Tandem Mass Spectrometry de novo Sequencing of the Skin Defense Peptides of the Central Slovenian Agile Frog Rana dalmatina.

Peptides released on frogs' skin in a stress situation represent their only weapon against micro-organisms and predators. Every species and even population of frog possesses its own peptidome being appropriate for their habitat. Skin peptides are considered potential pharmaceuticals, while the whole peptidome may be treated as a taxonomic characteristic of each particular population. Continuing the studies on frog peptides, here we report the peptidome composition of the Central Slovenian agile frog Rana dalmatina population. The detection and top-down de novo sequencing of the corresponding peptides was conducted exclusively by tandem mass spectrometry without using any chemical derivatization procedures. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), electron transfer dissociation (ETD) and combined MS3 method EThcD with stepwise increase of HCD energy were used for that purpose. MS/MS revealed the whole sequence of the detected peptides including differentiation between isomeric Leu/Ile, and the sequence portion hidden in the disulfide cycle. The array of the discovered peptide families (brevinins 1 and 2, melittin-related peptides (MRPs), temporins and bradykinin-related peptides (BRPs)) is quite similar to that of R. temporaria. Since the genome of this frog remains unknown, the obtained results were compared with the recently published transcriptome of R. dalmatina.

Diversity and phylogeography of Northeast Asian brown frogs allied to Rana dybowskii (Anura, Ranidae).

We investigated the species diversity and phylogeography of the Northeast Asian brown frogs allied to Rana dybowskii (the R. dybowskii species complex: R. dybowskii, R. pirica, and R. uenoi) using four mitochondrial and three nuclear loci. Phylogenetic analyses confirmed the existence of three distinct species in this complex; using extensive molecular data, we confirm the validity of Rana uenoi recognized as a distinct species, and infer R. dybowskii and R. pirica to be sister species. Also, we included populations from previously unsampled regions in Northeast China, and identified them to be R. dybowskii. While many species in Northeast Asia diverged due to Pleistocene glaciation, divergence-dating analyses inferred older, Miocene speciation in the R. dybowskii species complex. Ancestral area reconstruction identified the orogenic movement of the Changbai Mountain Range and the opening of the Sea of Japan/East Sea being major events influencing allopatric speciation.

Prevalence and intensity of Alaria alata (Goeze, 1792) in water frogs and brown frogs in natural conditions.

In the last 15 years, the mesocercariae of Alaria alata have frequently been reported in the wild boar during routine Trichinella inspections made compulsory for the trade of venison meat in Europe. If these studies have focused primarily on mesocercariae isolated from meat, few works have been done so far to understand the circulation of the parasite in natural conditions especially in the intermediate hosts. This study focuses on the second intermediate hosts of this parasite assessing the suitability of two amphibian groups-brown frogs and water frogs sensu lato-for mesocercarial infection on an area where A. alata has already been identified in water snails and wild boars. During this study, both groups showed to be suitable for mesocercarial infection, with high prevalence and parasite burdens. Prevalence was higher in the brown frog group (56.9 versus 11.54 % for water frogs) which would indicate that it is a preferential group for infection on the study area, though reasons for this remain to be investigated. No significant difference among prevalences was observed between tadpoles and frogs. This study, the first focusing on A. alata in these amphibians in Europe, provides further information on circulation of this parasite in natura.

Diverse families of antimicrobial peptides isolated from skin secretions of three species of East Asian frogs, Babina daunchina, Babina adenopleura, and Rana omeimontis (Ranidae).

Twenty-two novel cDNAs encoding 22 peptide precursors for 19 mature peptides including antimicrobial peptides (AMPs) were identified from East Asian frog species Babina daunchina, Babina adenopleura, and Rana omeimontis skin-derived cDNA libraries. Two atypical members of the brevinin-1 family AMPs, named brevinin-1AN1 (FLTGVLKLASKIPSVLCAVLKTC) and brevinin-1DN1(FLKGVINLASKIPSMLCAVLKTC), were purified from the skin secretions of B. adenopleura and B. daunchina, respectively. A member of the ranatuerin-2 family AMP named ranatuerin-2DN1 (GLFDSITQGLKDTAVKLLDKIKCKLSACPPA) was also purified from the skin secretion of B. daunchina. One AMP named japonicin-2OM1 (FIVPSIFLLKKAFCIALKKNC) was purified from the skin secretion of R. omeimontis. The antimicrobial tests showed that brevinin-1DN1, brevinin-1DN2, brevinin-1AN1, and japonicin-2OM1 possess higher antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria.

The complete mitochondrial genome of Rana coreana (Anura: Ranidae).

Rana coreana is a brown frog species native to the Korean Peninsula. We characterized the complete mitochondrial genome of the species. The mitochondrial genome sequence of R. coreana is 22,262 bp and comprises 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and two control regions (CRs). The CR duplication and gene organization were identical to those observed in Rana kunyuensis and Rana amurensis. A total of 13 protein-coding genes were used to examine the phylogenetic relationships between this species and the genus Rana. R. coreana living on the Korean Peninsula, formed a cluster with R. kunyuensis and R. amurensis, with R. coreana showing the closest phylogenetic affinity for R. kunyuensis.

The complete mitochondrial genome information of Rana uenoi (Amphibia, Anura, Ranidae) and the phylogenetic implication.

We determined the complete mitochondrial genome of Rana uenoi (Anura: Ranidae) for the first time. The whole sequences were 17,370 bp and included 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. The gene arrangement was completely identical to those observed from other Ranidae species. We used 11 protein-coding genes to examine the phylogenetic placement of this species in the genus Rana. Rana dybowskii was the closest sister species to R. uenoi. The clade of R. uenoi and R. dybowskii formed a cluster with Rana huarensis, which had a sister relationship with the group of Rana amurensis, Rana coreana, and Rana kunyuensis.

A new species of the genus Rana from Henan, central China (Anura, Ranidae).

A new species of brown frog Rana luanchuanensis Zhao & Yuan, sp. n. is described from Luanchuan County, western Henan, central China. The mitochondrial genealogy suggests that the new species is the sister taxon to the clade including R. amurensis and R. coreana, and is separated by uncorrected pairwise distances more than 12.5%. Morphologically, this new species differs from its congeners by a suite of characters. Analyses of partial sequences of cytochrome oxidase subunit I (COI) resolve the new species as a single matriline.

Radiocesium accumulation in the anuran frog, Rana tagoi tagoi, in forest ecosystems after the Fukushima Nuclear Power Plant accident.

Amphibians are key components in forest food webs. When examining radioactive contamination in anurans, it is important to understand how radiocesium transfer occurs from lower to higher trophic levels in forest ecosystems. We investigated the activity concentration of radiocesium ((134)Cs and (137)Cs) in Tago's brown frog (Rana tagoi tagoi) captured on the forest floor approximately 2.5 years after the Fukushima Nuclear Power Plant (FNPP) accident. We collected 66 R. tagoi tagoi at different distances from the FNPP. Radiocesium accumulation showed positive correlations with the air radiation dose rate and litter contamination but not with distance from the FNPP. Whole-body radioactivity showed no correlation with body mass or length. Our results suggest that differences in the available food items result in large variability in individual contamination. Contamination level monitoring in terrestrial and aquatic amphibian is necessary for clarifying the processes and mechanisms of radiocesium transfer through forest food webs.