Poison Frog Research Articles

Assessment of biosafety and toxicity of hydrophilic gel for implantation in experimental in vitro and in vivo models.

BackgroundThe assessment of biosafety of pharmacologically active substances is crucial for determining the feasibility of their medical use. There are controversial issues regarding the use of substances of different origins as implants.MethodsWe have conducted the comprehensive studies to determine the in vivo toxicity and in vitro genotoxicity of new generation of hydrophilic gel for implantation (production name of the substance “Activegel”) to detail its characteristics and assess its biosafety.ResultsIn vivo studies have shown the absence of clinical manifestations of intoxication in animals and no abnormalities in their physiological condition, general and biochemical blood tests. Evaluation of the site of the gel application showed no inflammatory reaction and evidenced on normal state of tissues of animal skin. The results of the genotoxicity test indicated that the gel did not affect the parameters of DNA comets and the formation of micronuclei, accordingly, had no genotoxic effect on human peripheral blood lymphocytes. When studying the effect of the gel on malignantly transformed cells in vitro, it was found that the gel for implantation did not change the proliferative activity and viability of human breast cancer cells.ConclusionsComprehensive in vitro and in vivo study using various experimental model systems showed that the hydrophilic gel for implantation “Activegel” is non-toxic.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40360-022-00577-3.

POISONOUS PLANTS IN LIVESTOCK ANIMALS 1: Astragalus spp., Oxytropis spp., Hypericum perforatum, Nerium oleander, Pteridium aquilinum

Poisoning of livestock animals with plants commonly occurs worldwide. Poisonous plants and their metabolites cause important economic losses for industry of livestock animals in the world. Poisoning of livestock animals with poisonous plants mostly develops when these plants are within hay or silage. In this paper context, it was dealt with poisonous effects of Astragalus spp. and Oxytropis spp., Hypericum perforatum, Nerium oleander, Pteridium aquilinum from plants leading to poisoning in livestock animals. Broad knowledge was presented about toxicity of poisonous plants and clinical signs that they cause in livestock animals. In addition, the brief knowledge was presented on the required measures for preventing poisoning of livestock animals with poisonous plants and treatment choices.

Phytochemical composition and biological activities of the plants of the genus Randia

Background: The genus Randia L. (Rubiaceae) is native to Americas and highly distributed in tropical areas. Some Randia species are used in traditional medicine in some countries to treat diverse illnesses/symptoms of kidney, circulatory system, lungs, diabetes, cancer, inflammation, and against the bites/stings of snakes and other poisonous animals.
 Questions: What are the phytochemical compounds previously identified in Randia ? What biological activities do they present?
 Data description: Twenty-eight studies on chemical composition and biological activities of Randia were reviewed. Species names were corroborated in Plants of the World Online and World Flora Online.
 The site and studied years: Studies of Randia of Americas from 1991 to 2021.
 Methods: Database reviewed were PubMed, Scopus, Scielo, BVS, DAOJ, Science Direct, Springer Link, Web of Science, and Google Scholar, employing the keywords Randia and its synonym Basanacantha .
 Results: Six species are the most studied ( R. aculeata, R. echinocarpa , R. ferox , R. hebecarpa , R. matudae, and R. monantha ). Ethnopharmacology information of 12 species was recovered. One hundred compounds in Randia have been identified (phenolic acids, terpenes, sterols, and others), and diverse biological activities reported in 24 studies ( e.g. , antimutagenic, antioxidant, and antivenom) have demonstrated for nine species.
 Conclusions: Biological activities found in some species of Randia support their traditional uses, but only the antivenom effect of Randia aculeata has been demonstrated. Randia species could be a source of bioactive compounds; however, knowledge must be expanded to demonstrate their traditional uses and contribute to the development of strategies for their preservation and rational use.

Development, Optimization, and Validation of Forensic Analytical Method for Quantification of Anticholinesterase Pesticides in Biological Matrices from Suspected Cases of Animal Poisoning.

Anticholinesterase pesticides are a main cause of the intentional or accidental poisoning of animals. Anticholinesterases include several substances that cause the overstimulation of both central and peripheral acetylcholine-dependent neurotransmission. Forensic analyses of poisoning cases require high levels of expertise, are costly, and often do not provide reliable quantitative information for unambiguous conclusions. The purpose of the present study was to develop and validate a method of high-performance liquid chromatography with diode array detector (HPLC–DAD) for the identification and quantitation of n-methyl carbamates, organophosphates and respective metabolites from biological samples of animals that were suspected of poisoning. HPLC–DAD is reliable, fast, simplistic and cost-effective. The method was validated for biological samples obtained from stomach contents, liver, vitreous humor and blood from four different animal species. The validation of the method was achieved using the following analytical parameters: linearity, precision, accuracy, selectivity, recovery, and matrix effect. The method showed linearity at the range of 25–500 μg/mL, and the correlation coefficient (r2) values were >0.99 for all matrices. Precision and accuracy were determined by the (a) coefficient of variation (CV), (b) relative standard deviation low-quality control (LQC), (c) medium-quality control (QCM), and (d) high-quality control (QCA). The indicated parameters were all less than 15%. The recovery of analytes ranged from 31 to 71%. The analysis of results showed no significant interfering peaks due to common xenobiotics or matrix effects. The abovementioned method was used to positively identify pesticide analytes in 44 of the 51 animal samples that were suspected of poisoning, demonstrating its usefulness as a forensic tool.

Correction to: Large, old trees define the vertical, horizontal, and seasonal distributions of a poison frog

Correction to: Large, old trees define the vertical, horizontal, and seasonal distributions of a poison frog

Spatial assessment of groundwater quality using CCME-WQI and hydrochemical indices: a case study from Talupula Mandal, Ananthapuramu district, South India

Fluoride contamination in groundwater is known from different villages of Talupula Mandal. In the present study in part of SoI toposheet no.57 J/8) of Ananthapuramu district, Andhra Pradesh, the magnitude and credible geogenic sources of the fluoride contamination in groundwater are studied. The present study carried out includes 30 groundwater samples covering an area of 442.89 sq km using (GIS) and (CCME-WQI) techniques. Problem is causally related to fluoride intoxication of human beings and animals through drinking waters. Well waters in the Talupula Mandal surrounded areas carry a high level of fluoride concentrations, much above the internationally decided safety limits. ‘Fluorosis’ is imminent as a result of prolonged intake of waters with more than 1 to 1.5 ppm of fluoride. Fluoride testing results of groundwater from borewell pump using fluoride test kit reveal that about 50% groundwater samples show fluoride concentration more than 4.0 mg/l, 30% samples show between 2.0 and 3.0 mg/l and 20% samples show fluoride concentration is 1–2 mg/l and above (BIS permissible limit: 1.5 mg/l). Agriculture indices are as follows: sodium percentage (Na%) was found between ranges (20.04–55.26), sodium absorption ratio varies from 0.86 to 3.91), Kelly's ratio (KR) varies from 0.22 to 1.16, residual sodium carbonate varies from − 4.12 to 20.66), permeability index (PI) varies from 39.00 to 82.28, magnesium hazard varies from 52.83 to 85.47, potential salinity varies from (0.71–3.15); results shown that the majority of the groundwater samples were suitable for irrigation uses. According to Gibbs’ ratio samples Gibbs ratio 1 (0.04–0.45) and Gibbs ratio 2 (0.44–0.81) fall in the rock dominance. Major ion compositions plotted on a Piper tri-linear diagram indicate that the water is of Ca-HCO3 type and is mixed Ca–Mg–Cl type. The present study indicates that the groundwater chemistry is mostly controlled by geogenic processes (weathering, dissolution and ion exchange) and some extent of anthropogenic activities.

Evaluation of the context of downstream N- and free N-glycomic alterations induced by swainsonine in HepG2 cells

Swainsonine (SWA), a potent inhibitor of class II α-mannosidases, is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. The mechanisms underlying SWA-induced animal poisoning are not fully understood. In this study, we analyzed the alterations that occur in N- and free N-glycomic upon addition of SWA to HepG2 cells to understand better SWA-induced glycomic alterations. After SWA addition, we observed the appearance of SWA-specific glycomic alterations, such as unique fucosylated hybrid-type and fucosylated M5 (M5F) N-glycans, and a remarkable increase in all classes of Gn1 FNGs. Further analysis of the context of these glycomic alterations showed that (fucosylated) hybrid type N-glycans were not the precursors of these Gn1 FNGs and vice versa. Time course analysis revealed the dynamic nature of glycomic alterations upon exposure of SWA and suggested that accumulation of free N-glycans occurred earlier than that of hybrid-type N-glycans. Hybrid-type N-glycans, of which most were uniquely core fucosylated, tended to increase slowly over time, as was observed for M5F N-glycans. Inhibition of swainsonine-induced unique fucosylation of hybrid N-glycans and M5 by coaddition of 2-fluorofucose caused significant increases in paucimannose- and fucosylated paucimannose-type N-glycans, as well as paucimannose-type free N-glycans. The results not only revealed the gross glycomic alterations in HepG2 cells induced by swainsonine, but also provide information on the global interrelationships between glycomic alterations.

Convergent evolution of toxin resistance in animals.

ABSTRACTConvergence is the phenomenon whereby similar phenotypes evolve independently in different lineages. One example is resistance to toxins in animals. Toxins have evolved many times throughout the tree of life. They disrupt molecular and physiological pathways in target species, thereby incapacitating prey or deterring a predator. In response, molecular resistance has evolved in many species exposed to toxins to counteract their harmful effects. Here, we review current knowledge on the convergence of toxin resistance using examples from a wide range of toxin families. We explore the evolutionary processes and molecular adaptations driving toxin resistance. However, resistance adaptations may carry a fitness cost if they disrupt the normal physiology of the resistant animal. Therefore, there is a trade‐off between maintaining a functional molecular target and reducing toxin susceptibility. There are relatively few solutions that satisfy this trade‐off. As a result, we see a small set of molecular adaptations appearing repeatedly in diverse animal lineages, a phenomenon that is consistent with models of deterministic evolution. Convergence may also explain what has been called ‘autoresistance’. This is often thought to have evolved for self‐protection, but we argue instead that it may be a consequence of poisonous animals feeding on toxic prey. Toxin resistance provides a unique and compelling model system for studying the interplay between trophic interactions, selection pressures and the molecular mechanisms underlying evolutionary novelties.

Colour, location and movement: what do models tell us about predation on colour morphs of a poison frog from eastern Amazonia?

Abstract Many dendrobatid frogs are known to be aposematic: brightly coloured and unpalatable to predators. To deceive predators, frog models used to test for predatory colour bias must be similar in size, colour, shape, and movement to frogs. We carried out an experiment with moving models of the species Adelphobates galactonotus, in two localities. A. galactonotus is a polytypic frog and each population of the species has a distinct colour. Birds and mammals were the vertebrates responsible for the marks on the models, but there was no difference in frequency of attacks on local-, non-local- and cryptic-colour models. Only invertebrates avoided cryptic models. Different populations of the species seem to be under different predation pressure, but colour differentiation in this species is probably related to other mechanisms, such as sexual selection.

Pseudomonas aeruginosa and Its Pathogenicity

Pseudomonas aeruginosa, belonging to the Pseudomonadaceae family, is Gram-negative, rod-shaped, motile, aerobic, endospore negative, oxidase and catalase positive. It is widely found in nature and isolated from soil, plants, water and animals. It can grow rapidly on the surface of the food and form oxidized products and mucous substances. P. aeruginosa, one of the leading foodborne pathogens, causes important concerns in food safety due to being a source of contamination, causing food poisoning and antimicrobial resistance in animals, forming biofilms and difficulties in preventing biofilms. In this review, information on history, microbiological, cultural and biochemical characteristics, virulence factors and pathogenicity of P. aeruginosa are given. In addition, infections caused by P. aeruginosa and its presence in food are described.

Aposematic patterns shift continuously throughout the life of poison frogs

AbstractColor and pattern are often dynamic traits that change throughout an individual's lifetime. Still, long‐term shifts in coloration have received limited attention. Dendrobatid poison frogs are a classical system in the study of color and pattern evolution in which both sexual selection and predation avoidance are thought to drive the evolution of color and pattern at the population and species level. Here, we highlight an overlooked axis of pattern diversity, within individual variation, using three species in the genus Dendrobates. We collected longitudinal photographs of individuals at the National Aquarium to test the hypothesis that patterns shift predictably throughout the lifetimes of individual frogs. In all three species, we found a consistent reduction in the relative area of aposematic color as individuals aged and that the rate of pattern shift did not differ between the sexes. Consequently, within individual variation in coloration may confound inferences from ecological studies that inherently assume individual pattern is static. Finally, we note that using simple and noninvasive photography protocols, animals in zoos and aquaria have the potential to deepen our understanding of how color and pattern change throughout the lifetimes of a wide range of species.

Repeatable Territorial Aggression in a Neotropical Poison Frog.

Intra-specific aggressive interactions play a prominent role in the life of many animals. While studies have found evidence for repeatability in boldness, activity, and exploration in amphibians, we know relatively little about consistent among-individual variation in aggressiveness, despite its importance for male-male competition and territoriality. Amphibians, and Neotropical poison frogs (Dendrobatidae) in particular, are highly suitable for investigating among-individual variation in aggressiveness, as most species exhibit strong territoriality in at least one of the sexes. In the present study, we aimed to fill this gap in knowledge, by investigating within- and between-individual variation in territorial aggression in a semi-natural population of the Neotropical poison frog Allobates femoralis (Dendrobatidae) in French Guiana. We conducted repeated, standardized behavioral tests to assess if the level of territorial aggression is consistent within and different between individuals. Further, we tested a possible link between body size and level of territorial aggression. We found moderate repeatability in territorial aggressiveness, but no link to age and/or body size. In conclusion, our study represents the first documentation of repeatable aggressive behavior in a territorial context in amphibians.

Androgen responsiveness to simulated territorial intrusions in Allobates femoralis males: Evidence supporting the challenge hypothesis in a territorial frog

Territoriality has been widely described across many animal taxa, where the acquisition and defence of a territory are critical for the fitness of an individual. Extensive evidence suggests that androgens are involved in the modulation of territorial behaviours in male vertebrates. Short-term increase of androgen following a territorial encounter appears to favour the outcome of a challenge. The “Challenge Hypothesis” proposed by Wingfield and colleagues outlines the existence of a positive feedback relationship between androgen and social challenges (e.g., territorial intrusions) in male vertebrates. Here we tested the challenge hypothesis in the highly territorial poison frog, Allobates femoralis, in its natural habitat by exposing males to simulated territorial intrusions in the form of acoustic playbacks. We quantified repeatedly androgen concentrations of individual males via a non-invasive water-borne sampling approach. Our results show that A. femoralis males exhibited a positive behavioural and androgenic response after being confronted to simulated territorial intrusions, providing support for the Challenge Hypothesis in a territorial frog.

Discrimination method of seven species pufferfish of the genus Takifugu using DNA chromatography

Identifying poisonous plants and animals is very important because they not only can cause food poisoning, but also can be eaten for the purpose of suicide. A pufferfish is a poisonous fish that contains tetrodotoxin. In Japan, 136 pufferfish poisoning cases occurred from 2015 to 2019, but in many cases, the specific species involved was unidentified. To address this, we focused on a rapid and simple DNA chromatography technology called Single-stranded Tag Hybridization (STH). We collected seven pufferfish species of the genus Takifugu and designed species-specific primers as target regions of cytochrome c oxidase subunit I (COI) and cytochrome b with a specific base sequence at the 3′ end. STH-PCR was performed in two separate reactions. After mixing the PCR products, a developing solution was added to perform chromatograph development and the results were visually analyzed. Specific lines were detected in all seven species. The pufferfish species could be properly determined using between 0.1 ng and 50 ng of template DNA. The PCR product length was 85–149 bp, making it very resistant to degradation. The species could be properly identified even in a mixture of multiple pufferfish species DNA. Furthermore, verification was performed using the supernatants of digested samples with artificial gastric juice and processed foods. Extracted DNA was obtained in all but the highly roasted fins, enabling discrimination. Overall, we applied a novel DNA chromatography detection system capable of discriminating seven species of the genus Takifugu, which have closely related DNA sequences.

EFFECT OF THE FEED ADDITIVE BUTASELMEVIT-PLUS ON THE ANTIOXIDANT STATUS OF THE RAT BODY DUE TO CADMIUM AND LEAD INTOXICATION

The relevance of the study subject is due to the need to create effective methods for preventing the poisoning of animals with heavy metals because lead and cadmium are among the environmental pollutants that negatively affect agriculture and are potentially dangerous to animal and human health. The purpose of the study was to establish the effect of the feed additive Butaselmevit-Plus on the antioxidant protection of the rat body under the chronic action of cadmium and lead. Experimental studies were performed on 2 groups of male rats, 6 animals each. In the control and experimental groups, rats were given a 16.6% aqueous solution of lead acetate at a dose of 100 mg/kg (0.6 ml/kg) of body weight and 0.029% aqueous solution of cadmium chloride at a dose of 2.0 mg/kg (6.9 ml/kg) of body weight. In the experimental group, the rats were additionally provided with food containing the feed additive Butaselmevit-Plus in the amount of 0.1 g per 100 g of body weight. Experimental lead-cadmium toxicosis in the blood serum of rats demonstrated a substantial decrease in the indicators of the antioxidant protection system (reduced glutathione – by 38.4%, superoxide dismutase – by 27.6%, catalase – by 22.7%). On the fourteenth day of the experiment, the lowest activity of the antioxidant protection system indicators in the blood of control rats was observed with the combined administration of heavy metals. Under the experimental load of lead and cadmium, the feed additive Butaselmevit-Plus demonstrated antioxidant properties, which is due to its chemical composition (milk thistle, selenium, methionine, and vitamins). The introduction of the feed additive Butaselmevit-Plus to the rats of the experimental group contributed to an increase in the activity of superoxide dismutase and catalase in the blood serum by 22.7 and 20.7%, respectively. When providing this feed additive to rats of the experimental group, an increase in the level of reduced glutathione was also identified, which reached its maximum value on the 28th day of the experiment. Thus, the results of the study confirm the effectiveness of using the supplement Butaselmevit-Plus to improve the antioxidant status of animals in conditions of chronic intoxication of the rat body with lead and cadmium. The practical value of the results obtained is to substantiate the feasibility of using the feed additive Butaselmevit-Plus in animal husbandry to prevent the negative impact of heavy metals on the animal body

A natural experiment identifies an impending ecological trap for a neotropical amphibian in response to extreme weather events.

Extreme weather events are predicted to increase as a result of climate change, yet amphibian responses to extreme disturbance events remain understudied, especially in the Neotropics. Recently, an unprecedented windstorm within a protected Costa Rican rainforest opened large light gaps in sites where we have studied behavioral responses of diurnal strawberry poison frogs (Oophaga pumilio) to ultraviolet radiation for nearly two decades. Previous studies demonstrate that O. pumilio selects and defends perches where ultraviolet radiation (UV‐B) is relatively low, likely because of the lethal and sublethal effects of UV‐B. In this natural experiment, we quantified disturbance to O. pumilio habitat, surveyed for the presence of O. pumilio in both high‐disturbance and low‐disturbance areas of the forest, and assessed UV‐B levels and perch selection behavior in both disturbance levels. Fewer frogs were detected in high‐disturbance habitat than in low‐disturbance habitat. In general, frogs were found vocalizing at perches in both disturbance levels, and in both cases, in significantly lower UV‐B levels relative to ambient adjacent surroundings. However, frogs at perches in high‐disturbance areas were exposed to UV‐B levels nearly 10 times greater than males at perches in low‐disturbance areas. Thus, behavioral avoidance of UV‐B may not reduce the risks associated with elevated exposure under these novel conditions, and similarly, if future climate and human‐driven land‐use change lead to sustained analogous environments.

Accumulation of heavy metal in spleen of gallus gallus within polluted areas in wasit province

With the continuation of oil extraction and manufacture of brick from brick factories in Wasit province, the heavy metals amounts emitted to the environments are steadily increasing. Hence, can result in high levels of accumulation of these contaminants and intoxication of humans and animals living nearby, that pose a hazard to ecosystems, animals and human health. In this study we detected the concentrations of lead (Pb), cadmium (Cd), zinc (Zn), iron (Fe) and cupper (Cu), in spleen tissues of Gallus gallus specimens being collected from two sites in Wasit province. All studied mineral parameters were significantly higher in samples taken from areas near Al-Hay brick factories than those taken from Al-Ahdeb oil fields, this indicates that areas near Al-Hay brick factories are highly polluted and it can be considered unsuitable for living due to the hazardous effect resulted from high rate of accumulation of toxic heavy menials.

Prevalence of exogenous intoxications in dogs and cats in the municipality of Fortaleza and metropolitan region

Animals with animal poisoning are seen with increasing frequency in the country's veterinary clinics and represent a clinical emergency in most cases. To determine the main toxic agents and the frequency of poisoning in dogs and cats in the city of Fortaleza and its metropolitan area, this epidemiological study focused on the cases of intoxication in dogs and cats that occurred between January 2015 and December 2020 and was carried out in 100 veterinary clinics with 100 veterinary medical professionals. The number of registered cases was 9,951. Of these, 7,039 were cases of intoxication in dogs (70.7%), and 2,533 were cases of intoxication in cats (25.5%). The main categories of intoxicating agents in decreasing order of prevalence were food (31.7%), pesticides (25.5%), medicines (16.2%), poisonous and venomous animals (14.5%), toxic plants (4.6%), cleaning products (3.7%) and drugs of abuse (2.9%). Therefore, we concluded that cases of exogenous intoxication remain frequent in medical clinics for small animals. In this context, it was possible to create an alert profile about cases of intoxication in dogs and cats, and the importance of practicing methods of preventing poisoning in domestic animals is emphasized.

The Ubiquitous Soil Terpene Geosmin Acts as a Warning Chemical.

Known as the smell of earth after rain, geosmin is an odorous terpene detectable by humans at picomolar concentrations. Geosmin production is heavily conserved in actinobacteria, myxobacteria, cyanobacteria, and some fungi, but its biological activity is poorly understood. We theorized that geosmin was an aposematic signal used to indicate the unpalatability of toxin-producing microbes, discouraging predation by eukaryotes. Consistent with this hypothesis, we found that geosmin altered the behavior of the bacteriophagous nematode Caenorhabditis elegans on agar plates in the absence of bacteria. Normal movement was restored in mutant worms lacking differentiated ASE (amphid neurons, single ciliated endings) neurons, suggesting that geosmin is a taste detected by the nematodal gustatory system. In a predation assay, geosmin and the related terpene 2-methylisoborneol reduced grazing on the bacterium Streptomyces coelicolor. Predation was restored by the removal of both terpene biosynthetic pathways or the introduction of C. elegans that lacked differentiated ASE taste neurons, leading to the apparent death of both bacteria and worms. While geosmin and 2-methylisoborneol appeared to be nontoxic, grazing triggered bacterial sporulation and the production of actinorhodin, a pigment coproduced with a number of toxic metabolites. In this system, geosmin thus appears to act as a warning signal indicating the unpalatability of its producers and reducing predation in a manner that benefits predator and prey. This suggests that molecular signaling may affect microbial predator-prey interactions in a manner similar to that of the well-studied visual markers of poisonous animal prey. IMPORTANCE One of the key chemicals that give soil its earthy aroma, geosmin is a frequent water contaminant produced by a range of unrelated microbes. Many animals, including humans, are able to detect geosmin at minute concentrations, but the benefit that this compound provides to its producing organisms is poorly understood. We found that geosmin repelled the bacterial predator Caenorhabditis elegans in the absence of bacteria and reduced contact between the worms and the geosmin-producing bacterium Streptomyces coelicolor in a predation assay. While geosmin itself appears to be nontoxic to C. elegans, these bacteria make a wide range of toxic metabolites, and grazing on them harmed the worms. In this system, geosmin thus appears to indicate unpalatable bacteria, reducing predation and benefiting both predator and prey. Aposematic signals are well known in animals, and this work suggests that metabolites may play a similar role in the microbial world.

Drymarchon corais(Colubridae) andCaiman crocodilus(Alligatoridae) use different feeding behaviors to consume poisonous toads

AbstractDrymarchon coraisandCaiman crocodilusare reported here using different feeding behaviors for consuming poisonous toads of the genusRhinella(Bufonidae).Drymarchon coraiswas observed feeding on specimens ofRhinella diptychawithout avoiding contact with the parotoid macroglands, suggesting that this snake species is immune to toad toxins.Caiman crocodiluswas recorded avoiding contact with the parotoid macroglands of specimens ofRhinella marinaby consuming only the front and rear legs, suggesting that this caiman may be sensitive to toad toxins. Further experimental and field studies are needed to better understand the feeding behavior ofD. coraisandC. crocodilusand the effects ofRhinellatoxins on these predators.Abstract in Portuguese is available with online material.