Evaluation of the in vitro Activity of Dermaseptin 01, a Cationic Antimicrobial Peptide, against Schistosoma mansoni

Schistosomiasis is a neglected tropical disease that remains a considerable public health problem worldwide. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we examined the in vitro effects of dermaseptin 01 (DS 01), an antimicrobial peptide found in the skin secretion of frogs of the genus Phyllomedusa, on Schistosoma mansoni adult worms. DS 01 at a concentration of 100 mg/ml reduced the worm motor activity and caused the death of all worms within 48 h in RPMI 1640 medium. At the highest sublethal concentration of antimicrobial peptide (75 mg/ml), a 100% reduction in egg output of paired female worms was observed. Additionally, DS 01 induced morphological alterations on the tegument of S. mansoni, and a quantitative analysis carried out by confocal microscopy revealed extensive destruction of the tubercles in a dose-dependent manner over the concentration range of 50-200 mu g/ml. It was the first time that an anthelmintic activity towards schistosomes has been reported for a dermaseptin.

The breathing pattern and the ventilatory response to aquatic and aerial hypoxia and hypercarbia in the frog Pipa carvalhoi

Anuran amphibians are known to exhibit an intermittent pattern of pulmonary ventilation and to exhibit an increased ventilatory response to hypoxia and hypercarbia. However, only a few species have been studied to date. The aquatic frog Pipa carvalhoi inhabits lakes, ponds and marshes that are rich in nutrients but low in O-2. There are no studies of the respiratory pattern of this species and its ventilation during hypoxia or hypercarbia. Accordingly, the aim of the present study was to characterize the breathing pattern and the ventilatory response to aquatic and aerial hypoxia and hypercarbia in this species. With this purpose, pulmonary ventilation (V-1) was directly measured by the pneumotachograph method during normocapnic normoxia to determine the basal respiratory pattern and during aerial and aquatic hypercarbia (5% CO2) and hypoxia (5% O-2). Our data demonstrate that P. carvalhoi exhibits a periodic breathing pattern composed of single events (single breaths) of pulmonary ventilation separated by periods of apnea. The animals had an enhanced V-1 during aerial hypoxia, but not during aquatic hypoxia. This increase was strictly the result of an increase in the breathing frequency. A pronounced increase in V-1 was observed if the animals were simultaneously exposed to aerial and aquatic hypercarbia, whereas small or no ventilatory responses were observed during separately administered aerial or aquatic hypercarbia. P. carvalhoi primarily inhabits an aquatic environment. Nevertheless, it does not respond to low O-2 levels in water, although it does so in air. The observed ventilatory responses to hypercarbia may indicate that this species is similar to other anurans in possessing central chemoreceptors. (C) 2012 Elsevier Inc. All rights reserved.

Molecular phylogeny and morphometric analyses reveal deep divergence between Amazonia and Atlantic Forest species of Dendrophryniscus

Dendrophryniscus is an early diverging clade of bufonids represented by few small-bodied species distributed in Amazonia and the Atlantic Forest. We used mitochondrial (414 bp of 12S, 575 bp of 16S genes) and nuclear DNA (785 bp of RAG-1) to investigate phylogenetic relationships and the timing of diversification within the genus. These molecular data were gathered from 23 specimens from 19 populations, including eight out of the 10 nominal species of the genus as well as Rhinella boulengeri. Analyses also included sequences of representatives of 18 other bufonid genera that were publically available. We also examined morphological characters to analyze differences within Dendrophryniscus. We found deep genetic divergence between an Amazonian and an Atlantic Forest clade, dating back to Eocene. Morphological data corroborate this distinction. We thus propose to assign the Amazonian species to a new genus, Amazonella. The species currently named R. boulengeri, which has been previously assigned to the genus Rhamphophryne, is shown to be closely related to Dendrophryniscus species. Our findings illustrate cryptic trends in bufonid morphological evolution, and point to a deep history of persistence and diversification within the Amazonian and Atlantic rainforests. We discuss our results in light of available paleoecological data and the biogeographic patterns observed in other similarly distributed groups. (C) 2011 Elsevier Inc. All rights reserved.

Biological invasions and the acoustic niche: the effect of bullfrog calls on the acoustic signals of white-banded tree frogs

Invasive species are known to affect native species in a variety of ways, but the effect of acoustic invaders has not been examined previously. We simulated an invasion of the acoustic niche by exposing calling native male white-banded tree frogs (Hypsiboas albomarginatus) to recorded invasive American bullfrog (Lithobates catesbeianus) calls. In response, tree frogs immediately shifted calls to significantly higher frequencies. In the post-stimulus period, they continued to use higher frequencies while also decreasing signal duration. Acoustic signals are the primary basis of mate selection in many anurans, suggesting that such changes could negatively affect the reproductive success of native species. The effects of bullfrog vocalizations on acoustic communities are expected to be especially severe due to their broad frequency band, which masks the calls of multiple species simultaneously.

Batrachochytrium dendrobatidis in amphibians confiscated from illegal wildlife trade and used in an ex situ breeding program in Brazil

This paper describes an outbreak of chytridiomycosis affecting a group of Dendrobates tinctorius, a Neotropical anuran species, confiscated from the illegal wildlife trade and housed in a private zoo in Brazil as part of an ex situ breeding program. We examined histological sections of the skin of 30 D. tinctorius and 20 Adelphobates galactonotus individuals. Twenty D. tinctorius (66.7%) and none of the A. galactonotus were positive for Batrachochytrium dendrobatidis (Bd). Multiple development stages of Bd infection were observed. The reasons for the interspecific difference in the rate of infection could not be determined, and further studies are advised. Because the examined population consisted of confiscated frogs, detailed epidemiological aspects could not be investigated, and the source of the fungus remains uncertain. The existence of ex situ amphibian populations is important for protecting species at higher risk in the wild, and ex situ amphibian conservation and breeding programs in Brazil may be established using confiscated frogs as founders. However, this paper alerts these programs to the urgency of strict quarantine procedures to prevent the introduction of potential pathogens, particularly Bd, into ex situ conservation programs.

From Amazonia to the Atlantic forest: Molecular phylogeny of Phyzelaphryninae frogs reveals unexpected diversity and a striking biogeographic pattern emphasizing conservation challenges

Documenting the Neotropical amphibian diversity has become a major challenge facing the threat of global climate change and the pace of environmental alteration. Recent molecular phylogenetic studies have revealed that the actual number of species in South American tropical forests is largely underestimated, but also that many lineages are millions of years old. The genera Phyzelaphryne (1 sp.) and Adelophryne (6 spp.), which compose the subfamily Phyzelaphryninae, include poorly documented, secretive, and minute frogs with an unusual distribution pattern that encompasses the biotic disjunction between Amazonia and the Atlantic forest. We generated >5.8 kb sequence data from six markers for all seven nominal species of the subfamily as well as for newly discovered populations in order to (1) test the monophyly of Phyzelaphryninae, Adelophryne and Phyzelaphryne, (2) estimate species diversity within the subfamily, and (3) investigate their historical biogeography and diversification. Phylogenetic reconstruction confirmed the monophyly of each group and revealed deep subdivisions within Adelophryne and Phyzelaphryne, with three major clades in Adelophryne located in northern Amazonia, northern Atlantic forest and southern Atlantic forest. Our results suggest that the actual number of species in Phyzelaphryninae is, at least, twice the currently recognized species diversity, with almost every geographically isolated population representing an anciently divergent candidate species. Such results highlight the challenges for conservation, especially in the northern Atlantic forest where it is still degraded at a fast pace. Molecular dating revealed that Phyzelaphryninae originated in Amazonia and dispersed during early Miocene to the Atlantic forest. The two Atlantic forest clades of Adelophryne started to diversify some 7 Ma minimum, while the northern Amazonian Adelophryne diversified much earlier, some 13 Ma minimum. This striking biogeographic pattern coincides with major events that have shaped the face of the South American continent, as we know it today. (C) 2012 Elsevier Inc. All rights reserved.

Vocal and territorial behavior in the Smith frog (Hypsiboas faber): Relationships with plasma levels of corticosterone and testosterone

The possible trade-off between the roles of glucocorticoids as facilitators of energy substrate mobilization and neural inhibitors of sexual behavior during breeding season is under debate. We studied the relationship between calling and territorial behavior with plasma levels of corticosterone (CORT) and plasma levels of testosterone (T) across the breeding season of Hypsiboas faber, a large and territorial Neotropical treefrog. We investigated these relationships through focal observations of males calling naturally, followed by blood sampling for hormonal radioimmunoassay. We additionally used an experimental approach, which consisted of broadcasting recorded advertisement calls for 10 min to simulate an invasion in the territory of the focal subjects, followed by behavioral observation and blood sampling for hormonal radioimmunoassay. Results showed a pattern of co-variation between CORT and T across the breeding season. Furthermore, individual variation in CORT and T was related to different aspects of behavior: individuals with higher CORT showed higher calling rates, and individuals with higher steroid levels, mainly T, showed higher responsivity to social stimulation by other males in the chorus. Experimental simulation of territorial intrusion by using playback of advertisement calls of this species did not elicit consistent changes in agonistic behavior and CORT, but decreased T in focal males. (C) 2012 Elsevier Inc. All rights reserved.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.