Phyllomedusa Rohdei Research Articles

Molecular cytogenetics and phylogenetic analysis of Brazilian leaf frog species of the genera Phyllomedusa and Phasmahyla (Hylidae: Phyllomedusinae)

In this study we describe the karyotypes and molecular phylogenetic relationships of the leaf frogs Phyllomedusa bahiana Lutz, 1925, Phyllomedusa burmeisteri Boulenger, 1882, Phyllomedusa nordestina Caramaschi, 2006, Phyllomedusa rohdei Mertens, 1926, Phyllomedusa hypochondrialis (Daudin, 1800), and Phasmahyla spectabilis Cruz, Feio and Nascimento, 2008. We analyzed the karyotypes using C-banding, fluorochrome staining, and fluorescence in situ hybridization of telomeric probe, and inferred phylogeny using nuclear tyrosinase and mitochondrial 16S rDNA sequences. Heterochromatin distribution in P. nordestina diverged from the other species, and P. bahiana and P. rohdei showed evident interstitial telomere sequences. Molecular analyzes confirmed the current taxonomic classification, grouping mitochondrial DNA sequences of each species, and usually without haplotype sharing in the nuclear gene. We also extended the distribution of P. burmeisteri to northern Bahia state and restricted P. bahiana to southern Bahia state, with a discrete extension of the range of P. bahiana to the north and northeast of Minas Gerais state. The tyrosinase haplotype network showed a haplotype shared between P. bahiana and P. burmeisteri from Porto Seguro, Bahia. Based on this, we suggest a new distribution in the overlapping area between these two species, which according to our data ranges between Porto Seguro and Camacan, in Bahia state.

Molecular cytogenetics of nucleolar organizer regions in Phyllomedusa and Phasmahyla species (Hylidae, Phyllomedusinae): a cytotaxonomic contribution

Chromosome numbers, morphology, and nucleolus organizer region (NOR) locations are useful cytological characters for taxonomic and evolutionary studies. In this study, we provide the first cytogenetic analysis of Phyllomedusa bahiana and Phasmahyla spectabilis, and report new cytogenetic data on variation in NOR numbers and positions in Phyllomedusa rohdei and Phyllomedusa nordestina using conventional staining, AgNOR-banding, and 45S rDNA fluorescence in situ hybridization. All 4 species showed 2n = 26 chromosomes. P. spectabilis and P. bahiana had only 1 pair of NOR-carrying chromosomes. P. bahiana showed an NOR length polymorphism, and a rare homomorphic self-compatibility for both NOR lengths in anurans. Variation in the number of NOR-bearing chromosomes was found between the sampled populations of P. nordestina, ranging from 3 to 4. This study also clarified previous conflicting results concerning the occurrence of inter- and intra-population NOR variation in P. rohdei. The variation, confirmed by 45S rDNA fluorescence in situ hybridization analysis, was congruent with results obtained from AgNOR-banding in all species.

PTM-driven differential peptide display: Survey of peptides containing inter/intra-molecular disulfide bridges in frog venoms

Amphibian defensive skin secretions are complex species-specific mixtures of biologically active molecules, including many uncharacterized peptides. Many of these peptides are post-translationally modified and amongst the modifications discovered so far on amphibian defense peptides, disulfide bonds are quite frequently encountered. The presence of this PTM often complicates the MS-based sequencing. Here we demonstrate a method to target peptides containing inter/intra-molecular S-S bonds applying a PTM-driven differential display. Upon reduction of the disulfide bond both molecular mass and retention time of a peptide are altered. Assembling the LC-MS data by plotting the m/z data against retention time generates a peptide display and overlaying peptide displays of untreated and DTT-reduced material yields a differential display. From such an overlay, peptides originally carrying a disulfide bond are recognized due to the shift in both retention time and m/z values, whereas non cystine containing peptides remain unaltered in the differential display. The success of this approach is demonstrated by the visualization of the cystines-containing peptides in the skin secretion of Odorrana schmackeri, Phyllomedusa burmeisteri, Phyllomedusa rohdei, Kassina senegalensis, and Bombina variegata. The venoms from these different species yield complicated differential displays, showing interesting peptides, allowing one to target them for more detailed structural characterization.

Isolation, structure determination and synthesis of a novel tryptophan‐containing heptapeptide (basic tryptophyllin) from the skin of Phyllomedusa rohdei

A novel tryptophyllin has been isolated from the methanol extracts of the skin of the South American frog Phyllomedusa rohdei. Its primary structure, as determined by chemical/enzymatic methods and by FAB mass spectrometry, is the following: Lys‐Pro‐Hyp‐Ser‐Trp‐Ile‐Pro‐NH2. This basic tryptophyllin has been synthesized in solid phase, in order to allow its complete pharmacological characterization.

Synthesis of TPH‐13, a new tridecapeptide from Phyllomedusa rohdei

The synthesis of TPH‐13 (Glp‐Glu‐Lys‐Pro‐Tyr‐Trp‐Pro‐Pro‐Pro‐Ile‐Tyr‐Pro‐Met‐OH), a tridecapeptide isolated from the skin of the South American frog Phyllomedusa rohdei, is described and alternative approaches are discussed.

Primary structure determination of a tryptophan‐containing tridecapeptide from Phyllomedusa rohdei

A new tryptophan‐containing peptide has been identified in the methanol extracts of the skin of Phyllomedusa rohdei. The primary structure proposed is the following: <Glu‐Glu‐Lys‐Pro‐Tyr‐Trp‐Pro‐Pro‐Pro‐Ile‐Tyr‐Pro‐Met‐OH.