Small Interfering RNA Mediated Messenger RNA Knockdown in the Amphibian Pathogen Batrachochytrium dendrobatidis.

RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the "knockdown" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis.

Genomic SNPs resolve the phylogeny of an ancient amphibian island radiation from the Seychelles.

Unusually for oceanic islands, the granitic Seychelles host multiple lineages of endemic amphibians. This includes an ancient (likely ca. 60 million years) radiation of eight caecilian species, most of which occur on multiple islands.These caecilians have a complicated taxonomic history and their phylogenetic inter-species relationships have been difficult to resolve. Double-digest RAD sequencing (ddRADseq) has been applied extensively to phylogeography and increasingly to phylogenetics but its utility for resolving ancient divergences is less well established. To address this, we applied ddRADseq to generate a genome-wide SNP panel for phylogenomic analyses of the Seychelles caecilians, whose phylogeny has so far not been satisfactorily resolved with traditional DNA markers. Based on 129,154 SNPs, we resolved deep and shallow splits, with strong support. Our findings demonstrate the capability of genome-wide SNPs for evolutionary inference at multiple taxonomic levels and support the recently proposed synonymy of Grandisonia Taylor, 1968 with Hypogeophis Peters, 1879. We revealed three clades of Hypogeophis (large-, medium- and short-bodied) and identify a single origin of the diminutive, stocky-bodied and pointy-snouted phenotype.

Glutathione-Mediated Metal Tolerance in an Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis).

The spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has resulted in amphibian declines and extinctions worldwide. Some susceptible amphibian species can persist in contaminated habitats, prompting the hypothesis that B. dendrobatidis might be sensitive to heavy metals. We tested a panel of 12 metals to rank their toxicity to B. dendrobatidis zoospores and zoosporangia during a 6-h exposure. To better understand the mechanism for metal detoxification, we also evaluated whether glutathione is required for metal tolerance by depleting cellular glutathione before metal exposure. In addition, we investigated whether prior exposure to low metal concentrations impacted tolerance of subsequent exposure, as well as identifying metal combinations that may act synergistically. Silver (Ag), cadmium (Cd), and copper (Cu) were particularly toxic to B. dendrobatidis, with zoospore minimum lethal concentration values of 0.01 mM (Ag), 0.025 mM (Cd), and 0.5 mM (Cu). These three metals along with zinc (Zn) were also inhibitory to zoosporangia, with minimum inhibitory concentration values of 0.005 mM (Ag), 0.04 mM (Cd), 0.075 mM (Cu), and 0.04 mM (Zn). The fungicidal effects of several metals was reduced when assays were conducted in nutrient medium compared with synthetic pond water, highlighting the need for careful in vitro assay design and interpretation. Glutathione depletion strongly influenced tolerance of Cd and Ag (85% and 75% less growth, respectively) and moderately influenced tolerance of Cu, Zn, and lead (37%, 18%, and 14% less growth, respectively), indicating the importance of glutathione for metal detoxification. In general, the minimum metal concentrations that inhibited growth of B. dendrobatidis far exceeded values detected in contaminated amphibian habitats in Australia, suggesting that metal contamination alone may not have a strong protective effect against chytridiomycosis. We discuss future research directions to futher understand the potential for dissolved metals to create chytrid refuges. Environ Toxicol Chem 2024;43:1583-1591. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Caspase-1 activates gasdermin A in non-mammals.

Gasdermins oligomerize to form pores in the cell membrane, causing regulated lytic cell death called pyroptosis. Mammals encode five gasdermins that can trigger pyroptosis: GSDMA, B, C, D, and E. Caspase and granzyme proteases cleave the linker regions of and activate GSDMB, C, D, and E, but no endogenous activation pathways are yet known for GSDMA. Here, we perform a comprehensive evolutionary analysis of the gasdermin family. A gene duplication of GSDMA in the common ancestor of caecilian amphibians, reptiles, and birds gave rise to GSDMA-D in mammals. Uniquely in our tree, amphibian, reptile, and bird GSDMA group in a separate clade than mammal GSDMA. Remarkably, GSDMA in numerous bird species contain caspase-1 cleavage sites like YVAD or FASD in the linker. We show that GSDMA from birds, amphibians, and reptiles are all cleaved by caspase-1. Thus, GSDMA was originally cleaved by the host-encoded protease caspase-1. In mammals the caspase-1 cleavage site in GSDMA is disrupted; instead, a new protein, GSDMD, is the target of caspase-1. Mammal caspase-1 uses exosite interactions with the GSDMD C-terminal domain to confer the specificity of this interaction, whereas we show that bird caspase-1 uses a stereotypical tetrapeptide sequence to confer specificity for bird GSDMA. Our results reveal an evolutionarily stable association between caspase-1 and the gasdermin family, albeit a shifting one. Caspase-1 repeatedly changes its target gasdermin over evolutionary time at speciation junctures, initially cleaving GSDME in fish, then GSDMA in amphibians/reptiles/birds, and finally GSDMD in mammals.

Estrogen contamination increases vulnerability of amphibians to the deadly chytrid fungus.

Aquatic contaminants and infectious diseases are among the major drivers of global amphibian declines. However, the interaction of these factors is poorly explored and could better explain the amphibian crisis. We exposed males and females of the Brazilian Cururu Toad, Rhinella icterica, to an environmentally relevant concentration of the estrogen 17-alpha-ethinylestradiol (an emerging contaminant) and to the chytrid infection (Batrachochytrium dendrobatidis), in their combined and isolated forms, and the ecotoxicity was determined by multiple biomarkers: cutaneous, hematological, cardiac, hepatic, and gonadal analysis. Our results showed that Cururu toads had many physiological alterations in response to the chytrid infection, including the appearance of cutaneous Langerhans's cells, increased blood leukocytes, increased heart contraction force and tachycardia, increased hepatic melanomacrophage cells, which in turn led to gonadal atrophy. The estrogen, in turn, increased the susceptibility of the toads to the chytrid infection (higher Bd loads) and maximized the deleterious effects of the pathogen: reducing leukocytes, decreasing the contraction force, and causing greater tachycardia, increasing hepatic melanomacrophage cells, and leading to greater gonadal atrophy, which were more extreme in females. The exposure to estrogen also revealed important toxicodynamic pathways of this toxicant, as shown by the immunosuppression of exposed animals, and the induction of the first stages of feminization in males, which corroborates that the synthetic estrogen acts as an endocrine disruptor. Such an intricate relationship is unprecedented and reinforces the importance of studying the serious consequences that multiple environmental stressors can cause to aquatic populations.

Amphibian-derived peptide RL-RF10 ameliorates paraquat-induced pulmonary fibrosis injury.

Pulmonary fibrosis is the result of dysfunctional repair after lung tissue injury, characterized by fibroblast proliferation and massive extracellular matrix aggregation. Once fibrotic lesions develop, effective treatment is difficult, with few drugs currently available. Here, we identified a short cyclic decapeptide RL-RF10 derived from frog skin secretions as a potential novel lead molecule for the amelioration of pulmonary fibrosis. In vivo experiments indicated that RL-RF10 treatment ameliorated lung histopathological damage and fibrogenesis after paraquat (PQ) induction in a concentration-dependent manner. On day 7, bronchoalveolar lavage fluid assays performed on mice showed that RL-RF10 exerted anti-inflammatory effects by decreasing the expression of inflammation-related factors, including transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α, in lung tissue. In addition, RL-RF10 down-regulated the levels of collagen I, collagen III, and vimentin, while increasing the expression of E-cadherin to inhibit epithelial-mesenchymal transition. Further research demonstrated that the SMAD2/3 signaling pathway, which is strongly linked to TGF-ß1, played a critical function in enhancing the pulmonary fibrosis relief achieved by RL-RF10. Both in vivo and in vitro assays showed that RL-RF10 treatment led to a significant reduction in the phosphorylation levels of SMAD2 and SMAD3 following PQ induction. Overall, we investigated the protective effects and underlying mechanisms of the RL-RF10 peptide against pulmonary fibrosis and demonstrated its potential as a novel therapeutic drug candidate for the treatment of pulmonary fibrotic diseases.

Glutathione is required for growth and cadmium tolerance in the amphibian chytrid fungus, Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd) is a lethal amphibian pathogen, partly due to its ability to evade the immune system of susceptible frog species. In many pathogenic fungi, the antioxidant glutathione is a virulence factor that helps neutralise oxidative stressors generated from host immune cells, as well as other environmental stressors such as heavy metals. The role of glutathione in stress tolerance in Bd has not been investigated. Here, we examine the changes in the glutathione pool after stress exposure and quantify the effect of glutathione depletion on cell growth and stress tolerance. Depletion of glutathione repressed growth and release of zoospores, suggesting that glutathione is essential for life cycle completion in Bd. Supplementation with <2 mM exogenous glutathione accelerated zoospore development, but concentrations >2 mM were strongly inhibitory to Bd cells. While hydrogen peroxide exposure lowered the total cellular glutathione levels by 42 %, glutathione depletion did not increase the sensitivity to hydrogen peroxide. Exposure to cadmium increased total cellular glutathione levels by 93 %. Glutathione-depleted cells were more sensitive to cadmium, and this effect was attenuated by glutathione supplementation, suggesting that glutathione plays an important role in cadmium tolerance. The effects of heat and salt were exacerbated by the addition of exogenous glutathione. The impact of glutathione levels on Bd stress sensitivity may help explain differences in host susceptibility to chytridiomycosis and may provide opportunities for synergistic therapeutics.

Into the danger zone: How the within-host distribution of parasites controls virulence.

Despite the importance of virulence in epidemiological theory, the relative contributions of host and parasite to virulence outcomes remain poorly understood. Here, we use reciprocal cross experiments to disentangle the influence of host and parasite on core virulence components-infection and pathology-and understand dramatic differences in parasite-induced malformations in California amphibians. Surveys across 319 populations revealed that amphibians' malformation risk was 2.7× greater in low-elevation ponds, even while controlling for trematode infection load. Factorial experiments revealed that parasites from low-elevation sites induced higher per-parasite pathology (reduced host survival and growth), whereas there were no effects of host source on resistance or tolerance. Parasite populations also exhibited marked differences in within-host distribution: ~90% of low-elevation cysts aggregated around the hind limbs, relative to <60% from high-elevation. This offers a novel, mechanistic basis for regional variation in parasite-induced malformations while promoting a framework for partitioning host and parasite contributions to virulence.

Do blood parasites increase immature erythrocytes and mitosis in amphibians? / ¿Causan los parásitos sanguíneos un aumento en los eritrocitos inmaduros y la mitosis en los anfibios?

Abstract Introduction: In amphibians, blood may act as a hematopoietic tissue. However, the knowledge concerning hematological features is scarce, there is not much information that allows an analysis about the possible explanations of this physiological feature. Objective: This study aimed to evaluate the relationship between immature red blood cells (RBCs) mitosis and the presence of blood parasites in amphibians. Methods: We sampled 116 amphibians (31 species) in six Colombian localities. Blood was taken by cardiac puncture or maxillary vein puncture. Smears were prepared, fixed, and Giemsa stained for microscopical analysis. The variables analyzed were the percentage of immature RBCs, mitotic cells in peripheral blood, and blood parasite infection. Data were analyzed using Wilcoxon's rank test and exact Fisher statistical tests. Results: Sixty-two individuals showed mitosis in peripheral blood, and these mitotic RBCs shared morphological features with immature RBCs. Overall, parasite prevalence was 30.1 %, distributed as follows: Trypanosoma (24.1 %), Hepatozoon-like (6 %), Dactylosoma (4.3 %), Karyolysus-like (0.9 %), and Filarioidea (2.6 %). A positive association between the percentage of immature RBCs and the presence of mitotic RBCs was found, and also between the blood parasite infection and the percentage of immature RBCs. Conclusions: In this study, we found that the presence of blood parasites, immature RBCs, and RBCs mitosis are frequent events in amphibians' peripheral blood, and our analysis suggests an association between those features. Thus, the release of immature RBCs and the mitosis of those cells in peripheral blood may be a physiological response to blood parasite infection. Further studies characterizing hematology in amphibians and wildlife, in general, are desirable.

Resumen Introducción: En anfibios, la sangre puede actuar como un tejido hematopoyético. Sin embargo, el conocimiento acerca de las características hematológicas es escaso y no hay información que permita un análisis acerca de las posibles explicaciones a este rasgo fisiológico. Objetivo: La intención de este estudio fue evaluar la relación entre la presencia de eritroblastos, mitosis de glóbulos rojos (GRs) y la infección por hemoparásito en sangre periférica de anfibios. Métodos: Se muestrearon 116 anfibios (31 especies) en seis localidades de Colombia. Se tomaron muestras de sangre mediante punción cardiaca o punción a la vena maxilar. Se prepararon extendidos sanguíneos, se fijaron y tiñeron con Giemsa para su posterior análisis por microscopía. Se analizaron variables como porcentaje de GRs inmaduros, células mitóticas en sangre periférica e infección por hemoparásitos. Los datos fueron analizados mediante el test de rango de Willcoxon y el test exacto de Fisher. Resultados: sesenta y dos individuos evidenciaron mitosis en sangre periférica y dichas mitosis compartían características morfológicas con GRs inmaduros. La prevalencia general de parásitos fue del 30.1 %, distribuido de la siguiente forma: Trypanosoma (24.1 %), Hepatozoon-like (6 %), Dactylosoma (4.3 %), Karyolysus-like (0.9 %), y Filarioidea (2. 6 %). Hay una asociación positiva entre el porcentaje de GRs inmaduros y la presencia de células mitóticas, también se encontró una relación entre la infección por hemoparásitos y el porcentaje de GRs inmaduros. Conclusiones: En este estudio encontramos que la presencia de parásitos sanguíneos, GRs inmaduros y mitosis de GRs son eventos frecuentes en sangre periférica de anfibios, y nuestros resultados sugieren una asociación entre dichas características. Por tanto, la liberación de GRs inmaduros y la mitosis de estas células en sangre periférica podría ser una respuesta fisiológica a infecciones parasitarias. Posteriores estudios que caractericen la hematología en anfibios y en vida silvestre en general, son deseables.

Atividade antimicrobiana in vitro de extratos metanólicos de secreções cutâneas de anfíbios amazônicos sobre fitopatógenos de interesse agrícola / n vitro antimicrobial activity of methanolic extracts from cutaneous secretions of Amazonian amphibians against phytopathogens of agricultural interest

Os mecanismos de defesa bioquímica dos anfíbios envolvem secreções cutâneas de moléculas bioativas com atividade antimicrobiana. Este estudo avaliou a atividade in vitro de extratos metanólicos da secreção cutânea de duas espécies de anfíbios da família Bufonidae, Rhaebo guttatus e Rhinella marina, no controle dos patógenos Fusarium udum, Fusarium solani, Colletotrichum truncatum, Aspergillus flavus, Rhizoctonia solani, Macrophomina phaseolina e Calonectria pseudometrosideri. O extrato de R. guttatus inibiu o crescimento micelial de F. udum, F. solani, A. flavus e M. phaseolina em algumas concentrações testadas. O extrato de R. marina inibiu o crescimento micelial de C. truncatum na concentração de 0,5 mg mL- ¹, e inibiu o crescimento micelial de A. flavus nas concentrações de 0,1 e 0,5 mg mL- ¹, que foi semelhante à inibição pelo controle positivo. O extrato de R. marina também diminuiu a produção de microescleródios de R. solani nas concentrações de 0,2 e 0,3 mg mL- ¹. Além disso, os extratos inibiram a esporulação e germinação de conídios em graus variados. A inibição da formação de apressórios em C. truncatum pelos extratos de R. guttatus e R. marina foi de 85%­99% e 63%­100%, respectivamente. Nossos resultados demonstraram que o tratamento com extratos da secreção cutânea de R. guttatus e R. marina apresentou atividade antifúngica sobre os fitopatógenos estudados.(AU)

Amphibians in a transition region between the Cerrado and the Atlantic Forest, west-central region of the state of São Paulo, Brazil / Anfíbios em uma região de transição entre o Cerrado e a Mata Atlântica, Região Centro-Oeste do estado de São Paulo, Brasil

Studies of the diversity and natural history of amphibians are extremely important and contribute with the knowledge about the patterns of geographical distribution and biology of the species, outlining the factors that influence this distribution, and contributing to the development of conservation projects of them. This study aimed to identify the richness, abundance, spatial and temporal distribution of amphibians in the Bauru region, state of São Paulo, Brazil, and compare the species composition with others regions in the São Paulo state located in the Cerrado and Atlantic Forest. Eight locations were sampled between the years 2009 and 2012. Thirty-seven amphibian species were recorded, which represents 15.61% of the species in the state of São Paulo. In comparison to other regions of the state, Bauru resemble with the north-western region of the state and with the Assis Ecological Station in species composition. The community of amphibians in the studied region is regulated to morphoclimatic conditions, environmental heterogeneity and the conservation of natural resources, which directly reflect on the composition, abundance and species' spatial and temporal distribution of the amphibians in this region. The richness of amphibians of the Bauru region is significant for the state of São Paulo and contributes with the maintenance of environments with conditions for the occurrence of Proceratophrys moratoi, which is considered in threat of extinction. This study contributed with the filling gap on the amphibian knowledge of the state of São Paulo.

Estudos sobre diversidade e história natural de anfíbios são de extrema importância para o conhecimento sobre os padrões de distribuição geográfica e biologia das espécies delineando os fatores que influenciam nessa distribuição, o que contribui para a elaboração de projetos de conservação mais realistas e efetivos. Este estudo visou identificar a riqueza, abundância, distribuição espacial e temporal das espécies de anfíbios da região de Bauru, estado de São Paulo, Brasil, e comparar a composição de espécies com outras regiões de Cerrado e Mata Atlântica do estado de São Paulo. Oito localidades foram amostradas entre os anos de 2009 e 2012. Foram registradas 37 espécies de anfíbios, as quais representam 15,61% das espécies do estado de São Paulo. Em comparação com outras regiões do estado, Bauru se assemelha à Região Noroeste do estado e à Estação Ecológica de Assis na composição de espécies. A comunidade de anfíbios da região estudada é regulada pelas condições morfoclimáticas, heterogeneidade ambiental e estado de conservação dos recursos naturais, que refletem diretamente na composição, na abundância e nos padrões de distribuição espacial e temporal das espécies de anfíbios presentes na região. A riqueza de anfíbios da região de Bauru é significativa para o estado de São Paulo e contribui com a manutenção de ambientes com condições para a ocorrência de Proceratophrys moratoi, considerada em risco de extinção. Este estudo contribui com o preenchimento de lacunas no conhecimento de anfíbios do estado de São Paulo.

Aspectos da ecologia reprodutiva de Trachycephalus cunauaru (Anura: Hylidae) na Amazônia meridional / Aspects of the reproductive ecology of Trachycephalus cunauaru (Anura: Hylidae) in the southern Amazon

Trachycephalus cunauaru é um hilídeo amazônico que utiliza fitotelmatas para se reproduzir. Existem relativamente poucas informações sobre a espécie, principalmente devido à dificuldade de acesso aos seus sítios reprodutivos. Nesse trabalho, reunimos dados de ecologia e história natural de T. cunauaru em uma área da Amazônia meridional, no estado de Mato Grosso, Brasil. Além de fitotelmatas naturais, utilizamos baldes instalados a uma altura de 10 m como fitotelmatas artificiais. Comparamos características físicas e químicas, bem como a presença e ausência de girinos, entre fitotelmatas naturais e artificiais. Também coletamos dados sobre o comportamento reprodutivo da espécie por meio de armadilhas fotográficas. Registramos uma densidade de 14,1 sítios reprodutivos por km2 . Os parâmetros ambientais diferiram significativamente entre fitotelmatas artificiais e naturais. Em sítios artificiais a presença de girinos esteve diretamente relacionada à árvores com maior diâmetro. Registramos, pela primeira vez, oofagia para a espécie e observamos que machos podem utilizar mais de um fitotelmata. Também registramos a presença de cobras nos sítios reprodutivos. Constatamos que sítios artificiais e armadilhas fotográficas representam uma alternativa satisfatória para registros comportamentais para T. cunauaru e, possivelmente, para outras espécies com hábitos similares. (AU)

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.

Quantity - but not diversity - of secreted peptides and proteins increases with age in the tree frog Pithecopus nordestinus

Amphibians inhabit the terrestrial environment, a conquest achieved after several evolutionary steps, which were still insufficient to make them completely independent of the aquatic environment. These processes gave rise to many morphological and physiological changes, making their skin (and cutaneous secretion) rich in bioactive molecules. Among the tree frogs, the secretion is composed mainly of peptides; but alkaloids, proteins and steroids can also be found depending on the species. The most known class of biologically active molecules is the antimicrobial peptides (AMPs) that act against bacteria, fungi and protozoans. Although these molecules are well-studied among the hylids, AMPs ontogeny remains unknown. Therefore, we performed peptidomic and proteomic analyses of Pithecopus nordestinus (formerly Phyllomedusa nordestina) in order to evaluate the peptide content in post-metamorphosed juveniles and adult individuals. Methods: Cutaneous secretion of both life stages of individuals was obtained and analyzed by LC-MS/MS after reduction and alkylation of disulfide bonds or reduction, alkylation and hydrolysis by trypsin. Results: Differences in the TIC profile of juveniles and adults in both treatments were observed. Moreover, the proteomic data revealed known proteins and peptides, with slight differences in the composition, according to the life stage and the treatment. AMPs were identified, and bradykinin-potentiating peptides were observed in trypsin-treated samples, which suggests a protein source of such peptide (cryptide). Conclusion: In general, skin secretion contents were similar between juveniles and adults, varying in quantity, indicating that the different stages of life are reflected in the number of molecules and not on their diversity.(AU)

Uso y selección de microhábitat en un ensamble de anuros del Chaco Serrano de Argentina / Microhabitat use and selection by an anuran assemblage from the Chaco Serrano of 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.

Experimental Frog Virus 3 infection using Brazilian strain: amphibians susceptibility / Infecção experimental utilizando estirpe brasileira de Frog Virus 3: suceptibilidade de anfíbios

An alarming number of global warnings concerning amphibian mortality outbreaks have been released in recent years. Emerging diseases stand out as the main potential causes. Ranavirus is a worldwide-spread highly infectious disease capable of affecting even other ectothermic animals such as fish and reptiles. One major issue regarding this pathology is the lack of clinical signs before it leads up to death. Aiming at having a better understanding of anurans susceptibility, this study analyzed bullfrog (Lithobates catesbeianus) survival rate, when challenged with three doses of a Brazilian strain of Frog Virus 3 (FV3). The qPCR analysis indicated a low infectivity rate in these animals both as larvae and as adults. To elucidate the results, the following hypothesis was performed: 1) The amount of inoculum used on the frogs was insufficient to trigger an infection; 2) For the FV3 to produce clinical signs in this species, there is the need for a cofactor; 3) The animals did undergo FV3 infection but recovered in the course of the experiment, and 4) The inoculum utilized might have been low-virulence. Finally, the presence of actual clinical signs of ranavirus is discussed, with the more likely hypothesis.(AU)

Um número alarmante de notificações globais sobre surtos de mortalidade de anfíbios tem sido realizado nos últimos anos. As doenças emergentes destacam-se como as principais causas potenciais. O ranavírus é uma doença altamente infecciosa disseminada em todo o mundo, capaz de afetar até outros animais ectotérmicos como peixes e répteis. Uma questão importante em relação a essa patologia é a falta de sinais clínicos antes de levar à morte. Com o objetivo de compreender melhor a suscetibilidade dos anuros, o presente trabalho analisou a taxa de sobrevivência de rãs-touro (Lithobates catesbeianus), desafiadas com três doses de uma estirpe brasileira do Frog virus 3 (FV3). A análise de qPCR indicou baixa taxa de infectividade nesses animais, tanto como larvas quanto como adultos. Procurando esclarecer os resultados, foram formuladas as seguintes hipóteses: 1) A quantidade de inóculo aplicada nas rãs foi insuficiente para desencadear uma infecção; 2) Para que o FV3 dê sinais clínicos nesta espécie, é necessário um cofator; 3) Os animais sofreram infecção por FV3, mas se recuperaram no decorrer do experimento, e 4) O inóculo utilizado pode ter sido de baixa virulência. Finalmente, foi discutida a presença de sinais clínicos reais de ranavírus e levantada a hipótese mais provável(AU)

Dieta de la rana de hojarasca Craugastor rhodopis (Anura: Craugastoridae): una especie abundante en la región montañosa del este de México / Diet of the leaf litter frog Craugastor rhodopis (Anura: Craugastoridae): an abundant species in the mountainous region of the east Mexico

Resumen La composición de la dieta en anfibios puede ser influenciada por diversos factores que causan su variación intraespecífica, como pueden ser la distribución geográfica, ontogenia, estacionalidad, y el sexo de los organismos. El objetivo de este estudio fue analizar la composición de la dieta en adultos de la Craugastor rhodopis (rana hojarasquera común) en la región montañosa del centro de Veracruz, México. Un total de 77 individuos adultos fueron colectados durante la época de lluvias de 2012, de los cuales 66 tuvieron contenido estomacal. Se realizó un análisis de la variación intersexual en la diversidad de la dieta, y el volumen, número de presas, y número de categorías de presa consumidas. La dieta de C. rhodopis estuvo compuesta por 20 categorías, de las cuales predominó Orthoptera en cuanto a número, volumen, frecuencia de ocurrencia e importancia relativa. Los machos consumieron presas de menor tamaño en relación a las hembras. Aunque ambos sexos consumieron similar número de presas, se encontró un efecto significativo de la interacción del sexo y el tamaño de las ranas sobre esta variable de la dieta, en la cual las hembras pequeñas consumen más presas que las grandes, y los machos grandes consumen más presas que los pequeños. El alto consumo de ortópteros (al menos en cuanto a volumen) es un fenómeno común dentro de la familia Craugastoridae y posiblemente está relacionado con el éxito de algunas especies en los ecosistemas donde habitan. A pesar de que la composición de la dieta fue similar entre sexos en C. rhodopis, se sugiere que la competencia intersexual por los recursos tróficos es mínima, dadas las diferencias en el tamaño de presas consumidas por machos y hembras.(AU)

Abstract Dietary composition in amphibians may be influenced by several factors that cause their intraspecific variation, such as geographic distribution, ontogeny, seasonality, and sex of organisms. The objective of this study was to analyze the composition of the diet in adults of the Polymorphic Robber Frog Craugastor rhodopis in the mountainous region of central Veracruz, Mexico. A total of 77 adult individuals were collected during the rainy season of 2012, of which 66 had stomach contents. We performed an analysis of the intersexual variation in diet diversity, volume, number of prey, and number of prey categories consumed. The diet of C. rhodopis was composed by 20 categories. Orthoptera predominated in terms of number, volume, frequency of occurrence, and relative importance. Males consumed smaller prey items relative to females. Although both sexes consumed similar numbers of prey, we found a significant effect of interaction of sex and size of frogs on this variable, with small females consuming more prey than the large ones, whereas in males the opposite occurred. High consumption of orthopterans (at least in volume) is a common phenomenon within Craugastoridae and is possibly related to the success of some species in the ecosystems where they live. Although the composition of the diet was similar between sexes in C. rhodopis, we suggest that intersexual competition for trophic resources is minimal, given differences in prey size consumed by males and females.(AU)

Threatened amphibians sheltered under the big cat's umbrella: conservation of jaguars Panthera onca (Carnivora: Felidae) and endemic herpetofauna in Central America / Anfibios amenazados se refugiaron bajo la sombrilla del gato grande: evaluación de la conservación del jaguar Panthera onca (Carnivora: Felidae) y la herpetofauna endémica en Centro América

Abstract The umbrella species concept posits that protection of a single, wide-ranging species may confer protection to a large number of sympatric species. Due to their large home ranges, widespread distribution in the Mesoamerican Biodiversity Hotspot (MBH), and status as the focal species of numerous conservation initiatives, the jaguar Panthera onca is an ideal species to evaluate the umbrella strategy. After ground-truthing jaguar corridors from 2009-2016, we tested the umbrella value of jaguars for endemic herpetofauna (Amphibia, Reptilia) in Nuclear Central America (NCA), a ~ 370 000 km² sub-region of the MBH. NCA contains the greatest density of threatened reptiles in the Western Hemisphere and harbors extraordinary high diversity of amphibians, the most threatened class of vertebrate worldwide. Of the 304 regional endemics in NCA, the distributions of 187 (61.5 %) species of amphibians and reptiles overlapped ground-truthed jaguar range. The distributions of 14 reptiles, including a critically endangered Bothriechis spp. and two endangered Norops spp., occur exclusively within jaguar distribution. Similarly, the distributions of 19 amphibians, including four critically endangered Craugastor spp. and two critically endangered Plectrohyla spp. occur entirely within jaguar distribution. Our results indicate greater effectiveness of ground-truthed jaguar distribution than modeled and randomly selected networks in overlapping the distributions of endemic herpetofauna, especially threatened amphibians, in NCA. Substantiation of multi-taxa dependence on habitat in jaguar distribution would strengthen justification for wider application of the umbrella strategy beyond NCA and aid conservation planning in the MBH.

Resumen Las especies sombrilla son aquellas cuya conservación confiere protección a un gran número de especies simpátricas. Debido a sus ámbitos hogareños extensos, a una distribución generalizada en el Hotspot de Biodiversidad de Mesoaméricana (MBH), y su estatus como especie focal de numerosas iniciativas de conservación, el jaguar Panthera onca es una especie ideal para evaluar la estrategia sombrilla. Evaluamos el valor de los jaguares como estrategia sombrilla para la herpetofauna endémica en Centro América Nuclear (NCA), una subregión de ~ 370 000 km² que se encuentra dentro del MBH. La NCA contiene la mayor densidad de reptiles amenazados en el hemisferio occidental y alberga una diversidad extraordinaria de anfibios, que es la clase de vertebrados más amenazada del mundo. De las 304 especies endémicas regionales presentes en el NCA, las distribuciones de 187 (61.5 %) se sobrepusieron al área de distribución del jaguar verificada. Las distribuciones de 14 reptiles, incluyendo una Bothriechis spp. (en Peligro Crítico) y dos Norops spp. (en Peligro de Extinción), se dan exclusivamente dentro del rango del jaguar. Similarmente, 19 especies de anfibios, incluidas cuatro Craugastor spp. (en Peligro Crítico) y dos Plectrohyla spp. (en Peligro Crítico) están presentes exclusivamente dentro del área de distribución del jaguar. Nuestros resultados indican que los corredores verificados para el jaguar coinciden mejor con las distribuciones de la herpetofauna endémica dentro del NCA, especialmente anfibios, en comparación con los corredores modelados y seleccionados al azar. La confirmación del traslape de la distribución del hábitat del jaguar con la de múltiples taxones fortalece la justificación de una aplicación más amplia de la estrategia sombrilla, aún más allá del NCA, contribuyendo también en la selección de áreas de conservación previamente subestimadas dentro del MBH.

Geographical context of forgotten amphibians: Colombian "Data Deficient species" sensu IUCN / Contexto geográfico de los anfibios olvidados: especies colombianas con "Datos Insuficientes" según la UICN

Abstract Whereas more than 10 % of global amphibian richness is known to occur in Colombia, almost 16 % of these species are currently classified as Data Deficient according to the IUCN. These estimates suggest that the available data for a large portion of amphibians occurring in Colombia is insufficient to assess extinction risk. Here we aim to (1) review the available information on the distribution of the Colombian Data Deficient (DD hereafter) amphibians, (2) analyze their geographic distribution, and (3) evaluate the relationship between anthropogenic impact and their current conservation status. For this, we first compiled geographical records for the DD amphibian species using primary sources. Geographical records were obtained mainly from taxonomic descriptions and non-systematic surveys. We then estimated the geographical range and inferred the potential distribution for each species using letsR and MaxEnt, respectively. We quantified the human footprint for each species and tested the relationship between spatial distribution and anthropogenic change across populations. Analyses are here based on 128 of the 129 DD amphibian species that occur in Colombia. We found that most of these species were recently described and have small geographic ranges. A large proportion of these DD amphibians inhabit the Colombian Andes, and their populations have been strongly affected by human activities. Overall, the spatial clustering suggests that many of these species have faced similar environmental and anthropogenic pressures that have contributed to their rareness. We also suggest that the conservation status for several of the analyzed DD amphibians should be changed to account for the threats they face. Rev. Biol. Trop. 66(3): 1272-1281. Epub 2018 September 01.

Resumen A pesar de que más del 10 % de la riqueza global de anfibios se encuentra en Colombia, cerca del 16 % de estas especies es actualmente clasificada con Datos Deficientes según la IUCN. Estas estimaciones sugieren que los datos disponibles para esta gran porción de anfibios que habitan en Colombia, son insuficientes para evaluar su riesgo de extinción. En este documento nosotros (1) revisamos la información disponible sobre la distribución de los anfibios colombianos con Datos Deficientes (ó DD), (2) analizamos su distribución geográfica, e (3) hipotetizamos sobre la relación entre el impacto antropogénico y su estado de conservación. Para esto, compilamos los registros geográficos para las especies de anfibios DD usando referencias primarias. Los registros geográficos fueron obtenidos principalmente a partir de descripciones taxonómicas y búsquedas no sistemáticas. Para estimar la distribución geográfica e inferir la distribución potencial de cada especie usamos letsR y MaxEnt, respectivamente. Cuantificamos la huella humana para cada especie y evaluamos la relación entre la distribución espacial y el cambio antropogénico entre poblaciones. Los análisis fueron basados en 128 de las 129 especies de anfibios que se encuentran en Colombia y actualmente son clasificadas como DD. Encontramos que la mayoría de estas especies fueron descritas recientemente, y presentan una distribución geográfica reducida. Una gran proporción de estas especies de anfibios DD habitan los Andes colombianos, y sus poblaciones han sido fuertemente afectadas por las actividades humanas. Este agrupamiento geográfico sugiere que muchas de estas especies enfrentan similares presiones ambientales y antropogénicas que contribuyen a su rareza. Sugerimos además que el estado de conservación para muchas de las especies de anfibios DD aquí analizados podría ser reevaluado para considerar las amenazas que enfrentan.