Pachymedusa Research Articles

Recent habitat modification of a tropical dry forest hotspot drives population genetic divergence in the Mexican leaf frog: a landscape genetics approach.

Tropical dry forests (TDF) are among the ecosystems with the highest deforestation and transformation rates. Because of habitat loss and fragmentation, modified landscapes can impose resistance to the movement of individuals, with important genetic consequences. One of the most affected taxa due to habitat alteration are amphibians, which currently face extreme population declines globally. Here, we used single nucleotide polymorphisms (SNPs) to evaluate genetic diversity, genetic structure, and the effect of landscape elements on genetic connectivity of the Mexican tree frog (Agalychnis dacnicolor) in a TDF biodiversity hotspot in Mexico. We collected samples of 96 individuals from 16 sites located within fragmented areas of TDF and within continuous forest in the Chamela-Cuixmala region. Sampling sites from the fragmented forest showed slightly lower genetic diversity and effective population size compared to those in the continuous forest. We detected three admixed genetic groups, in which most of the sites within the fragmented forest were differentiated from the sites within continuous forest. Although these analyses suggest historical gene flow, we did not detect significant recent migration among the three genetic groups. While original vegetation (TDF + tropical evergreen forest), and in some areas, agriculture facilitated genetic connectivity, open-areas (grasslands + human settlements + exposed soil), and agriculture in other areas limited genetic connectivity in A. dacnicolor. This study helps to understand the factors shaping contemporary population divergence in highly modified complex landscapes, and highlights the importance to maintain connectivity in a rapidly changing ecosystem.

Peptide DMS-DA6 have immunomodulatory effects in a mouse model of actinomicetoma by N. brasiliensis

Abstract Actinomycetoma is an infectious disease recognized by the World Health Organization as a neglected tropical disease. In the Americas, the most common etiologic agent is the Gram-positive bacteria, Nocardia brasiliensis. Current treatment is prolonged, toxic and resistant strains have emerged. A promising solution is the use of host defense peptides, which are recognized for their bactericidal activity and ability to control infection by modulating the host immune system. Recently, the peptide DMS-DA6, a dermaseptin from the skin of the frog Pachymedusa dacnicolor, was reported to be bactericidal against Gram-positive bacteria. However, the function of the DMS-DA6 in host defense against infection remains unknown. In this study, we investigated the in vivo properties and effects of DMS-DA6 in a mouse model of actinomicetoma by N. brasiliensis. After peptide administration, a significant reduction in the volume of the affected limb was observed; furthermore, flow citometry showed that DMS-DA6 modified systemic leukocyte populations, such as monocytes/macrophages, neutrophils and CD8+ T lymphocytes were significantly reduced in spleen and peripheral blood. Interestingly, the combination of DMS-DA6 with the antibiotic linezolid had the opposite effect, significantly inducing CD4+ and CD8+ T lymphocyte influx into the spleen. These results suggest that the peptide DMS-DA6 is a promising alternative approach and a valuable addition to the current anti-infective strategy.

Characterization of a New Immunosuppressive and Antimicrobial Peptide, DRS-DA2, Isolated from the Mexican Frog, Pachymedusa dacnicolor.

Inflammatory and antimicrobial diseases constitute a major burden for society, and fighting them is a WHO strategic priority. Most of the treatments available to fight inflammatory diseases are anti-inflammatory drugs, such as corticosteroids or immunomodulators that lack cellular specificity and lead to numerous side effects. In addition to suppressing undesired inflammation and reducing disease progression, these drugs lessen the immune system protective functions. Furthermore, treating infectious diseases is more and more challenging due to the rise of microbial resistance to antimicrobial drugs. Thus, controlling the inflammatory process locally without compromising the ability to combat infections is an essential feature in the treatment of inflammatory diseases. We isolated three forms (DRS-DA2N, DRS-DA2NE, and DRS-DA2NEQ) of the same peptide, DRS-DA2, which belongs to the dermaseptin family, from the Mexican tree frog Pachymedusa dacnicolor. Interestingly, DRS-DA2N and DRS-DA2NEQ exhibit a dual activity by inducing the death of leukocytes as well as that of Gram-negative and Gram-positive bacteria, including multiresistant strains, without affecting other cells such as epithelial cells or erythrocytes. We showed that the death of both immune cells and bacteria is induced rapidly by DRS-DA2 and that the membrane is permeabilized, leading to the loss of membrane integrity. We also validated the capacity of DRS-DA2 to regulate the pool of inflammatory cells in vivo in a mouse model of noninfectious peritonitis. After the induction of peritonitis, a local injection of DRS-DA2N could decrease the number of inflammatory cells locally in the peritoneal cavity without inducing a systemic effect, as no changes in the number of inflammatory cells could be detected in blood or in the bone marrow. Collectively, these data suggest that this peptide could be a promising tool in the treatment of inflammatory diseases, such as inflammatory skin diseases, as it could reduce the number of inflammatory cells locally without suppressing the ability to combat infections.

Metal Bioaccumulation and Assemblage Attributes for Anurans in the Sierra de Huautla Biosphere Reserve, Mexico

Background and Research Aims: Morphological and physiological effects of anthropogenic metals have been the focus of several studies in the Sierra de Huautla Biosphere Reserve (REBIOSH, Mexico) but little is known about how they affect anurans in the area. We evaluated metal bioaccumulation in anurans from streams in the REBIOSH and examined anuran communities in five sites at different positions relative to abandoned mine tailings.Methods: Three and two sites were located upstream and downstream from mine tailings, respectively. We collected anuran community information and obtained sediment, water and liver samples for metal quantification from each site.Results: Concentrations of Cu, Pb, Zn and Mn in water, and Zn, Mn, Fe and Cr in sediment, were significantly different among sites. We found bioaccumulation of Pb, Zn and Fe in livers of Smilisca baudinii, Lithobates spectabilis, L. zweifeli and Rhinella horribilis, species with different life traits. We found similar among-site species richness and diversity. Communities were dominated by L. zweifeli, R. horribilis and S. baudinii. Dominance was highest in sites distant from mine tailings. Agalychnis dacnicolor, Tlalocohyla smithii and Hypopachus variolosus were only present in sites located closest to mine tailings.Conclusion: We did not detect an effect of metal bioacummulation on anuran communities, but our findings suggest a potential effect on certain species in the protected area. Our results form a baseline for future explorations of the impacts of metals in the region.Implications for Conservation: Toxic metal bioaccumulation has potential for reducing anuran genetic variability and altering sexual proportions and fecundity, potentially leading to anuran extirpations. We provide first evidence for metal bioaccumulation in anurans in the protected area, and one of few studies on its anuran communities. Our evidence can serve as guidance to ameliorate effects of legacy metal mining in this biodiversity hotspot.

Locomotion and Biomechanical Adaptations in Hind Limb Muscles of Three Mexican Anuran Species

Anurans are anatomically built for jumping, requiring the muscles to contract to generate sufficient mechanical force to launch their bodies. A lot of energy is needed in the hind limbs, where the majority of the force is produced, whereas the front legs and both girdles serve mainly to support the frog's weight at rest as well as to absorb the shock of landing. Different species of anurans have different locomotion mechanisms, evidenced by comparing the mechanical contractile properties of hind limb muscles. In this study, we compared two muscles in arboreal (Agalychnis dacnicolor), terrestrial (Incilius marmoreus), and aquatic (Lithobates sierramadrensis) species of Mexican anurans. We performed low frequency in situ stimulations of the muscles and found that A. dacnicolor showed higher stresses than L. sierramadrensis and I. marmoreus in the two studied muscles, the cruralis and the gastrocnemius. Regarding the cruralis, the stress generated by the aquatic and terrestrial anurans was up to 23% less than that generated by the arboreal anuran. There was also a difference of up to 30% in the ratio of performance between low frequencies and twitch in L. sierramadrensis compared to the other anurans. These differences in muscle physiology suggest adaptive differences among species, although more comprehensive studies are needed to corroborate this hypothesis.

Husbandry and captive reproduction of the giant Mexican leaf frog Agalychnis dacnicolor

The giant Mexican leaf frog, Agalychnis dacnicolor, is a large arboreal frog endemic to Mexico. This species was previously under-represented in European zoological collections and specific techniques for reproductive management under vivarium conditions little known. A group of four males and one female A. dacnicolor were maintained in captivity at Chester Zoo (Great Britain). To bring them into reproductive condition, they were subjected to three simulated environmental phases that differed in temperature, humidity and feeding regime. This proved successful so that two clutches of spawn, each containing 150-300 eggs, were deposited on leaves overhanging water. Tadpoles hatched from 4 days following oviposition with approximately 80 % success rate. They were reared at a water temperature of 27 °C to 29 °C and displayed no negative effects from living in high density. All tadpoles metamorphosed successfully and froglets with resorbing tails left water after about 32 days. Their tails were resorbed in a further 4 to 5 days at which time they began to feed; at least in the case of males, sexual maturity was reached after 10 months. This methodology will enable zoological collections in Europe to breed this species for potential conservation, research and educational purposes.

Functional connectivity of an endemic tree frog in a highly threatened tropical dry forest in Mexico

ABSTRACT The increase in anthropogenic activities that lead to fragmentation and habitat loss, could result in a reduction of connectivity among habitat patches of terrestrial species. We used ecological niche models, circuit and graph theories to evaluate functional connectivity among home-range patches and suitable habitat patches of the Mexican Leaf Frog (Agalychnis dacnicolor), in a heterogeneous landscape of tropical dry forest (TDF) in central-western Mexico. We found high connectivity among home-range patches within the Chamela-Cuixmala Biosphere Reserve (CCBR) and among those surrounding the CCBR. Similarly, suitable habitat patches along the Pacific slope (except those in the South) were well-connected. Conversely, we detected weak connectivity in the southern and eastern parts of the study area, which is in accordance with the poor habitat quality and fragmentation that characterize that zone. Suitable habitat patches with the largest areas of TDF were the most important in maintaining functional connectivity, but only one patch was within a natural protected area. Our results highlight the importance of conserving large and continuous patches of habitat in a very threatened landscape to maintain connectivity in A. dacnicolor and probably in other anurans.

Peptidoglycan potentiates the membrane disrupting effect of the carboxyamidated form of DMS-DA6, a Gram-positive selective antimicrobial peptide isolated from Pachymedusa dacnicolor skin

The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.

Distribution of corticotropin-releasing factor (CRF) receptor binding in the mouse brain using a new, high-affinity radioligand, [125 I]-PD-Sauvagine.

The corticotropin-releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G-protein coupled receptors, CRF1 and CRF2 . Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF-related peptide, termed PD-sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high-affinity ligand for CRF receptor studies. A radiolabeled analog, [125 I]-PD-sauvagine, with high signal-to-noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor-deficient mice and subtype-specific antagonists, CRF1 and CRF2 binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [125 I]-PD-sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF1 expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF2 in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD-sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.

Efecto de los estadios sucesionales del bosque tropical seco sobre el microhabitat usado por Agalychnis dacnicolor (Anura: Phyllomedusidae) y Smilisca fodiens (Anura: Hylidae)

Habitat disturbance can modify microhabitats and therefore have a significant effect on the abundance and distribution of anurans species, particularly those associated to specific microhabitats. In this study we evaluated changes in the use of microhabitat by two syntopic species of tree frogs, Agalychnis dacnicolor and Smilisca fodiens along a successional gradient of Tropical Dry Forest in the region of the Biosphere Reserve of Chamela-Cuixmala at the coast of Jalisco, Mexico. We hypothesized that because these two species are habitat generalists, microhabitat use would change in response to secondary forest succession and to the climatic season of the year. Although both species were registered in both secondary and mature forests, A. dacnicolor was associated to mature forest, whereas S. fodiens was more associated to disturbed sites. Neither species showed differences in biomass among successional stages (ES) or between climatic seasons. Unlike S. fodiens, A. dacnicolor presented larger sizes during the rainy season. Body temperature of both species varied between ES and climatic season. S. fodiens presented a more constant temperature among individuals in pastures, while A. dacnicolor in mature forests. Environmental and structural habitat variables that influenced the presence of both species in ES were substrate temperature, height of tree or shrub where the frog was perching and percentage cover of litter, grasses, shrubs, trees and bare soil. The variables that best explained biomass, snout-vent length (LHC) and body temperature of S. fodiens were cover of dry branches and substrate temperature, whereas biomass of A. dacnicolor was better explained by cover of herbs, LHC, height of the tree or shrub where the frog was perching, body temperature and the substrate temperature. Both species used differentially environmental and structural variables of microhabitats along the gradient of initial to late ES. The variables that separated the species were the height of the shelter or perch and the percentage of trees in different ES. Whereas A. dacnicolor was more associated to the lowest strata of vegetation, S. fodiens showed plasticity in microhabitat use, especially in height of shelter or perch. Our results suggested that even when both species are considered habitat generalists, in anthropic landscapes, A. dacnicolor is specialist in microhabitat use, whereas S. fodiens is a species with greater ecological plasticity in microhabitat use. These differences in microhabitat use in response to habitat disturbance may help explain the success of these species in these new landscapes, as well as the importance of secondary forests in different stages of succession to maintain adequate microhabitats for the permanence of species in anthropogenic landscapes.

Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor.

The dermaseptin antimicrobial peptide family contains members of 27–34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5′-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested.

Characterization of a Pachymedusa dacnicolor-Sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies.

The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [(125)ITyr(0)-Glu(1), Nle(17)]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor-expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.

Pachymodulin, a new functional formyl peptide receptor 2 peptidic ligand isolated from frog skin has Janus-like immunomodulatory capacities.

Recruitment of leukocytes is essential to fight infections or to heal injuries; however, excessive and/or prolonged responses favor the development of major inflammatory pathologies, such as cardiovascular or neurodegenerative diseases. Thus, it is of great interest to seek novel compounds that can regulate leukocyte recruitment depending on the degree of inflammation. We have isolated and characterized, by different chromatographic techniques, mass spectrometry, and Edman sequencing, a new hexapeptide (SSLSKL) from the Mexican frog Pachymedusa dacnicolor, which we named pachymodulin. In vitro, pachymodulin promotes the migration of leukocytes through the binding and activation of the human and mouse N-formyl peptide receptor 2 (huFPR2). In vivo, it exhibits opposite biological activities: under homeostatic conditions, pachymodulin induces the recruitment of leukocytes, whereas under inflammatory conditions, it inhibits this process. Therefore, pachymodulin represents an interesting template in the quest to design new immunomodulatory drugs in the therapy of immune-related diseases.

Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides

Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides

Bradykinin-related peptides (BRPs) from skin secretions of three genera of phyllomedusine leaf frogs and their comparative pharmacological effects on mammalian smooth muscles

Bradykinin-related peptides (BRPs) from skin secretions of three genera of phyllomedusine leaf frogs and their comparative pharmacological effects on mammalian smooth muscles

PsT-1: A new tryptophyllin peptide from the skin secretion of Waxy Monkey Leaf Frog, Phyllomedusa sauvagei

PsT-1: A new tryptophyllin peptide from the skin secretion of Waxy Monkey Leaf Frog, Phyllomedusa sauvagei

Medusins: A new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs

Medusins: A new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs

A stereochemical switch in the aDrs model system, a candidate for a functional amyloid

A stereochemical switch in the aDrs model system, a candidate for a functional amyloid

PD-sauvagine: a novel sauvagine/corticotropin releasing factor analogue from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor

Sauvagine is a potent and broad-spectrum biologically active peptide of 40 amino acid residues originally isolated from the skin of the South American frog, Phyllomedusa sauvagei. Since its discovery, no additional sauvagine structures have been reported. Following the discovery of sauvagine, peptides with similar primary structures/activities were identified in mammalian brain [corticotropin-releasing factor (CRF) and urocortin]. Here, we report the identification of a second sauvagine from the Mexican giant leaf frog, Pachymedusa dacnicolor, which displays primary structural features of both sauvagine and CRF. A cDNA encoding the peptide precursor was "shotgun" cloned from a cDNA library constructed from lyophilised skin secretion by 3'- and 5'-RACE reactions. From this, the primary structure of a 38-mer peptide was deduced and this was located in reverse phase HPLC fractions of skin secretion and both its mass and structure were confirmed by mass spectrometry. The biological activities of synthetic replicates of PD-sauvagine and sauvagine were compared using two different mammalian smooth muscle preparations and the novel peptide was found to be more potent in both. Bioinformatic analyses of PD-sauvagine revealed that it shared different regional sequence identities with both sauvagine and CRF.

Analysis of sulfated peptides from the skin secretion of the Pachymedusa dacnicolor frog using IMAC‐Ga enrichment and high‐resolution mass spectrometry

Immobilized metal ion affinity chromatography (IMAC) has been widely used for the enrichment of phosphopeptides, whereas no report exists describing the use of IMAC columns for the enrichment of sulfopeptides. In this study, we used IMAC-Ga microcolumns for the enrichment of sulfopeptides from a complex mixture of peptides, extracted from skin secretions of the Pachymedusa dacnicolor frog. The enriched fraction obtained by IMAC-Ga was analyzed by liquid chromatograpy/electrospray ionization mass spectrometry (LC/ESI-MS) in an Orbitrap XL and by matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF/TOF) in an ABI 4800 instrument. From this fraction, different sulfated and non-sulfated peptides belonging to the caerulin and bradykinin families were structurally characterized. Other interesting negatively charged groups, such as phosphate adducts of dermaseptins and pyridoxal phosphate attached to a protease inhibitor, were also characterized. Unexpectedly, some dermaseptin antimicrobial peptides were also enriched by IMAC-Ga and a Sauvatine-like peptide was also fully sequenced. Furthermore, neutral loss of sulfated peptides and their fragmentation patterns in the gas phase were also compared using collision-induced dissociation (CID) and high-energy collision dissociation (HCD). Our present study provides evidence that IMAC-Ga enrichment is a fast, useful and promising method for high-throughput analysis of sulfated-peptides, since high-resolution mass spectrometers can be used for this purpose.