Development of a novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF)

Cystic fibrosis (CF) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. The black yeast, Exophiala dermatitidis (synonym Wangiella dermatitidis) has been found with increasing frequency in sputum specimens of CF patients, with reported isolation rates ranging from 1.1 to 15.7%. At present, no diagnostic PCR exists to aid with the clinical laboratory detection and identification of this organism. A novel species-specific PCR-based assay was developed for the detection of E. dermatitidis, based on employment of rDNA operons and interspacer (ITS) regions between these rDNA operons. Two novel primers, (designated ExdF & ExdR) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of Exophiala, as well as with other commonly described yeasts and filamentous fungi within CF sputum, including Candida. Aspergillus and Scedosporium. An amplicon of approximately 455 by was generated, spanning the partial ITS I region - the complete 5.8S rDNA region - partial ITS2 region, employing ExdF (forward primer [16-mer], 5'-CCG CCT ATT CAG GTC C-3' and ExdR (reverse primer [16-mer], 5'-TCT CTC CCA CTC CCG C-3', was employed and optimised on extracted genomic DNA from a well characterised culture of E. dermatitidis, as well as with high quality genomic DNA template from a further 16 unrelated fungi, including Candida albicans, C. dubliniensis, C. parapsilosis, C. glabrata, Scedosporium apiospermum, Penicillium sp., Aspergillus fumigatus, Aspergillus versicolor, Pichia guilliermondii, Rhodotorula sp., Trichosporon sp., Aureobasidium pullulans, Fusarium sp., Mucor hiemalis, Bionectria ochroleuca, Gibberella pulicaris. Results demonstrated that only DNA from E. dermatitidis gave an amplification product of the expected sire, whilst none of the other fungi were amplifiable. Subsequent employment of this primer pair detected this yeast from mycological cultures from 2/50 (4%) adult CF patients. These two patients were the only patients who were previously shown to have a cultural history of E. dermatitidis from their sputum. E. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan- and multiresistant bacterial pathogens from sputum. namely Pseudomonas aeruginosa and Burkholderia cepacia complex organisms, hence this species-specific PCR assay may help detect this organism from CF sputum more specifically and rapidly. Overall, employment of this novel assay nay help in the understanding of the occurrence. aetiology and epidemiology of E. dermatitidis, as an emerging fungal agent in patients with CF. (C) 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Nitrogen fluxes from treefrogs to tank epiphytic bromeliads: an isotopic and physiological approach

Diverse invertebrate and vertebrate species live in association with plants of the large Neotropical family Bromeliaceae. Although previous studies have assumed that debris of associated organisms improves plant nutrition, so far little evidence supports this assumption. In this study we used isotopic ((15)N) and physiological methods to investigate if the treefrog Scinax hayii, which uses the tank epiphytic bromeliad Vriesea bituminosa as a diurnal shelter, contributes to host plant nutrition. In the field, bromeliads with frogs had higher stable N isotopic composition (delta(15)N) values than those without frogs. Similar results were obtained from a controlled greenhouse experiment. Linear mixing models showed that frog feces and dead termites used to simulate insects that eventually fall inside the bromeliad tank contributed, respectively, 27.7% (+/- 0.07 SE) and 49.6% (+/- 0.50 SE) of the total N of V. bituminosa. Net photosynthetic rate was higher in plants that received feces and termites than in controls; however, this effect was only detected in the rainy, but not in the dry season. These results demonstrate for the first time that vertebrates contribute to bromeliad nutrition, and that this benefit is seasonally restricted. Since amphibian-bromeliad associations occur in diverse habitats in South and Central America, this mechanism for deriving nutrients may be important in bromeliad systems throughout the Neotropics.

Hemoparasites of the genus Trypanosoma (Kinetoplastida: Trypanosomatidae) and hemogregarines in Anurans of the São Paulo and Mato Grosso do Sul States - Brazil

Os animais silvestres estão expostos a inúmeros patógenos,dentre eles estão os hemoparasitas. Podem-se destacar espécies do gênero Trypanosoma e do grupo das hemogregarinas,que ocorrem com freqüência parasitando anuros (rãs, pererecas e sapos). Normalmente, a descrição destes hemoparasitas é feita através da morfologia dos estágios observados nosangue periférico do hospedeiro e as pesquisas sobre o ciclobiológico desses hemoparasitas são escassas. Os objetivos dopresente estudo foram avaliar a presença de hemogregarinas eTrypanosoma spp. em anuros capturados nos Estados de São Paulo e Mato Grosso do Sul e fazer a caracterização morfológica e morfométrica dos seus hemoparasitas. As espécies deanuros examinadas foram: Dendropsophus nanus, D. minutus, Leptodactylus chaquensis, L. podicipinus, L. labyrinthicus, L. fuscus, Bufo granulosus, B. schneideri, Phyllomedusahypocondrialis, Trachicephalus venulosus, Scinax fuscovarius e Hypsiboas albopunctatus. Dos 40 animais estudados, foramencontrados quatro (10%) positivos para hemogregarinas e oito(20%) positivos para Trypanosoma spp. Foram observadosgamontes de hemogregarinas com morfologia variável e, alémdas formas intraeritrocíticas, também foram observados gamontes fora das hemácias. As formas de Trypanosoma encontradas eram muito polimórficas, conforme é descrito na literatura, sendo na sua maioria, larga e oval.

On the identity of Hyla strigilata Spix, 1824 (Anura : Hylidae): redescription and neotype designation for a ghost taxon

Recent field work in Atlantic Rain Forest patches in the southern region of the State of Bahia, Brazil, resulted in the discovery of some populations of an unidentified species of the Scinax catharinae group. An extensive literature review, along with the examination of specimens and distribution patterns of all known species of this group, showed that Hyla strigilata Spix, 1824, a long confused species with lost type material, is an available name for the specimens from Bahia. In order to clarify the taxonomic problems surrounding this taxon, the nomenclatural history of Hyla strigilata is reviewed and a neotype is designated, described, and figured. The association of this name to extant populations from southern Bahia and its consequent stabilization is considered important since it is the type species of the genus Ololygon, a name available for the clade of Scinax catharinae. Data on habits, habitat, and geographic distribution are also presented.

Comparative cytogenetic analysis on four tree frog species (Anura, Hylidae, Hylinae) from Brazil

A comparative cytogenetic analysis was carried out on four Hylinae tree frogs from Brazil (Aparasphenodon brunoi, Corthomantis greeningi, Osteocephalus langsdorffi and Scinax fuscovarius) using Giemsa staining, BrdU replication banding, Ag-NOR staining, C-banding, DAPI and CMA(3) fluorochrome staining, and fluorescence in situ hybridization (FISH) with an rDNA probe. All the species share closely similar 2n = 24 karyotypes, almost indistinguishable by standard staining. The technique of BrdU incorporation allowed the identification of each pair of homologs and the establishment of extensive homeology for the great majority of the chromosomes, mainly of A. brunoi, C greeningi, and O. langsdorffii. Despite highly conserved replication banding patterns, the use of the other banding techniques disclosed some minor differsences, which reinforces the importance of extensive cytogenctic analyses for the karyotypic characterization of Anuran species. The present cytogenctic data confirm the closer proximity of A. brunoi, C greeningi, and O. langsdorfjii, whereas S. fuscovarius is phylogenetically more distant. Copyright (C) 2003 S. Karger AG, Basel

Amphibians and reptiles from a highly diverse area of the Caatinga domain: composition and conservation implications

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diet of the colubrid snake, Thamnodynastes strigatus (Günther, 1858) from Paraná State, Brazil, with field notes on anuran predation

São apresentados dados da dieta de Thamnodynastes strigatus (Serpentes: Colubridae), a partir da dissecção de 44 espécimes. No tubo digestivo dos exemplares examinados foram encontrados anfíbios anuros (71,4% da amostra), roedores (14,3%), peixes (10,7%) e lagartos (3,6%). A maioria das espécies de anuros (Bufo sp., Leptodactylus sp., Physalaemus cuvieri e Scinax fuscovarius) encontradas no exame de conteúdo estomacal de T. strigatus, utiliza o solo ou o nível d'água como sítio de vocalização. Também são apresentados dados sobre a observação de eventos de predação na natureza.

Novos registros na distribuição geográfica de anuros na floresta com araucária e considerações sobre suas vocalizações

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Nitrogen fluxes from treefrogs to tank epiphytic bromeliads: an isotopic and physiological approach

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytochemistry and polarization microscopy of amphibian sperm cell nuclei presumed to differ in basic protein composition

The pattern of availability of free DNA phosphates, and the kind of DNA-protein complex arrangement, both induced by nuclear basic proteins, and the richness in arginine residues in these proteins were investigated cytochemically and cytophysically in spermatozoa of the South-American Hylidae species, Hyla fuscovaria and Hyla biobeba. The aim was to demonstrate differences at the level of sperm histones in two species of Hyla until recently considered to be congeneric. The results indicated differences in the spermatozoal nuclear basic proteins and DNA-protein complexes when the two species were compared. The spermatozoa of Hyla biobeba were assumed to be likely to contain a Bloch's ''type 3'' protein type (intermediate sperm basic protein), similarly to Hyla species of North and Central America. on the other hand, the data obtained for the spermatozoa of Hyla fuscovaria indicated that they contain a protamine or protamine-like protein, differing from Hyla biobeba and Hyla species of North and Central America. It is suggested that the differences reported here may be genus-specific, since Hyla fuscovaria has recently been reclassified as Scinax fuscovaria based on parameters other than sperm histone types. These findings are in agreement with the general view of a wide variability in sperm nuclear proteins in the Anura group.

Visual communication in Brazilian species of anurans from the Atlantic forest

Visual communication is widespread among several anuran families, but seems to be more common than currently thought. We investigated and compared visual communication in six species of an anuran community in the Brazilian Atlantic forest. Four are nocturnal species: Hyalinobatrachium uranoscopum (Centrolenidae), Hyla albomarginata, Hyla sp. (aff. ehrhardti), and Scinax eurydice (Hylidae), and two are diurnal species: Hylodes phyllodes and Hylodes asper ( Leptodactylidae). For H. uranoscopum, H. albomarginata, S. eurydice, and H. phyllodes, this is the first record of visual communication. Observations were made at Nucleo Picinguaba, Parque Estadual da Serra do Mar, in the Municipality of Ubatuba, State of São Paulo, Brazil. Descriptions of behaviour were based on individuals observed in the field, using sequence sampling with continuous tape recording for behavioural observations. Eight new behaviours are described: body wiping, face wiping, jump display, leg kicking, limb lifting, mouth opening, toe flagging, and vocal sac display. of the 42 anuran species known from Nucleo Picinguaba, at least six ( approximately 14%) display visual communication. The evolution of visual signals in these species may be related to the availability of ambient light, the structural complexity of the habitat, and/or the ambient noise. They may also have evolved to aid in the location of the individual, to avoid physical combat, and/or may be a by-product of seismic communication.

WATER BALANCE and SPATIAL DISTRIBUTION of AN ANURAN COMMUNITY FROM BRAZIL

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Produção de biomassa por fungos Filamentosos em meio de vinhaça de cana-de-açúcar suplementado com melaço

Three species of filamentous fungi, Aspergillus niger, Penicillium fellutanum and Mucor hiemalis, were selected and cultivated in vinasse media with different addition of molasses, pasteurized to 85°C for 30 minutes and with pH = 5.0. The microorganisms, previously adapted to the respective medium for 48 hours, from a solution of 107 spores.ml-1, were cultivated in pure and mixed cultures in Erlenmeyer vessel of 500ml, to 30°C, with constant agitation of 170 rpm, for 24, 48 and 72 hours, with four repetition for each samples. The biomass was separated by vacuum filtration in filter Whatman #1 and dried in oven at 105°C until right weight, the obtained liquid was submited to COD analysis. The datas were statistically analysed using a response surface methodology, to improve the effect on the molasses proportion and culture time, in the biomass production by microorganism in research. According to the obtained results (5.02% of molasses, 55.59h, 70% of spores solution of A. niger and 30% of spores solution of P. fellutanum), cultivating was carried out in Microferm Fermentor New Brunswick for 48 hours at 300 rpm, aired at 1v/v/m, using 5 liters of medium added with 5.0% of molasses on the conditions above described. The average of the results obtained (6.81g.l-1) was higher than the confidence interval (5.937 ; 6.369) and was inside the prediction interval (4.471 ; 7.834) both of them significant at 95% by the statistical test employed.

Systematic review of the frog family Hylidae, with special reference to Hylinae: Phylogenetic analysis and taxonomic revision

Hylidae is a large family of American, Australopapuan, and temperate Eurasian treefrogs of approximately 870 known species, divided among four subfamilies. Although some groups of Hylidae have been addressed phylogenetically, a comprehensive phylogenetic analysis has never been presented. The first goal of this paper is to review the current state of hylid systematics. We focus on the very large subfamily Hylinae (590 species), evaluate the monophyly of named taxa, and examine the evidential basis of the existing taxonomy. The second objective is to perform a phylogenetic analysis using mostly DNA sequence data in order to (1) test the monophyly of the Hylidae; (2) determine its constituent taxa, with special attention to the genera and species groups which form the subfamily Hylinae, and c) propose a new, monophyletic taxonomy consistent with the hypothesized relationships. We present a phylogenetic analysis of hylid frogs based on 276 terminals, including 228 hylids and 48 outgroup taxa. Included are exemplars of all but 1 of the 41 genera of Hylidae (of all four nominal subfamilies) and 39 of the 41 currently recognized species groups of the species-rich genus Hyla. The included taxa allowed us to test the monophyly of 24 of the 35 nonmonotypic genera and 25 species groups of Hyla. The phylogenetic analysis includes approximately 5100 base pairs from four mitochondrial (12S, tRNA valine, 16S, and cytochrome b) and five nuclear genes (rhodopsin, tyrosinase, RAG-1, seventh in absentia, and 28S), and a small data set from foot musculature. Concurring with previous studies, the present analysis indicates that Hemiphractinae are not related to the other three hylid subfamilies. It is therefore removed from the family and tentatively considered a subfamily of the paraphyletic Leptodactylidae. Hylidae is now restricted to Hylinae, Pelodryadinae, and Phyllomedusinae. Our results support a sister-group relationship between Pelodryadinae and Phyllomedusinae, which together form the sister taxon of Hylinae. Agalychnis, Phyllomedusa, Litoria, Hyla, Osteocephalus, Phrynohyas, Ptychohyla, Scinax, Smilisca, and Trachycephalus are not monophyletic. Within Hyla, the H. albomarginata, H. albopunctata, H. arborea, H. boons, H. cinerea, H. eximia, H. geographica, H. granosa, H. microcephala, H. miotympanum, H. tuberculosa, and H. versicolor groups are also demonstrably nonmonophyletic. Hylinae is composed of four major clades. The first of these includes the Andean stream-breeding Hyla, Aplastodiscus, all Gladiator Frogs, and a Tepuian clade. The second clade is composed of the 30-chromosome Hyla, Lysapsus, Pseudis, Scarthyla, Scinax (including the H. uruguaya group), Sphaenorhynchus, and Xenohyla. The third major clade is composed of Nyctimantis, Phrynohyas, Phyllodytes, and all South American/West Indian casque-headed frogs: Aparasphenodon, Argenteohyla, Corythomantis, Osteocephalus, Osteopilus, Tepuihyla, and Trachycephalus. The fourth major clade is composed of most of the Middle American/Holarctic species groups of Hyla and the genera Acris, Anotheca, Duellmanohyla, Plectrohyla, Pseudacris, Ptychohyla, Pternohyla, Smilisca, and Triprion. A new monophyletic taxonomy mirroring these results is presented where Hylinae is divided into four tribes. Of the species currently in Hyla, 297 of the 353 species are placed in 15 genera; of these, 4 are currently recognized, 4 are resurrected names, and 7 are new. Hyla is restricted to H. femoralis and the H. arborea, H. cinerea, H. eximia, and H. versicolor groups, whose contents are redefined. Phrynohyas is placed in the synonymy of Trachycephalus, and Pternohyla is placed in the synonymy of Smilisca. The genus Dendropsophus is resurrected to include all former species of Hyla known or suspected to have 30 chromosomes. Exerodonta is resurrected to include the former Hyla sumichrasti group and some members of the former H. miotympanum group. Hyloscirtus is resurrected for the former Hyla armata, H. bogotensis, and H. larinopygion groups. Hypsiboas is resurrected to include several species groups - many of them redefined here - of Gladiator Frogs. The former Hyla albofrenata and H. albosignata complexes of the H. albomarginata group are included in Aplastodiscus. New generic names are erected for (1) Agalychnis calcarifer and A. craspedopus; (2) Osteocephalus langsdorffii; the (3) Hyla aromatica, (4) H. bromeliacia, (5) H. godmani, (6) H. mixomaculata, (7) H. taeniopus, (8) and H. tuberculosa groups; (9) the clade composed of the H. pictipes and H. pseudopuma groups; and (10) a clade composed of the H. circumdata, H. claresignata, H. martinsi, and H. pseudopseudis groups. Copyright © American Museum of Natural History 2005.

Organização celular dos testículos em Hylidae e leptodactylidae, no Pantanal (Estado do Mato Grosso do Sul, Brasil)

The objective of this work was to make a comparative analysis of germ cell organization at different stages of cellular differentiation in adult males of Dendropsophus nanus (Boulenger, 1889), Pseudis limellum (Cope, 1862), P. paradoxa (Linnaeus, 1758), and Scinax acuminatus (Cope, 1862), belonging to the family Hylidae; and Leptodactylus chaquensis (Cei, 1950) and L. podicipinus (Cope, 1862), belonging to the family Leptodactylidae, collected in the Pantanal and in Serra da Bodoquena, Mato Grosso do Sul State, Brazil. The testes were removed and fixed, dehydrated in a graded series of ethanol and embedded in methacrylate glycol resin (Historesin Leica®). The sections were stained by 1% toluidine blue and observed under light microscope. It was detected that all individual of the Hylidae family show, throughout the year, the presence of all germ cell types of spermatogenesis. However, all Leptodactylidae family individuals only show the presence of all germ cell types during the rainy season. The variations of characteristics in seminiferous epithelium organization, as well as the evident difference in the amount of spermatozoa inside the tubules, are evidence that the anurans in this work show different forms of spermatogenesis development throughout the year: the cycle is continuous for the Hylidae family, and discontinuous with explosive release of spermatozoa for the Leptodactylidae family.