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Development of the nasal chemosensory organs in two terrestrial anurans: the directly developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), and the metamorphosing toad, Bufo americanus (Anura: Bufonidae).

Nearly all vertebrates possess an olfactory organ but the vomeronasal organ is a synapomorphy for tetrapods. Nevertheless, it has been lost in several groups of tetrapods, including aquatic and marine animals. The present study examines the development of the olfactory and vomeronasal organs in two terrestrial anurans that exhibit different developmental modes. This study compares the development of the olfactory and vomeronasal organs in metamorphic anurans that exhibit an aquatic larva (Bufo americanus) and directly developing anurans that have eliminated the tadpole (Eleutherodactylus coqui). The olfactory epithelium in larval B. americanus is divided into dorsal and ventral branches in the rostral and mid-nasal regions. The larval olfactory pattern in E. coqui has been eliminated. Ontogeny of the olfactory system in E. coqui embryos starts to vary substantially from the larval pattern around the time of operculum development, the temporal period when the larval stage is hypothesized to have been eliminated. The nasal anatomy of the two frogs does not appear morphologically similar until the late stages of embryogenesis in E. coqui and the terminal portion of metamorphosis in B. americanus. Both species and their respective developing offspring, aquatic tadpoles and terrestrial egg/embryos, possess a vomeronasal organ. The vomeronasal organ develops at mid-embryogenesis in E. coqui and during the middle of the larval period in B. americanus, which is relatively late for neobatrachians. Development of the vomeronasal organ in both frogs is linked to the developmental pattern of the olfactory system. This study supports the hypothesis that the most recent common ancestor of tetrapods possessed a vomeronasal organ and was aquatic, and that the vomeronasal organ was retained in the Amphibia, but lost in some other groups of tetrapods, including aquatic and marine animals.

Animals↗

Histological examination of the effects of corticosterone in larvae of the western toad, Bufo boreas (Anura: Bufonidae), and the Oriental fire-bellied toad, Bombina orientalis (Anura: Discoglossidae).

The effects of corticosterone (CORT)-treatment on various tissues were examined in two species of anuran larvae, the discoglossid Bombina orientalis, and the bufonid Bufo boreas. Corticosterone was administered directly into aquarium water for 15 days. After treatment, histological analyses were conducted on skin, gut, spleen, thymus, and neural and muscle tissue. Corticosterone treatment prevented sloughing of the skin, which resulted in a build-up of stratum corneum, and inhibited the development of gland nests and the subsequent formation of dermal granular and mucous glands in both species. Corticosterone treatment also decreased epithelial folding in the gut and caused vesiculation of the gut epithelial cells. The thymus of CORT-treated animals was significantly reduced in size (P < .05) and cell density (P < .05), and the spleen of CORT-treated animals was completely involuted. The brain and pituitary of CORT-treated animals had a decreased cell density (P < .05) and many pyknotic cells. An examination of muscle revealed that muscle fibers of CORT-treated animals had a decreased cross-sectional area (P < .05). The dose of CORT used (1.1 microM) was within the range used in other studies in the literature and resulted in tissue levels within the range experienced by larvae at metamorphic climax. Thus, this study is appropriate to address the histological effects of CORT in experimental manipulations and the role of increasing CORT at metamorphic climax. The data suggest that increasing endogenous CORT at metamorphosis may be involved in degeneration of larval tissue, prior to regeneration, which is stimulated by thyroid hormones.

Animals↗

On the arrangement of chromosomes in the elongated sperm nuclei of Anura (Amphibia).

The position of specific constitutive heterochromatic chromosome regions within the elongated sperm nuclei of eight species of Anura was examined with Q- and C-banding. These species differ widely with regard to the number, size and position of the brightly fluorescing heterochromatic regions. The empirical frequency distributions determined for the heterochromatic regions relative to the longitudinal axis of the sperm nuclei were compared with random frequency distributions calculated on the basis of two spatial models. None of the specifically stained heterochromatic regions occupy any definite preferential position within the sperm nuclei. In two instances, a specific sequence of the heterochromatic regions within the sperm nuclei could be excluded. The type of chromosomal arrangement within the elongated sperm nuclei of Anura is discussed on the basis of the distribution patterns obtained.

Amphibians↗

On the diversity of sperm histones in the vertebrates: IV. Cytochemical and amino acid analysis in Anura.

The variability of sperm histones in frogs has been studied by cytochemical and amino acid analyses. Cytochemically, Rana sperm proteins fall into Bloch's ('69, '76) type 4 somatic-like histone category, while Xenopus and Bufo have type 3 intermediate sperm histones. Extractability in 5% trichloroacetic acid (TCA) at different temperatures splits this type 3 category into two groups: type 3B intermediate sperm histones of Bufo are extractable at 85-90 degrees C, while Xenopus intermediate type 3A sperm histones require temperatures of 95-100 degrees C for extraction. Amino acid analysis confirms that Rana sperm histones are of the nucleosomal type, with a testis-specific, very lysine-rich H1 histone. The sperm protein in Bufo is richer in arginine than the proteins in Xenopus. Both of these genera contain lysine and histidine as well as arginine in their sperm proteins. These results confirm earlier electrophoretic data (Kasinsky et al., '78) and indicate that sperm histones in the order Anura can vary markedly between different genera.

Amino Acids↗

Insulin and proglucagon-derived peptides from the horned frog, Ceratophrys ornata (Anura:Leptodactylidae).

Insulin and peptides derived from the processing of proglucagon have been isolated from an extract of the pancreas of the South American horned frog, Ceratophrys ornata (Leptodactylidae). Ceratophrys insulin is identical to the insulin previously isolated from the toad, Bufo marinus (Bufonidae). Ceratophrys glucagon was isolated in two molecular forms with 29- and 36-amino acid residues in approximately equal amounts. Glucagon-29 is identical to glucagon from B. marinus and from the bullfrog, Rana catesbeiana (Ranidae) and contains only 1 amino acid substitution (Thr29 --> Ser) compared with glucagon from Xenopus laevis (Pipidae). Glucagon-36 comprises glucagon-29 extended from its C-terminus by Lys-Arg-Ser-Gly-Gly-Met-Ser. This extension is structurally dissimilar to the C-terminal octapeptide of mammalian oxyntomodulin and resembles more closely that found in C-terminally extended glucagons isolated from fish pancreata. Ceratophrys glucagon-like peptide-1 (GLP-1) (His-Ala-Asp-Gly-Thr-Tyr-Gln-Asn-Asp-Val10-Gln-Gln-Phe-Leu-Glu- Glu-Lys-Ala-Ala-Lys20-Glu-Phe-Ile-Asp-Trp-Leu-Ile-Lys-Gly- Lys30-Pro-Lys-Lys-Gln-Arg-Leu-Ser) contains 3 amino acid substitutions compared with the corresponding peptide from B. marinus, 8 substitutions compared with GLP-1 from R. catesbeiana, and between 4 and 11 substitutions compared with the three GLP-1 peptides identified in X. laevis proglucagon. GLP-2 was not identified in the extract of Ceratophrys pancreas. The data indicate that, despite its importance in the regulation of glucose metabolism, the primary structure of GLP-1 has been very poorly conserved during evolution, even among a single order such as the Anura.

Amino Acid Sequence↗

Neuron-specific enolase-like immunoreactivity in the vertebrate retina: selective labelling of Müller cells in Anura.

Neuron-specific enolase (NSE) immunocytochemistry was carried out in retinae of goldfish, axolotl, clawed frog, cane toad, lizard, chick, guinea-pig, rabbit, rat, cat and human. With the exception of Anura, strong immunoreactivity was seen in the large ganglion, amacrine cells and horizontal cells of the retina in all of the other species. Photoreceptors were found to be labelled in the rat and human retina and only one cone type in rabbit. Photoreceptor pedicles and ellipsoids were stained in the goldfish and the somata and inner segments of some photoreceptors in axolotl. In the axolotl retina, besides neurons, Müller cells (MCs) were also immunolabelled. In the retina of the cane toad and the clawed frog MCs were the only stained elements. Similarly in other parts of the central nervous system of the cane toad, glial elements of the optic tectum and spinal cord were immunoreactive. In contrast, in the peripheral nervous system, neurons of the 1st sympathetic ganglion and the 2nd dorsal root ganglion were labelled. In double-labelling experiments, glial fibrillary acidic protein and NSE showed colocalisation both in the glial elements of the optic tectum and spinal cord and in MCs of the retina of the cane toad.

Ambystoma↗

Methylation of ribosomal cistrons in diploid and tetraploid Odontophrynus americanus (Amphibia, Anura).

Odontophyrynus americanus (Amphibia, Anura) genomic DNA from diploid and tetraploid specimens was treated with restriction enzymes sensitive to cytosine and adenine methylation (5 meC and 6 meA). In both diploids and tetraploids a high proportion of the total DNA was not cleaved by 5 meC-sensitive enzymes as observed on agarose gels stained with ethidium bromide. The DNAs were transferred to nitrocellulose filters and hybridized with cloned fragments containing sequences of Xenopus laevis 28S and 18S ribosomal DNA (rDNA). A high level of methylation of the ribosomal repeat units was revealed by 5 meC-sensitive enzymes in blood, liver, kidney and testis tissues. Adenine was methylated to a lesser degree and similarly in the rDNA from both germinative and somatic tissues. Comparison of the results obtained with DNA of diploids and tetraploids showed that methylation of ribosomal genes was increased in tetraploid genomes of adult frogs, but exact quantitative determinations could not be performed by this methodology. Cloning of the 28S region of the rDNA repeat unit was performed in the lambda gtWES lambda C vector. Restriction patterns obtained with methylation-sensitive enzymes using diploid and tetraploid derived clones confirmed the high level of methylation of the corresponding region of the ribosomal repeat unit in genomic DNAs. The implications of these results in the regulation of expression of the ribosomal genes in diploids and tetraploids are discussed.

Adenine↗

The organization of ribosomal genes in diploid and tetraploid species of the genus Odontophrynus (Amphibia, Anura).

The organization of the repeat unit of the ribosomal genes was determined in populations of Odontophrynus americanus 2n, 4n, O. occidentalis 2n and O. barrioi 2n (Amphibia, Anura) from South America. HindIII, EcoRI and BamHI restriction patterns of rDNA from single individuals were probed with two rDNA clones from Xenopus laevis. Variations in length and sequence were found among different species, populations and specimens. A general pattern could be established from the most frequent type of repeat observed in each case, so that diploid populations could be distinguished from tetraploid ones. The molecular evolution of variant repeats is discussed. It is suggested that the reduced size of the non-transcribed spacers in the tetraploids from São Paulo could play a role in the control of rDNA transcription. Distinct restriction maps were tentatively correlated to the varying localization of active rDNA clusters on metaphase chromosomes of corresponding populations.

Animals↗

Comparative metabolism of histamine in two Amphibia anura.

The metabolism of histamine has been compared in Rana ridibunda and Discoglossus pictus, both Amphibia anura. An intralymphatic injection of 14C-histamine (5 microCi) was administered to the animals and the total radioactivity excreted in the urine was measured 24, 48, and 72 hours later. The metabolites excreted in the urine at 24 hours were analyzed by thin-layer chromatography and subsequent autoradiography. In both species approximately 60--70% of the injected dose was found in the urine by 72 hours. In Rana ridibunda two histamine metabolites were found: imidazoleacetic and methylimidazoleacetic acid. The present results suggest that the catabolism of histamine differs in the two species.

Amphibians↗

Phylogenetic relationships of discoglossid frogs (Amphibia:Anura:Discoglossidae) based on complete mitochondrial genomes and nuclear genes.

The complete nucleotide sequence of the mitochondrial (mt) genome was determined for three species of discoglossid frogs (Amphibia:Anura:Discoglossidae), representing three of the four recognized genera: Alytes obstetricans, Bombina orientalis, and Discoglossus galganoi. The organization and size of these newly determined mt genomes are similar to those previously reported for other vertebrates. Phylogenetic analyses (maximum likelihood, Bayesian inference, minimum evolution, and maximum parsimony) of mt protein-coding genes at the amino acid level were performed in combination with already published mt genome sequence data of three species of Neobatrachia, one of Pipoidea, and four of Caudata. Phylogenetic analyses based on the deduced amino acid sequences of all mt protein-coding genes arrived at the same topology. The monophyly of Discoglossidae is strongly supported. Within the Discoglossidae, Alytes is consistently recovered as sister group of Discoglossus, to the exclusion of Bombina. The three species representing Neobatrachia exhibited extremely long branches irrespective of the phylogenetic inference method used, and hence their relative position with respect to Discoglossidae and Xenopus may be artefactual due to a severe long branch attraction effect. To further investigate the phylogenetic intrarelationships of discoglossids, nucleotide sequences of four nuclear protein-coding genes (CXCR4, RAG1, RAG2, and Rhodopsin) with sequences available for the three discoglossid genera and Xenopus were retrieved from GenBank, and together with a concatenated nucleotide sequence data set containing all mt protein-coding genes except ND6 were subjected to separate and combined phylogenetic analyses. In all cases, a sister group relationship between Alytes and Discoglossus was recovered with high statistical support.

Amino Acid Sequence↗

The complete mitochondrial genome of a tree frog, Polypedates megacephalus (Amphibia: Anura: Rhacophoridae), and a novel gene organization in living amphibians.

In this study, we have determined the complete sequence of the mitochondrial genome of an Old World tree frog Polypedates megacephalus (Anura: Rhacophoridae) by using a long polymerase chain reaction (PCR) technique and shotgun strategy of sequencing. The entire mtDNA sequence is 16,473 nt long with a novel mitogenomic gene organization in amphibians. Unlike other neobatrachian frogs, the transfer ribonucleic acid (tRNA)-Leu(CUN) and tRNA-Thr genes exchange their positions in P. megacephalus and form a Thr-Leu(CUN)-Pro-Phe tRNA gene tetrad. Moreover, we found that the ATP8 gene was replaced by a noncoding sequence of 853 nt long and that the ND5 gene was absent in the new mitogenome. These peculiar features of P. megacephalus mtDNA were further studied among related anuran species by PCR amplification. The new sequence data was used to assess the phylogenetic relationships of the three living amphibian orders using neighbor-joining, maximum likelihood, and Bayesian methods. In agreement with most morphological studies, phylogenetic analyses of a whole mitochondrial genome data set suggest a close relationship between salamanders and frogs. Moreover, using a molecular clock-independent Bayesian approach for inferring dating information from molecular phylogenies, we have provided a rough timescale for living amphibian evolution. This timescale provides a working framework for future paleontological researches on amphibian evolution and improves our understanding of the evolutionary history of modern amphibians.

Animals↗

The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia: Anura): a new gene order in the vertebrate mtDNA.

The mitochondrial DNA of the rice frog, Fejervarya limnocharis (Amphibia, Anura), was obtained using long-and-accurate polymerase chain reaction (LA-PCR) combining with subcloning method. The complete nucleotide sequence (17,717 bp) of mitochondrial genome was determined subsequently. This mitochondrial genome is characterized by four distinctive features: the translocation of ND5 gene, a cluster of rearranged tRNA genes (tRNAThr, tRNAPro, tRNA(Leu CUN)), a tandem duplication of tRNAMet gene, and eight large 89-bp tandem repeats in the control region, as well as three short noncoding regions containing two repeated motifs existing in the gene cluster of ND5/tRNAThr/tRNAPro/tRNALeu/tRNAPhe. The tandem duplication of gene regions followed by deletions of supernumerary genes can be invoked to explain the shuffling of tRNAMet and a cluster of tRNA and ND5 genes, as observed in this study. Both ND5 gene translocation and tandem duplication of tRNAMet were first observed in the vertebrate mitochondrial genomes.

Animals↗

Mitochondrial phylogeny of Anura (Amphibia): a case study of congruent phylogenetic reconstruction using amino acid and nucleotide characters.

We explore whether phylogenetic analyses of the same sequence data set at the amino acid and nucleotide level are able to recover congruent topologies, as well as the advantages and limitations of both alternative approaches. As a case study, mitochondrial protein-coding genes were used to discern among competing hypotheses on the phylogenetic relationships of major anuran amphibian lineages. To properly address this phylogenetic question, the complete nucleotide sequences of the mitochondrial genomes of two archaeobatrachian species, Ascaphus truei and Pelobates cultripes, were determined anew. Bayesian and maximum likelihood phylogenetic inferences of the same sequence data set were performed based on both amino acid and nucleotide characters, with the latter analysed either as codons or as a reduced data set of first+second (P12) codon positions. In addition, likelihood-based ratio tests were performed to evaluate the support of alternative topologies. The different data sets arrived at congruent and highly supported topologies, suggesting a similar phylogenetic resolving power of the two character types provided that correctly selected sites and appropriate evolutionary models are used. The reconstructed anuran mitochondrial phylogeny supports the paraphyly of Archaeobatrachia, with Ascaphus as sister group to all the remaining anurans, and Pelobates as sister group of Neobatrachia. However, the employed tree reconstruction methods and likelihood-based ratio tests seemed to be negatively affected by the fast evolving sequences of neobatrachians, suggesting that the phylogeny of Anura here presented is not definitive, and needs further investigation using an extended taxon sampling.

Amphibian Proteins↗

An antimicrobial peptide from the skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae).

A 25 amino-acid-residue, C-terminally alpha-amidated peptide with antimicrobial activity, which has been termed fallaxin, was isolated in high yield from the norepinephrine-stimulated skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae). The amino acid sequence of the peptide (Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Ile-Ala-Gly-His-Leu-Ala-Ser-Lys-Val-Met-Asn-Lys-Leu.NH2) shows structural similarity with members of the ranatuerin-2 family previously isolated from the skins of frogs of the genus Rana that are only distantly related to the Leptodactylidae. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different families. Fallaxin inhibited the growth of reference strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) but with relatively low potency (MIC> or =20 microM) and was inactive against the Gram-positive bacterium (Staphylococcus aureus) and the yeast Candida albicans. The hemolytic activity of fallaxin was very low (HC50>200 microM). A second peptide, comprising residues (1-22) of fallaxin, was also isolated from the skin secretions but this component was inactive against the microorganisms tested.

Amino Acid Sequence↗

Evolution of Bombina bombina and Bombina variegata (Anura: Discoglossidae) in the Carpathian Basin: a history of repeated mt-DNA introgression across species.

The structure and geographic location of hybrid zones change through time. Current patterns result from present and historical population-environment interactions that act on each of the hybridizing taxa. This is particularly evident for species involved in complex hybrid zones, such as that formed by the toad species Bombina bombina and Bombina variegata (Anura: Discoglossidae), which interact along extensive areas in Central Europe. We used data on external morphology and partial sequences of the cytochrome oxidase I (cox1) and nicotinamide adenine dinucleotid dehydrogenase subunit 4 (nad4) mitochondrial DNA (mt-DNA) genes to analyze the current patterns of genetic structure shown by both species of Bombina along their contact zone in Hungary. Phylogenetic, phylogeographic, and historical demography analyses were applied to 1.5kb mt-DNA obtained from 119 individuals representing 24 populations from Hungary and additional specimens from Slovakia, Albania, and Bosnia-Herzegovina. We use these data to infer the evolutionary history of the isolated populations of B. variegata in Hungary and to discriminate between competing biogeographic scenarios accounting for the historical interactions between species in this region. Results from the inferred phylogenetic branching pattern and sequence divergence among species and populations support the following: (i) recent population expansion has occurred in Hungarian populations of B. bombina, which are genetically very homogeneous; (ii) the Hungarian populations of B. variegata correspond to two distinct mitochondrial lineages (Carpathian and Alpine, respectively); average maximum-likelihood-corrected sequence divergence between these lineages is 8.96% for cox1 and 10.85% for nad4; (iii) mt-DNA divergence among the three isolated western populations of B. variegata from Transdanubia is low, with four closely related haplotypes, which suggests that the isolation between these populations is the result of a recent process, possibly mediated by the invasion of B. bombina; and (iv) we have detected discordances between morphology and mt-DNA data in the Transdanubia region (Bakony Mountains, Mecsek Mountains, Orség area), suggesting mt-DNA introgression across species in this regions. These results are discussed with reference to previous biogeographic hypotheses.

Animals↗

A linkage map for the hybridising toads Bombina bombina and B. variegata (Anura: Discoglossidae).

Stable hybrid zones in which ecologically divergent taxa give rise to a range of recombinants are natural laboratories in which the genetic basis of adaptation and reproductive isolation can be unraveled. One such hybrid zone is formed by the fire-bellied toads Bombina bombina and B. variegata (Anura: Discoglossidae). Adaptations to permanent and ephemeral breeding habitats, respectively, have shaped numerous phenotypic differences between the taxa. All of these are, in principle, candidates for a genetic dissection via QTL mapping. We present here a linkage map of 28 codominant and 10 dominant markers in the Bombina genome. In an F2 cross, markers that were mainly microsatellites, SSCPs or allozymes were mapped to 20 linkage groups. Among the 40 isolated CA microsatellites, we noted a preponderance of compound and frequently interleaved CA-TA repeats as well as a striking polarity at the 5' end of the repeats.

Animals↗

Changes in numbers and dimensions of glomeruli during metamorphosis of Pelobates syriacus (Anura; Pelobatidae).

Changes in glomerular numbers and volume were followed throughout five ontogenetic stages of Pelobates syriacus (Anura, Pelobatidae). The number of glomeruli increased markedly between the 2-legged and the 4-legged tadpole stages. In the post-metamorphic stages the number of glomeruli was about twice their number during the tadpole stages. Glomerular volume peaked during the 2-legged tadpole stage, when the tadpole reached its maximal size, and dropped thereafter. There is some evidence to indicate that the number of glomeruli in post-metamorphic P. syriacus increased during growth thus indicating perhaps an increase in their functional importance. The number was still lower than that reported for some other anurans, more similar to the numbers given for representative urodeles, however, the latter have considerably larger glomeruli. Two main factors appear to be involved in regulating numbers and volume of glomeruli in frogs. The ontogenetic changes during metamorphosis affect both glomerular numbers and volume, whereas post-metamorphic growth affected only the glomerular numbers.

Animals↗

Chromosome banding in Amphibia. XXI. Inversion polymorphism and multiple nucleolus organizer regions in Agalychnis callidryas (Anura, Hylidae).

Cytogenetic analyses were performed on several populations of the Central American tree frog Agalychnis callidryas, using conventional methods and banding techniques. The karyotype of this species is distinguished by an inversion polymorphism in chromosome 9, which is either submetacentric or telocentric. The populations examined are in Hardy-Weinberg equilibrium with respect to the two alternative morphs of chromosome 9. This is the first report of the occurrence of an intrapopulational chromosomal inversion polymorphism in the order Anura. In male meiosis, the two chromosomes 9 form a bivalent exhibiting a ring-like pairing configuration with terminal chiasmata in both arms, regardless of whether the paired homologs are heteromorphic or homomorphic. Furthermore, individual specimens of A. callidryas exhibit one or two unexpected 18S + 28S ribosomal RNA gene clusters, in addition to the standard nucleolus organizers. The chromosomal localization of these extra nucleolus organizers is identical in all metaphases from the same specimen and shows a specific intraindividual pattern. The karyotype evolution in the phyllomedusine hylids, the structure of the various classes of heterochromatin, and the occurrence and possible origin of the rare inversion polymorphisms and multiple nucleolus organizers in A. callidryas and a few other amphibian species are discussed.

Animals↗