Localization of hematopoietic cells in the bullfrog (Lithobates catesbeianus).

Amphibians represent the first phylogenetic group to possess hematopoietic bone marrow. However, adult amphibian hematopoiesis has only been described in a few species and with conflicting data. Bone marrow, kidney, spleen, liver, gut, stomach, lung, tegument, and heart were therefore collected from adult Lithobates catesbeianus and investigated by light microscopy and immunohistochemical methods under confocal laser microscopy. Our study demonstrated active hematopoiesis in the bone marrow of vertebrae, femur, and fingers and in the kidney, but no hematopoietic activity inside other organs including the spleen and liver. Blood cells were identified as a heterogeneous cell population constituted by heterophils, basophils, eosinophils, monocytes, erythrocytic cells, lymphocytes, and their precursors. Cellular islets of the thrombocytic lineage occurred near sinusoids of the bone marrow. Antibodies against CD34, CD117, stem cell antigen, erythropoietin receptor, and the receptor for granulocyte colony-stimulating factor identified some cell populations, and some circulating immature cells were seen in the bloodstream. Thus, on the basis of these phylogenetic features, we propose that L. catesbeianus can be used as an important model for hematopoietic studies, since this anuran exhibits hematopoiesis characteristics both of lower vertebrates (renal hematopoiesis) and of higher vertebrates (bone marrow hematopoiesis).

Propagule pressure and hunting pressure jointly determine genetic evolution in insular populations of a global frog invader.

Islands are often considered to be more susceptible to biological invasions and to suffer greater impacts from invaders than mainland areas, and this difference is generally attributed to differences in species introductions, ecological factors or human activities between islands and mainland areas. Genetic variation, as a good estimate of evolutionary potential, can influence the invasion process and impacts of alien species. However, few studies have compared the genetic diversity of alien species between islands and a corresponding mainland. Here, we examined the genetic variation and differentiation in feral populations (30 sampled individuals/population) of a globally invasive species (the American bullfrog, Lithobates catesbeianus) that was extensively farmed on 14 islands in the Zhoushan Archipelago of China and in three nearby regions on the mainland. We quantified the relative importance of propagule pressure and hunting pressures on the genetic variation of bullfrog populations and found that insular populations have greater genetic variation than their mainland counterparts. Although genetic differentiation between the populations was observed, no evidence of recent bottlenecks or population expansion in any of the tested population was found. Our results suggest that the propagule pressures of bullfrogs escaping from farms, multiple releases and hunting pressure influence the genetic variation among bullfrog populations. These results might have important implications for understanding the establishment and evolution of alien species on islands and for the management of invasive species.

Development and characterization of 27 new microsatellite markers for the Indian bullfrog Hoplobatrachus tigerinus and its congeneric species.

The Indian bullfrog Hoplobatrachus tigerinus and its four congeneric species are common frog species and distributed throughout South Asia. Due to recent human activity, they are facing a changing environment and reduction in natural population size. For effective conservation and molecular ecological studies, we therefore isolated and characterized microsatellite loci for these frogs. We obtained genomic data using an Ion Torrent PGM sequencer and designed 54 primer sets for candidate loci. By screening for polymorphic loci in individuals of H. tigerinus and its congeneric species, we isolated 27 loci as highly polymorphic microsatellite loci. Eight of these loci were commonly applicable for all species except H. chinensis. Within two populations of H. tigerinus, the total number of alleles per locus and expected heterozygosity ranged from 2 to 18 and 0.271 to 0.938, respectively. No significant linkage disequilibrium was observed across all loci, and five showed a significant deviation from Hardy-Weinberg equilibrium in some populations after Bonferroni correction. Consequently, our findings suggest that these novel markers will be applicable for conservation genetic studies across varying scales from inter-population to inter-individual.

Cathelicidins from the bullfrog Rana catesbeiana provides novel template for peptide antibiotic design.

Cathelicidins, a class of gene-encoded effector molecules of vertebrate innate immunity, provide a first line of defense against microbial invasions. Although cathelicidins from mammals, birds, reptiles and fishes have been extensively studied, little is known about cathelicidins from amphibians. Here we report the identification and characterization of two cathelicidins (cathelicidin-RC1 and cathelicidin-RC2) from the bullfrog Rana catesbeiana. The cDNA sequences (677 and 700 bp, respectively) encoding the two peptides were successfully cloned from the constructed lung cDNA library of R. catesbeiana. And the deduced mature peptides are composed of 28 and 33 residues, respectively. Structural analysis indicated that cathelicidin-RC1 mainly assumes an amphipathic alpha-helical conformation, while cathelicidin-RC2 could not form stable amphipathic structure. Antimicrobial and bacterial killing kinetic analysis indicated that the synthetic cathelicidin-RC1 possesses potent, broad-spectrum and rapid antimicrobial potency, while cathelicidin-RC2 exhibited very weak antimicrobial activity. Besides, the antimicrobial activity of cathelicidin-RC1 is salt-independent and highly stable. Scanning electron microscopy (SEM) analysis indicated that cathelicidin-RC1 kills microorganisms through the disruption of microbial membrane. Moreover, cathelicidin-RC1 exhibited low cytotoxic activity against mammalian normal or tumor cell lines, and low hemolytic activity against human erythrocytes. The potent, broad-spectrum and rapid antimicrobial activity combined with the salt-independence, high stability, low cytotoxic and hemolytic activities make cathelicidin-RC1 an ideal template for the development of novel peptide antibiotics.

Estudio morfológico, morfométrico e histoquímico de los epitelios de revestimiento y glandular de la lengua de la rana toro, rana catesbeiana / Morphological, morphometrical and histochemical study of the lining and glandular epithelia of the tongue of the tullfrog rana catesbeiana

La superficie dorsal de la lengua de la rana toro, Rana catesbeiana, presenta un epitelio simple cilíndrico, constituido por células caliciformes y raras células ciliadas. El dorso de la lengua posee numerosas papilas filiformes y algunas fungiformes. Las primeras poseen un epitelio simple cilíndrico, con células secretoras, mientras que las segundas poseen en la región apical, un disco sensorial con epitelio estratificado cilíndrico, con células basales, periféricas, glandulares y receptoras. A lo largo del dorso de la lengua existen numerosas glándulas tubulares, que penetran en profundidad, entremezclándose con las fibras musculares. El epitelio glandular es simple cilíndrico, con células secretoras y de sostén. Las primeras son las únicas en la base de la glándula y las segundas solo se encuentran en número escaso en el tercio superior. La superficie ventral de la lengua posee un epitelio estratificado, con células caliciformes y, entre éstas, células ciliadas. La morfometría de las glándulas mostró que son más cortas en la región anterior de la lengua (330 um) que en la región posterior (450 um). Las células secretoras de las glándulas linguales anteriores son menores (1457,7 um3) que en las posteriores (2645,9 um3). Lo mismo ocurre con los núcleos celulares: 130,0 um3 en las glándulas anteriores y 202,3 um3 en las posteriores. Las células secretoras de las glándulas linguales sintetizan producto rico en proteínas y mucopolisacáridos neutros, pudiendo caracterizarse como seromucoso. Las células caliciformes de las superficies dorsal y ventral secretan proteínas y mucopolisacáridos neutros, clasificándose como del tipo G1, mientras que las células de sostén de las glándulas superficiales de las papilas fungiformes secretan moco rico en mucopolisacáridos neutros, sulfomucinas y sialomucinas.

The dorsal surface of the tongue of the bullfrog, Rana catesbeiana, has simple columnar epithelium with a few ciliated cells and goblet cells. The entire surface is covered with numerous filiform papillae and few fungiform. Filiform papillae have a simple columnar epithelium with secretory cells, while the fungiform have a sensory disc on their upper surface the lined by a stratified columnar epithelium with basal, peripheral, glandular and receptor cells. Over the dorsal lingual surface there are numerous winding tubular glands, which penetrate deeply into the muscle of the tongue, mingling with the fibers. The gland epithelium is cylindrical with secretory and supporting cells. The first are absolute on the basis of the gland and the latter are rare in the upper third. The ventral surface of the tongue is lined by a stratified epithelium, with the presence of goblet cells, with ciliated cells among them. Morphometrically, lingual glands varies in length, according to their location: shorter in the anterior region of the tongue (330 um) than in the posterior region (450 um). Secretory cells of the anterior lingual glands are smaller (1457.7 mm3) than the posterior ones (2645.9 um3). The same can be said of the cell nuclei, 130.0 um3 for the anterior glands and 202.3 um3 for the posterior ones. Secretory cells of the lingual glands contain substances rich in protein and neutral mucopolysaccharides, which characterize the seromucous type. Goblet cells of the dorsal and ventral surface epithelia secrete neutral mucopolysaccharides and proteins, and can be characterized as type G1 cells, and the supporting cells of the superficial glands of the fungiform papillae secrete a mucus rich in neutral mucopolysaccharides, sulfomucins and sialomucins.

A phylogeny for the African treefrog family Hyperoliidae based on mitochondrial rDNA.

Mitochondrial 12S rRNA sequences were used to construct a phylogeny for the African treefrog family, Hyperoliidae. The sequences were aligned using the well-established mt 12S rRNA secondary structure as a map to designate stem and loop positions. Conservation of secondary structure permits a more accurate basis for assessing homologies than does primary sequence alone. The molecular trees showed many similarities to the morphological trees constructed previously. Species within a genus always grouped together. The genera Hyperolius, Heterixalus, Afrixalus, and Kassina were resolved, but not with statistical significance. Leptopelis was the basal group. The analysis shows clearly that Phylyctimantis groups with Kassina as it did in Drewes' morphological tree and that Tachycnemis groups closely with Heterisxalus, a relationship not suggested by the morphological data. Character weighting (including compensatory base changes), mitochondrial trees vs gene trees, and the biogeography of the group are discussed.