Eight new microsatellite loci for the critically endangered fire‐bellied toad Bombina bombina and their cross‐species applicability among anurans

We describe eight new microsatellite loci for the critically endangered fire‐bellied toad, Bombina bombina. Seven of them are polymorphic with two to seven alleles per locus, an expected heterozygosity between 0.41 and 0.8, and an observed heterozygosity between 0.27 and 0.7. The yield of new loci was relatively low, presumably due to mildly repetitive sequence motifs in microsatellite flanking regions. As typical for anurans, cross‐species amplification was limited (here, to congeners Bombina orientalis and Bombina variegata). Combining these new loci with those already available provides a reasonable number of loci for population studies and pedigree analysis in Bombina.     

Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog,Rana catesbeiana

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine-and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.     

NPY- and CGRP-like immunoreactive nerve fibers in the testis and mesorchium of the toad (Bufo arenarum)

The presence and distribution of peptidergic nerve fibers were studied in the testis and mesorchium of the toad by means of immunohistochemistry. Cryostat sections of the testis and whole-mount preparations of mesorchia were immunostained with antisera to calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY). After leaving the mesorchium CGRP-immunoreactive (IR) fibers were seen predominantly running in between the seminiferous tubules. In addition, a small population of CGRP-IR nerve fibers formed thin plexuses around blood vessels. Conversely, NPY-like immunoreactivity predominated in nerve fibers that formed dense plexuses around vessels both in the mesorchium and testis. Additionally, some single NPY-IR nerve fibers could be seen in both structures studied. The functional significance of these peptidergic systems in the testis of the toad remains to be analyzed.     

Evolutionary history of Scinax treefrogs on land‐bridge islands in south‐eastern Brazil

Aim We investigated how Pleistocene refugia and recent (c. 12,000 years ago) sea level incursions shaped genetic differentiation in mainland and island populations of the Scinax perpusillus treefrog group. Location Brazilian Atlantic Forest, São Paulo state, south‐eastern Brazil. Methods Using mitochondrial and microsatellite loci, we examined population structure and genetic diversity in three species from the S. perpusillus group, sampled from three land‐bridge islands and five mainland populations, in order to understand the roles of Pleistocene forest fragmentation and sea level incursions on genetic differentiation. We calculated metrics of relatedness and genetic diversity to assess whether island populations exhibit signatures of genetic drift and isolation. Two of the three island populations in this study have previously been described as new species based on a combination of distinct morphological and behavioural characters, thus we used the molecular datasets to determine whether phenotypic change is consistent with genetic differentiation. Results Our analyses recovered three distinct lineages or demes composed of northern mainland São Paulo populations, southern mainland São Paulo populations, and one divergent island population. The two remaining island populations clustered with samples from adjacent mainland populations. Estimates of allelic richness were significantly lower, and estimates of relatedness were significantly higher, in island populations relative to their mainland counterparts. Main conclusions Fine‐scale genetic structure across mainland populations indicates the possible existence of local refugia within São Paulo state, underscoring the small geographic scale at which populations diverge in this species‐rich region of the Atlantic Coastal Forest. Variation in genetic signatures across the three islands indicates that the populations experienced different demographic processes after marine incursions fragmented the distribution of the S. perpusillus group. Genetic signatures of inbreeding and drift in some island populations indicate that small population sizes, coupled with strong ecological selection, may be important evolutionary forces driving speciation on land‐bridge islands.     

Cryopreservation and hormonal induction of spermic urine in a novel species: The smooth-sided toad (Rhaebo guttatus)

Global amphibian declines have fueled an increased interest in amphibian assisted reproductive technologies. Within the genus Rhaebo, half of the species are experiencing decreasing population trends; however, insufficient information is available on many of these species’ reproductive biology. Using the smooth-sided toad, Rhaebo guttatus, we present effective methods for collecting and cryopreserving an example of Rhaebo sperm. Specifically, our findings show that administering 10 IU/g body weight of hCG (human chorionic gonadotropin) yields the most motile and concentrated sperm and that cryopreserving spermic urine in a solution of 5% DMFA (N,N-Dimethylformamide) and 10% trehalose returns sperm with a 33 ± 3% average post-thaw motility. These findings may represent an important step forward in developing techniques that can be safely applied to other, more vulnerable species within the Rhaebo genus.     

Somatostatin-immunoreactive cells in the gastrointestinal tract of the frog Rana esculenta

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.Fellow of the Alexander von Humboldt Foundation, Bonn, Germany     

Distribution of gonadotropin-releasing hormone-like peptides in the brain during development of juvenile male Rana esculenta

Summary The distribution and density of cell bodies and fibers immunoreactive to GnRH-like peptides were investigated in the brain of male juvenile frogs (Rana esculenta) during postmetamorphic development. An immunohistochemical technique was used, involving antisera raised against 4 variants of GnRH: mammalian GnRH, chicken GnRH-I, chicken GnRH-II and salmon GnRH. A comparison of the immunohistochemical distribution at 8 different developmental stages shows that the maximum density of immunoreactive-GnRH elements, and the full distributional complexity of this system, is attained at the completion of spermatogenesis. Immunoreactive-GnRH cell bodies first appear in the anterior preoptic area during the metamorphic climax, and then appear sequentially in the medial septal area, tegmentum and, lastly, in the retrochiasmatic area and olfactory bulb when immunoreactive-fibers also reach the cerebellum. The GnRH system reacts positively to antisera for all 4 GnRH variants, but immunoreactivity for chicken GnRH-I is the weakest.