performed on the localization and expression of exosome subunits during gametogenesis, process during which gene expression is largely controlled at the posttranscriptional level. RESULTS: We report the identification of exosome subunits in Lithobates catesbeianus and analysis of the differential subcellular localization of RNA exosome core and catalytic subunits in testis cells. In addition, we show seasonal differences in the expression levels of four exosome subunits in different organs. In addition to being part of the RNA exosome complex, its subunits might participate independently of the complex in the control of gene expression during seasonal variation in bullfrog tissues. These results may be relevant for other eukaryotic species.

Seasonal acclimatization and thermal acclimation induce global histone epigenetic changes in liver of bullfrog (Lithobates catesbeianus) tadpole.

The American bullfrog (Lithobates catesbeianus) is a eurythermal amphibian that is naturally distributed from subarctic to subtropical areas. The tadpoles of this species overwinter, in water, in cold environments. Therefore, they may have adapted to a wide range of temperatures in an active state. To understand the adaptation mechanisms to cope with low or high temperatures, we investigated global epigenetic modifications, histone variants, transcript levels of related genes, and the cellular acetyl coenzyme A (acetyl-CoA) and free CoA (CoA-SH) levels, in the livers of tadpoles collected in summer and winter and of those acclimated to 4 °C and 21 °C. Among epigenetic marks tested, the levels of acetylated histones and the histone variant H2A.Z were influenced by different temperature conditions. Histone acetylation levels were higher in summer than in winter and increased within 3 days of warm acclimation, whereas histone H2A.Z levels were higher in winter than in summer and decreased within 2 weeks of warm acclimation. Transcript analysis revealed that decreased expression of histone H2A.Z in warm acclimation was regulated at the transcriptional level. Acetyl-CoA levels were not correlated with those of the acetylated histones, indicating that cellular acetyl-CoA levels may not directly influence the state of histone acetylation in the tadpole liver. Such epigenetic and metabolic changes in the tadpole liver may contribute to the maintenance of energy balance during seasonal acclimatization and thermal acclimation.

Sub-lethal effects induced by a mixture of different pharmaceutical drugs in predicted environmentally relevant concentrations on Lithobates catesbeianus (Shaw, 1802) (Anura, ranidae) tadpoles.

The increasing consumption of medications by humans has negative effects such as the increased disposal of these compounds in the environment. Little is known about how the disposal of a "drug mix" (DM) in aquatic ecosystems can affect their biota. Thus, we evaluated whether the exposure of Lithobates casteibeianus tadpoles to a DM composed of different medication classes (antibiotic, anti-inflammatory, antidepressant, anxiolytic, analgesic, and antacid drugs)-at environmentally relevant concentrations-may change their oral morphology, trigger behavioral disorders, and have mutagenic effects on erythrocyte cells. Based on our data, animals exposed to the DM showed changes in mandibular sheath pigmentation, dentition, and swimming activity, as well as atypical behavior in the social aggregation test [with co-specific and interspecific (Physalaemus cuvieri) individuals] and antipredatory defensive response deficit (chemical stimulus from Odonata larvae), after 15 exposure days. The mutagenic analysis revealed higher frequency of nuclear abnormalities in the erythrocytes of tadpoles exposed to the DM (e.g., multilobulated, blebbed, kidney-shaped, notched nucleus, binuclear, and micronucleated erythrocytes). Given the chemical complexity of the DM, we assumed that several organic functions may have been affected, either by the isolated, synergistic, antagonistic, or additive action of DM compounds. Finally, our study confirms the toxicological potential of DM in L. catesbeianus tadpoles, with emphasis to impacts that can affect the fitness of individuals and their natural populations. Thus, we suggest that more attention should be given to the disposal of medications in the environment and reinforce the need of improving water and sewage treatment systems.