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.

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.

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

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

Trophic eggs in the foam nests of Leptodactylus labyrinthicus (Anura, Leptodactylidae): An experimental approach

The South American pepper frog, Leptodactylus labyrinthicus, is a large species that lays eggs in foam nests in holes dug out of the banks of different bodies of water. Recently, it was reported that only 6-10% of eggs are fertilized in foam nests of L. labyrinthicus and the remaining unfertilized eggs are consumed by the tadpoles inside the nest. Here we tested experimentally the influence of the ingestion of trophic eggs on the survivorship and growth of L. labyrinthicus tadpoles. Tadpoles fed on trophic eggs and subsequently fed on dry fish food grew larger than those fed only on dry fish food, and this suggests that the ingestion of trophic eggs is an adaptation to improve tadpole growth. The ingestion of trophic eggs also seems to be important for the maintenance of tadpoles in environments with unpredictable rainfall, as they were able to survive for about 70 days feeding only on these trophic eggs and one tadpole managed to complete metamorphosis feeding on trophic eggs only. Details of the spawning behaviour observed in the field, occurrence of multiple mating, and predation on eggs by terrestrial invertebrates and vertebrates are also reported.

Cytogenetic and random amplified polymorphic DNA analysis of Leptodactylus species from rural and urban environments (Anura, Amphibia)

Cytogenetic and random amplified polymorphic DNA analyses carried out in the species Leptodactylus podicipinus, L. ocellatus, L. labyrinthicus, and L. fuscus from rural and urban habitats of the northwest region of São Paulo State, Brazil, showed that the karyotypes (2n = 22), constitutive heterochromatin distribution and nucleolus organizer region (NOR) location did not differ between the populations from the two environments. The in situ hybridization with an rDNA probe confirmed the location of the NORs on chromosome 8 revealing an in tandem duplication of that region in one of the chromosomes of L. fuscus. DAPI showed that part of the C-band-positive heterochromatin is rich in AT, including that in the proximity the NORs in L. podicipinus and L. ocellatus. The molecular analyses showed that the two populations (urban and rural) of L. podicipinus and L. fuscus are similar from a genetic point of view. The urban and rural populations of species L. ocellatus and L. labyrinthicus showed differences in genetic structures, probably due to urbanization which interferes with the dispersion of those frogs. The marked differences observed between the two populations of L. ocellatus can be representing the cryptic condition of the species. Unweighted pair-group method of analysis and genetic distance analysis detected the genetic proximity between L. ocellatus and L. fuscus. The results indicate that there was no reduction in the genetic diversity in the populations from the urban environment; however, the survival of these frogs would not be guaranteed in the case of an increase in human impact especially for populations of L. labyrinthicus and L. ocellatus. ©FUNPEC-RP.

Marcadores citológicos no cariótipo de espécies de Leptodactylus (Amphibia, Anura, Leptodactylidae) analisado com técnicas de citogenética clássica e molecular

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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

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

Flexible feeding kinematics of a tropical carnivorous anuran tadpole

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cytogenetic analyses of eight species in the genus Leptodactylus Fitzinger, 1843 (Amphibia, Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Chromosomal studies on five species of the genus Leptodactylus Fitzinger, 1826 (Amphibia, Anura) using differential staining

Cytogenetic studies were carried out on five species of Leptodactylus, namely L. fuscus, L, notoaktites, L. labyrinthicus, L. ocellatus, and L. podicipinus, after standard staining, Ag-NOR and C-banding as well as BrdU incorporation for three of them. The species had 2n = 22 chromosomes and two basic karyotype patterns. Chromosome 8 was a marker bearing a secondary constriction. In all species, this secondary constriction corresponded to the Ag-NOR site. The species had centromeric C-bands in all chromosomes of the complement, but some interstitial or telomeric bands seemed to differentiate some karyotypes, either at the species or the population level. In L. ocellatus, the C-banding pattern confirmed the occurrence of a heteromorphic pericentric inversion in chromosome 8 in specimens from one of the populations. The BrdU incorporation technique showed no detectable difference in the replication patterns of the major bands in the chromosomes of L. noroaktites, L. labyrinthicus, and L. ocellatus.

Study on macroscopic, microscopic and hormonal changes in gonads of the crayfish Astacus leptodactylus in Aras Dam

This study was carried out to seasonal determination of some morphological characteristics, Seasonal fecundity, Seasonal fluctuations of vertebrate-type steroids and seasonal analysis of gonadal histology in both female and male sexes of freshwater crayfish (Astacus leptodactylus Eschscholtz 1823) in the area of Aras dam Lake. Crayfish were collected respectively in June, August, November (2011) and January (2012). The average length and weight of male crayfish was higher than that of females. %GSI of females fluctuated within an extended range (between 0.6 and 13.5% from June to January). Both of synchronous and asynchronous ovaries were seen in August sampled ovaries; however asynchronous form was higher than another. The annual reproductive cycle of male A. leptodactylus was surveyed by study on the seasonal changes of the external appearance of the testes and vasa deferentia, fluctuations in the gonadosomatic index (GSI%) and the histological analysis of the male reproductive system. Based on the histological differentiation of testis, spermatogenisis devided to 5 separated stages. The findings suggested asynchronous testis in the species A.leptodactylus. The presence of primary spermatophore layer may help keeping spermatozoa alive while the secondary spermatophore layer may produces spermatophore or synthesize of acellular material which forms spermatophre. Pleopodal fecundity was 37.3%lower than ovarian fecundity observed. The significantly higher number of eggs attached to 3rd and 4th pairs of pleopods. The egg number and gonadosomatic index increased with female size while egg weight and egg diameter didn’t increase with female size. Hemolymph levels of 17β-estradiol and progesterone followed a similar fluctuation pattern with % GSI in females, while testosterone didn’t follow the mentioned pattern. The testis of November sampled crayfish presented significantly higher gonadosomatic (%GSI) index (P < 0.05).The most observed gonadosomaticindices were 13.5%(forfemales) and 1.21% (for males, in autumn. Althogh the lowest GSI was (0.50%) formales in spring and (0.26%0 for spent females in January. Testosterone which followed a similar pattern with %GSI in males increased remarkably in November. 17β-estradiol increased strictly in January. The strictly enhancement of the three estroid hormones in January in both male and female sexes could bedue totheir stimulating role in in spermatophre and egg lying in the mating season (In January). Most of the ovaries followed the asynchoronous growth pattern. Also the testes presented asynchoronous growth pattern in autumn.

Effect of diets containing various protein and energy levels on some growth parameters, survival and the production of Astacus leptodactylus in different salinities

A factorial experiment was conducted for 60 days to determine of the response of Narrow clawed crayfish Astacus leptodactylus (average weight of 17±2.3 g) to diets containing various protein and energy levels. Nine diets containing three levels of protein (30, 35 and 40 %) and three levels of energy (300,370 and 450 kcal/100g) were formulated and prepared in this trial. Each diet also was used in two levels of salinity include 0 (fresh water) and 12 ppt(Caspian sea water). So this study was conducted with 18 treatments and triplicates random group of 5 crayfish per each 110-litre tank. Weight Gain, Feed conversion ratio (FCR), Protein Efficiency Ratio (PER), Net Protein Utilization (NPU), Daily Food Consumption (DFC), Survival (SVR) and body composition of tail-muscle meat of animal were determined. Comparing the growth parameters in response to interaction between protein, energy and salinity levels demonstrated that all growth parameters have difference between them significantly (p<0.05). Comparing between survival in fresh and Caspian Sea water showed difference significantly. Compare the body composition results indicate the greatest amount of protein absorption in diet number 2(30/370) on fresh water condition. Results from this study indicate that narrow clawed crayfish can be fed a practical diet containing 30% protein and 370 Kcal/100g on non-salinity water which is the optimize CP percentage for their producer’s profits.

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.