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.

Pronephros and mesonephros characterization during the embryonic development of the giant South American river turtle, Podocnemis expansa (Podocnemididae: Testudines).

This study aimed to describe pronephros and mesonephros morphology during the embryonic development of Podocnemis expansa. Eggs were collected on an artificial beach at Balbina, Amazonas, Brazil, during the entire incubation period (mean of 59 days). The kidney-gonad complex was processed using light microscopy and the mesonephros using transmission electron microscopy. The pronephros was present for the first time on stage 4, composed of external glomeruli devoid of a capsule, protruding into the coelomic cavity, and internally composed of a capillary network. The pronephros degenerated after development stage 15. The first sign of the appearance of the mesonephros occurred around stage 8, indicated by the early formation of renal corpuscles. The mesonephros comprised an renal corpuscles, neck segment, proximal tubule, intermediate segment, distal tubule, collector tubule, and collector duct. Ultrastructural analysis of the mesonephros brush border was done in the proximal tubule, and the presence of cells with structural characters indicative of secretory activity was detected in the juxtatubular region. Renal corpuscles and proximal tubules were the main components that underwent morphological alterations during mesonephros degeneration. The pronephros is a transient kidney, and the mesonephros became the functional embryonic kidney in P. expansa. Mesonephros degeneration occurs in the cranial-caudal direction, and histologically, the degeneration is identified by changes in the morphology of the renal corpuscle and proximal tubule. However, the mesonephros is still present after hatching.

The peptide secreted at the water to land transition in a model amphibian has antioxidant effects.

In addition to the morphophysiological changes experienced by amphibians during metamorphosis, they must also deal with a different set of environmental constraints when they shift from the water to the land. We found that Pithecopus azureus secretes a single peptide ([M + H]+ = 658.38 Da) at the developmental stage that precedes the onset of terrestrial behaviour. De novo peptide and cDNA sequencing revealed that the peptide, named PaT-2, is expressed in tandem and is a member of the tryptophyllins family. In silico studies allowed us to identify the position of reactive sites and infer possible antioxidant mechanisms of the compounds. Cell-based assays confirmed the predicted antioxidant activity in mammalian microglia and neuroblast cells. The potential neuroprotective effect of PaT-2 was further corroborated in FRET-based live cell imaging assays, where the peptide prevented lipopolysaccharide-induced ROS production and glutamate release in human microglia. In summary, PaT-2 is the first peptide expressed during the ontogeny of P. azureus, right before the metamorphosing froglet leaves the aquatic environment to occupy terrestrial habitats. The antioxidant activity of PaT-2, predicted by in silico analyses and confirmed by cell-based assays, might be relevant for the protection of the skin of P. azureus adults against increased O2 levels and UV exposure on land compared with aquatic environments.

A Staging Table of Post-Ovipositional Development for the South American Collared Lizard Tropidurus torquatus (Squamata: Tropiduridae).

The mouse, chicken, African clawed frog, and zebrafish are considered ¨model organisms¨ due to their extensive embryological and genetic characterization. However, they are far from representative of known diversity, impairing phylogenetic analyses of developmental patterns. Since squamates have historically received limited attention in developmental studies, we here describe the developmental sequence for Tropidurus torquatus, and provide the first post-ovipositional developmental series for the lizard family Tropiduridae. Fifteen developmental stages are described based on morphological traits such as the eye and accessory visual structures, pharyngeal arches, fusion of facial primordia, limb development, pigmentation, and scales. Organogenesis is already in progression at oviposition (Stage 28), with embryos continuing to develop at the incubation temperature of 30°C ± 1°C, and hatching after 75 ± 5 days, at Stage 42. Comparisons with other lizards show a conserved embryonic sequence, however developmental timing differences were found in features such as the pharyngeal arches, endolymphatic sacs, pigmentation and scales. The development of the phallic and cranial lip of the cloaca anlages are compared with that of other lizards. The order of T. torquatus fore- and hindlimb formation differs from that most commonly observed in lizards. The abundance and close association of this species with urban environments, as well as the ease of capturing and managing females, makes T. torquatus an attractive source of developmental data for future experimental and ontogenetic studies. Anat Rec, 300:277-290, 2017. © 2016 Wiley Periodicals, Inc.

Osteologia de Melanosuchus niger (Crocodylia: Alligatoridae) e a evidência evolutiva / Osteology of Melanosuchus niger (Crocodylia: Alligatoridae) and the evolutionary evidence

O objetivo foi realizar a descrição anatômica do esqueleto de Melanosuchus niger, com o intuito de contribuir com informações evolutivas sobre a espécie. Utilizaram-se três espécimes adultos de M. niger, com comprimento médio de 2,40m, provenientes da coleção biológica do Lapas-UFU. Na cintura peitoral, a escápula é maior do que o coracóide. Já nos elementos da cintura pelvina, o púbis não participa da formação do acetábulo, o contato com o ilío, ocorre por ligamentos, e sua articulação com o ísquio, permite movimentos dorso-ventrais. Nos membros torácicos, o úmero figura como elemento do estilopódio, a ulna e rádio como elementos do zeugopódio. No carpo há o ulnar do carpo, fusão do radial+intermédio, fusão dos distais do carpo 3+4+5 e o pisiforme; possui cinco metacarpos, numerados lateromedialmente e a fórmula falângica 2:3:4:3:2. Nos membros pelvinos, o estilopódio é formado pelo fêmur e o zeugopódio pela tíbia e fíbula. No tarso há a fusão do intermédio+central, fibular do tarso, distal do tarso 3, distal do tarso 4; possui quatro metatarsos longos I, II, III e IV, sendo os metatarsos II e III maiores que os demais. O metatarso V é um osso bastante reduzido e o pé possui a fórmula falângica 2:3:4:4. No crânio, a abertura nasal é única, o palatino, vômer, pterigóide, pré-maxila e maxila formam a estrutura óssea do palato secundário; o osso parietal é o único elemento no teto craniano. No esqueleto pós- axial em pares de costelas distintas que se articulam com as vértebras cervicais, dorsais, lombares, sacrais e caudais. A gastrália é formada por sete fileiras de ossos finos localizados entre o púbis e a região caudal do esterno.(AU)

The objective was an anatomical description of the skeleton of Melanosuchus niger, in order to contribute with evolutionary information about the species. Three adult specimens of M. niger with an average length of 2.40m were used, originating from the biologic collection of Lapas-UFU. In the forelimb, the scapula is bigger than the coracoid. Regarding the hindlimbs, the pubic does not participate in the formation of the acetabulum; the contact with the ilium is made by ligaments, and its articulation with the rump allows dorsal-ventral movements. Regarding the forelimbs, the humerus is a stylopodium element, and the ulna and radius a zeugopodium element. The carpus exibits the ulnar-radial+intermedium fusion, fusion of the distal carpals 3+4+5, and the pisiform. It has five metacarpals, numbered lateromedially as metacarpal 1, 2, 3, 4, and 5. The phalangeal formula is 2:3:4:3:2. Regarding the pelvic limbs, the stylopodium is formed by the femur, and the zeugopodium by tibia and fibula. In tarsus has four bones: fusion of the intermedium+centrale, fibulare, distal tarsal 3, and distal tarsal 4. It has four long metatarsals I, II, III and IV, with metatarsal II and III being relatively longer than the others. Metatarsal V is a very small bone; the feet have the phalangeal formula 2:3:4:4. At the skull, the nasal opening is only the palatine bones, vomer, pterygoid, premaxilla and maxilla forming the bone structure of the secondary palate; the parietal bone is the only element on the cranial roof. In the pos- axial skeleton ist distinct pairs of ribs which articulate with the cervical, dorsal, lumbar, sacral and caudal vertebrae. The gastralia consists of seven rows of fine bone located between the pubic bone and caudal part of the sternum.(AU)

Ontogeny of the Appendicular Skeleton in Melanosuchus niger (Crocodylia: Alligatoridae).

The objective of the present study was to analyze chondrogenesis and the ossification pattern of the limbs of Melanosuchus niger in order to contribute with possible discussions on homology and the fusion pattern of autopodial elements and phylogeny. In the Reserva Extrativista do Lago Cuniã, Rondônia, Brazil, six nests were marked and two eggs removed from each nest at 24-hour intervals until hatching. Embryos were cleared using KOH; bone tissue was stained with alizarin red S and cartilage with Alcian blue. Routine staining with HE was also performed. In the pectoral girdle, the scapula showed ossification centers before the coracoid process. In the pelvic girdle, the ilium and the ischium were condensed as a single cartilage, although ossification took place through two separate centers, forming distinct elements in the adult. The pubis developed from an independent cartilaginous center with free end, which reflects its function in breathing. In the initial stages, the stylopodium and the zeugopodium developed from the condensation of a Y-shaped cartilage in the limbs, and differentiation of the primary axis and digital arch were observed. The greatest changes were observed in the mesopodia. In their evolution, Crocodylia underwent a vast reduction in the number of autopodial elements as a consequence of fusions and ossification of some elements. This study shows that the chondrogenesis and ossification sequences are dissociated. Moreover, the differences between M. niger and other species show clear variation in the patterns for these events in Alligatoridae.

Osteologia do neurocrânio de Iguana iguana iguana (Squamata: Iguanidae) / Neurocranium osteology of Iguana iguana iguana (Squamata: Iguanidae)

O crânio representa o segmento com conspícuas adaptações que, nos lagartos, podem ser conservativas ou impulsionadas por pressões seletivas. Objetivando subsidiar o conhecimento morfológico dos répteis, fornecemos uma descrição detalhada dos ossos que formam o neurocrânio de Iguana iguana iguana com base na análise de três esqueletos secos de espécimes adultos. O crânio da referia espécie possui características basais entre os lagartos sem o fechamento das aberturas cranianas e formato geral triangular. As estruturas ósseas que formam a base craniana apresentam muitas fusões, principalmente no assoalho. Na face caudal o exoccipital e o opistótico estão fundidos e formam o otoccipital, que contribui para a formação dos terços laterais do côndilo occipital. A parte central do côndilo é formada pelo supraoccipital. Fusões e estruturas esqueléticas presentes em Iguana são similares aos demais lagartos. Não foram descritas autapomorfias no neurocrânio para esta espécie.(AU)

Skull represents the segment with conspicuous adaptations that, in lizards, may be conservative or promoted by selective pressures. The aim of assisting the morphological knowledge of reptiles, we provide a detailed description of the neurocranium of Iguana iguana iguana based on analysis of three dried adult skeletons. The skull of this species has basal characteristics in lizards without closure of cranial openings and general triangular shape. Bony structures that form the caudal base have many fusions, especially on the floor. In the caudal face the exoccipital and the opisthotic are fused and form the otooccipital, which contributes to the formation of the lateral part of the condyle. The central part is formed by the condyle supraocciopital. Fusions and skeletal structures in Iguana are similar to other lizards. There are no autopomorphies in the neurocranium for this species.(AU)

Hylin a1, the first cytolytic peptide isolated from the arboreal South American frog Hypsiboas albopunctatus ("spotted treefrog").

RP-HPLC fractionation of the electrically stimulated skin secretion of the arboreal South American frog Hypsiboas albopunctatus ("spotted treefrog") led to the isolation of a cytolytic C-terminally amidated peptide. This novel peptide, named hylin a1 (Hy-a1), consists of 18 amino acid residues (IFGAILPLALGALKNLIK-NH(2)). The alpha-helical structure of the synthetic hylin a1 peptide was confirmed by CD spectroscopy in the presence of 60% (v/v) TFE. The synthetic peptide displayed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa and also against fungi (Candida albicans, C. krusei, C. parapsilosis and Cryptococcus neoformans). Hylin a1 was also able to disrupt human erytrocytes (HC(50)=18 microM). Similarity analysis using PSI-BLAST revealed 50-44% of identity to maximins Hv, H16, H15 and H10 from Bombina maxima and also to hylins b1 and b2 (Hy-b1 and Hy-b2) from Hypsiboas lundii (synonym: Hyla biobeba).

Uso de animais em pesquisa: o fim justifica os meios / Using animals in research: the ends justify the means

A utilização de animais em pesquisa é um procedimento antigo, sendo de extrema importância no desenvolvimento de novos fármacos e medicamentos. Os princípios éticos que norteiam essa prática estão em constante mudança. Atualmente a utilização de animais no ensino e/ou pesquisa está regulamentada pela Lei n.º 11.794/2008. O objetivo deste artigo é apresentar uma breve revisão da atual legislação e da importância que assumem os comitês de ética no uso animal (CEUA). Com a vigência da nova lei, a implementação dos CEUAs passou a ter maior respaldo para a análise dos projetos de aulas práticas e de pesquisas. O Ministério da Ciência e Tecnologia passará também a credenciar as instituições que pretendem criar e alojar esses animais, aumentando o controle sobre os cuidados dispensados a eles. A partir desse novo referencial, a utilização de animais terá maior controle e racionalidade com garantia do seu bem-estar.

The medical testing in animals is an old procedure and it is a very importance practice related to new drugs development. The ethical principles used as a reference to the practice are changing continuously. The animals used during the classes or researches are regulated by the Law n.º 11.794/2008. The objective of this article is to present a brief review of the legislation and the importance of the ethical committees of research with animals (CEUAs). The implementation of news laws provide to CEUAs greater support to perform the ethical review of the research projects and the practical classes. The Brazilian Ministry of Science and Technology is initiating the accreditation of the institutions which wants to raise laboratory animals, increasing control over the care provided to them. These measures will provide rational care for the experiments including animals as subjects.

Purification, characterization and homology analysis of ocellatin 4, a cytolytic peptide from the skin secretion of the frog Leptodactylus ocellatus.

Neobatrachia is the amphibian suborder with the largest number of species and a most important source of bioactive peptides from frog skin secretions. However, 90% of the studies on this subject have been focused on the frog families Hylidae and Ranidae, while very little is known about peptides of other families, like Leptodactylidae. Our work reports for the first time the isolation and characterization of ocellatin 4 (GLLDFVTGVGKDIFAQLIKQI-NH(2)), a cytolytic peptide from the skin secretion of the South American frog Leptodactylus ocellatus. While most cytolytic amphibian skin peptides are selective for microorganisms and harmless for mammalian cells, the HC(50) of ocellatin 4 against human erythrocytes is 14.3muM. The interaction between ocellatin 4 and zwitterionic phospholipids in mammalian plasma membranes may be favored by its neutral charge at pH 7.0. Ocellatin 4 also shows some antibacterial activity (MICs(E. coli)(and)(S. aureus)=64muM) and its sequence shares similarities with the only six leptodactylid peptides previously known and with four peptides from Australian hylid frogs of the genus Litoria.

Hylins: bombinins H structurally related peptides from the skin secretion of the Brazilian tree-frog Hyla biobeba.

Two hemolytic peptides were isolated from the skin secretion of the Brazilian Hylidae frog Hyla biobeba, using reversed-phase chromatographic procedures. Hylins b1 and b2 exhibit short linear polypeptide chains, a large number of hydrophobic residues, amidated C termini and hemolytic properties. These two novel peptides are the first examples of bombinins H-like peptides isolated from anurans species not related to Bombina species.

Further report of the occurrence of tetrodotoxin and new analogues in the Anuran family Brachycephalidae.

Tetrodotoxin (TTX) is one of the most potent toxin already isolated, which occurs in a wide range of marine as well as terrestrial animals such as in newts and anurans. In this work, the occurrence of TTX and analogues was examined in three brachycephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix using LC-FLD and LC-MS/MS. In toxicity assay (intra-peritonial injection in mice) B. nodoterga extracts were non-toxic, while B. pernix extract exhibit the highest toxicity among the studied species. Skin showed the highest toxic, followed by the liver. Retention time data in the LC-FLD system indicated the presence of TTX, 4-epiTTX, 4,9-anhydroTTX and TDA, SIM data confirmed the presence of these compounds and revealed other analogs such as 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX, 6-epiTTX. Two new components were also identified by mass spectrometry (348 and 330Da). These unknown compounds have daughter ions similar to TTX, suggesting new putative TTX analogues.

Main alkaloids from the Brazilian dendrobatidae frog Epipedobates flavopictus: pumiliotoxin 251D, histrionicotoxin and decahydroquinolines.

Epipedobates flavopictus, Dendrobatidae, is a small aposematic frog found in Brazilian Cerrado bioma. In the present work, we isolated and characterized chemically the most abundant alkaloids present in the cutaneous extract of E. flavopictus. The specimens were collected in Pirenópolis (Goiás, Brazil), their skins were removed and extracted with methanol, and submitted to purification by HPLC and identification by gas chromatography and mass spectrometry. Pumiliotoxin 251D, histrionicotoxin 285Da and two decahydroquinolines, 219A and 243A, were identified. The pumiliotoxin 251D was tested on isolated frog sciatic nerve and on isolated guinea pig ileum muscle. The pumiliotoxin 251D slightly reduced the action potentials amplitude of frog sciatic nerve. The crude skin extract of E. flavopictus and the pumiliotoxin 251D produced rhythmic contractions and increased the muscular tension on isolated guinea pig ileum.

Ocellatins: new antimicrobial peptides from the skin secretion of the South American frog Leptodactylus ocellatus (Anura: Leptodactylidae).

The emergence, in recent years, of microbial resistance to commonly used antibiotics has aroused a search for new naturally occurring bactericidal and fungicidal agents that may have clinical utility. In the present study, three new antimicrobial peptides were purified from the electrical-stimulated skin secretion of the South American frog Leptodactylus ocellatus by reversed-phase chromatographic procedures. Ocellatin 1 (1GVVDILKGAGKDLLAHLVGKISEKV25-CONH2), ocellatin 2 (1GVLDIFKDAAKQILAHAAEKQI25-CONH2) and ocellatin 3 (1GVLDILKNAAKNILAHAAEQI21-CONH2) are structurally related peptides. These peptides present hemolytic activity against human erythrocytes and are also active against Escherichia coli. Ocellatins exhibit significant sequence similarity to other amphibian antimicrobial peptides, mainly to brevinin 2ED from Rana esculenta.

The occurrence of 11-oxotetrodotoxin, a rare tetrodotoxin analogue, in the brachycephalidae frog Brachycephalus ephippium.

11-oxoTTX is an analogue 4-5 times more toxic than TTX itself, been rare even in marine animals. Two ions at m/z 320 and 336 corresponding to TTX and 11-oxoTTX (M+H(+)), respectively, were detected in the Brachycephalidae frog Brachycephalus ephippium extracts. The fragment ion pattern of 11-oxoTTX is similar to that TTX, although its possible to verify some specific fragments.

Antimicrobial peptides from anurans skin secretions.

This article is an overview of antimicrobial peptides found in anurans skin secretions. These molecules constitute an initial barrier against microbial infections because of their activity against a large array of microorganisms. These peptides hold remarkable pharmaceutical and technological interest since they selectively kill microorganisms and are unlikely to induce resistance in pathogens. Also, outstanding synergism occurs when these peptides are combined with classic antibiotics and other antimicrobial peptides.

Purification and primary structure determination of Tf4, the first bioactive peptide isolated from the venom of the Brazilian scorpion Tityus fasciolatus.

In the present study Tityus fasciolatus crude venom toxicity was evaluated and we also report the purification and characterization of a 6.6 kDa neurotoxin isolated from T. fasciolatus venom. This new toxin, named Tf4, has a molecular mass of 6614Da and its primary structure is homologous to TbIT-I from T. bahiensis and TsTX-VI and TsNTxP from T. serrulatus. Tf4 delays frog sodium channel inactivation reversibly, but it is non-toxic to mammals or crustaceans. An attempt to identify the residues responsible for the partial loss of toxicity in Tf4 was carried out based on homology modeling and sequence comparison.

Composition of indolealkylamines of Bufo rubescens cutaneous secretions compared to six other Brazilian bufonids with phylogenetic implications.

The composition of indolealkylamines of Bufo rubescens cutaneous secretions was compared to those from six other Brazilian bufonids. Skin, parotoid and tibial gland secretions were obtained for analysis by thin-layer chromatography. A triple-quadrupole mass spectrometer was used to confirm the indolealkylamines standards (serotonin, 5-HT; bufotenin, BTN; dehydrobufotenin, DHB and bufotenidin, BTD). We observed clear variation in the composition of indolealkylamines of the cutaneous secretions studied and also between those found in the skin and parotoid gland secretions of the same species. We discuss the utility of indolealkylamines to the phylogeny of this group of toads.

Occurrence of tetrodotoxin and its analogues in the Brazilian frog Brachycephalus ephippium (Anura: Brachycephalidae).

Brachycephalus ephippium is a diurnal frog, that shows aposematic colouration and inhabits Atlantic forest leaf litter in south-eastern Brazil. The presence of tetrodotoxin (TTX) in the skin, liver and ovaries of B. ephippium was demonstrated. The skin (260 M.U./g) exhibited the highest toxicity followed by liver (177 M.U./g). TTX and its analogues, tetrodonic acid, 4-epitetrodotoxin and 4,9 anhydrotetrodotoxin were isolated and identified by HPLC followed by fluorimetric analysis. TTX and 11-nortetrodotoxin-6(S)-ol had their presence confirmed by mass spectrometry (MALDI-TOF). The results confirm Brachycephalidae as a fourth family of anurans containing TTX.