Testicular morphological changes during the seasonal reproductive cycle in the bullfrog Rana catesbeiana

Spermatogenesis and steroidogenesis undergo seasonal variations during the reproductive cycle in amphibians. Testicular morphological and morphometric seasonal variations as well as interstitial lipidic inclusions and intralobular glycoconjugates were evaluated during seasonal cycle of Rana catesbeiana. Testes of frogs collected during the annual seasons were weighed for calculation of GSI (Gonadosomatic index). Seminiferous lobule diameters (DSL) and volume densities of seminiferous lobules (VvSL), excretory ducts (VvED), and interstitial tissue (VvIT) were analyzed. Semithin sections were submitted to Periodic Acid-Schiff (PAS) and Alcian Blue (AB) methods for detection of glycoconjugates, while lipidic inclusions were detected by Sudan Black B. GSI showed no significant variations during the year. Since VvED and VvIT increased significantly during summer and were inversely proportional to VvSL, a compensatory effect between the testicular compartments may be related to the maintenance of GSI. During autumn/winter, larger lobular diameters were observed in comparison to spring/summer when spermiogenesis and spermiation were commonly observed. The increased VvIT and the numerous lipidic inclusions in the interstitial cells during summer suggest a relationship between spermiogenesis and steroidogenesis. Besides the structural stability variations occurring in the IT and SL, a possible paracrine interaction between ED and IT should be also involved in the IT development during summer. The presence of PAS and AB-positive globular structures were observed in the seminiferous lobules and excretory ducts. These structures containing acid glycoconjugates appear to be Sertoli cell apical portions, which are accumulated in the lumen of the seminiferous lobules mainly during spermiation. © 2004 Wiley-Liss, Inc.

Size-fecundity relationships and reproductive investment in female frogs in the Pantanal, south-western Brazil

The ovarian complement of anurans exhibiting different reproductive modes is highly diverse, and intraspecific variation in reproductive output of anurans is generally related to variation in female properties and/or environmental conditions. The size-fecundity relationships, reproductive investment, and correlation between ovary mass and fat body mass were investigated for females of an anuran assemblage in the Pantanal, Brazil. Female body size was positively correlated with clutch size among seven of the eight species analyzed. However, these results seem to be influenced by seasonal variation in fecundity. Interspecific size-fecundity relationships revealed that female SVL was positively correlated with clutch size and egg size regardless of reproductive mode. Among 11 species analyzed, the reproductive investment (RI: ovary mass relative to body mass) varied from 5.5 to 18%, and there were no differences among reproductive modes and activity patterns (explosive/prolonged). RI correlated negatively with female size. Among three Leptodactylus species examined, negative correlation between ovary mass and fat body mass was verified for two species. Variations in the reproductive strategies are closely related to the reproductive activity patterns and reproductive modes exhibited by individual species, but are also influenced by environmental conditions. However, regardless of reproductive mode or activity pattern, each species seems to exhibit a reproductive strategy that allows them to respond differently to the same environmental restrictions.

Feeding habits and habitat use in Bothrops pubescens (Viperidae, Crotalinae) from southern Brazil

Bothrops pubescens is a member of the neuwiedi complex that occurs in southern Brazil and Uruguay. We studied the ecology of B. pubescens from a field site (at Santa Maria, Rio Grande do Sul, Brasil) and based on preserved specimens from the state of Rio Grande do Sul, Brasil. In Santa Maria, individuals were collected during visual encounter surveys (VES), in pitfall traps with drift fences and during incidental encounters. Most snakes found in the field were on the ground, mainly on leaf litter, in mosaics of light and shadow or in completely shaded areas. In disturbed areas, snakes were usually associated with country houses and agricultural fields. Snakes were found much more frequently in forests and forest edges than in open habitats. The diet of B. pubescens comprised small mammals (56.2% of individual prey found), anurans (21.2%), lizards (7.5%), snakes (7.5%), birds (5.0%), and centipedes (2.5%). Prey predator mass ratios ranged from 0.002-0.627, and larger snakes tended to consume larger prey. Bothrops pubescens seems to be able to survive in disturbed areas, mainly those close to forests, and this ability may be facilitated by its generalized feeding habits. Copyright 2005 Society for the Study of Amphibians and Reptiles.

Amphibian secretions for drug discovery studies: A search for new antiparasitic and antifungal compounds

The antiparasitic and antifungal activities of nine amphibian skin secretions were studied in different in vitro models. Seven secretions presented a considerable antiprotozoan activity and one showed promising results against Candida sp. These results can be the basis for the development of new drugs, especially for neglected parasitic diseases. © 2007 Bentham Science Publishers Ltd.

Predation on Scinax littoralis (Anura: Hylidae) by another treefrog, Hypsiboas albomarginatus (Anura: Hylidae)

Herein we report predation by the hylid anuran Hypsiboas albomarginatus on another hylid anuran Scinax littoralis, observed in a remnant of Atlantic Forest in the municipality of Santos, southeastern Brazil.

Reinforcing and expanding the predictions of the disturbance vicariance hypothesis in Amazonian harlequin frogs: A molecular phylogenetic and climate envelope modelling approach

The disturbance vicariance hypothesis (DV) has been proposed to explain speciation in Amazonia, especially its edge regions, e. g. in eastern Guiana Shield harlequin frogs (Atelopus) which are suggested to have derived from a cool-adapted Andean ancestor. In concordance with DV predictions we studied that (i) these amphibians display a natural distribution gap in central Amazonia; (ii) east of this gap they constitute a monophyletic lineage which is nested within a pre-Andean/western clade; (iii) climate envelopes of Atelopus west and east of the distribution gap show some macroclimatic divergence due to a regional climate envelope shift; (iv) geographic distributions of climate envelopes of western and eastern Atelopus range into central Amazonia but with limited spatial overlap. We tested if presence and apparent absence data points of Atelopus were homogenously distributed with Ripley's K function. A molecular phylogeny (mitochondrial 16S rRNA gene) was reconstructed using Maximum Likelihood and Bayesian Inference to study if Guianan Atelopus constitute a clade nested within a larger genus phylogeny. We focused on climate envelope divergence and geographic distribution by computing climatic envelope models with MaxEnt based on macroscale bioclimatic parameters and testing them by using Schoener's index and modified Hellinger distance. We corroborated existing DV predictions and, for the first time, formulated new DV predictions aiming on species' climate envelope change. Our results suggest that cool-adapted Andean Atelopus ancestors had dispersed into the Amazon basin and further onto the eastern Guiana Shield where, under warm conditions, they were forced to change climate envelopes. © 2010 The Author(s).

Herpetofauna, municipality of Porto Murtinho, Chaco region, state of Mato Grosso do Sul, Brazil

Among the physiographic regions included in the central South American diagonal of open formations, the Chaco, with some endemic species, represents the southernmost dry area. In Brazil, the Chaco is found in southwestern corner of Mato Grosso do Sul state, mostly in the municipality of Porto Murtinho along the Paraguay and Apa rivers. From February 2008 to December 2009, we carried out an inventory of amphibians and reptiles in Porto Murtinho, using pitfall traps, time-limited searches, and occasional encounters. A total of 34 amphibian and 39 reptile species were registered. Although some typical Chacoan species were found, most of the species are open area dwellers that also occur in other open biomes, such as the Cerrado and Caatinga. © 2010 Check List and Authors.

Development of microsatellite markers for the Neotropical endemic Brazilian Guanabara frog, Euparkerella brasiliensis, through 454 shotgun pyrosequencing

The new-generation 454 GS-FLX Titanium pyrosequencing was used to isolate microsatellite markers for the Brazilian Guanabara frog, Euparkerella brasiliensis, an Atlantic forest endemic species. Three multiplex polymerase chain reaction sets were optimized for genotyping of 11 polymorphic (di- and tetranucleotide) microsatellite markers. Genetic diversity was assessed in 21 individuals from a population (Reserva Ecológica de Guapiaçu, REGUA) locatedin the central region of the Rio de Janeiro State, in Brazil. The mean number of alleles per locus ranged from 3 to 12. Observed and expected heterozygosities ranged from 0.095 to 0.905 and from 0.094 to 0.904, respectively. After using the Bonferroni correction for multiple tests, there was no evidence of linkage disequilibrium between pairs of loci but deviations for Hardy-Weinberg equilibrium were found in 4 loci. We found no evidence for allele dropouts or stuttering, but we detected the presence of null alleles at loci Eb10 and Eb36. These markers will be useful for analyses of fine-scale population structure and determination of relative effects of habitat loss and fragmentation on population genetic variability within species. © FUNPEC-RP.

Biochemical biomarkers in Scinax fuscovarius tadpoles exposed to a commercial formulation of the pesticide fipronil

One of the main pesticides used in the cultivation of sugarcane in São Paulo State, Brazil, is Regent®800WG, the main active compound of which is fipronil. Fipronil is a potent insecticide that eliminates pests, including insects resistant to pyrethroids, organophosphates (OP) and carbamates (CA). There is little known on the toxic effects of fipronil on non-target organisms, such as tadpoles of frogs. It is possible that this compound carries a high toxicity for these organisms, since the pesticide can be incorporated into aquatic environments during the rainy season, a time which coincides with the time of amphibian reproduction and the occurrence of tadpoles in the aquatic environment in this region. Thus, the pesticide could be contributing to the decline of amphibians in the northwest region of São Paulo state due to its wide use. This study aimed to test the influence of Regent®800WG on some biochemical systems of tadpoles (such as antioxidant defense systems) at different stages of development. The results of analysis from in vivo exposures demonstrated that only a few parameters in the groups exposed to fipronil responded to exposure to Regent®800WG, results which indicate that the pesticide instigates biochemical responses in tadpoles. Although catalase and glucose-6-phosphate dehydrogenase (G6PDH) were unchanged during the experiments, glutathione-S-transferase (GST) was inhibited in tadpoles, and the activity of glutathione reductase (GR) varied according to the exposure period and pesticide concentration. This data demonstrated the influence of the fipronil formulation on the metabolism of tadpoles, and showed that it can increase their susceptibility to environmental contaminants. © 2013 Elsevier Ltd. All rights reserved.

From widespread to microendemic: Molecular and acoustic analyses show that Ischnocnema guentheri (Amphibia: Brachycephalidae) is endemic to Rio de Janeiro, Brazil

Many species of tropical amphibians are restricted to very small ranges, and this microendemism coupled with ongoing habitat loss and susceptibility to emerging pathogens imperils the long-term persistence of these species. Incomplete taxonomic and distributional knowledge may obscure conservation assessment, particularly in putatively widespread species that are typically considered to be of Least Concern in Red List assessments, but that in fact may constitute complexes of partly microendemic species. Such is the case in the Steindachner's Robber Frog, Ischnocnema guentheri which, together with the recently recognized Ischnocnema henselii, is thought to occupy most of the Brazilian Atlantic Forest. To test whether these taxa may constitute a species complex of range-restricted and thus potentially threatened species, we analyzed 160 samples of I. guentheri and/or I. henselii for two molecular markers, 16S rRNA (16S) and recombination activation gene 1 (RAG1). To verify the monophyly of the complex, closely related species were also included in the 16S analysis. Congruent evidence from the molecular data and from analyses of advertisement calls support the existence of six distinct species within the complex: I. guentheri and I. henselii as well as four candidate new species. The lineages are distributed as a mosaic in the Atlantic Forest and are sympatric at some localities without indication of admixture. Their phylogeographical pattern partially agrees with paleo-models for the Atlantic Forest, but also suggests the existence of micro-refugia in less stable areas. I. guentheri, previously considered to be widespread, was found only in its type locality, a reserve within the urban area of Rio de Janeiro city. Although none of the species studied appears highly threatened with extinction, we recommend their IUCN threat status to be re-evaluated carefully for the next comprehensive update of the Red List of Brazil's amphibians. © 2013 Springer Science+Business Media Dordrecht.

Comparative feeding kinematics of tropical hylid tadpoles

Anuran larvae, which are otherwise simple in shape, typically have complex keratinized mouthparts (i.e. labial teeth and jaw sheaths) that allow them to graze upon surfaces. The diversity in these structures among species presumably reflects specializations that allow for maximal feeding efficiency on different types of food. However, we lack a general understanding of how these oral structures function during feeding. We used high-speed digital imaging (500 Hz) to observe tadpoles of six species from the anuran family Hylidae grazing on a standardized food-covered substrate. Tadpoles of these species vary in the number of labial tooth rows, belong to two different feeding guilds (benthic and nektonic), and inhabit ponds and streams. We confirmed that the labial teeth in these species serve two functions: anchoring the mouth to the substrate and raking material off of the substrate. In general, tadpoles with a larger maximum gape or those with fewer labial tooth rows opened and closed their mouths slower than tadpoles with smaller gape or more tooth rows. Nektonic feeding tadpoles released each of their tooth rows proportionally earlier in the gape cycle compared with benthic feeding tadpoles. Lastly, we found some support for the idea that deformation of the jaw sheaths during a feeding cycle is predictable based on tadpole feeding guild. Collectively, our data show that anatomical (e.g. number of labial teeth) and ecological features (e.g. feeding guild) of tadpoles significantly influence how tadpoles open and close their mouths during feeding. © 2013. Published by The Company of Biologists Ltd.

The pioneering use of ISSR (Inter Simple Sequence Repeat) in Neotropical anurans: Preliminary assessment of genetic diversity in populations of Physalaemus cuvieri (Amphibia, Leiuperidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

From doubly labelled water to half-life; validating radio-isotopic rubidium turnover to measure metabolism in small vertebrates

The doubly labelled water method (DLW) is widely used to measure field metabolic rate (FMR), but it has some limitations. Here, we validate an innovative technique for measuring FMR by comparing the turnover of isotopic rubidium (86Rb kb) with DLW depletion and the rate of CO2 production (V·co2) measured by flow-through respirometry (FTR) for two dunnart species (Marsupialia: Dasyuridae), Sminthopsis macroura (17 g) and Sminthopsis ooldea (10 g). The rate of metabolism as assessed by V·co2 (FTR) and 86Rb kb was significantly correlated for both species (S. macroura, r2 = 0·81, P = 1·19 × 10-5; S. ooldea, r2 = 0·63, P = 3·84 × 10-4), as was V·co2 from FTR and DLW for S. macroura (r2 = 0·43, P = 0·039), but not for S. ooldea (r2 = 0·29, P = 0·168). There was no relationship between V·co2 from DLW and 86Rb kb for either species (S. macroura r2 = 0·22, P = 0·169; S. ooldea r2 = 0·21, P = 0·253). We conclude that 86Rb kb provided useful estimates of metabolic rate for dunnarts. Meta-analysis provided different linear relationships between V·co2 and 86Rb kb for endotherms and ectotherms, suggesting different proportionalities between metabolic rate and 86Rb kb for different taxa. Understanding the mechanistic basis for this correlation might provide useful insights into the cause of these taxonomic differences in the proportionality. At present, it is essential that the relationship between metabolic rate and 86Rb kb be validated for each taxon of interest. The advantages of the 86Rb technique over DLW include lower equipment requirements and technical expertise, and the longer time span over which measurements can be made. The 86Rb method might be particularly useful for estimating FMR of groups for which the assumptions of the DLW technique are compromised (e.g. amphibians, diving species and fossorial species), and groups that are practically challenging for DLW studies (e.g. insects). © 2013 British Ecological Society.

Comparative cytogenetic analysis of some species of the Dendropsophus microcephalus group (Anura, Hylidae) in the light of phylogenetic inferences

Background: Dendropsophus is a monophyletic anuran genus with a diploid number of 30 chromosomes as an important synapomorphy. However, the internal phylogenetic relationships of this genus are poorly understood. Interestingly, an intriguing interspecific variation in the telocentric chromosome number has been useful in species identification. To address certain uncertainties related to one of the species groups of Dendropsophus, the D. microcephalus group, we carried out a cytogenetic analysis combined with phylogenetic inferences based on mitochondrial sequences, which aimed to aid in the analysis of chromosomal characters. Populations of Dendropsophus nanus, Dendropsophus walfordi, Dendropsophus sanborni, Dendropsophus jimi and Dendropsophus elianeae, ranging from the extreme south to the north of Brazil, were cytogenetically compared. A mitochondrial region of the ribosomal 12S gene from these populations, as well as from 30 other species of Dendropsophus, was used for the phylogenetic inferences. Phylogenetic relationships were inferred using maximum parsimony and Bayesian analyses.Results: The species D. nanus and D. walfordi exhibited identical karyotypes (2n = 30; FN = 52), with four pairs of telocentric chromosomes and a NOR located on metacentric chromosome pair 13. In all of the phylogenetic hypotheses, the paraphyly of D. nanus and D. walfordi was inferred. D. sanborni from Botucatu-SP and Torres-RS showed the same karyotype as D. jimi, with 5 pairs of telocentric chromosomes (2n = 30; FN = 50) and a terminal NOR in the long arm of the telocentric chromosome pair 12. Despite their karyotypic similarity, these species were not found to compose a monophyletic group. Finally, the phylogenetic and cytogenetic analyses did not cluster the specimens of D. elianeae according to their geographical occurrence or recognized morphotypes.Conclusions: We suggest that a taxonomic revision of the taxa D. nanus and D. walfordi is quite necessary. We also observe that the number of telocentric chromosomes is useful to distinguish among valid species in some cases, although it is unchanged in species that are not necessarily closely related phylogenetically. Therefore, inferences based on this chromosomal character must be made with caution; a proper evolutionary analysis of the karyotypic variation in Dendropsophus depends on further characterization of the telocentric chromosomes found in this group. © 2013 Medeiros et al.; licensee BioMed Central Ltd.

Visceral colouration of Eupemphix nattereri (Anura: Leiuperidae): effects of NDP-α-melanocyte stimulating hormone

Amphibians have melanin-containing cells in visceral organs that are similar to pigmentary cells from the epidermis. Both of them are derived from the ectodermal neural crest. Epidermal cells respond to α-melanocyte stimulating hormone (α-MSH), which is associated to the dispersion of melanin granules within melanocytes. Therefore, our aim was to test whether a non-degradable analogue of the α-MSH changes the superficial colouration of organs of Eupemphix nattereri. The hormone rapidly increases (within 12 hours) the colouration on the surface of the pericardium, heart, testes, nerves of the lumbar plexus, and lumbosacral parietal peritoneum. Colouration increased late (after 24 hours) in the kidneys and mesentery following hormone administration. However, this hormone did not change colouration of intestine, rectum and lungs. Our findings could be explained by the similarities between epidermal and visceral melanocytes, since both cells have a common embryonic origin. Furthermore, the increase in visceral colouration may be related to the dispersion of melanosomes within melanocytes, which causes the darkening of organs. Our results demonstrate for the first time that the visceral colouration is responsive, thereby altering the internal pattern of organs' colouration in anurans. © 2013 Copyright 2013 Unione Zoologica Italiana.