Larvicidal activity of the skin secretion of Rhinella marina and Rhaebo guttatus (Anura: Bufonidae) against the Brazilian malaria vector, Anopheles darlingi (Diptera: Culicidae)

@#Malaria, a mosquito-borne disease, is caused by protozoa of the genus Plasmodium and constitutes a serious public health problem. Because current insecticides used to control malaria face resistance due to continuous use, new alternatives are prompted. Considering this context, and the insecticidal potential of vertebrate venoms/secretions, crude and methanolic extracts from two frog species were tested as larvicides against Anopheles darlingi. Skin secretions of Rhinella marina and Rhaebo guttatus were obtained by manual stimulation. Then, methanol was added to obtain steroidal fractions from both venoms. Mosquitos were captured in suburban areas of Porto Velho and An. darlingi females were later fed with blood and stimulated to oviposit. The larvae were fed with fish food until the 3rd and 4th instars. For the larvicidal assays, crude secretions and methanolic fractions of both frog species were evaluated, and larvae mortality was recorded after 48 hours. Crude extracts and steroidal fractions from both species had larvicidal effects, with an LC50 of 127.5 and 133 ppm for the crude extract and steroidal fraction of R. marina, and an LC50 of 37.5 and 35.8 ppm for the crude extract and steroidal secretion of R. guttatus, respectively. The present work reports for the first time the larvicidal effects of the skin secretions from bufonid species occurring in the western Amazon region. Further studies should be carried out to investigate the purified components responsible for the observed activity.

Uso y selección de microhábitat en un ensamble de anuros del Chaco Serrano de Argentina

Introducción: El estudio de las estrategias de uso y partición del recurso espacial resulta importante para entender los mecanismos de coexistencia de especies de anfibios simpátricas, más aún cuando existen variaciones temporales en la disponibilidad de hábitats. Objetivo: Conocer los patrones de uso de microhábitat, sus variaciones estacionales y la posible influencia de la filogenia en un ensamble de anuros del Chaco Serrano de Argentina. Métodos: Los muestreos se realizaron en el área protegida Parque Natural Valle Fértil, Departamento Valle Fértil, San Juan, entre el 2012 y 2017. En campo, se registró el microhábitat donde se encontró cada ejemplar y se midieron variables del sitio. Además, se determinó la disponibilidad de microhábitats. Los datos se analizaron mediante el índice de selectividad de Manly. Para evaluar variaciones temporales en el uso de microhábitats se utilizaron modelos lineales generalizados (GLM). Resultados: Las especies evaluadas fueron: Rhinella arenarum arenarum, Leptodactylus latrans, Pleurodema tucumanum y Odontophrynus occidentalis. Todas las especies mostraron diferencias en las frecuencias de uso: R. a. arenarum mostró preferencias hacia los sitios rocosos y acuáticos, P. tucumanum exhibió preferencias hacia los sitios con suelo desnudo y rocas, L. latrans y O. occidentalis presentaron mayores preferencias hacia los sitios rocosos y acuáticos. Todas las especies excepto Odontophrynus exhibieron variaciones estacionales en la selección y uso de los microhábitats. Odontophrynus occidentalis presentó diferencias en las proporciones de uso entre microhábitats. Conclusiones: Este estudio muestra la plasticidad en el uso de microhábitats como un determinante importante en la distribución espacial de anuros, sin restricciones aparentes impuestas por la competencia, el espacio ni la filogenia. Cuando la actividad de las especies es coincidente, el recurso espacial puede distribuirse de manera que se reduzca el solapamiento entre especies.

Introduction: Research on spatial resource usage and partition strategies is important to understand the mechanisms of coexistence in sympatric amphibian species, even more when there are temporal variations in habitat availability. Objective: To learn about the patterns of microhabitat use, its seasonal variations and the possible influence of phylogeny on an anuran assembly of the Chaco Serrano in Argentina. Methods: Samplings were carried out in the Valle Fértil Natural Park protected area, Valle Fértil Department, San Juan, between 2012 and 2017. In the field, we recorded the microhabitat where each specimen was found, and we also measured site variables. In addition, microhabitats availability was determined. Data were analyzed using Manly's selectivity index. Generalized linear models (GLM) were used to assess temporal variations in microhabitat use. Results: The species evaluated were: Rhinella arenarum arenarum, Leptodactylus latrans, Pleurodema tucumanum and Odontophrynus occidentalis. All species showed differences in usage frequencies: R. a. arenarum showed preferences for rocky and aquatic sites, P. tucumanum showed preferences towards bare soil and rocky sites, L. latrans and O. occidentalis showed greater preferences for rocky and aquatic sites. All species but Odontophrynus exhibited seasonal variations in microhabitat selection and usage. Odontophrynusoccidentalis showed differences in usage proportions among microhabitats. Conclusions: This study shows plasticity in microhabitat usage as an important determinant of anuran spatial distribution, without apparent restrictions imposed by space competition or phylogeny. When species activity is coincident, the space resource may be distributed in a way that species overlap is reduced.

Toxicidade aguda (LC50) e efeitos comportamentais e morfológicos de formulado comercial com princípio ativo glifosato em girinos de Physalaemus cuvieri (Anura, Leptodactylidae) e Rhinella icterica (Anura, Bufonidae) / Acute Toxicity (CL50) and behavioral and morphological effects of a commercial formulation with glyphosate active ingredient in tadpoles of Physalaemus cuvieri (Anura, Leptodactylidae) and Rhinella icterica (Anura, Bufonidae)

RESUMO O declínio de anfíbios no mundo está cada vez maior e a perda de riqueza, muitas vezes, está associada a áreas agrícolas. O uso de agrotóxicos, em especial daqueles à base de glifosato, é cada vez maior e novas formulações estão sendo desenvolvidas. O presente trabalho avaliou a toxicidade aguda de um composto comercial com o princípio ativo glifosato e os efeitos comportamentais e morfológicos em girinos de Physalaemus cuvieri e Rhinella icterica. O primeiro mostrou-se mais sensível em relação ao formulado comercial do que o segundo e não houve diferenças significativas em relação às mudanças comportamentais e o aumento das concentrações do formulado entre as espécies, porém houve alterações morfológicas de peso e comprimento dos girinos de ambas espécies.

ABSTRACT The decline of amphibians in the world is increasing and the loss of wealth is often associated with agricultural areas. The use of pesticides, especially those based on glyphosate is increasing and new formulations are being developed. This study evaluated the acute toxicity of a commercial compound with glyphosate and behavioral and morphological effects on Physalaemus cuvieri and Rhinella icterica tadpoles; the former was more sensitive to the commercially formulated compound than R. icterica and there were no significant differences in relation to behavioral changes and increase compound concentrations among species, but there were morphological changes of weight and length of tadpoles of both species.

Assessment of neuropharmacological potential of low molecular weight components extracted from Rhinella schneideri toad poison

Studies on toad poison are relevant since they are considered a good source of toxins that act on different biological systems. Among the molecules found in the toad poison, it can be highlighted the cardiotonic heterosides, which have a known mechanism that inhibit Na+/K+-ATPase enzyme. However, these poisons have many other molecules that may have important biological actions. Therefore, this work evaluated the action of the low molecular weight components from Rhinella schneideri toad poison on Na+/K+-ATPase and their anticonvulsive and / or neurotoxic effects, in order to detect molecules with actions of biotechnological interest. Methods: Rhinella schneideri toad (male and female) poison was collected by pressuring their parotoid glands and immediately dried and stored at -20 °C. The poison was dialysed and the water containing the low molecular mass molecules (< 8 kDa) that permeate the dialysis membrane was collected, frozen and lyophilized, resulting in the sample used in the assays, named low molecular weight fraction (LMWF). Na+/K+ ATPase was isolated from rabbit kidneys and enzyme activity assays performed by the quantification of phosphate released due to enzyme activity in the presence of LMWF (1.0; 10; 50 and 100 µg/mL) from Rhinella schneideri poison. Evaluation of the L-Glutamate (L-Glu) excitatory amino acid uptake in brain-cortical synaptosomes of Wistar rats was performed using [3H]L-glutamate and different concentration of LMWF (10-5 to 10 µg/µL). Anticonvulsant assays were performed using pentylenetetrazole (PTZ) and N-methyl-D-aspartate (NMDA) to induce seizures in Wistar rats (n= 6), which were cannulated in the lateral ventricle and treated with different concentration of LMWF (0.25; 0.5; 1.0; 2.0; 3.0 and 4.0 µg/µL) 15 min prior to the injection of the seizure agent. Results: LMWF induced a concentration-dependent inhibition of Na+/K+-ATPase (IC50% = 107.5 μg/mL). The poison induces an increased uptake of the amino acid L-glutamate in brain-cortical synaptosomes of Wistar rats. This increase in the L-glutamate uptake was observed mainly at the lowest concentrations tested (10-5 to 10-2 µg/µL). In addition, this fraction showed a very relevant central neuroprotection on seizures induced by PTZ and NMDA. Conclusions: LMWF from Rhinella schneideri poison has low molecular weight compounds, which were able to inhibit Na+/K+-ATPase activity, increase the L-glutamate uptake and reduced seizures induced by PTZ and NMDA. These results showed that LMWF is a rich source of components with biological functions of high medical and scientific interest.(AU)

First record of Ornithodoros faccinii (Acari: Argasidae) on toads of genus Rhinella (Anura: Bufonidae) in Brazil / Primeiro registro de Ornithodoros faccinii (Acari: Argasidae) em sapos do gênero Rhinella no Brasil

Abstract Although a group of soft ticks (Argasidae) associated with amphibians was recently discovered in Brazilian rainforests, parasitism by these ticks on cold-blooded animals remains less common than on mammal and bird species. In this study, we identified ticks that were collected from toads that had been caught in December 2016 and January 2017, at Itinguçú waterfall (22°54'05" S; 43°53'30" W) in the municipality of Itaguaí, state of Rio de Janeiro. Tick specimens were identified using a morphological and molecular approach. In total, twelve larvae of Ornithodoros ticks were collected from three individuals of Rhinella ornata and were identified as Ornithodoros faccinii. Our results include a longer 16S rRNA mitochondrial sequence for O. faccinii that supports its phylogenetic relatedness to Ornithodoros saraivai, and we report this tick species parasitizing Rhinella toads for the first time in Brazil.

Resumo Embora um grupo de carrapatos moles (Argasidae) associado a anfíbios tenha sido recentemente descoberto nas florestas brasileiras, o parasitismo por esses carrapatos em animais de sangue frio permanece menos comum do que nas espécies de mamíferos e aves. Neste estudo, identificamos carrapatos que foram coletados de sapos capturados em dezembro de 2016 e janeiro de 2017, na cachoeira de Itinguçú (22°54'05" S; 43°53'30" W) no município de Itaguaí, estado do Rio de Janeiro. Os espécimes de carrapatos foram identificados usando uma abordagem morfológica e molecular. No total, doze larvas de carrapatos Ornithodoros foram coletadas de três indivíduos de Rhinella ornata e foram identificadas como Ornithodoros faccinii. Nossos resultados incluem uma maior seqüência mitocondrial 16S rRNA para O. faccinii que suporta sua relação filogenética com Ornithodoros saraivai e relatamos esta espécie de carrapato parasitando sapos Rhinella pela primeira vez no Brasil.

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. Results: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.(AU)

Patterns of parasitism by Amblyomma rotundatum (Ixodidae) on Rhinella schneideri (Bufonidae) in islands of São Francisco River, Minas Gerais, Brazil / Parasitismo por Amblyomma rotundatum (Ixodidae) em Rhinella schneideri (Bufonidae) em duas ilhas do Rio São Francisco, próximas à represa de Três Marias, Minas Gerais

We sampled ticks from specimens of the rococo toad Rhinella schneideriby flannel dragging on two Islands located in the São Francisco River near the Três Marias hydroelectric dam, southeastern Brazil. A total of 120 toads was examined, of which 63 (52.5%) were parasitized only by Amblyomma rotundatumtotaling 96 larvae, 163 nymphs and 134 females. The burden of parasitism ranged from one to 43 ticks, with a mean intensity of infestation of 6.2±5.5 ticks per host. The tick A. rotundatumexhibited highly aggregated distribution. Peak abundance of larvae and nymphs occurred in the dry season (May to September), whereas peak abundance of females occurred in the wet season (October to April). We collected most ticks near the head and hind limbs of R. schneideri. The finding of two engorged A. rotundatumnymphs in the same resting places of two toads and the absence of this species in the dragged areas suggest a nidicolous behavior at the studied site.

Sapos da espécie Rhinella schneideri foram capturados e examinados para coleta das fases parasitárias, assim como arrasto de flanela para coleta das fases de vida livres de carrapatos em duas ilhas localizadas no rio São Francisco , próximas à represa Três Marias, região sudeste do Brasil. No total, 120 indivíduos foram examinados, dos quais 63 (52,5%) estavam parasitados por Amblyomma rotundatum totalizando 96 larvas, 163 ninfas e 134 fêmeas. A abundância do parasitismo variou de 1 a 43 carrapatos, com uma intensidade média de infestação de 6,2±5,5 carrapatos/hospedeiro. A infestação por A. rotundatumapresentou uma distribuição altamente agregada. O pico de abundância de larvas e ninfas ocorreu na estação seca (maio a setembro), enquanto o pico de abundância de fêmeas ocorreu na estação chuvosa (outubro a abril). A maioria dos carrapatos foi coletada na região da cabeça e membros posteriores. A presença de duas ninfas ingurgitadas de A. rotundatum nos mesmos lugares de descanso de dois sapos e a ausência desta espécie na coleta por arrasto de flanela sugere um comportamento nidicola no local estudado.

Biomarcadores hematológicos del sapo común Bufo (Rhinella) arenarum en ecosistemas alterados de la provincia de Córdoba / Hematologic biomarkers of the common toad Bufo arenarum in altered ecosystem of Córdoba province

El uso de biomarcadores hematológicos es importante para poder evaluar riesgos ambientales y la exposición a sustancias químicas potencialmente tóxicas y así poder desarrollar medidas que sirvan como señales de alarma temprana en ambientes contaminados. El objetivo de este trabajo fue determinar biomarcadores hematológicos en el sapo común Bufo (Rhinella) arenarum que proporcionen información para el diagnóstico de la salud de distintos ecosistemas de la provincia de Córdoba. Un total de 93 individuos adultos de Bufo (Rhinella) arenarum fueron capturados en cuatro sitios de la provincia de Córdoba y distintas variables ambientales del agua fueron medidas en cada sitio. Las muestras de sangre se obtuvieron mediante la punción de la vena angularis. Luego fueron teñidas con la tinción diferencial May Grünwald-Giemsa y fueron observadas mediante microscopio Zeiss Primo Star iLED para realizar el conteo de glóbulos blancos. Las variables ambientales del agua mostraron diferencias estadísticamente significativas entre las poblaciones, se registraron valores menores en “Alpa Corral”, mientras que “Villa Dálcar” presentó los mayores valores para dichas variables. No se encontraron diferencias estadísticamente significativas entre localidades en el recuento de glóbulos blancos (cada 1000 eritrocitos); sin embargo, en “Villa Dálcar” se registraron los ejemplares con mayor cantidad de leucocitos en el conteo de glóbulos blancos (425,48 ± 481,34). Mientras que para la fórmula leucocitaria, los linfocitos fueron las células más abundantes para los ejemplares de todos los sitios; encontrándose diferencias estadísticamente significativas entre cada localidad y “Alpa Corral”. Los ejemplares de “Villa Dálcar” presentaron diferencias estadísticamente significativas en relación a los monocitos y basófilos, registrándose altos valores para ambas células en los ejemplares de este sitio; mientras que en los ejemplares de “Cultivo” las diferencias se encontraron en eosinófilos y la relación neutrófilos/linfocitos. Estos resultados revelaron que de las cuatro localidades analizadas, “Villa Dálcar” representa la más afectada por las actividades antrópicas. Estas variaciones registradas en los parámetros hematológicos podrían considerarse evidencia de la posible presencia de agentes contaminantes en el lago “Villa Dálcar”.

Hematologic biomarkers are important to assess the environmental and health risks of exposure to potentially toxic chemicals, and for developing measures that serve as early warning signals in polluted areas. The aim of this study was to determine hematologic biomarkers of common toad Bufo (Rhinella) arenarum that provide information for the diagnosis of the health of ecosystems of Córdoba province. A total of 93 adult individuals of Bufo (Rhinella) arenarum were collected from four sites of Córdoba Province and hydric environmental variables were measured on each site. Blood samples were obtained by angularis vein puncture, smears of fresh blood were stained with May Grünwald-Giemsa and observed by using a microscope Carl Zeiss Primo Star (Pack 5), for white blood cell count. Water environmental variables showed statistically significant differences among localities, with the lowest values in “Alpa Corral” and highest values in “Villa Dálcar”. There were no statistically significant differences among individuals of different sites for white blood cells count. However, individuals from "Villa Dálcar" showed the highest number of white cells (425.48 ± 481.34). In the leukocyte formula, lymphocytes were most abundant white blood cells in individuals from all study sites. The blood parameters of toads from all sites showed statistically significant differences with individuals from “Alpa Corral”. "Villa Dálcar" Individuals exhibited significant differences in relation to monocytes and basophils with the highest values for both cell types. “Cultivo” locality showed significant differences for eosinophils and for the relationship neutrophils/ lymphocytes. These results revealed that “Villa Dálcar” represent the site most affected by human activities. In particular, changes recorded in hematological parameters could be giving evidence of the possible presence of contaminants in Lake “Villa Dálcar”.

First serine protease inhibitor isolated from Rhinella schneideri poison

Background Toad secretions are a source of molecules with potential biotechnological application on a wide spectrum of diseases. Toads from theRhinella family have two kinds of poisonous glands, namely granular and mucous glands. Rhinella schneideritoads produce granular secretions that comprise a great number of molecules, including serine proteases inhibitors. Serine proteases, such as trypsin, chymotrypsin and elastase, are enzymes that have a serine amino acid into its catalytic site and can be found in a large number of vertebrate species and pathogenic microorganisms. Therefore, the present work aims to purify a serine protease inhibitor from Rhinella schneiderigranular secretions.Findings This study presents the protocol used to purify a serine protease inhibitor from the Rhinella schneideri poison. The granular secretion was submitted to dialysis in order to separate the low molecular weight compounds, which were submitted to a reversed phase-fast protein liquid chromatography fractionation step in a C2C18 column. The major fractions were tested over trypsin, chymotrypsin and elastase through colorimetric assay. The inhibition tests were performed with the enzyme in absence (positive control) and presence of fractions, denatured enzyme (negative control) and the respective chromogenic substrate. Rs20 was the compound with the major inhibitory activity over chymotrypsin, inducing a delay in the formation of the chromogenic enzymatic product. The structure characterization of Rs20 was performed by high resolution electronspray ionization-mass spectrometry (HRESI-MS) and gas chromatography coupled with mass spectrometry (GC-MS). HRESI showed an intense signal suggesting the presence of bufadienolide with less than 10 ppm error. In addition, it was observed a low intense signal at m/z399 that could be lithocholic acid, a biosynthetic precursor of bufadienolide. Finally, GC-MS analysis applying NIST library identification reinforced this hypothesis.Conclusions The current study have isolated and partially characterized the function and structure of the first bufadienolide with inhibitory action over chymotrypsin.(AU)

Biological characterization of compounds from Rhinella schneideri poison that act on the complement system

Background The skin secretions of toads of the family Bufonidae contain biogenic amines, alkaloids, steroids (bufotoxins), bufodienolides (bufogenin), peptides and proteins. The poison of Rhinella schneideri, formerly classified as Bufo paracnemis, presents components that act on different biological systems, including the complement system. The aim of this study was to isolate and examine the activity ofRhinella schneideri poison (RsP) components on the complement system.Methods The components active on the complement system were purified in three chromatographic steps, using a combination of cation-exchange, anion-exchange and gel filtration chromatography. The resulting fractions were analyzed by SDS-PAGE and screened for their activity in the hemolytic assay of the classical/lectin complement pathways. Fractions active on the complement system were also assessed for their ability to generate C3 fragments evaluated by two dimensional immunoelectrophoresis assay, C3a and C5a by neutrophil chemotaxis assay and SC5b-9 complex by ELISA assay.Results The fractionation protocol was able to isolate the component S5 from theRsP, as demonstrated by SDS-PAGE and the RP-FPLC profile. S5 is a protein of about 6000 Da, while S2 presents components of higher molecular mass (40,000 to 50,000 Da). Fractions S2 and S5 attenuated the hemolytic activity of the classical/lectin pathways after preincubation with normal human serum. Both components stimulated complement-dependent neutrophil chemotaxis and the production of C3 fragments, as shown by two-dimensional immunoelectrophoresis. S2 showed a higher capacity to generate the SC5b- 9 complex than the other fractions. This action was observed after the exposure of normal human serum to the fractions.Conclusions This is the first study to examine the activity of RsP components on the complement system. Fractions S2 and S5 reduced the complement hemolytic activity, stimulated complement-dependent neutrophil chemotaxis and stimulated the production of C3 fragments, indicating that they were able to activate the complement cascade. Furthermore, fraction S2 was also able to generate the SC5b-9 complex. These components may be useful tools for studying dysfunction of the complement cascade.(AU)

Biological characterization of compounds from Rhinella schneideri poison that act on the complement system

Background The skin secretions of toads of the family Bufonidae contain biogenic amines, alkaloids, steroids (bufotoxins), bufodienolides (bufogenin), peptides and proteins. The poison of Rhinella schneideri, formerly classified as Bufo paracnemis, presents components that act on different biological systems, including the complement system. The aim of this study was to isolate and examine the activity ofRhinella schneideri poison (RsP) components on the complement system.Methods The components active on the complement system were purified in three chromatographic steps, using a combination of cation-exchange, anion-exchange and gel filtration chromatography. The resulting fractions were analyzed by SDS-PAGE and screened for their activity in the hemolytic assay of the classical/lectin complement pathways. Fractions active on the complement system were also assessed for their ability to generate C3 fragments evaluated by two dimensional immunoelectrophoresis assay, C3a and C5a by neutrophil chemotaxis assay and SC5b-9 complex by ELISA assay.Results The fractionation protocol was able to isolate the component S5 from theRsP, as demonstrated by SDS-PAGE and the RP-FPLC profile. S5 is a protein of about 6000 Da, while S2 presents components of higher molecular mass (40,000 to 50,000 Da). Fractions S2 and S5 attenuated the hemolytic activity of the classical/lectin pathways after preincubation with normal human serum. Both components stimulated complement-dependent neutrophil chemotaxis and the production of C3 fragments, as shown by two-dimensional immunoelectrophoresis. S2 showed a higher capacity to generate the SC5b- 9 complex than the other fractions. This action was observed after the exposure of normal human serum to the fractions.Conclusions This is the first study to examine the activity of RsP components on the complement system. Fractions S2 and S5 reduced the complement hemolytic activity, stimulated complement-dependent neutrophil chemotaxis and stimulated the production of C3 fragments, indicating that they were able to activate the complement cascade. Furthermore, fraction S2 was also able to generate the SC5b-9 complex. These components may be useful tools for studying dysfunction of the complement cascade.

First serine protease inhibitor isolated from Rhinella schneideri poison

Background Toad secretions are a source of molecules with potential biotechnological application on a wide spectrum of diseases. Toads from theRhinella family have two kinds of poisonous glands, namely granular and mucous glands. Rhinella schneideritoads produce granular secretions that comprise a great number of molecules, including serine proteases inhibitors. Serine proteases, such as trypsin, chymotrypsin and elastase, are enzymes that have a serine amino acid into its catalytic site and can be found in a large number of vertebrate species and pathogenic microorganisms. Therefore, the present work aims to purify a serine protease inhibitor from Rhinella schneiderigranular secretions.Findings This study presents the protocol used to purify a serine protease inhibitor from the Rhinella schneideri poison. The granular secretion was submitted to dialysis in order to separate the low molecular weight compounds, which were submitted to a reversed phase-fast protein liquid chromatography fractionation step in a C2C18 column. The major fractions were tested over trypsin, chymotrypsin and elastase through colorimetric assay. The inhibition tests were performed with the enzyme in absence (positive control) and presence of fractions, denatured enzyme (negative control) and the respective chromogenic substrate. Rs20 was the compound with the major inhibitory activity over chymotrypsin, inducing a delay in the formation of the chromogenic enzymatic product. The structure characterization of Rs20 was performed by high resolution electronspray ionization-mass spectrometry (HRESI-MS) and gas chromatography coupled with mass spectrometry (GC-MS). HRESI showed an intense signal suggesting the presence of bufadienolide with less than 10 ppm error. In addition, it was observed a low intense signal at m/z399 that could be lithocholic acid, a biosynthetic precursor of bufadienolide. Finally, GC-MS analysis applying NIST library identification reinforced this hypothesis.Conclusions The current study have isolated and partially characterized the function and structure of the first bufadienolide with inhibitory action over chymotrypsin.

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action.Conclusions This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.(AU)

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action...

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.

Demografía poblacional de Rhinella arenarum (Anura: Bufonidae) y Physalaemus biligonigerus (Anura: Leiuperidae) en agroecosistemas de la provincia de Córdoba, Argentina / Population demography in Rhinella arenarum (Anura: Bufonidae) and Physalaemus biligonigerus (Anura: Leiuperidae) in agroecosystems in the province of Córdoba, Argentina

The advancing agricultural frontier has led to an important loss of natural habitats, with significant consequences for biodiversity. The demography for two species of anurans, Physalaemus biligonigerus and Rhinella arenarum, both associated with agricultural systems in the central region of the Córdoba Province, were analyzed and compared in this study. Four sites were sampled: three agroecosystems with different alteration degrees (C1, C2 and SM1) and a fourth site not cultivated (SM2). The sampling was conducted during two reproductive periods, from September 2008 to April 2009 and September 2009 to April 2010. Individuals were captured using live pitfall traps for the metamorphic, juveniles and adults; and visual encounter survey, for the capture of eggs and larvae. With the abundance data, the survival for each age class was estimated using the KNM method (Kiritani Nakasuki Manly). With survival rates and fertility population, Leslie matrices were elaborated to obtain a quantitative projection of the population size. Altered environments showed lower eggs and larvae survival. Population pro-jections were favorable in the site SM2 and were less favorable and a tendency to extinction, in sites dominated by crops. This study showed that the agroecosystems of this region are possibly inhospitable environments for reproduction and survival of the species studied. The aquatic stages in the life cycle of both species would be the more affected, since water bodies deterioration is present or may occur in those areas. We can recognize species-specific effects of agricultural ecosystems; P. biligonigerus was the most affected species, possibly because of their life histories and habitat requirements. We suggested that environmental degradation caused by the cropland in the central region of Argentina would impact on the demographics of the anuran populations in the area.

El crecimiento de la agricultura produce pérdida del hábitat natural, con consecuencias para la biodiversidad de los anfibios. Se analizó la demografía poblacional de dos especies de anfibios en agroecosistemas de la provincia de Córdoba, Rhinella arenarum y Physalaemus biligonigerus. Los muestreos se realizaron en tres agroecosistemas y en un cuarto sitio, no afectado por cultivos. Los individuos se capturaron utilizando trampas de caída viva y relevamientos visuales. Con los datos de abundancia se estimó la supervivencia para cada clase de edad y con las tasas de supervivencia y fertilidad poblacional, se construyeron matrices de Leslie para obtener una proyección cuantitativa del tamaño poblacional. Las proyecciones poblacionales fueron favorables para el sitio SM2 y desfavorables para los sitios cultivados. Los agroecosistemas son ambientes posiblemente inhóspitos para la reproducción y supervivencia de las especies estudiadas. Particularmente se ven afectados aquellos estadios acuáticos del ciclo de vida de estas especies, debido al deterioro de los cuerpos de agua en estas áreas. Se reconocieron efectos especie-específicos de los ecosistemas agrícolas, ya que P. biligonigerus resultó la especie más afectada. Se sugiere un deterioro ambiental producido por cultivos extensivos de soja, lo que impactaría la demografía de las poblaciones de anuros.

Ecología trófica en larvas de Rhinella arenarum (Anura: Bufonidae) en agroecosistemas y sus posibles implicaciones para la conservación / Trophic ecology in tadpoles of Rhinella arenarum (Anura: Bufonidae) in agroecosystems and their possible implications for conservation

Trophic ecology in tadpoles of Rhinella arenarum (Anura: Bufonidae) in agroecosystems and their possible implications for conservation. The progress of the agriculture border has led an important loss of natural habitats, with significant consequences for biodiversity. In this sense, the studies in anuran amphibian tadpoles inhabiting these environments are relevant, because the larval stage is a phase of population regulation. The aim of this study was to analyze the body condition and diet in Rhinella arenarum, tadpoles, an anuran species widely distributed in South America and that inhabit agroecosystems. Three sites were sampled, two agroecosystems with different alteration degrees (C1 and C2) and an uncultured (SM) third place. The captured tadpoles were anesthetized, fixed and preserved in formaldehyde (10%). Subsequently, body measurements were made and the complete intestine was removed and analyzed for food items under a binocular microscope. The diet in R. arenarum tadpoles has a dominance of algae Bacillariophyceae, followed by Cyanophyceae. In particular, the class Bacillariophyceae, due to the presence of the genus Navicula, Nitzschia, Gomphonema and Hantzschia, was important in the diet of the anurans in those agroecosystems. Class Cyanophyceae, mainly represented by genus Oscillatoria and Euglenophyceae represented by Euglena and Strombomonas, were predominant in the diet of the anurans in SM. Some differences in the total items consumed by tadpole were observed between the studied sites. Tadpoles that inhabit the modified sites (C1 and C2) recorded a significantly smaller amount of food. Moreover, the tadpoles that inhabit these sites showed a lower body condition. The presence of certain algae associated with eutrophic environments, could indicate some pollution in agroecosystems (C1 and C2). Food resources would be lesser in places with strong agricultural activity, possibly with a greater degree of eutrophication. A smaller food amount could have consequences at population level for the short and long time terms, because of its impact on individual growth. Larval diet is suggested as a potential bioindicator of environmental health for these areas. Rev. Biol. Trop. 60 (2): 771-779. Epub 2012 June 01.

El crecimiento de la agricultura produce perdida del habitat natural, con consecuencias para la biodiversidad de los anfibios. Se analizo la dieta y condición corporal de larvas de anuros de Rhinella arenarum que habitan agroecosistemas. Los muestreos fueron realizados en dos agroecosistemas y en un tercer sitio, no afectado por cultivos. Las larvas capturadas fueron anestesiadas, fijadas y preservadas en formaldehido, se realizaron medidas corporales y se analizo la dieta de las larvas. Se registro diversidad en la oferta alimenticia proporcionada por los distintos ambientes. La dieta tuvo una predominancia de algas Bacillarophyceae, seguidas por Cyanophyceae. Particularmente, los géneros Navicula, Nitzschia, Hantzschia y Gomphonema (clase Bacillarophyceae), fueron importantes en los agroecosistemas. Los géneros Osillatoria, Euglena y Strombomonas (clases Cyanophyceae y Euglenophyceae), predominaron en la dieta de las larvas de anuros en el sitio menos alterado. Las larvas de los sitios más alterados consumen menor cantidad de alimento y registran una menor condición corporal. La presencia de determinadas algas, indicarían un mayor grado de contaminación en los agroecosistemas. La eutrofización de lagunas podría alterar la disponibilidad de alimento para larvas de anuros que podría tener consecuencias poblacionales negativas. Se sugiere el análisis de dietas larvarias como un potencial bioindicador de salubridad ambiental.

Phototoxic and modulatory effects of natural products from the skin of Rhinella jimi (Stevaux, 2002)

The skin of amphibians possesses a large diversity of biologically active compounds that are associated with the natural defenses of these animals against pathogens. Five different extracts and fractions were obtained from the skin of Rhinella jimi: methanol extract (ME), methanol fractions (MF), chloroform extract of methanol extract (CF), aqueous alkaloid fraction (AAF) and aqueous non-alkaloid fraction (ANAF). All fractions were evaluated with respect to their antibiotic modifying activity in standard bacterial strains and multiresistant clinical isolates. Antagonism was detected with kanamycin and gentamicin when combined with substances obtained from the skin of R. jimi. Phototoxic activity was observed in the methanol and chlorophorm fractions, as well as the aqueous non-alkaloid fraction. The antagonistic action was apparently associated with the protection afforded by the bacterial populations that inhabit the skin of this amphibian, preventing colonization by pathogenic fungi. The phototoxic activity demonstrated by natural products from the skin of R. jimi showed an interruption of the bacterial growth after UV exposure. This could indicate an antibacterial effect activated by the UV light, opening a path for carrying the attack by pathogenic fungi, causing the disease related with the amphibian decline.

Ornithophagy in Rhinella icterica / Ornitofagia em Rhinella icterica

In this work we report an observation of an ornitophagy event on Certhiaxis cinnamomeus by Rhinella icterica occurred in the central region of Rio Grande do Sul, Brazil.

Reportamos um evento de ornitofagia por Rhinella icterica sobre Certiaxis cinnamomeus ocorrido em 2010, na região central do Estado do Rio Grande do Sul (29°41S, 53°48W), Brasil.