Leptodactylus Labyrinthicus Research Articles

Phylogenetic position of Gorgoderina parvicava Travassos, 1922 (Digenea: Gorgoderidae), a parasite of Leptodactylus labyrinthicus (Spix, 1824) (Anura: Leptodactylidae) in Brazil.

During a parasite survey in Brazilian amphibians from São Paulo state, Brazil, Gorgoderina parvicava Travassos, 1922 was found in the urinary bladder (11 adult worms) and (five juvenile worms) in the kidneys of the pepper-frog Leptodactylus labyrinthicus (Spix, 1824). Parasites were examined by microscopy and 28S rDNA and COI gene were sequenced and analyzed for the molecular study. The phylogenetic reconstructions resulted in identical topologies with highly supported values in the nodes in most clades using Maximum likelihood and Bayesian inference methods and positioned G. parvicava in the subclade formed by species of subfamily Gorgoderinae parasitizing the urinary bladder of amphibians. Molecular phylogenetic data showed that this species is related to other species of Gorgoderina. In addition, new molecular data and the analyses of genetic distances provide extra comparative data, which can be applied in further investigations on the taxonomic status and the diversity among Gorgoderina spp. and host-parasite relationships.

New insights on the diversity of Brazilian anuran blood parasites: With the description of three new species of Hepatozoon (Apicomplexa: Hepatozoidae) from Leptodactylidae anurans.

The Amphibia are considered the most threatened vertebrate class globally, yet in Brazil they are also one of the more diverse and species rich groups. Although, in recent years there has been strong focus on amphibian related research, their parasites have not received the same attention. In Brazil, only a single species of Hepatozoon, namely H. leptodactyli (Lesage, 1908) Pessoa, 1970, has been described from anuran hosts. The present study aimed to describe three new species of Hepatozoon parasitising Leptodactylus labyrinthicus and Leptodactylus latrans from Mato Grosso State, Brazil. From 66 anurans screened for haemogregarines, four belonging to the Leptodactylidae were found positive for species of Hepatozoon. Based on the morphological analysis of peripheral blood gamonts and spleen and liver tissue meronts, three different morphotypes of Hepatozoon spp. were identified. Morphotype 1 (M1) and morphotype 2 (M2) in L. labyrinthicus and morphotype 3 (M3) in L. latrans. Molecular data based on partial 18S rDNA sequences revealed an interspecific divergence, between the species ranging from 0.43% to 1.16%. Phylogenetic analysis recovered isolates from the present study monophyletic with other isolates from Brazilian reptile and anuran hosts, sister to a clade comprising species isolated from African, North American and European reptile and anuran host species. Thus, using morphological and molecular analysis three new species infecting Brazilian Leptodactylidae anurans were identified and described. This study increases the knowledge of Brazilian anurans blood parasites and demonstrates the importance of using integrative approaches for diagnosis of hemoparasites.

NMR structures in different membrane environments of three ocellatin peptides isolated from Leptodactylus labyrinthicus

The peptides ocellatin-LB1, −LB2 and −F1 have previously been isolated from anurans of the Leptodactylus genus and the sequences are identical from residue 1–22, which correspond to ocellatin-LB1 sequence (GVVDILKGAAKDIAGHLASKVM-NH2), whereas ocellatin-LB2 carries an extra N and ocellatin-F1 extra NKL residues at their C-termini. These peptides showed different spectra of activities and biophysical investigations indicated a direct correlation between membrane-disruptive properties and antimicrobial activities, i.e. ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. To better characterize their membrane interactions, we report here the detailed three-dimensional NMR structures of these peptides in TFE-d2:H2O (60:40) and in the presence of zwitterionic DPC-d38 and anionic SDS-d25 micellar solutions. Although the three peptides showed significant helical contents in the three mimetic environments, structural differences were noticed. When the structures of the three peptides in the presence of DPC-d38 micelles are compared to each other, a more pronounced curvature is observed for ocellatin-F1 and the bent helix, with the concave face composed mostly of hydrophobic residues, is consistent with the micellar curvature and the amphipathic nature of the molecule. Interestingly, an almost linear helical segment was observed for ocellatin-F1 in the presence of SDS-d25 micelles and the conformational differences in the two micellar environments are possibly related to the presence of the extra Lys residue near the peptide C-terminus, which increases the affinity of ocellatin-F1 to anionic membranes in comparison with ocellatin-LB1 and -LB2, as proved by isothermal titration calorimetry. To our knowledge, this work reports for the first time the three-dimensional structures of ocellatin peptides.

Perspectives on invasive amphibians in Brazil.

Introduced species have the potential to become invasive and jeopardize entire ecosystems. The success of species establishing viable populations outside their original extent depends primarily on favorable climatic conditions in the invasive ranges. Species distribution modeling (SDM) can thus be used to estimate potential habitat suitability for populations of invasive species. Here we review the status of six amphibian species with invasive populations in Brazil (four domestic species and two imported species). We (i) modeled the current habitat suitability and future potential distribution of these six focal species, (ii) reported on the disease status of Eleutherodactylus johnstonei and Phyllodytes luteolus, and (iii) quantified the acoustic overlap of P. luteolus and Leptodactylus labyrinthicus with three co-occurring native species. Our models indicated that all six invasive species could potentially expand their ranges in Brazil within the next few decades. In addition, our SDMs predicted important expansions in available habitat for 2 out of 6 invasive species under future (2100) climatic conditions. We detected high acoustic niche overlap between invasive and native amphibian species, underscoring that acoustic interference might reduce mating success in local frogs. Despite the American bullfrog Lithobates catesbeianus being recognized as a potential reservoir for the frog-killing fungus Batrachochytrium dendrobatidis (Bd) in Brazil, we did not detect Bd in the recently introduced population of E. johnstonei and P. luteolus in the State of São Paulo. We emphasize that the number of invasive amphibian species in Brazil is increasing exponentially, highlighting the urgent need to monitor and control these populations and decrease potential impacts on the locally biodiverse wildlife.

Ocellatin peptides from the skin secretion of the South American frog Leptodactylus labyrinthicus (Leptodactylidae): characterization, antimicrobial activities and membrane interactions

BackgroundThe availability of antimicrobial peptides from several different natural sources has opened an avenue for the discovery of new biologically active molecules. To the best of our knowledge, only two peptides isolated from the frog Leptodactylus labyrinthicus, namely pentadactylin and ocellatin-F1, have shown antimicrobial activities. Therefore, in order to explore the antimicrobial potential of this species, we have investigated the biological activities and membrane interactions of three peptides isolated from the anuran skin secretion.MethodsThree peptide primary structures were determined by automated Edman degradation. These sequences were prepared by solid-phase synthesis and submitted to activity assays against gram-positive and gram-negative bacteria and against two fungal strains. The hemolytic properties of the peptides were also investigated in assays with rabbit blood erythrocytes. The conformational preferences of the peptides and their membrane interactions have been investigated by circular dichroism spectroscopy and liposome dye release assays.ResultsThe amino acid compositions of three ocellatins were determined and the sequences exhibit 100% homology for the first 22 residues (ocellatin-LB1 sequence). Ocellatin-LB2 carries an extra Asn residue and ocellatin-F1 extra Asn-Lys-Leu residues at C-terminus. Ocellatin-F1 presents a stronger antibiotic potential and a broader spectrum of activities compared to the other peptides. The membrane interactions and pore formation capacities of the peptides correlate directly with their antimicrobial activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. All peptides acquire high helical contents in membrane environments. However, ocellatin-F1 shows in average stronger helical propensities.ConclusionsThe obtained results indicate that the three extra amino acid residues at the ocellatin-F1 C-terminus play an important role in promoting stronger peptide-membrane interactions and antimicrobial properties. The extra Asn-23 residue present in ocellatin-LB2 sequence seems to decrease its antimicrobial potential and the strength of the peptide-membrane interactions.

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus.

BackgroundRabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms.MethodsSkin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus – PV) strain was used for virological assays based on rapid fluorescent focus inhibition test.ResultsHerein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. < IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the virus’s glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide.ConclusionsThis novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s40409-015-0048-1) contains supplementary material, which is available to authorized users.

Introduction of Leptodactylus labyrinthicus (Spix, 1824) (Anura: Leptodactylidae) in central Amazonia, Brazil

This note reports new records of Leptodactylus labyrinthicus in central Amazonia (Manaus, Brazil). We found a small population on the banks of a small polluted tributary of the Negro river. The knowledge about the natural species range suggests that the population was introduced, probably carried to Manaus for commercial production.

The crude skin secretion of the pepper frog Leptodactylus labyrinthicus is rich in metallo and serine peptidases.

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.

Avaliação do comportamento interespecífico de rã, Leptodactylus labyrinthicus (rã pimenta) e Lithobates catesbeianus (rã-touro) em diferentes ambientes. DOI: 10.7902/ecb.v2i1.23

&lt;span&gt;O experimento foi realizado no Ran&amp;aacute;rio Experimental da Universidade Federal de Vi&amp;ccedil;osa, no per&amp;iacute;odo de 7 a 8 de novembro de 2010, com o objetivo de avaliar o comportamento interespec&amp;iacute;fico de 2 esp&amp;eacute;cies de r&amp;atilde;,&amp;nbsp; Leptodactylus labyrinthicus (r&amp;atilde;-pimenta) e Lithobates catesbeianus (r&amp;atilde;-touro)&amp;nbsp;&amp;nbsp; em diferentes ambientes. Na baia 1 (piso de alvenaria) foram acondicionados 5 exemplares de r&amp;atilde;-pimenta e 5 de r&amp;atilde;-touro com peso m&amp;eacute;dio de 171,6 &amp;plusmn; 2g e 157,8 &amp;plusmn; 2g respectivamente. Na baia 2 (piso de terra) foram acondicionados 5 exemplares de r&amp;atilde;-pimenta e 5 de r&amp;atilde;-touro com peso m&amp;eacute;dio de 169,1&amp;plusmn; 2g&amp;nbsp; e 157,8 &amp;plusmn; 2g&amp;nbsp; respectivamente. Concluiu-se que o ritmo circadiano das esp&amp;eacute;cies envolvidas n&amp;atilde;o sofreu altera&amp;ccedil;&amp;atilde;o e que a presen&amp;ccedil;a de popula&amp;ccedil;&amp;otilde;es diferentes n&amp;atilde;o interferiu no comportamento dos animais das baias 1 e 2. Sendo assim a r&amp;atilde;-pimenta e a r&amp;atilde;-touro podem ocupar o mesmo h&amp;aacute;bitat.&lt;/span&gt;

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

Further notes on the natural history of the South American pepper frog, Leptodactylus labyrinthicus (Spix, 1824) (Anura, Leptodactylidae)

Leptodactylus labyrinthicus tadpoles reach a large size in the nest through consumption of trophic eggs. We previously suggested that the trophic eggs are laid just after amplexus has finished, but our new data do not support this hypothesis. We also present further details on the natural history of the species with regard to breeding activity, spawning site, retreats and the ability of tadpoles in preying upon fully-growth heterospecific tadpoles. We also show that the tadpoles are mainly nocturnal and take diurnal refuges. We collected the data in Brazil in three localities within the Cerrado Biome. We examined burrows used by L. labyrinthicus males, verified if females still contained mature eggs just after released from amplexus, and tested the ability of tadpoles in preying fully-growth heterospecific tadpoles. Field observations and experiments were conducted on tadpole activity time, hiding behaviour and level of susceptibility to predation by the bird leaf-scrapers in four sheltering situations. Reproduction could start before the first rains; this may be advantageous by allowing the tadpoles to exploit eggs of other frogs. We found one floating nest built in a temporary pool. The nest of the species is normally circumscribed in an excavated basin beside the water body. Adult males were found during the day with their head-out of the entrance of underwater burrows, which were perforations through dense root mats beside calling/spawning sites. Probably, these burrows in permanently water-filled soil are actively excavated by males. Females released all their eggs during the amplexus, so trophic eggs are not produced by the currently-accepted mechanism. Fully-grown heterospecific tadpoles were not preyed upon by L. labyrinthicus tadpoles, which can prey only slow-moving newly hatched ones. Field tadpoles took shelter under mud/dead leaves during daylight and became exposed on the bottom at night. Free-ranging leaf-scrapers removed dead leaves from a pool with their beaks and preyed upon tadpoles. In the experiments, the tadpoles sheltered under gravel/leaves during daylight, but they were exposed at night. Leaf-scrapers ate all exposed tadpoles, but no tadpole of the gravel/leaves trays was consumed. Hence the nocturnal habits and use of diurnal refuges may protect the tadpoles from visual predators, such as the leaf-scrapers.

Morfologia bucal interna dos girinos de Leptodactylus labyrinthicus Spix, 1824 (Amphibia: Anura: Leptodactylidae)

A microanatomia do disco oral e da cavidade bucal da larva de Leptodactylus labyrinthicus é descrita e comparada com outras espécies do gênero pertencentes ao grupo de L. pentadactylus. Trinta e cinco girinos entre os estágios 25 e 46 foram dissecados e analisados em microscopia eletrônica de varredura. A descrição da morfologia oral foi feita utilizando larvas entre os estágios 36 e 39. A morfologia oral da larva de L. labyrinthicus é similar às descritas para outros girinos com adaptação a macrofagia, com forte queratinização das coberturas das mandíbulas, redução das estruturas bucais internas e presença de uma glote descoberta, propícias às dietas carnívoras. As larvas do grupo de L. pentadactylus compartilham essa tendência à redução das estruturas de seleção e captura de partículas, apresentando redução no número de papilas da arena do assoalho e do teto bucal, redução ou ausência das papilas anteriores às bolsas bucais, apenas duas papilas pós-nasais, pequenas papilas laterais à crista mediana e zonas glandulares restritas a estreitas faixas no bordo velar. Nos girinos do gênero Leptodactylus a morfologia larvária reflete os grupos atuais propostos para o gênero.

Diversidade e distribuição espaço-temporal de anuros em região com pronunciada estação seca no sudeste do Brasil

As atividades antrópicas têm alterado profundamente os ambientes naturais e muitas vezes afetado a diversidade e distribuição dos anuros. O objetivo deste estudo foi investigar as seguintes questões: (1) qual a composição da anurofauna em uma região de pastagem com clima marcadamente sazonal no extremo noroeste paulista? (2) como adultos e girinos das espécies se distribuem temporal e espacialmente? (3) a riqueza de espécies está correlacionada com descritores da heterogeneidade dos hábitats de reprodução? Na área estudada foram registradas 20 espécies de anuros, distribuídas em 11 gêneros de quatro famílias: Leptodactylidae (9), Hylidae (8), Microhylidae (2) e Bufonidae (1). Destas, Chaunus schneideri (Werner, 1894), Physalaemus centralis Bokermann, 1962 e Physalaemus fuscomaculatus (Steindachner, 1864) foram registradas apenas por coleta de girinos, enquanto Dendropsophus minutus (Peters, 1872) e Leptodactylus labyrinthicus (Spix, 1824) ocorreram somente em corpos d'água próximos aos selecionados. As espécies registradas são conhecidas por sua ampla distribuição geográfica e por colonizarem áreas alteradas em outras localidades. Não houve correlação entre a riqueza de espécies e a complexidade estrutural dos corpos d'água. Entretanto, a maior riqueza de espécies foi registrada nos corpos d'água de longa duração. As poças temporárias de hidroperíodo instável foram colonizadas inicialmente por leptodactilídeos, enquanto que as poças permanentes ou temporárias estáveis foram colonizadas por hilídeos. A atividade de vocalização e de reprodução da maioria das espécies foi restrita ao período quente e chuvoso do ano, um padrão típico de ambientes tropicais sazonais. Cinco espécies [Chaunus schneideri, Dendropsophus nanus (Boulenger, 1889), Hypsiboas albopunctatus Spix, 1824, Leptodactylus podicipinus (Cope, 1862) e Pseudopaludicola aff. saltica (Cope, 1887)] vocalizaram durante a estação seca e chuvosa, mas apenas C. schneideri e H. albopunctatus se reproduziram durante o período seco. A fraca partilha espacial e temporal, registrada para adultos e girinos, não foi suficiente para explicar o isolamento reprodutivo entre as espécies. Outros fatores, como a partilha acústica e a segregação dos sítios de vocalização, podem ter maior importância para explicar a coexistência das espécies. A severidade climática (extensa e pronunciada estação seca, imprevisibilidade e inconstância das chuvas no início da estação chuvosa), juntamente com o elevado grau de conversão do hábitat natural em áreas de cultivo são, provavelmente, os fatores responsáveis pelo predomínio de espécies conhecidas por colonizar com sucesso áreas antrópicas em outras regiões do país.

Short-Term Movement and Retreat Sites of Leptodactylus labyrinthicus (Anura: Leptodactylidae) during the Breeding Season: A Spool-and-Line Tracking Study

Capture/recapture studies significantly increase our knowledge of the natural history of anuran amphibians. Many different methods have been employed in these studies, but a number of new techniques still require experimental validation. During two reproductive seasons in a Cerrado remnant in southeastern Brazil, we investigated the movement patterns and habitat use of the pepper frog, Leptodactylus labyrinthicus, using a spool-and-line device. This low-cost device did not appear to interfere with the activities of the frogs and allowed for constant monitoring, showing precise routes of movement and great predictability of relocations. Both sexes were active at night. During the day, males and females made use of retreat sites under vegetation or in burrows constructed by small- and medium-sized mammals. Males and females did not use standardized routes; there were no significant differences between their movements, and movements were not correlated with body size or environmental conditions. Individuals are able to move further than 100 m per day, a characteristic that may enable this species to colonize or recolonize open areas.

From Foam to Free-living: Ecology of LarvalLeptodactylus Labyrinthicus

Members of the genus Leptodactylus deposit eggs in foam nests; however, species vary in nest deposition sites, which may be aquatic or terrestrial, and the amount of time larvae remain in the nest. Life history characteristics are hypothesized to have a phylogenetic basis, but data to evaluate this idea are lacking for most species of Leptodactylus. We studied the ecology of tadpoles of Leptodactylus labyrinthicus in the Brazilian Cerrado to gain insight into the reproductive ecology of this species. Foam nests of L. labyrinthicus were deposited on land generally along the periphery of small, shallow ponds. Internal nest temperatures were lower, less variable, and less influenced by the environment than nest surface temperatures. Only a small percentage of eggs in each nest were fertilized, and tadpoles grew and developed within the foam while feeding on unfertilized eggs. Heavy rainfall washed the majority of tadpoles out of nests and into ponds. Tadpoles collected from the ponds had eggs in their guts, and feeding experiments confirmed that free-living tadpoles of L. labyrinthicus eat eggs of other frog species. The reproductive mode of L. labyrinthicus is intermediate between two previously described modes for closely related species. This mode may be adaptive in the unpredictable and seasonal environment in which this species occurs.Todos os membros do gênero Leptodactylus depositam ovos em ninhos de espuma; se os ninhos são aquáticos ou terrestres e até qual estágio as larvas desenvolvem-se neles, contudo, varia entre espécies. Acredita-se que os modos reprodutivos de anuros têm uma base filogenética, mas para a maioria das espécies não existem dados suficientes para avaliar essa hipótese. Nós estudamos a ecologia dos girinos de Leptodactylus labyrinthicus no Cerrado brasileiro com o intuito de tentar compreender melhor sua ecologia reprodutiva. Os ninhos de espuma de L. labyrinthicus foram depositados sobre o chão ao longo da periferia de poças pequenas e rasas. As temperaturas internas dos ninhos foram mais baixas e variaram menos do que as temperaturas externas. Apenas uma pequena porcentagem dos ovos em cada ninho foi fertilizada e os girinos permaneceram dentro da espuma, crescendo e desenvolvendo-se através da ingestão de ovos. Chuvas fortes dissolveram os ninhos e lavaram os girinos, carregando-os para as poças. Girinos coletados nas lagoas possuiam ovos em seus intestinos, e experimentos confirmaram que girinos de L. labyrinthicus alimentam-se dos ovos de outras espécies após abandonarem os ninhos. O modo reprodutivo de L. labyrinthicus é intermediário a dois outros modos descritos para espécies próximas. Esse modo pode ser adaptativo em ambientes sazonais e imprevisiveis onde essa espécie ocorre.

Reproductive activity and vocalizations of Leptodactylus labyrinthicus (Anura: Leptodactylidae) in southeastern Brazil

Vocalizations and reproductive activity of two Leptodactylus labyrinthicus populations were studied from Jun/2001 to Feb/2003 in the State of São Paulo, Brazil. Observations began at dusk and ended around 2300 h. Occasionally individuals were monitored throughout the night. Data on reproductive period, calling sites, adult snout-vent length (SVL), oviposition sites, and oviposition period was collected. Leptodactylus labyrinthicus had an extended breeding period associated mainly with rainfall. Males called from the edge of temporary or permanent ponds, began vocalization activity at dusk, and finished around 2300 or 2400h. During the peak of the vocalization period (Dec- Jan), calling activity could extend up to 0400 or 0500h. Three types of vocalizations associated with reproduction were recorded: advertisement call, territorial call, and courtship call. The advertisement call was the most common vocalization. Males and females showed no sexual dimorphism in SVL. However, the males of one population were significantly larger than those of the other population studied. This fact could be explained by frog-hunting in one of the areas, which could wipe out the larger males of the population. Foam nests were recorded mainly in Oct-Nov 2001/2002 in depressions at the edge of temporary ponds, always protected by vegetation. A mean of 6.5% of the eggs present in the foam were fertilized and the other 93.5% possibly are used as a food source by the tadpoles. Mean diameter of the foam nest was 25.4 cm and mean height was 11.4 cm.

Allosteric effect of protons and adenosine triphosphate on hemoglobins from aquatic amphibia

The analysis of hemoglobins from two anurans, one semi-aquatic (Leptodactylus labyrinthicus) and the other aquatic (Pipa carvalhoi), showed several components isolated by CM-cellulose chromatography. The two major components (Hb II and Hb III) ofL. labyrinthicus and the major components (Hb III and Hb IV) ofP. carvalhoi possess functional properties as follows: i.P. carvalnoi Hb III and Hb IV andL. labyrinthicus Hb II had very small Bohr effects (−0.05) and a substantial heterotropic effect with polyphosphates. ii.L. labyrinthicus Hb III produced a normal Bohr effect of −0.17, with no influence of polyphosphates.

Separation and functional characterization of Leptodactylus labyrinthicus hemoglobin components

1. 1. Some functional and structural properties of Leptodactylus labyrinthicus hemoglobin were studied. 2. 2. Three distinct components were isolated by CM-cellulose chromatography corresponding to 10, 60 and 30% of the total hemolysate. 3. 3. The whole hemolysate as well as the major component presented no Bohr effect, although the 30% component exhibited a normal alkaline Bohr effect, and all of them showed an heterotropic effect of phosphates on the oxygen affinity. 4. 4. The globins of the two major components were obtained and their two chains were identified by polyacrylamide gel disc electrophoresis.

Morphological and Histochemical Study of Mast Cells in Lizards and Frogs, with Special Reference to the So-Called "Summer Cells" or "Stilling Cells" of Amphibians

Morphological and histochemical studies were performed on mast of three species of lizards: Ameiva ameiva Linnaeus; Tupinambis teguixin Linnaeus; and Tropidurus torguatus Wied. The greatest amounts of mast were found in the tongue, stomach, small and large intestines and mesentery. Smaller amounts were observed in other organs. The cytoplasmic granules of mast of these animals were amphoteric. Besides being metachromatic, the granules were PAS positive; PAS positive granules and Alcian blue positive granules could be seen intermingled within the same cell. For a comparative study, Rana catesbiana Shaw and Leptodactylus labyrinthicus Spix were used. The amphoteric character was also observed here, where the cytoplasmic granules of mast presented acidophilia and/or basophilia. A weak positivity to PAS and a few Alcian blue positive granules could be observed. It may be assumed that the acidophilia of mast cell granules may be a normal property for lower vertebrates and the so-called summer cells or Stilling cells which have been observed in the interstitial tissue of adrenal gland of some amphibians are actually mast and not mast derivatives, as proposed by some authors.