Novel Kazal-type Protease inhibitors from the skin secretion of the Splendid leaf frog, Cruziohyla calcarifer

Peptidase inhibitors have an important role controlling a variety of biological processes. Here, we employed a peptidomic approach including molecular cloning, tandem mass spectrometry and enzymatic assays to reveal 7 Kazal-type proteinase inhibitors (CCKPs) (18 variants) in the skin secretion of the unexplored frog, Cruziohyla calcarifer. All 18 proteins shared the Kazal pattern C-X(7)-C-X(6,7)-C-X(6,7)-Y-X(3)-C-X(2)-C-X(15-21)-C and 3 disulphide bridges. Based on structural comparative analysis, we deemed trypsin and chymotrypsin inhibitory activity in CCKP-1, 4 and CCKP 2, 5, 7, respectively. These peptidase inhibitors presumably play a role to control the balance between other functional peptides produced in the amphibian skin secretions

Bats, Trypanosomes, and Triatomines in Ecuador: New Insights into the Diversity, Transmission, and Origins of Trypanosoma cruzi and Chagas Disease

The generalist parasite Trypanosoma cruzi has two phylogenetic lineages associated almost exclusively with bats—Trypanosoma cruzi Tcbat and the subspecies T. c. marinkellei. We present new information on the genetic variation, geographic distribution, host associations, and potential vectors of these lineages. We conducted field surveys of bats and triatomines in southern Ecuador, a country endemic for Chagas disease, and screened for trypanosomes by microscopy and PCR. We identified parasites at species and genotype levels through phylogenetic approaches based on 18S ribosomal RNA (18S rRNA) and cytochrome b (cytb) genes and conducted a comparison of nucleotide diversity of the cytb gene. We document for the first time T. cruzi Tcbat and T. c. marinkellei in Ecuador, expanding their distribution in South America to the western side of the Andes. In addition, we found the triatomines Cavernicola pilosa and Triatoma dispar sharing shelters with bats. The comparisons of nucleotide diversity revealed a higher diversity for T. c. marinkellei than any of the T. c. cruzi genotypes associated with Chagas disease. Findings from this study increased both the number of host species and known geographical ranges of both parasites and suggest potential vectors for these two trypanosomes associated with bats in rural areas of southern Ecuador. The higher nucleotide diversity of T. c. marinkellei supports a long evolutionary relationship between T. cruzi and bats, implying that bats are the original hosts of this important parasite

Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador

Abstract Some South American poison frogs (Dendrobatidae) are chemically defended and use bright aposematic colors to warn potential predators of their unpalatability. Aposematic signals are often frequency‐dependent where individuals deviating from a local model are at a higher risk of predation. However, extreme diversity in the aposematic signal has been documented in poison frogs, especially in Oophaga. Here, we explore the phylogeographic pattern among color‐divergent populations of the Little Devil poison frog Oophaga sylvatica by analyzing population structure and genetic differentiation to evaluate which processes could account for color diversity within and among populations. With a combination of PCR amplicons (three mitochondrial and three nuclear markers) and genome‐wide markers from a double‐digested RAD (ddRAD) approach, we characterized the phylogenetic and genetic structure of 199 individuals from 13 populations (12 monomorphic and 1 polymorphic) across the O. sylvatica distribution. Individuals segregated into two main lineages by their northern or southern latitudinal distribution. A high level of genetic and phenotypic polymorphism within the northern lineage suggests ongoing gene flow. In contrast, low levels of genetic differentiation were detected among the southern lineage populations and support recent range expansions from populations in the northern lineage. We propose that a combination of climatic gradients and structured landscapes might be promoting gene flow and phylogenetic diversification. Alternatively, we cannot rule out that the observed phenotypic and genomic variations are the result of genetic drift on near or neutral alleles in a small number of genes

Frog Skin LCMS

Little Devil poison frog (Oophaga sylvatica) skin alkaloids were analyzed by liquid chromatography/mass spectrometry. Ants and mites collected from the stomachs of these same frogs were also analyzed (see other data files)

Oophaga sylvatica transcriptome

Transcriptome from Oophaga sylvatica. Sequenced from skin, liver, and intestines from a single individual. Full description of sample processing and sequencing can be found in associated manuscript

Data from: Molecular physiology of chemical defenses in a poison frog

Poison frogs sequester small molecule lipophilic alkaloids from their diet of leaf litter arthropods for use as chemical defenses against predation. Although the dietary acquisition of chemical defenses in poison frogs is well-documented, the physiological mechanisms of alkaloid sequestration has not been investigated. Here, we used RNA sequencing and proteomics to determine how alkaloids impact mRNA or protein abundance in the Little Devil Frog (Oophaga sylvatica) and compared wild caught chemically defended frogs to laboratory frogs raised on an alkaloid-free diet. To understand how poison frogs move alkaloids from their diet to their skin granular glands, we focused on measuring gene expression in the intestines, skin, and liver. Across these tissues, we found many differentially expressed transcripts involved in small molecule transport and metabolism, as well as sodium channels and other ion pumps. We then used proteomic approaches to quantify plasma proteins, where we found several protein abundance differences between wild and laboratory frogs, including the amphibian neurotoxin binding protein saxiphilin. Finally, because many blood proteins are synthesized in the liver, we used thermal proteome profiling as an untargeted screen for soluble proteins that bind the alkaloid decahydroquinoline. Using this approach, we identified several candidate proteins that interact with this alkaloid, including saxiphilin. These transcript and protein abundance patterns suggest the presence of alkaloids influences frog physiology and that small molecule transport proteins may be involved in toxin bioaccumulation in dendrobatid poison frogs

Oophaga sylvatica Thermal Proteome Profiling

This assay was performed to identify proteins that bind to alkaloids. The assay relies on thermodynamic principles of protein-ligand binding, where a protein that has bound its small molecule target has increased thermal stability, a shift detectable by tandem mass spectrometry. A single laboratory reared frog was used for this analysis

Osylvatica reference proteome

Oophaga sylvatica proteome generated from mRNA-based protein reference database for peptide matching, using the PHROG tool (Proteomic Reference with Heterogeneous RNA Omitting the Genome)

Oophaga sylvatica Plasma Proteomics (part 2 of 3)

Tandem Mass Tag (TMT; Thermo Scientific, Waltham, MA, USA) labeling was performed according to manufacturer's instructions. TMT is a chemical label that allows the quantification of proteins from pooled samples by adding slight variations to the molecular mass of proteins. Full description available in the associated manuscript

Oophaga sylvatica Plasma Proteomics (part 3 of 3)

Tandem Mass Tag (TMT; Thermo Scientific, Waltham, MA, USA) labeling was performed according to manufacturer's instructions. TMT is a chemical label that allows the quantification of proteins from pooled samples by adding slight variations to the molecular mass of proteins. Full description available in the associated manuscript