The cardiorespiratory system of miniature frogs.

Anurans of the genus Brachycephalus are among the smallest vertebrates in the world, due to an extreme process of miniaturization. As an example of this process, Brachycephalus species show loss of fingers, loss of the eardrum and middle ear, bone fusions, and the presence of paravertebral plates and parotic plaque. However, no studies addressing the consequences of miniaturization on internal organs, such as the lungs and heart, are currently available. Thus, this study aimed to investigate if overall small body size has affected the cardiorespiratory system. We investigated, via dissections, individuals of four Brachycephaloidea species: Brachycephalus rotenbergae, B. pitanga, Eleutherodactylus johnstonei, and Ischnocnema parva. We observed that B. rotenbergae and B. pitanga present a reduction of the atrial septum and absence of the carotid body. On the other hand, despite being a member of the sister genus to Brachycephalus (both genera belong to the Brachycephalidae), individuals of Ischnocnema present a heart with a complete septum and carotid body; this is also observed in E. johnstonei (Eleutherodactylidae). We observed that B. rotenbergae and B. pitanga have thin skin with a one to two cell thick germ layer, and their lungs likely exhibit lower blood supply when compared to individuals of the E. johnstonei and I. parva species. Based on the observed structures, we suggest that in species of Brachycephalus, respiration is performed mainly through the skin, and their lungs may have a reduced respiratory function.

Synchrotron X-ray biosample imaging: opportunities and challenges.

Synchrotron radiation phase-contrast microtomography is sensitive to low attenuating tissues, giving an alternative visualisation of the sample and being useful for investigating microstructure inside biological specimens without staining them with a contrast medium. The phase-contrast technique has been widely used in the scientific community, as it is a technique associated with radiography and microscopy and able to enhance contrast in soft tissues, specifically at the edges, showing details that could not be seen by the absorption technique. This work aims to show the ability of synchrotron-based phase-contrast microtomography for the visualisation of soft tissues and hard internal structures of millimetre-sized biological organisms. Case studies of the anatomy of Rhodnius prolixus head and Thoropa miliaris tadpole are presented to illustrate the imaging technique.

The dispersal between Amazonia and Atlantic Forest during the Early Neogene revealed by the biogeography of the treefrog tribe Sphaenorhynchini (Anura, Hylidae).

The Amazonia and the Atlantic Forest, separated by the diagonal of open formations, are two ecoregions that comprise the most diverse tropical forests in the world. The Sphaenorhynchini tribe is among the few tribes of anurans that occur in both rainforests, and their historical biogeographic have never been proposed. In this study, we infer a dated phylogeny for the species of the Sphaenorhynchini and we reconstructed the biogeographic history describing the diversification chronology, and possible patterns of dispersion and vicariance, providing information about how orogeny, forest dynamics and allopatric speciation affected their evolution in South America. We provided a dated phylogeny and biogeography study for the Sphaenorhynchini tribe using mitochondrial and nuclear genes. We analyzed 41 samples to estimate the ancestral areas using biogeographical analysis based on the estimated divergence times and the current geographical ranges of the species of Sphaenorhynchini. We recovered three characteristic clades that we recognize as groups of species (S. lacteus, S. planicola, and S. platycephalus groups), with S. carneus and G. pauloalvini being the sister taxa of all other species from the tribe. We found that the diversification of the tribe lineages coincided with the main climatic and geological factors that shaped the Neotropical landscape during the Cenozoic. The most recent common ancestor of the Sphaenorhynchini species emerged in the North of the Atlantic Forest and migrated to the Amazonia in different dispersion events that occurred during the connections between these ecoregions. This is the first large-scale study to include an almost complete calibrated phylogeny of Sphaenorhynchini, presenting important information about the evolution and diversification of the tribe. Overall, we suggest that biogeographic historical of Sphaenorhynchini have resulted from a combination of repeated range expansion and contraction cycles concurrent with climate fluctuations and dispersal events between the Atlantic Forest and Amazonia.

Performance evaluation of segmentation methods for assessing the lens of the frog Thoropa miliaris from synchrotron-based phase-contrast micro-CT images.

PURPOSE: In the context of synchrotron microtomography using propagation-based phase-contrast imaging (XSPCT), we evaluated the performance of semiautomatic and automatic image segmentation of soft biological structures by means of Dice Similarity Coefficient (DSC) and volume quantification. METHODS: We took advantage of the phase-contrast effects of XSPCT to provide enhanced object boundaries and improved visualization of the lenses of the frog Thoropa miliaris. Then, we applied semiautomatic segmentation methods 1 and 2 (Interpolation and Watershed, respectively) and method 3, an automatic segmentation algorithm using the U-Net architecture, to the reconstructed images. DSC and volume quantification of the lenses were used to quantify the performance of image segmentation methods. RESULTS: Comparing the lenses segmented by the three methods, the most pronounced difference in volume quantification was between methods 1 and 3: a reduction of 4.24%. Method 1, 2 and 3 obtained the global average DSC of 97.02%, 95.41% and 89.29%, respectively. Although it obtained the lowest DSC, method 3 performed the segmentation in a matter of seconds, while the semiautomatic methods had the average time to segment the lenses around 1 h and 30 min. CONCLUSIONS: Our results suggest that the performance of U-Net was impaired due to the irregularities of the ROI edges mainly in its lower and upper regions, but it still showed high accuracy (DSC = 89.29%) with significantly reduced segmentation time compared to the semiautomatic methods. Besides, with the present work we have established a baseline for future assessments of Deep Neural Networks applied to XSPCT volumes.

The First Brazilian Thesis of Evolution: Haeckel's Recapitulation Theory and Its Relations with the Idea of Progress.

The aim of this work is to present the thesis "On the Ontogenetic Evolution of the Human Embryo in its Relations with Phylogenesis," by Affonso Regulo de Oliveira Fausto (1866-1930), published in Brazil in 1890. To our knowledge, it was one of the first Brazilian academic works focused specifically on evolution. It was also the first doctoral thesis that addressed the topic of recapitulation in order to analyze what was then called the progressive evolution of the human species in tandem with the embryological development of the individuals that would constitute the Brazilian "type." In the present work, we analyze the author's thesis in relation to its sources, concepts, as well as the country's political context at the time of its publication. Fausto's text, in which he explicitly recognized the influence of Ernst Haeckel's (1834-1919) recapitulation theory, represents a window to understand better a concept of nation based on science and on the idea of inexorable progress that was accepted in Brazil at the end of the nineteenth century.

Risk factors associated with Leishmania exposure among dogs in a rural area of Ilha Solteira, SP, Brazil.

INTRODUCTION: We sought to determine risk factors (RFs) associated with the presence of antibodies against Leishmania in dogs from a rural area of Ilha Solteira, SP, Brazil. METHODS: Serum samples were collected from 250 dogs and tested using indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody tests (IFATs). Data concerning dogs, their environment, and their owners' knowledge of leishmaniasis were collected using a questionnaire. To determine RFs for contact with the parasite, univariate statistical analysis based on chi-squared and Fisher's exact tests, followed by logistic regression, was used. RESULTS: It was found that 79/250 (31.6%) of the dogs were positive by IFAT, and 72/250 (28.8%) by ELISA. A total of 82/250 dogs (32.8%) were positive in at least one test. The RFs associated with occurrences of Leishmania exposure were large body size (OR = 2.25; 95% CI = 1.26-4.04; p = 0.003), presence of chickens (OR = 1.94; 95% CI = 1.05-3.65; p = 0.023), and lack of knowledge about Leishmania among dog owners (OR = 1.74; 95% CI = 0.96-3.21; p = 0.049). After multivariate analysis, the RFs for occurrence of Leishmania exposure in dogs that remained significantly associated were the dog's size (large dogs) (OR = 1.2; 95% CI = 1.06-1.35; p = 0.003) and presence of chickens on the properties (small farms) (OR = 1.15; 95% CI = 1.02-1.30; p = 0.023). CONCLUSIONS: These results may be useful for improving preventive practices to reduce the incidence of Leishmania exposure among dogs in rural areas.

Risk factors associated with Leishmania exposure among dogs in a rural area of Ilha Solteira, SP, Brazil

Abstract INTRODUCTION: We sought to determine risk factors (RFs) associated with the presence of antibodies against Leishmania in dogs from a rural area of Ilha Solteira, SP, Brazil. METHODS: Serum samples were collected from 250 dogs and tested using indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody tests (IFATs). Data concerning dogs, their environment, and their owners' knowledge of leishmaniasis were collected using a questionnaire. To determine RFs for contact with the parasite, univariate statistical analysis based on chi-squared and Fisher's exact tests, followed by logistic regression, was used. RESULTS: It was found that 79/250 (31.6%) of the dogs were positive by IFAT, and 72/250 (28.8%) by ELISA. A total of 82/250 dogs (32.8%) were positive in at least one test. The RFs associated with occurrences of Leishmania exposure were large body size (OR = 2.25; 95% CI = 1.26-4.04; p = 0.003), presence of chickens (OR = 1.94; 95% CI = 1.05-3.65; p = 0.023), and lack of knowledge about Leishmania among dog owners (OR = 1.74; 95% CI = 0.96-3.21; p = 0.049). After multivariate analysis, the RFs for occurrence of Leishmania exposure in dogs that remained significantly associated were the dog's size (large dogs) (OR = 1.2; 95% CI = 1.06-1.35; p = 0.003) and presence of chickens on the properties (small farms) (OR = 1.15; 95% CI = 1.02-1.30; p = 0.023). CONCLUSIONS: These results may be useful for improving preventive practices to reduce the incidence of Leishmania exposure among dogs in rural areas.

A new species of spotted leaf frog, genus Phasmahyla (Amphibia, Phyllomedusidae) from Southeast Brazil.

Based on concordant differences in male advertisement call, tadpole morphology, and absence of haplotype sharing in the barcoding 16S mitochondrial DNA, we describe here a new species of spotted leaf frog of the genus Phasmahyla from Atlantic Forest, State of Rio de Janeiro, Southeast Brazil. The new species is most similar to P. cochranae (type locality) and P. spectabilis (type locality). It differs from these species by the size of the calcar, moderate-sized body (snout-vent length 30.4-34.4 mm in adult eight males), and in the advertisement call. The tadpoles of Phasmahyla lisbella sp. nov. differ from P. exilis, P. spectabilis, P. timbo, P. guttata and P. jandaia because they do not have row of teeth in the anterior part; differ from P. cruzi by the shape of the anterior end of the oral disc. Through genetic data (phylogenetic distance and haplotype genealogy) we diagnosed the new species where the genetic divergences among its congeners is about 3-6% in a fragment of the 16S rRNA gene, which is above the threshold typically characterizing distinct species of anurans. However, the new species can be distinguished from other congeneric species based on an integrative approach (molecular, bioacoustics, larval, and adult morphology).

Frog size on continental islands of the coast of Rio de Janeiro and the generality of the Island Rule.

Island Rule postulated that individuals on islands tend to dwarfism when individuals from mainland populations are large and to gigantism when mainland populations present small individuals. There has been much discussion about this rule, but only few studies were carried out aiming to reveal this pattern for anurans. Our study focused on measuring the size of individuals on islands and to find a possible pattern of size modification for insular anurans. Individuals were collected on continental islands, measured and compared to mainland populations. We selected four species with different natural history aspects during these analyses. Island parameters were compared to size of individuals in order to find an explanation to size modification. Three of the four species presented size shifting on islands. Ololygon trapicheiroi and Adenomera marmorata showed dwarfism, Boana albomarginata showed gigantism and in Thoropa miliaris there was no evident size modification. Allometric analysis also revealed differential modification, which might be a result of different selective pressures on islands in respect of mainland populations. Regression model explained most of the size modification in B. albomarginata, but not for the other species. Our results indicate that previous assumptions, usually proposed for mammals from older islands, do not fit to the anurans studied here. We support the assumption that size modification on islands are population-specific. Hence, in B. albomarginata some factor associated to competition, living area and isolation time might likely be responsible for gigantism on islands.

On the type series of Scinax perpusillus (Lutz & Lutz, 1939) (Anura: Hylidae).

The literature regarding the type series of Scinax perpusillus (Lutz & Lutz, 1939) presents two distinct numbers for the holotype. In 1973, thirty-four years after the description, Bertha Lutz associated numbers to the type series. This numbering has been followed by subsequent reviewers of Scinax ever since, with exception of Peixoto (1987) who assigned another number to the holotype. Our reevaluation of the literature and examination of the type series allowed us to access that, in both cases, the numbers were incorrectly assigned to the specimens and determine the proper museum numbers for this series.

On the diagnosis and conservation of the poorly known bromeligenous Scinax arduous Peixoto, 2002 (Amphibia; Anura; Hylidae).

Since Scinax arduous description, many other populations belonging to the Scinax perpusillus group have been recorded for the States of Espírito Santo and Minas Gerais, Southeastern Brazil. Both in collections and publications most of these new specimens are identified as S. arduous, Scinax cf. arduous, Scinax cf. perpusillus, Scinax gr. perpusillus, S. perpusillus and S. v-signatus. Such state of affairs may be due to the lack of information on the original description of S. arduous. Only two individuals [the holotype (female) and the paratype (male)] were used in the original description and diagnosis, therefore, information on variation and distribution were not available. Furthermore, in S. arduous description, the section on coloration in life was based only on two juveniles raised in laboratory. Herein we redefine Scinax arduous based on 44 males and 17 females from the type locality, the Municipality of Santa Teresa, in the State of Espírito Santo. In addition, we provide information on its conservation status, distribution, natural history, vocalization, tadpoles, and detailed photographs of both adults and larvae.

The Gondwana Breakup and the History of the Atlantic and Indian Oceans Unveils Two New Clades for Early Neobatrachian Diversification.

The largest anuran diversity belongs to the Neobatrachia, which harbor more than five thousand extant species. Here, we propose a new hypothesis for the historical aspects of the neobatrachian evolution with a formal biogeographical analysis. We selected 12 genes for 144 neobatrachian genera and four archaeobatrachian outgroups and performed a phylogenetic analysis using a maximum likelihood algorithm with the rapid bootstrap test. We also estimated divergence times for major lineages using a relaxed uncorrelated clock method. According to our time scale, the diversification of crown Neobatrachia began around the end of the Early Cretaceous. Our phylogenetic tree suggests that the first split of Neobatrachia is related to the geological events in the Atlantic and Indian Oceans. Hence, we propose names for these clades that indicate this connection, i.e., Atlanticanura and Indianura. The Atlanticanura is composed of three major neobatrachian lineages: Heleophrynidae, Australobatrachia and Nobleobatrachia. On the other hand, the Indianura consists of two major lineages: Sooglossoidea and Ranoides. The biogeographical analysis indicates that many neobatrachian splits occurred as a result of geological events such as the separation between South America and Africa, between India and the Seychelles, and between Australia and South America.

Patterns of parasitism by Amblyomma rotundatum (Ixodidae) on Rhinella schneideri (Bufonidae) in islands of São Francisco River, Minas Gerais, Brazil / Parasitismo por Amblyomma rotundatum (Ixodidae) em Rhinella schneideri (Bufonidae) em duas ilhas do Rio São Francisco, próximas à represa de Três Marias, Minas Gerais

We sampled ticks from specimens of the rococo toad Rhinella schneideriby flannel dragging on two Islands located in the São Francisco River near the Três Marias hydroelectric dam, southeastern Brazil. A total of 120 toads was examined, of which 63 (52.5%) were parasitized only by Amblyomma rotundatumtotaling 96 larvae, 163 nymphs and 134 females. The burden of parasitism ranged from one to 43 ticks, with a mean intensity of infestation of 6.2±5.5 ticks per host. The tick A. rotundatumexhibited highly aggregated distribution. Peak abundance of larvae and nymphs occurred in the dry season (May to September), whereas peak abundance of females occurred in the wet season (October to April). We collected most ticks near the head and hind limbs of R. schneideri. The finding of two engorged A. rotundatumnymphs in the same resting places of two toads and the absence of this species in the dragged areas suggest a nidicolous behavior at the studied site.

Sapos da espécie Rhinella schneideri foram capturados e examinados para coleta das fases parasitárias, assim como arrasto de flanela para coleta das fases de vida livres de carrapatos em duas ilhas localizadas no rio São Francisco , próximas à represa Três Marias, região sudeste do Brasil. No total, 120 indivíduos foram examinados, dos quais 63 (52,5%) estavam parasitados por Amblyomma rotundatum totalizando 96 larvas, 163 ninfas e 134 fêmeas. A abundância do parasitismo variou de 1 a 43 carrapatos, com uma intensidade média de infestação de 6,2±5,5 carrapatos/hospedeiro. A infestação por A. rotundatumapresentou uma distribuição altamente agregada. O pico de abundância de larvas e ninfas ocorreu na estação seca (maio a setembro), enquanto o pico de abundância de fêmeas ocorreu na estação chuvosa (outubro a abril). A maioria dos carrapatos foi coletada na região da cabeça e membros posteriores. A presença de duas ninfas ingurgitadas de A. rotundatum nos mesmos lugares de descanso de dois sapos e a ausência desta espécie na coleta por arrasto de flanela sugere um comportamento nidicola no local estudado.

Occurrence of Lutzomyia longipalpis (Phlebotominae) and canine visceral leishmaniasis in a rural area of Ilha Solteira, SP, Brazil.

This study aimed to investigate the occurrence of Lutzomyia longipalpis and also the canine visceral leishmaniasis (CVL) in a rural area of Ilha Solteira, state of São Paulo. Blood samples were collected from 32 dogs from different rural properties (small farms) and were analyzed by ELISA and the indirect immunofluorescence antibody test (IFAT) in order to diagnose CVL. From these serological tests, 31.25% of the dogs were positive for CVL and these were distributed in 66.7% (8/12) of the rural properties, which were positive for L. longipalpis. CDC (Center for Disease Control and Prevention) light traps were installed in 12 properties (one per property) and insects were caught on three consecutive days per month for one year. L. longipalpis was present on 100% of the rural properties visited, at least once during the twelve-month interval, totaling 64 males and 25 females. The insects were more numerous after the peak of the rain, but the association between prevalence of peridomestic vectors and the climatic data (precipitation, relative air humidity and temperature) and the occurrences of CVL among dogs on each rural property were not statistical significant (p <0.05). However, the occurrence of CVL cases in dogs and the presence of L. longipalpis indicate that more attention is necessairy for the control of this disease in the rural area studied.

Occurrence of Lutzomyia longipalpis (Phlebotominae) and canine visceral leishmaniasis in a rural area of Ilha Solteira, SP, Brazil / Ocorrência de Lutzomyia longipalpis (Phlebotominae) e leishmaniose visceral canina em uma área rural de Ilha Solteira, SP, Brasil

This study aimed to investigate the occurrence of Lutzomyia longipalpis and also the canine visceral leishmaniasis (CVL) in a rural area of Ilha Solteira, state of São Paulo. Blood samples were collected from 32 dogs from different rural properties (small farms) and were analyzed by ELISA and the indirect immunofluorescence antibody test (IFAT) in order to diagnose CVL. From these serological tests, 31.25% of the dogs were positive for CVL and these were distributed in 66.7% (8/12) of the rural properties, which were positive for L. longipalpis. CDC (Center for Disease Control and Prevention) light traps were installed in 12 properties (one per property) and insects were caught on three consecutive days per month for one year. L. longipalpis was present on 100% of the rural properties visited, at least once during the twelve-month interval, totaling 64 males and 25 females. The insects were more numerous after the peak of the rain, but the association between prevalence of peridomestic vectors and the climatic data (precipitation, relative air humidity and temperature) and the occurrences of CVL among dogs on each rural property were not statistical significant (p <0.05). However, the occurrence of CVL cases in dogs and the presence of L. longipalpis indicate that more attention is necessairy for the control of this disease in the rural area studied.

O objetivo desse trabalho foi o estudo da prevalência de Lutzomyia longipalpis e da leishmaniose visceral canina (LVC) em uma área rural do município de Ilha Solteira do estado de São Paulo. Amostras de sangue foram coletadas de 32 cães provenientes de pequenas propriedades rurais e analisadas por meio dos métodos sorológicos ELISA (imunoensaio enzimático indireto) e RIFI (reação de imunofluorescência indireta) para o diagnóstico da LVC. Pelos exames sorológicos, dos 32 cães avaliados, 31,25% foram diagnosticados positivos para LVC, os quais estavam diostribuídos em 66,67% (8/12) das propriedades positivas para Lutzomyia longipalpis. Armadilhas luminosas do tipo CDC (Center for Disease Control and Prevention) foram instaladas em 12 propriedades, sendo uma por propriedade, e as coletas dos insetos foram realizadas três dias consecutivos a cada mês, durante um ano. O inseto L. longipalpis foi encontrado em 100% das propriedades visitadas, pelo menos uma vez no ano, totalizando 65 machos e 25 fêmeas. A maior quantidade de insetos foi observada principalmente após a ocorrência dos maiores picos de precipitação pluvial, mas a associação entre a prevalência dos vetores peridomiciliares e os dados climáticos (precipitação, umidade relativa do ar e temperatura) assim como a ocorrência da CVL em cães em cada propriedade não foi estatisticamente significante (p<0.05). No entanto, alerta-se que pela presença dos casos de LVC nos cães amostrados e também de L. longipalpis, maior atenção deve ser dada durante as investigações epidemiológicas para o controle dessa doença nessa área rural estudada.

Predictive-like distribution mapping using Google Earth: reassessment of the distribution of the bromeligenous frog, Scinax v-signatus (Anura: Hylidae).

The hylid frog Scinax perpusillus species group comprises 13 species that share, in addition to a few morphological features, reproduction that occurs exclusively associated with bromeliads. Among the species in the group, Scinax v-signatus (Lutz, 1968) is one of the few with a relatively large geographic distribution, occurring in association with bromeliads growing on granitic outcrops above 800 m along the Serra dos Órgãos (a local designation of Serra do Mar) in the Atlantic forest, State of Rio de Janeiro. Here we demonstrate that previous assessment of the distribution of this species was overestimated, and reevaluate the available data on its occurrence. The distributional data analyzed was based on three levels of evidence. First, we assessed the distribution of the bromeliad, Alcantarea imperialis (Carrière) Harms, which is used by S. v-signatus at the type locality. We plotted potential occurrence data for this plant using Google Earth (GE) by visually inspecting GE images in search of indications of granitic outcrops where groups and large individual bromeliads could be identified. Second, we plotted the distribution of these plants and that of the frog based on locality data taken from the literature and voucher specimens in natural history collections and checked for congruence between these sets of data. Third, as a second test of accuracy of this methodology we visited four new localities indicated by the bromeliad-occurrence GE prediction map and searched for the occurrence of both the frog and the bromeliad. This simple process has proven efficient and accurate in finding new collecting sites and determining the distribution of the two involved taxa. We discuss this and other possibilities of using Google Earth as a tool for mapping and discovering the distribution of organisms and habitats. Furthermore, this study has shed light on a more accurate and realistic estimate of the distribution of Scinax v-signatus with implications for the assessment of its conservation status.

Life cycle and behavior of Amblyomma rotundatum (Acari: Ixodidae) under laboratory conditions and remarks on parasitism of toads in Brazil.

The life cycle and behavior of Amblyomma rotundatum were evaluated under laboratory conditions. The experiment started with four engorged females collected from toads (Rhinella schneideri) naturally infested at the Pirapitinga Ecological Station in the state of Minas Gerais, Brazil. Developmental periods of free-living stages were assessed in an incubator at 27 ± 1 °C, >80 % RH and darkness. The complete life cycle, including pre-attachment periods for each parasitic stage, ranged from 126 to 228 days. The pre-attachment, feeding and molting periods increased as the life cycle progressed from larva to adult female. Oviposition lasted about 20 days, with the peak occurring on days 4 and 5. Longevity of nymphs and adult females was quite similar (approximately 250 and 240 days, respectively) and slightly longer than that of larvae. Lesions caused by tick feeding are discussed and a list of known hosts, including new host records for A. rotundatum, is offered.

Leptodactylus ocellatus (Amphibia): mechanism of defense in the skin and molecular phylogenetic relationships.

Amphibian antimicrobial peptides have been known for many decades and several of them have already been isolated. However, the number of species investigated is still small. Herein, we report on the skin secretions of Leptodactylus ocellatus, which were extracted by mild electrical stimulation and its semi-preparative reverse-phase chromatography was resolved in more than 30 fractions. Among these fractions, two novel antimicrobial peptides were isolated and their amino acid sequences determined by de novo sequencing. The ocellatins-5 and -6 (21 and 22 amino acid residues, respectively) are amidated at the C-terminus. Ocellatins inhibited the growth of reference strains of both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) with minimal inhibition concentration values in the range of 32-128 microg/mL. The amino acid sequence of the peptides shows structural similarity with members of the antimicrobial peptides found in the skin secretion of other leptodactylid frogs. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different species.