Effect of glyphosate and ciprofloxacin exposure on enteric bacteria of tadpoles / Efecto de la exposición a glifosato y ciprofloxacina en bacterias entéricasde renacuajos

Abstract The high load of agrochemicals and antibiotics present in agricultural aquatic environments represents a risk for wildlife. Since enteric bacteria, which play a key role in the physiological functioning of their hosts, are sensitive to a wide variety of pollutants, their study allows to evaluate the health of organisms. This study aimed to evaluate the effects of commercial formulations of a glyphosate-based herbicide (GBH) and the antibiotic ciprofloxacin (CIP), individually and in mixture, on the bacterial diversity of the intestinal content of common toad (Rhinella arenarum) tadpoles. The diversity of cultivable fast-growing bacteria with low nutritional requirements was evaluated using classic microbiological tests and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identification. Bacterial diversity varied among treatments. Taxa diversity increased in the GBH-treated group but decreased in the CIP-treated group. Remarkably, Yersinia spp. and Proteus spp. were only found in the GBH-treated group. The prevalence of Klebsiella spp. and Pseudomonas spp. decreased in the intestinal microbiota of the GBH-CIP-treated group. To our knowledge, this is the first report on the alteration of cultivable enteric bacteria of autochthonous tadpoles due to two pollutants of emerging concern. Our results demonstrate that R. arenarum tadpoles can be used as non-conventional model organisms for environmental pollution monitoring. Our preliminary findings would contribute to understanding how the presence of GBH and CIP in freshwaters may represent a threat to wildlife and human health by causing enteric dysbiosis of part of the bacterial community.

Resumen La alta carga de agroquímicos y antibióticos en los ambientes acuáticos y los agroe-cosistemas representa un riesgo para la vida silvestre. Dado que la microbiota intestinal juega un papel fundamental en el funcionamiento de su hospedador y es sensible a una amplia variedad de contaminantes, su estudio permite evaluar la salud de los organismos. En este trabajo estudiamos los efectos de formulaciones comerciales de un herbicida a base de glifosato (GBH) y del antibiótico ciprofloxacina (CIP), por separado y en mezcla, sobre la diversidad de bacterias intestinales de renacuajos del sapo común (Rhinella arenarum). El estudio de la diversidad de bacterias entéricas cultivables de rápido crecimiento y bajo requerimiento nutricional se llevó a cabo utilizando pruebas microbiológicas clásicas e identificación por espectrometría de masas de tiempo de vuelo por desorción/ionización láser asistida por matriz (MALDI-TOF). La microbiota entérica fue diferente según el tratamiento. El GBH indujo un aumento de la diversidad bacteriana, mientras que la CIP produjo una reducción. Entre estos cambios, destaca la presencia de Yersinia spp. y Proteus spp. solo en el tratamiento con GBH. Además, en el tratamiento GBH-CIP se encontró una disminución en la prevalencia de Klebsiella spp. y Pseudomonas spp. en la microbiota intestinal de los renacuajos. Este es el primer informe sobre la alteración del contenido bacteriano intestinal de renacuajos de R. arenarum producido por dos contaminantes emergentes de preocupación. Demostramos que el renacuajo del sapo común se puede utilizar como un organismo modelo no convencional para el monitoreo de la contaminación ambiental. Estos hallazgos constituyen el primer paso para comprender cómo la presencia de GBH y CIP en aguas dulces puede representar una amenaza para la vida silvestre y la salud humana a través de la disbiosis entérica asociada al efecto sobre la comunidad bacteriana.

Action of heparin and acetylcholine modulators on the neurotoxicity of the toad Rhinella schneideri (Anura: Bufonidae) in Brazil / Acción de la heparina y moduladores de acetilcolina en la neurotoxicidad del sapo Rhinella schneideri (Anura: Bufonidae) en Brasil

Abstract Introduction: Rhinella schneideri is a toad widely distributed in South America and its poison is characterized by inducing cardiotoxicity and neurotoxicity. Objective: In this work, we investigated pharmacological strategies to attenuate the peripheral neurotoxicity induced by R. schneideri poison in avian neuromuscular preparation. Methods: The experiments were carried out using isolated chick biventer cervicis preparation subjected to field stimulation for muscle twitches recordings or exposed to acetylcholine and potassium chloride for contracture responses. Results: Poison (10 μg/ml) produced complete neuromuscular blockade in chick biventer cervicis preparation within approximately 70 min incubation (times for 50 and 90 % blockade: 15 ± 3 min and 40 ± 2 min, respectively; P < 0.05, N= 5); contracture responses to exogenous acetylcholine and KCl were unaffected by poison indicating no specificity with postsynaptic receptors or myotoxicity, respectively. Poison (10 μg/ml)-induced neuromuscular blockade was not prevented by heparin (5 and 150 IU/ml) under pre- or post-treatment conditions. Incubation at low temperature (23-25 °C) abolished the neuromuscular blockade; after raising the temperature to 37 °C, the complete neuromuscular blockade was slightly slower than that seen in preparations directly incubated at 37 °C (times for 50 and 90 % blockade: 23 ± 2 min and 60 ± 2.5 min, respectively; P < 0.05, N= 4). Neostigmine (3.3 μM) did not reverse the neuromuscular blockade in BC preparation whereas 3,4-diaminopyridine (91.6 μM) produced a partial and sustained reversal of the twitch responses (29 ± 7.8 % of maximal reversal reached in approximately 40 min incubation; P < 0.05, N= 4). Conclusions: R. schneideri poison induces potent peripheral neurotoxicity in vitro which can be partially reversible by 3,4-diaminopyridine.

Resumen Introducción: Rhinella schneideri está ampliamente distribuida en Suramérica y su veneno es caracterizado por inducir cardiotoxicidad y neurotoxicidad. Objetivo: En este trabajo, investigamos estrategias farmacológicas para atenuar la neurotoxicidad periférica inducida por el veneno de R. schneideri en preparaciones neuromusculares de aves. Métodos: Los experimentos fueron realizados usando preparaciones de biventer cervicis de pollos sometidas a estimulación de campo para el registro de las contracciones musculares o expuestas a la acetilcolina y al cloruro de potasio para la respuesta contractural. Resultados: El veneno (10 µg/ml) provocó un bloqueo neuromuscular completo en las preparaciones después de aproximadamente 70 min de incubación (tiempos para 50 y 90 % de bloqueo: 15 ± 3 min y 40 ± 2 min, respectivamente; P < 0.05, N = 5); las contracturas en respuesta a la acetilcolina y el KCl exógenos no fueron afectadas por el veneno, indicando que no hay una interacción especifica con receptores postsinápticos o miotoxicidad respectivamente. El bloqueo neuromuscular causado por el veneno (10 µg/ml) no fue prevenido por la heparina (5 y 150 UI/ml) bajo condiciones pre y post-tratamiento. La incubación a bajas temperaturas (23-25 ºC) abolió el bloqueo neuromuscular; después de aumentar la temperatura a 37 ºC, el bloqueo neuromuscular total fue levemente más lento que el visto en preparaciones directamente incubadas a 37 ºC (tiempos para 50 y 90 % de bloqueo: 23 ± 2 min y 60 ± 2.5 min, respectivamente; P < 0.05, N= 4). Neostigmina (3.3 µM) no revirtió el bloqueo neuromuscular, mientras que 3.4-diaminopiridina (91.6 µM) produjo una reversión parcial y sostenida de las respuestas neuromusculares (29 ± 7.8 % de la reversión máxima alcanzada en aproximadamente 40 min de incubación; P < 0.05, N = 4). Conclusiones: El veneno de R. schneideri indujo neurotoxicidad periférica potente in vitro, el cual puede ser revertido por 3.4-diaminopiridina.

Immunohistochemical localization of vascular factors in tooth germ of amphibian (Cynops pyrrhogaster) / Localización inmunohistoquímica de factores vasculares en germen dentario de anfibios (Cynops pyrrhogaster)

SUMMARY: Vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2, are known to regulate blood vessel endothelium growth. They play important role in human and rodents teeth development. In newt jaws, there are sequential developmental teeth germs following behind the mature teeth. We examined the immunohistochemical localization of VEGF and its receptor and showed the specific expression pattern of VEGF and VEGF receptor in Cynops pyrrhogaster sequential tooth development. The intensity of immunoreactivity for VEGF in the inner enamel epithelium was weaker than that in the outer enamel epithelium in the dentine matrix formation and mineralization stages. Finally, at the enameloid maturation and enamel-like matrix formation stage, immunoreactivity for VEGF in inner enamel epithelium was stronger than in the outer enamel epithelium. The intensity of immunoreactivity for VEGFR-2 was positive for the outer enamel epithelium throughout tooth development. The crown sides of the odontoblasts were stained especially strongly for VEGF and VEGFR-2 during the dentine matrix formation and mineralization stage of the enameloid maturation and enamel- like matrix formation stage. We postulate that the expression of VEGF in the inner enamel epithelium and odontoblast widely effects tooth development in newts, as well as in human and rodents.

RESUMEN: Se sabe que el factor de crecimiento endotelial vascular (VEGF) y su receptor, VEGFR-2, regulan el crecimiento del endotelio de los vasos sanguíneos. Desempeñan un papel importante en el desarrollo de los dientes humanos y de los roedores. En las mandíbulas de tritón, hay gérmenes dentales de desarrollo secuenciales que siguen a los dientes maduros. Examinamos la localización inmunohistoquímica de VEGF y su receptor y mostramos el patrón de expresión específico de VEGF y receptor de VEGF en el desarrollo secuencial de dientes de Cynops pyrrhogaster. La intensidad de la inmunorreactividad para VEGF en el epitelio interno del esmalte era más débil que en el epitelio externo del esmalte en las etapas de formación y mineralización de la matriz de dentina. Finalmente, en la etapa de maduración del esmalte y de formación de la matriz similar al esmalte, la inmunorreactividad para VEGF en el epitelio interno del esmalte fue más fuerte que en el epitelio externo del esmalte. La intensidad de la inmunorreactividad para VEGFR- 2 fue positiva para el epitelio externo del esmalte durante el desarrollo del diente. Los márgenes de la corona de los odontoblastos se tiñeron especialmente para VEGF y VEGFR-2 durante la etapa de formación de la matriz de dentina y mineralización de la etapa de maduración del esmalte y la etapa de formación de la matriz similar al esmalte. Postulamos que la expresión de VEGF en el epitelio interno del esmalte y odontoblastos afecta ampliamente el desarrollo de los dientes en tritones, así como en humanos y roedores.

Celiotomia e gastrotomia para remoção cirúrgica de corpos estranhos em axolote (Ambystoma mexicanum): relato de caso / Celiotomy and gastrotomy for surgical removal of foreign bodies in axolotl (Ambystoma mexicanum): case report

A ingestão de corpos estranhos em aquários artificiais é uma ocorrência frequente observada na clínica cirúrgica de anfíbios criados como pets, o que inclui os axolotes (Ambystoma mexicanum). O presente relato descreve um caso de ingestão de sete corpos estranhos em um axolote macho, de cinco meses de idade, com histórico de abaulamento irregular e irredutível da superfície corpórea ventral, de consistência firme. Na anamnese foi informado que o animal habitava um aquário com substrato de cascalhos. Ao exame físico, o axolote apresentou bom estado geral e parâmetros vitais dentro da normalidade para a espécie. Os materiais deglutidos foram identificados em região gástrica por meio de exame radiográfico corpóreo total, e suas características condiziam com o substrato utilizado no aquário do paciente. Como protocolo anestésico, foi priorizada a imersão em Isofluorano e gás oxigênio, com o objetivo de atingir a via branquial e, ocasionalmente, transdérmica. A remoção cirúrgica foi feita através de celiotomia e gastrotomia em ambiente aquático com temperatura, pH e coleção bacteriana controlados, conforme literatura disponível e tendo em consideração a natureza, diâmetro e localização dos corpos estranhos. Após cinco e quinze dias do procedimento, acompanhou-se a cicatrização da ferida cirúrgica, sendo possível constatar bom restabelecimento da continuidade dos tecidos e bom estado geral do paciente.

The ingestion of foreign bodies in artificial aquariums is a frequent occurrence observed in the surgical clinic of amphibians raised as pets, which includes axolotls (Ambystoma mexicanum). The present report describes a case of ingestion of seven foreign bodies in a male axolotl, five months old, with a history of irregular and irreducible bulging of the ventral body surface, with firm consistency. In the anamnesis, it was reported that the animal inhabited an aquariums with gravel substrate. On physical examination, the axolotl showed good general condition and vital parameters within the normal range for the species. The swallowed materials were identified in the gastric region by means of total body radiographic examination, and their characteristics were consistent with the substrate used in the patient's aquarium. As anesthetic protocol, immersion in Isofluorane and oxygen gas was prioritized, in order to reach the branchial and, occasionally, transdermal route. Surgical removal was performed through celiotomy and gastrotomy in an aquatic environment with controlled temperature, pH and bacterial collection, according to available literature and taking into account the nature, diameter and location of foreign bodies. After five and fifteen days of the procedure, the healing of the surgical wound was monitored, showing a good restoration of tissue continuity and a good general condition of the patient.

Celiotomia e gastrotomia para remoção cirúrgica de corpos estranhos em axolote (Ambystoma mexicanum): relato de caso / Celiotomy and gastrotomy for surgical removal of foreign bodies in axolotl (Ambystoma mexicanum): case report

A ingestão de corpos estranhos em aquários artificiais é uma ocorrência frequente observada na clínica cirúrgica de anfíbios criados como pets, o que inclui os axolotes (Ambystoma mexicanum). O presente relato descreve um caso de ingestão de sete corpos estranhos em um axolote macho, de cinco meses de idade, com histórico de abaulamento irregular e irredutível da superfície corpórea ventral, de consistência firme. Na anamnese foi informado que o animal habitava um aquário com substrato de cascalhos. Ao exame físico, o axolote apresentou bom estado geral e parâmetros vitais dentro da normalidade para a espécie. Os materiais deglutidos foram identificados em região gástrica por meio de exame radiográfico corpóreo total, e suas características condiziam com o substrato utilizado no aquário do paciente. Como protocolo anestésico, foi priorizada a imersão em Isofluorano e gás oxigênio, com o objetivo de atingir a via branquial e, ocasionalmente, transdérmica. A remoção cirúrgica foi feita através de celiotomia e gastrotomia em ambiente aquático com temperatura, pH e coleção bacteriana controlados, conforme literatura disponível e tendo em consideração a natureza, diâmetro e localização dos corpos estranhos. Após cinco e quinze dias do procedimento, acompanhou-se a cicatrização da ferida cirúrgica, sendo possível constatar bom restabelecimento da continuidade dos tecidos e bom estado geral do paciente.

The ingestion of foreign bodies in artificial aquariums is a frequent occurrence observed in the surgical clinic of amphibians raised as pets, which includes axolotls (Ambystoma mexicanum). The present report describes a case of ingestion of seven foreign bodies in a male axolotl, five months old, with a history of irregular and irreducible bulging of the ventral body surface, with firm consistency. In the anamnesis, it was reported that the animal inhabited an aquariums with gravel substrate. On physical examination, the axolotl showed good general condition and vital parameters within the normal range for the species. The swallowed materials were identified in the gastric region by means of total body radiographic examination, and their characteristics were consistent with the substrate used in the patient’s aquarium. As anesthetic protocol, immersion in Isofluorane and oxygen gas was prioritized, in order to reach the branchial and, occasionally, transdermal route. Surgical removal was performed through celiotomy and gastrotomy in an aquatic environment with controlled temperature, pH and bacterial collection, according to available literature and taking into account the nature, diameter and location of foreign bodies. After five and fifteen days of the procedure, the healing of the surgical wound was monitored, showing a good restoration of tissue continuity and a good general condition of the patient.

Assessment of Computer Assisted Amphibian Lab

Computer-Aided Amphibian Laboratory (CAAL) means that experiments can be teached any time the computer is accessible; no laboratory technical help is required to set up the lab. The learning potential using computer simulation is tremendous. The Computer Assisted Amphibian Lab (CAAL) has the technology, classes and knowledgeable staff to help students in our Developmental Education. Various Software programs sharpen their skills. Various videos related to frog dissection and rabbit gut experiments provide a better understanding of experiments. Many medical institutes have shifted to techniques which minimize sacrifice of animals. MCI has given liberal permission to use these programs and has also invited faculty to build such programs for future use. OBJECTIVES:- The objectives of this study were to assess the benefits of computer assisted learning in amphibian lab and to compare the computer assisted teaching with other teaching methodologies. Methods: Total 70 students of MBBS from Glocal Medical College, Glocal University, Saharanpur were provided a self made questionnaire regarding the feedback about Computer Assisted Amphibian Lab. All students were taken from MBBS second prof. batch 2016. Questionnaire consisted of 10 questions out of which 9 were according to Likert scale and one multiple choice question. Data was analysed manually. Results: 1) When asked about the most beneficial method for teaching in CAAL lab; 36.45% students answered that they prefer online videos related to experiment shown on computer via website. 27% students answered that they prefer chalk and talk while 25% students preferred Computer assisted learning with predesigned softwares and only 9.37% students preferred teaching with powerpoint presentation. Conclusion: CAAL is a better alternative than mere teaching in the absence of animal experiments.

Hiponatremia grave secundaria a la exposición a veneno de Phyllomedusa Bicolor (Rana Kambó): caso clínico / Severe hyponatremia secondary to Phyllomedusa bicolor (Kambó frog) poisoning: report of one case

Phyllomedusa bicolor or Kambo is a frog that lives in the Amazon rainforest. It can release through its skin a substance used in healing rituals that are common among South-American tribes, as well as in urban people of America and Europe. We report a 41-year-old female patient who, during a healing ritual consumed ayahuasca (a drink obtained from the mixture of Banisteriopsis caapi, Psychotria viridis and Mimosa hostilis) and 12 hours later received the poison of Kambo Frog (Phyllomedusa bicolor) on superficial right shoulder skin burns. The ritual included a minimum of six-liter water intake over a few hours period. She evolved with clouding of sensorium, motor agitation, frequent vomiting, and generalized tonic-clonic seizures. She presented lethargic to the emergency room, with a weak pupillary light reflex, generalized stiffness, moving all four limbs. Laboratory showed severe hyponatremia (120 mEq/L) and a creatine kinase level of 8,479 UI/L, that increased 107,216 IU/L within few days. An admission CT Brain scan was normal. The toxicological screening did not identify the presence of other substances. During hospitalization the patient developed severe psychomotor agitation controlled by a dexmedetomidine infusion, hyponatremia, low plasma osmolality (248 mOsm/kg), and disproportionately high urinary osmolality (448 mOsm/kg), suggestive of inappropriate antidiuretic hormone secretion syndrome (SIADH). With correction of hyponatremia, the patient gradually recovered consciousness. Rhabdomyolysis was assumed to be secondary to seizure and managed by volume and bicarbonate infusions with a positive response.

Antibacterial activity of the skin secretion of Phyllomedusa azurea (Anura: Hylidae) from the Central Brazil Cerrado / Actividad antibacterial de secreciones de la piel de Phyllomedusa azurea (Anura: Hylidae) en Cerrado, Brasil centro

Abstract Phyllomedusa azurea is a frog species well distributed geographically in South America, including Brazilian biomes as Pantanal and Cerrado. Compared with other anurans from the Phyllomedusinae family, there are few reports on the bioactive potential of skin-derived molecules from this species. In this perspective, the aim of the present study was to evaluate the in vitro antibacterial activity of skin secretion of P. azurea by detection of Minimum Inhibitory Concentration (MIC) of the growth of bacterial indicator strains and to determine if occurs a changing in the bacterial cell envelope permeability. The MIC determination was carried out by the microdilution plate method. The absorbance was measured and analyzed statistically using the t-test to compare two groups (0.05 % of significance). The impact of the crude extract on cell envelope permeability of Staphylococcus aureus ATCC 25923 was conducted by the crystal violet assay, and the absorbance was measured spectrophotometry followed by the calculation of the crystal violet uptake percentage. The specific MIC for S. aureus ATCC 25923 and Escherichia coli ATCC 25922 was 31.25 µg/mL, while for Bacillus subtilis ATCC 6633 was 125 µg/mL and Pseudomonas aeruginosa ATCC 27853 was 250 µg/mL. The treatment with crescent concentrations of frog skin secretion increased the crystal violet uptake by S. aureus ATCC 25923 cells, suggesting an action on the cell plasma membrane. The results demonstrated that the skin secretion of P. azurea presents antibacterial activity and merit further investigations to characterize the bioactive molecules.(AU)

Resumen P. azurea es una especie de rana bien distribuida geográficamente en América del Sur, que incluye biomas brasileños como Pantanal y Cerrado. En comparación con otros anuros de Phyllomedusinae, existen pocos informes sobre el potencial bioactivo de las moléculas derivadas de la piel de esta especie. En esta perspectiva, el objetivo del presente estudio fue evaluar la actividad antibacteriana in vitro de la secreción de la piel de P. azurea mediante la detección de la Concentración Inhibitoria Mínima (CIM) del crecimiento de cepas indicadoras bacterianas y determinar si ocurre un cambio en la permeabilidad de la envoltura celular bacteriana. La determinación de MIC se llevó a cabo mediante el método de la placa de microdilución. La absorbancia se midió y se analizó estadísticamente mediante la prueba t para comparar dos grupos (0.05 de significancia). El impacto del extracto crudo sobre la permeabilidad de la envoltura celular de Staphylococcus aureus ATCC 25923 se realizó mediante el ensayo de cristal violeta, y se midió la absorbancia mediante espectrofotometría seguida del cálculo del porcentaje de absorción de violeta cristal. La CIM específica para S. aureus ATCC 25923 y Escherichia coli ATCC 25922 fue 31.25 μg / ml, mientras que para Bacillus subtilis ATCC 6633 de 125 μg / ml y Pseudomonas aeruginosa ATCC 27853 de 250 μg / ml. El tratamiento con concentraciones crecientes de secreción de piel de rana aumentó la absorción de violeta cristal por las células de S. aureus ATCC 25923, sugiriendo una acción sobre la membrana plasmática de la célula. Los resultados demostraron que la secreción de la piel de P. azurea presenta actividad antibacteriana y amerita más investigaciones para caracterizar las moléculas bioactivas.(AU)

Protein identification from the parotoid macrogland secretion of Duttaphrynus melanostictus

Bufonid parotoid macrogland secretion contains several low molecular mass molecules, such as alkaloids and steroids. Nevertheless, its protein content is poorly understood. Herein, we applied a sample preparation methodology that allows the analysis of viscous matrices in order to examine its proteins. Methods: Duttaphrynus melanostictus parotoid macrogland secretion was submitted to ion-exchange batch sample preparation, yielding two fractions: salt-displaced fraction and acid-displaced fraction. Each sample was then fractionated by anionic-exchange chromatography, followed by in-solution proteomic analysis. Results: Forty-two proteins could be identified, such as acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, galectin and histone. Moreover, de novo analyses yielded 153 peptides, whereas BLAST analyses corroborated some of the proteomic-identified proteins. Furthermore, the de novo peptide analyses indicate the presence of proteins related to apoptosis, cellular structure, catalysis and transport processes. Conclusions: Proper sample preparation allowed the proteomic and de novo identification of different proteins in the D. melanostictus parotoid macrogland secretion. These results may increase the knowledge about the universe of molecules that compose amphibian skin secretion, as well as to understand their biological/physiological role in the granular gland.(AU)

Spatiotemporal variation of roadkills show mass mortality events for amphibians in a highly trafficked road adjacent to a national park, Costa Rica / Variación espacio-temporal de atropellos de vertebrados muestra eventos de mortalidad masiva de anfibios en una carretera altamente transitada adyacente a un parque nacional, Costa Rica

Abstract Roads have detrimental impacts on wildlife populations around the world. Specifically, roads pose direct and indirect threats to wildlife by limiting dispersal movements or through vehicle-related mortality. The rate of wildlife mortality varies both in time and space depending on the landscape composition and the type and use of road infrastructure. The objective of this study was to investigate spatiotemporal variation of vertebrate mortality in a 4 km segment of the 34 national road, adjacent to Carara National Park, Costa Rica. We conducted 81 roadkill surveys by car and bicycle from June 2010 to May 2011, georeferenced the locations of the kills and identified them to the lowest possible taxonomic level. We recorded a total of 4 709 road-killed animals of at least 58 species of vertebrates during the whole study. Amphibians accounted for 93.5 % of all the vertebrate losses and showed strong spatiotemporal variation of mass mortality events. Reptiles, especially snakes, were the second most affected taxon followed by mammals and birds. Relative mortality per day in the 4 km segment was 125.4 amphibians, 4.6 reptiles, 2.7 mammals, 1 bird and 0.46 undetermined. Road proximity to the border of the park, traffic volumes and lack of enforcement of speed limits may influence the high rate of roadkills found. We suggest the reinforcement of speed limits, wildlife crossing signage and the retrofitting of the existing culverts as under passes for animals to minimize vertebrate mortality at the road adjacent to Carara National Park.

Resumen Las carreteras tienen impactos perjudiciales en las poblaciones de vida silvestre en todo el mundo. Específicamente, los caminos representan amenazas directas e indirectas a la vida silvestre limitando los movimientos de dispersión, o debido a la mortalidad por atropellos por vehículos. La tasa de mortalidad de la fauna varía tanto en el tiempo como en el espacio, dependiendo de la composición del paisaje y del tipo y uso de la infraestructura vial. El objetivo de este estudio fue investigar la variación espaciotemporal de la mortalidad de vertebrados en un tramo de 4 km de la carretera nacional 34, adyacente al Parque Nacional Carara, Costa Rica. Llevamos a cabo 81 censos de vertebrados atropellados utilizando un vehículo y una bicicleta entre junio 2010 y mayo 2011, georreferenciamos las ubicaciones de los cuerpos y los identificamos al nivel taxonómico más bajo posible. Registramos un total de 4 709 animales muertos por carretera de al menos 58 especies de vertebrados durante todo el estudio. Los anfibios representaron el 93.5 % de todos los vertebrados y mostraron una fuerte variación espaciotemporal en eventos de mortalidad masiva. Los reptiles, especialmente las serpientes, fueron el segundo taxón más afectado seguido por los mamíferos y las aves. La mortalidad relativa por día en el segmento de 4 km fue de 125.4 anfibios, 4.6 reptiles, 2.7 mamíferos, 1 ave y 0.46 indeterminado. La proximidad de la carretera al límite del parque, los volúmenes de tránsito y la falta de cumplimiento de los límites de velocidad pueden influir en la alta tasa de mortalidad registrada. Sugerimos la reducción en los límites de velocidad, la señalización de cruce de vida silvestre y la readecuación de las alcantarillas existentes como pasos para los animales para minimizar la mortalidad de los vertebrados en la carretera adyacente al Parque Nacional Carara.

Looking for attitudes related to amphibian species decline: how are peer-reviewed publications of education activities compared to ecological research?

ABSTRACT Biodiversity decline has been the focus of discussions in the last decade, especially on the amphibian species decline. After a scientometric analysis using international databases, we found that the number of peer-reviewed articles considering education practices related to the theme increased along with the number of ecological researches. However, the increase in ecological researches is much higher than the increase in publications of education practices. Studies suggest that conservation attitudes are important and that education practices are an important tool for improving human perceptions on this subject. In this sense, increase the publication of projects and programs results related to local education practices in international journals could help the dissemination of efficient methods for conservation, as well as facilitating access to information internationally, since species decline, especially for amphibians, is a global concern. Then, we suggest that educational practices, at least when related to conservation, should follow a more standardized protocol, and be published in international journals, as the efficiency of such practices should be evaluated and methods once published could help other nations to improve their ecological literacy.

Antiviral activity of animal venom peptides and related compounds

Viruses exhibit rapid mutational capacity to trick and infect host cells, sometimes assisted through virus-coded peptides that counteract host cellular immune defense. Although a large number of compounds have been identified as inhibiting various viral infections and disease progression, it is urgent to achieve the discovery of more effective agents. Furthermore, proportionally to the great variety of diseases caused by viruses, very few viral vaccines are available, and not all are efficient. Thus, new antiviral substances obtained from natural products have been prospected, including those derived from venomous animals. Venoms are complex mixtures of hundreds of molecules, mostly peptides, that present a large array of biological activities and evolved to putatively target the biochemical machinery of different pathogens or host cellular structures. In addition, non-venomous compounds, such as some body fluids of invertebrate organisms, exhibit antiviral activity. This review provides a panorama of peptides described from animal venoms that present antiviral activity, thereby reinforcing them as important tools for the development of new therapeutic drugs.(AU)

Antiviral activity of animal venom peptides and related compounds

Abstract Viruses exhibit rapid mutational capacity to trick and infect host cells, sometimes assisted through virus-coded peptides that counteract host cellular immune defense. Although a large number of compounds have been identified as inhibiting various viral infections and disease progression, it is urgent to achieve the discovery of more effective agents. Furthermore, proportionally to the great variety of diseases caused by viruses, very few viral vaccines are available, and not all are efficient. Thus, new antiviral substances obtained from natural products have been prospected, including those derived from venomous animals. Venoms are complex mixtures of hundreds of molecules, mostly peptides, that present a large array of biological activities and evolved to putatively target the biochemical machinery of different pathogens or host cellular structures. In addition, non-venomous compounds, such as some body fluids of invertebrate organisms, exhibit antiviral activity. This review provides a panorama of peptides described from animal venoms that present antiviral activity, thereby reinforcing them as important tools for the development of new therapeutic drugs.

Two novel antimicrobial peptides from skin venoms of spadefoot toad Megophrys minor / 中国天然药物

Amphibian skin contains rich bioactive peptides. Especially, a large amount of antimicrobial peptides have been identified from amphibian skin secretions. Antimicrobial peptides display potent cytolytic activities against a range of pathogenic bacteria and fungi and play important defense roles. No antimicrobial peptides have been reported from toads belonging to the family of Pelobatidae. In this work, two novel antimicrobial peptides (Megin 1 and Megin 2) were purified and characterized from the skin venoms of spadefoot toad Megophrys minor (Pelobatidae, Anura, Amphibia). Megin 1 had an amino acid sequence of FLKGCWTKWYSLKPKCPF-NH2, which was composed of 18 amino acid residues and contained an intra-molecular disulfide bridge and an amidated C-terminus. Megin 2 had an amino acid sequence of FFVLKFLLKWAGKVGLEHLACKFKNWC, which was composed of 27 amino acid residues and contained an intra-molecular disulfide bridge. Both Megin 1 and Megin 2 showed potential antimicrobial abilities against bacteria and fungi. The MICs of Megin 1 against Escherichia coli, Bacillus dysenteriae, Staphylococcus aureus, Bacillus subtilis, and Candida albicans were 25, 3, 6.25, 3, and 50 μg·mL(-1), respectively. The corresponding MICs for Megin 2 were 6.25, 1.5, 12.5, 1.5, and 12.5 μg·mL(-1), respectively. They also exerted strong hemolytic activity against human and rabbit red cells. The results suggested that megin peptides in the toad skin of M. minor displayed toxic effects on both eukaryotes and prokaryotes. This was the first report of antimicrobial peptides from amphibians belonging to the family of Pelobatidae.

Helminth parasite communities of two Physalaemus cuvieri Fitzinger, 1826 (Anura: Leiuperidae) populations under different conditions of habitat integrity in the Atlantic Rain Forest of Brazil / Comunidade de helmintos parasitos de duas populações de Physalaemus cuvieri Fitzinger, 1826 (Anura: Leiuperidae) sob diferentes condições de integridade de habitat da Mata Atlântica, Brasil

Abstract Adults of Physalaemus cuvieri were collected and necropsied between November 2009 and January 2010. This was carried out in order to report and compare the helminth fauna associated with two populations of this anuran species from the Brazilian Atlantic rain forest under different conditions of habitat integrity. The hosts from the disturbed area were parasitized with five helminth taxa: Cosmocerca parva, Aplectana sp., Physaloptera sp., Rhabdias sp., Oswaldocruzia subauricularis (Nematoda) and Polystoma cuvieri (Monogenea) while those from the preserved area had four helminth taxa: C. parva, Aplectana sp., Physaloptera sp., Rhabdias sp., and Acanthocephalus saopaulensis (Acanthocephala). Prevalence, mean intensity of infection, mean abundance, mean richness, importance index and dominance frequency of helminth component communities were similar in both areas. The helminth community associated with anurans from the disturbed area had higher diversity than that from the preserved area. This study is the first to report on the acanthocephalan parasites of Ph. cuvieri, and the similarity between helminth fauna composition of two host populations under different selective pressures.

Resumo Adultos de Physalaemus cuvieri foram coletados e necropsiados entre Novembro de 2009 e Janeiro de 2010. Este estudo foi realizado com o objetivo de informar e comparar a helmintofauna associada a duas populações desta espécie de anuro proveniente da Mata Atlântica sob duas condições de integridade de hábitat. Os hospedeiros da área perturbada estavam parasitados por cinco taxa de helmintos: Cosmocerca parva, Aplectana sp., Physaloptera sp., Rhabdias sp., Oswaldocruzia subauricularis (Nematoda) e Polystoma cuvieri (Monogenea), enquanto aqueles da área preservada apresentaram quatro taxa de helmintos: C. parva, Aplectana sp., Physaloptera sp., Rhabdias sp., e Acanthocephalus saopaulensis (Acanthocephala). Prevalência, intensidade média de infecção, abundância média, riqueza média, índice de importância específica e freqüência de dominância da comunidade componente dos helmintos foram similares em ambas as áreas. A comunidade helmíntica associada aos anuros da área perturbada apresentou-se mais diversa do que a encontrada naqueles da área preservada. Este estudo é o primeiro a relatar parasitas acantocéfalos em Ph. cuvieri, e a semelhança entre a composição da helmintofauna de duas populações hospedeiras sob diferentes pressões seletivas.

Differential success in sampling of Atlantic Forest amphibians among different periods of the day / Sucesso diferenciado na amostragem de anfíbios da Mata Atlântica entre diferentes períodos do dia

In general, anurans tend to be nocturnal, though diurnal activity is characteristic of some groups. Studies show that frog activity may be inferred based on the number of individuals collected at different periods of the day, during large-scale field surveys. We investigated the best period of the day to conduct amphibian sampling in nine Atlantic Rainforest areas in southeastern Brazil, based on intensive field surveys. At each locality we employed similar sampling effort during diurnal, crepuscular and nocturnal searches (totaling 704.5 sampling hours). We pooled data from all localities for each period and estimated the proportion of frogs of each species active at each period based on the total number of individuals and on the number of species found during all surveys for that period. We recorded a total of 817 individual frogs from 69 species. Species richness was highest at night (median = 12 species), intermediate at dusk (median = 8), and lowest during the day (median = 4). The percentage of the total number of individual frogs found (pooled species) was highest during the night (ca. 53%) and lowest during the day (ca. 14%). Analyzing each species separately, the number of individuals recorded was consistently higher at dusk and night for most species. Our study evidences a trend for nocturnal activity for most Atlantic Rainforest frogs, with few species having primarily diurnal habits. Those results may favor future studies and conservation efforts for amphibian species.

Em geral, anuros tendem a ser noturnos, apesar de a atividade diurna ser característica de alguns grupos. Estudos mostram que a atividade de anuros pode ser inferida com base no número de indivíduos coletados em diferentes períodos do dia durante pesquisas de campo de larga escala. Nós avaliamos qual o melhor período do dia para conduzir amostragens de anfíbios em nove áreas da Mata Atlântica no sudeste do Brasil, com base em amostragens de campo intensivas. Em cada localidade nós empregamos esforço de amostragem similar durante amostragens diurnas, crepusculares e noturnas (totalizando 704,5 horas de amostragem). Nós agrupamos dados de todas as localidades para cada período e estimamos a proporção de anuros de cada espécie ativa em cada período com base no número total de indivíduos e no número de espécies encontradas durante todas as buscas naquele período. Nós registramos um total de 817 indivíduos pertencentes a 69 espécies. A riqueza de espécies foi maior à noite (mediana = 12 espécies), intermediária ao crepúsculo (mediana = 8) e menor durante o dia (mediana = 4). A porcentagem do número total de indivíduos encontrados (todas as espécies agrupadas) foi maior durante a noite (ca. 53%) e menor durante o período diurno (ca. 14%). Analisando cada espécie separadamente, o número de indivíduos registrados foi consistentemente maior ao crepúsculo e à noite para a maioria das espécies. Nosso estudo evidencia uma tendência para atividade noturna para a maioria dos anuros da Mata Atlântica, com poucas espécies tendo hábitos primariamente diurnos. Esses resultados podem favorecer futuros estudos e esforços de conservação para as espécies de anfíbios.

MORFOHISTOLOGÍA TESTICULAR DE Hypsiboas pulchellus (AMPHIBIA, HYLIDAE)DURANTE LA ESTACIÓN REPRODUCTIVA

En este estudio se aportan datos sobre la morfohistología testicular de Hypsiboas pulchellus durante el período reproductivo con el fin de conocer aspectos de su biología reproductiva. Los testículos, ovoides y amarillentos de 4,64 ± 0,48 mm de largo por 2,05 ± 0,23 mm de ancho, están rodeados por la túnica albugínea que mide 5,60 ± 0,82 µm. Internamente presentan lóculos seminíferos de 257,47 ± 58,25 µm de diámetro, distinguiéndose en ellos cistos con células espermatogénicas asociadas a células de Sertoli. Existe escaso tejido intersticial. Las espermatogonias I son las células más grandes de la serie germinal (14,34 ± 1,74 µm), estas originan a las espermatogonias II (10,14 ± 1,33 µm). Los espermatocitos I presentan la cromatina levemente condensada midiendo 9,34 ± 0,32 µm. Los espermatocitos II miden 8,12 ± 1,07 µm. Las espermátidas I son esféricas, miden 7,61 ± 1,45 µm, y se agrupan en cistos redondeados. Las espermátidas II, en cambio, son alargadas, no hallándoselas dentro de cistos (4,09 ± 0,51 µm). Los espermatozoides, alargados y flagelados, se hallan libres en el centro del lóculo. La morfohistología de los testículos analizados muestra características similares a las observadas en otros anfibios neotropicales, y presentan todas las células del linaje espermatogénico en un mismo lóculo.

In this study, data concerning to the morphological testicular features of Hypsiboas pulchellus during the breeding season is provided, in order to know aspects of their reproductive biology. The testis, ovoid and yellowish with 4.64 ± 0.48 mm length and 2.05 ± 0.23 mm wide, are surrounded by the tunica albuginea which measures 5.60 ± 0.82 µm. The seminiferous locules, placed internally, measure 257.47 ± 58.25 µm in diameter, and cysts with spermatogenic cells associated with Sertoli cells are distinguished in them. The interstitial tissue is scarce. The biggest cells of the germinal series are the spermatogonia I (14.34 ± 1.74 µm), from which spermatogonia II (10.14 ± 1.33 µm) originate. Spermatocytes I measure 9.34 ± 0.32 µm and have slightly condensed chromatin. The spermatocytes II are 8.12 ± 1.07 µm long. The spermatids I are spherical, grouped in rounded cysts, and measure 7.61 ± 1.45 µm. On the other hand, spermatids II are elongated (4.09 ± 0.51 µm) and not within the cysts. Towards the center of the loculus, the free, lengthened and flagellated spermatozoids are located. The morphohistology of the analyzed testicles show similarities with those observed in other neotropical amphibians, being all the cells of the spermatogenic lineage in the same locule.

Copper toxicity for Scinax ruber and Rhinella granulosa (Amphibia: Anura) of the Amazon: Potential of Biotic Ligand Model to predict toxicity in urban streams / Toxicidade do cobre em Scinax ruber e Rhinella granulosa (Amphibia: Anura): Potencial do Modelo do Ligante Biótico para predizer a toxicidade em igarapés urbanos

In the last years many populations of anurans have declined and extinctions have been recorded. They were related to environmental pollution, changes of land use and emerging diseases. The main objective of this study was to determine copper sensitivity of the anuran of the Amazon Rhinella granulosa and Scinax ruber tadpoles at stage 25 and Scinax ruber eggs exposed for 96 h to copper concentrations ranging from 15 µg Cu L-1 to 94 µg Cu L-1. LC50 at 96 h of Rhinella granulosa Gosner 25, Scinax ruber Gosner 25 and Scinax ruber eggs in black water of the Amazon were 23.48, 36.37 and 50.02 µg Cu L-1, respectively. The Biotic Ligand Model was used to predict the LC50 values for these species and it can be considered a promising tool for these tropical species and water conditions. Copper toxicity depends on water physical-chemical composition and on the larval stage of the tadpoles. The Gosner stage 19-21 (related to the appearance of external gills) is the most vulnerable and the egg stage is the most resistant. In case of contamination by copper, the natural streams must have special attention, since copper is more bioavailable.

Nos últimos anos foram registrados muitas extinções e declínios de populações de anuros. Eles estavam relacionados com a poluição do ambiente, a mudanças no uso da terra e ao surgimento de doenças. O principal objetivo deste estudo foi determinar a sensibilidade dos anuros amazônicos ao cobre. Os girinos de Scinax ruber e Rhinella granulosa no estadio 25 e os ovos de Scinax ruber foram expostos por 96 horas a concentrações de cobre entre 15 µg Cu L-1 a 94 µg Cu L-1. A CL50 -96 h dos girinos de Rhinella granulosa, dos girinos de Scinax ruber e dos ovos de Scinax ruber em águas pretas da Amazônia foram 23,48; 36,37 e 50,02 µg Cu L-1, respectivamente. O modelo do ligante biótico foi usado para prever os valores de CL50 para essas duas espécies e pode ser considerado uma ferramenta promissora para essas espécies tropicais e para essas condições de água. A Toxicidade de cobre depende da composição físico-química da água e do estagio larval dos girinos. O estadio 19-21 de Gosner (relacionados ao aparecimento das brânquias externas) são os mais vulnerável e o estagio de ovo é o mais resistente. Em caso de contaminação por cobre, os igarapés naturais devem ter uma atenção especial, uma vez que o cobre é mais biodisponível nesse ambiente.

Characteristics of hemolytic activity induced by skin secretions of the frog Kaloula pulchra hainana

The hemolytic activity of skin secretions obtained by stimulating the frog Kaloula pulchra hainana with diethyl ether was tested using human, cattle, rabbit, and chicken erythrocytes. The skin secretions had a significant concentration-dependent hemolytic effect on erythrocytes. The hemolytic activity of the skin secretions was studied in the presence of osmotic protectants (polyethylene glycols and carbohydrates), cations (Mg2+, Ca2+, Ba2+, Cu2+, and K+), or antioxidants (ascorbic acid, reduced glutathione, and cysteine). Results Depending on their molecular mass, osmotic protectants effectively inhibited hemolysis. The inhibition of skin hemolysis was observed after treatment with polyethylene glycols (1000, 3400, and 6000 Da). Among divalent cations, only 1 mM Cu2+ markedly inhibited hemolytic activity. Antioxidant compounds slightly reduced the hemolytic activity. Conclusions The results suggested that skin secretions of K. pulchra hainana induce a pore-forming mechanism to form pores with a diameter of 1.36-2.0 nm rather than causing oxidative damage to the erythrocyte membrane.

Characteristics of hemolytic activity induced by skin secretions of the frog Kaloula pulchra hainana

Background: Previous works had shown that scorpion venom induced neurotransmitter elevation and an inflammatory response associated with various anatomo-pathological modifications. The most dangerous scorpions species in Algeria responsible for these effects are Androctonus australis hector (Aah) and Androctonus amoreuxi (Aam). Results: Comparison of the physiopathological effects induced by the two venoms showed differences in the kinetic of cytokine release and in lung injury. The lung edema was only observed in response to Aah venom and it was correlated with cell infiltration. In order to better understand the involved mechanism in inflammatory response, we used two antagonists, atropine (non-selective muscarinic antagonist) and propranolol (ß adrenergic antagonist), which lead to a decrease of cell infiltration but has no effect on edema forming. Conclusion: These results suggest another pathway in the development of lung injury following envenomation with Aam or Aah venom.(AU)