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.

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.

Water balance and spatial distribution of an anuran community from Brazil

Terrestrial amphibians may dehydrate when exposed to low humidity, representing an important factor affecting spatial distribution and community composition. In this study we investigated whether rates of dehydration and rehydration are able to explain the spatial distribution of an anuran community in a Restinga environment at the northern coast of the State of Bahia, Brazil, represented by 11 species distributed in 27 sample units. The environmental data set containing 20 variables was reduced to a few synthetic axes by principal component analysis (PCA). Physiological variables measured were rates of dehydration, rehydration from water, and rehydration from a neutral substrate. Multiple regression analyses were used to test the null hypothesis of no association between the environmental data set (synthetic axes of PCA) and each axis representative of a physiological variable, which was rejected (P < 0.001). Of 15 possible partial regressions only rehydration rate from neutral substrate vs. PC1. and PC2, rehydration rate from water vs. PC1, and dehydration rate vs. PC2 were significant. Our analysis was influenced by a gradient between two different groups of sample units: a beach area with high density of bromeliads and an environment without bodies of water with low density of bromeliads. Species of very specific natural history and morphological characters occur in these environments: Phyllodytes melanomystax and Scinax auratus, species frequently occurring in terrestrial bromeliads, and Ischnocnema paulodutrai, common along the northern coast of Bahia and usually found in forest remnants within environments with low number of bodies of water. In dry environments species with lower rates of dehydration were dominant, whereas species showing greater rates of dehydration were found predominantly in microhabitats with greater moisture or abundance of bodies of water.

Do Higher Temperatures Increase Growth in the Nocturnal Gecko Homonota darwini (Gekkota: Phyllodactylidae)? A Skeletochronological Assessment Analyzed at Temporal and Geographic Scales

Recent studies predict that several lineages of tropical animals are at particular risk given current estimates of global climate change. Yet, much uncertainty exists on the effects of climate shifts in ectothermic species from cool temperate regions such as Patagonia. In this study, we focus on the impact of environmental temperature on growth, age at sexual maturity, and life-span of the Patagonian gecko Homonota darwini. Skeletochronological methods were used to assess the bone growth rates Of individuals from three populations at different geographic and temporal scales: two populations from Chubut (warm site; 1941 and 2010) and one population from Rio Negro (cold site; 1997-1998). Populations displayed similar bone arrangement and the growth patterns fit a von Bertalanffy curve. Three populations attained reproductive size at a minimum age of 3 yr, but at the cold site two specimens were shown to mature in 4 yr. We found no differences in juvenile growth rates in body size or bone zone width between juveniles of 1 to 3 yr of age from the 1941 warm site and the 2010 warm site. However, these traits appeared to be higher at these two warm sites than at the cold site, which is consistent with the climatic differences among the three localities. Our results suggest that higher temperatures positively affect growth, denoting that global warming might benefit H. darwini, especially the southern populations.

Life History Aspects of Oxyrhopus trigeminus (Serpentes: Dipsadidae) from Two Sites in Southeastern Brazil

We studied the reproduction, sexual dimorphism, and diet of Oxyrhopus trigeminus from two sites in southeastern Brazil. Oxyrhopus trigeminus from Irape Power Plant (IPP) contained vitellogenic follicles and eggs in both rainy and dry seasons and clutch size was not correlated with female snout vent length (SVL). Sexual dimorphism was evident. Females attain larger SVL but males have longer tails. We found three females from Santa Clara Power Plant (SPP) with vitellogenic follicles, all of them collected in the dry season. Mean SVLs of adult females from IPP and SPP were 717.7 mm and 786 mm, respectively. Mean SVL of adult males from IPP was 553.4 mm and the single adult male from SPP was 507 mm. The diet of O. trigeminus from IPP included rodents (46.7%), lizards (33.3%), and birds (20%). The volume of individual prey items was not correlated with snake SVL. The diet of O. trigeminus from SPP included rodents (37.5%), lizards (37.5%), birds (12.5%), and marsupials (12.5%). It seemed that an ontogenetic shift may occur in individuals of this snake species from IPP.

Taxonomy, Hemipenial Morphology, and Natural History of Two Poorly Known Species of Anadia (Gymnophthalmidae) from Northern South America

Anadia pariaensis Rivas, La Marca, and Oliveros, 1999, and Anadia steyeri Nieden, 1914, are two particularly rare and poorly known lizards described from single specimens. In the case of A. pariaensis, it remains known from the holotype, whereas A. steyeri is known from three additional specimens reported in the literature after the original description of the species. A single new specimen of A. pariaensis and five of A. steyeri, including the first adult males recorded for both species, make possible a more representative description of both species, including descriptions of the hemipenes. Despite both species presenting some similar morphological characteristics, the examination of the hemipenial morphology revealed very different organs. The hemipenis of A. steyeri presents some characteristics that resemble the organs of two species from the Santa Marta Mountain Range in the "bitaeniata-group" (Anadia pulchella and Anadia altaserrania). On the other hand, the hemipenes of A. pariaensis are unique morphologically and cannot be associated with the hemipenes known from other species in the genus. We describe variation within both species, and we comment on possible sexual dimorphism (number and arrangement of the femoral pores), natural history, and the known geographic distribution of the species. We also comment on Anadia bumanguesa Rueda-Almonacid and Caicedo 2004 based on a new specimen, the second known. This species may be a synonym of A. steyeri.

The Panama microplate, island studies and relictual species of Melipona (Melikerria) (Hymenoptera: Apidae: Meliponini)

The endemic stingless honey-making bee Melipona (Melikerria) insularissp.n. on Coiba and Rancheria Islands in Pacific Panama is described, together with the proposed sister species, M. ambigua sp.n. from northeast Colombia. The Coiba Island group and Panama mainland were surveyed, yielding one meliponine endemic (M. insularissp.n.) and six meliponine genera and species. The poor Coiba fauna of amphibians and birds corresponds to the poor social bee fauna and suggests habitat barriers generally precluded recolonization from the mainland during glacial periods. Many animals became extinct, yet some remain as relicts. Melipona insularissp.n. was isolated on accreted terranes of Coiba rainforest in the Panama microplate. Morphology suggests that M. insularissp.n. is not a direct descendant of the San Blas-E. Panama endemic Melikerria, M. triplaridis. A phylogenetic hypothesis corroborates disjunct distributions. Rainforest endemics such as Peltogyne purpurea (Fabaceae) and Ptilotrigona occidentalis (Apidae, Meliponini) also occur as relictual, disjunct populations in Central and South America. These may have been isolated before accelerated biotic exchange began 2.4 Ma. Our work supports the geological findings of both a volcanic arc and the San Blas massif providing a substantial bridge for Melikerria from Colombia and Panama in Eocene to Miocene times. We suggest there have been taxon cycles permitting recolonization during glaciations, whereby colonies of M. insularissp.n. were able to recolonize Rancheria, a 250 ha island, 2 km from Coiba. However, rafting colonies nesting in trees, carried on vegetation mats, may have produced founding populations of Melipona in Central America and on oceanic islands such as Coiba.

The occurrence of defensive alkaloids in non-integumentary tissues of the Brazilian red-belly toad Melanophryniscus simplex (Bufonidae)

The red-belly toads (Melanophryniscus) of southern South America secrete defensive alkaloids from dermal granular glands. To date, all information on Melanophryniscus alkaloids has been obtained by extraction from either skins or whole organisms; however, in other amphibians, tetrodotoxins, samandarines, and bufadienolides have been detected in both skin and other organs, which raise the possibility that lipophilic alkaloids may occur in non-integumentary tissues in Melanophryniscus as well. To test this hypothesis, we studied the distribution of alkaloids in the skin, skeletal muscle, liver, and mature oocytes of the red-belly toad M. simplex from three localities in southern Brazil. Gas chromatography and mass spectrometry of skin extracts from 11 individuals of M. simplex resulted in the detection of 47 alkaloids (including isomers), 9 unclassified and 38 from 12 known structural classes. Each alkaloid that was present in the skin of an individual was also present in the same relative proportion in that individual's skeletal muscle, liver, and oocytes. The most abundant and widely distributed alkaloids were the pumiliotoxins 251D, 267C, and 323A, 5,8-disubstituted indolizidines 207A and 223D, 5,6,8-trisubstituted indolizidine 231B, 3,5-disubstituted pyrrolizidines cis-223B and cis- and trans-251K, and izidine 211C. We report the first record of piperidines in Melanophryniscus, bringing the total number of alkaloid classes detected in this genus to 16. Alkaloid composition differed significantly among the three study sites. The functional significance of defensive chemicals in non-integumentary tissues is unknown.

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.

Antimicrobial capacity of plasma from anurans of the Atlantic Forest

The viability and interpretation of techniques for the evaluation of immunocompetence of animals in their natural environment has been largely debated. One of these methods is based on testing the antimicrobial capacity of the blood and/or plasma in vitro, which could rapidly and effectively assess the immunological conditions of natural populations. We tested the applicability of the antimicrobial capacity of plasma (ACP) assay in anuran amphibians from the Atlantic Forest. The assay was performed by measuring both the turbidity (in a spectrophotometer) and the colony forming units (CFU) of the remaining bacteria (Escherichia coli) following exposure to amphibian plasma. Although both assays were correlated, the ACP assay by spectrophotometry showed 10 times lower intra-assay variation. We also found interspecific variation in ACP, as well as the maintenance of ACP values in males from the same population, collected in different breeding seasons. Thus, the estimation of ACP by spectrophotometry provides a convenient and accurate method for evaluating innate immunocompetence in comparative and ecophysiological studies of anuran amphibians.