Immune-complex-trapping cells in the spleen of the oriental fire-bellied toad,Bombina orientalis

Summary The spleen of the oriental fire-bellied toad, Bombina orientalis, consists of well-developed white pulp, separated from the lymphocytic marginal zone by the connective tissue boundary layer. Injection of peroxidase-conjugated rabbit anti-peroxidase revealed that these immune complexes were localized on the surface of acid-phosphatase-positive and non-specific-esterasepositive cells in the white pulp. The majority of immunecomplex-trapping cells were present around the blood vessels. Cell processes of some of these cells penetrated into the wall of blood vessels. The significance of the present findings is discussed with respect to the evolution of immune-complex-trapping cells in the spleen.     

Bradykinins and their precursor cDNAs from the skin of the fire-bellied toad (Bombina orientalis)

Bradykinin and (Thr(6))-bradykinin have been identified in the defensive skin secretion of the fire-bellied toad, Bombina orientalis. The homologous cDNAs for both peptides were cloned from a skin library using a 3'- and 5'-RACE strategy. Kininogen-1 (BOK-1) contained an open-reading frame of 167 amino acid residues encoding four repeats of bradykinin, and kininogen-2 (BOK-2) contained an open-reading frame of 161 amino acid residues encoding two repeats of (Thr(6))-bradykinin. Alignment of both precursor nucleotide and amino acid sequences revealed a high degree of structural similarity. These amphibian skin kininogens/preprobradykinins are not biologically analogous to mammalian kininogens.     

Multisensory signals trigger approach behaviour in the fire-bellied toad Bombina orientalis: sex differences and call specificity

Animal communication often involves multimodal signals, and interactions between sensory modalities can trigger unique responses in receivers. Response to social signals was investigated in fire-bellied toads by exposing them to playback of male calls (advertisement and release calls) and a video clip of a male conspecific in the laboratory. The cues were presented in isolation and as a combined bimodal stimulus, and approach frequency, latency to approach and time spent around the stimulus source were measured. No positive phonotaxis was observed toward the advertisement call, both during the day and during a phonotaxis trial performed at night. However, females, but not males, approached with greater frequency, lower latency, and spent more time near the source of the bimodal stimulus in an experiment involving the advertisement call. Female response was specific to the advertisement call, as approach was not increased when the release call was used. Males, on the other hand, did not show increased approach in the advertisement call experiment, but approached with greater frequency the bimodal stimulus involving the release call within the first minute of stimulus presentation. The findings suggest that females orient toward calling males and that males eavesdrop on release calls, but in both cases a visual stimulus is also needed to trigger a response. Social approach in Bombina orientalis is thus dependent on multisensory cues, and the nature of the interaction between sensory modalities depends on receiver sex and call type.     

Cranial neural-crest migration and chondrogenic fate in the oriental fire-bellied toad Bombina orientalis: Defining the ancestral pattern of head development in anuran amphibians

We assess cranial neural-crest cell migration and contributions to the larval chondrocranium in the phylogenetically basal and morphologically generalized anuran Bombina orientalis (Bombinatoridae). Methods used include microdissection, scanning electron microscopy, and vital dye labeling, in conjunction with confocal and fluorescence microscopy. Cranial neural-crest cells begin migrating before neural-fold closure and soon form three primary streams. These streams contribute to all cranial cartilages except two medial components of the hyobranchial skeleton (basihyal and basibranchial cartilages), the posterior portion of the trabecular plate, and the otic capsule, the embryonic origin of which is unknown. Chondrogenic fate is regionalized within the cranial neural folds, with the anterior regions contributing to anterior cartilages and the posterior regions to posterior cartilages. A neural-crest contribution also was consistently observed in several cranial nerves and the connective tissue component of many cranial muscles. Notwithstanding minor differences among species in the initial configuration of migratory streams, cranial neural-crest migration and chondrogenic potential in metamorphosing anurans seem to be highly stereotyped and evolutionarily conservative. This includes a primary role for the neural crest in the evolutionary origin of the paired suprarostral and infrarostral cartilages, two prominent caenogenetic features of the rostral skull unique to anuran larvae. Our results provide a model of the ancestral pattern of embryonic head development in anuran amphibians. This model can serve as a basis for examining the ontogenetic mechanisms that underlie the diversity of cranial morphology and development displayed by living frogs, as well as the evolutionary consequences of this diversity. © 1996 Wiley-Liss, Inc.     

Variation and timing of the cranial ossification sequence of the oriental fire-bellied toad,Bombina orientalis (Amphibia,Discoglossidae)

The sequence of appearance of the 17 different skull bones in the oriental fire-bellied toad, Bombina orientalis, is described. Data are based primarily on samples of ten or 11 laboratory-reared specimens of each of 11 Gosner developmental stages (36–46) representing middle through late metamorphosis. Ossification commences as early as stage 37 (hind limb with all five toes distinct), but the full complement of adult bones is not attained until stage 46 (metamorphosis complete). Number of bones present at intermediate stages is poorly correlated with external morphology. As many as four Gosner developmental stages elapse before a given bone is present in all specimens following the stage at which it may first appear. The modal ossification sequence is frontoparietal, exoccipital, parasphenoid, septomaxilla, premaxilla, vomer, nasal, maxilla, angulosplenial, dentary, squamosal, quadratojugal, pterygoid, prootic, interfrontal, sphenethmoid, and mentomeckelian. Most specimens are consistent with this sequence, despite the poor correlation between cranial ossification and external development as assayed by Gosner stage. The timing of cranial ossification in Bombina orientalis differs in many respects from that described for two other, distantly related anurans, the leopard frog (Rana pipiens) and the western toad (Bufo boreas). These include the total number and sequence of appearance of bones, and the timing of ossification relative to the development of external morphology. Interspecific variation may reflect differences in the timing of the tissue interactions known to underlie skeletal differentiation and evolution.     

Identification and molecular cloning of novel trypsin inhibitor analogs from the dermal venom of the Oriental fire-bellied toad (Bombina orientalis) and the European yellow-bellied toad (Bombina variegata)

The structural diversity of polypeptides in amphibian skin secretion probably reflects different roles in dermal regulation or in defense against predators. Here we report the structures of two novel trypsin inhibitor analogs, BOTI and BVTI, from the dermal venom of the toads, Bombina orientalis and Bombina variegata. Cloning of their respective precursors was achieved from lyophilized venom cDNA libraries for the first time. Amino acid alignment revealed that both deduced peptides, consisting of 60 amino acid residues, including 10 cysteines and the reactive center motif, -CDKKC-, can be affirmed as structural homologs of the trypsin inhibitor from Bombina bombina skin.     

The mating call of hybrids of the fire-bellied toad and yellow-bellied toad (Bombina bombina (L.), Bombina v. variegata (L.), discoglossidae,anura)

Summary Male hybrids obtained by crossing Bombina bombina with Bombina v. variegata displayed calling behavior when treated with hormones at the age of about 12 months. The mating calls of the hybrids are intermediate with respect to rate, duration, inter-call interval, and fundamental frequency between the calls of the two purebred species.     

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 μM) 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. © 1995 Wiley-Liss, Inc.     

Sequence of chondrocranial development in the oriental fire bellied toad Bombina orientalis

The vertebrate head as a major novelty is directly linked to the evolutionary success of the vertebrates. Sequential information on the embryonic pattern of cartilaginous head development are scarce, but important for the understanding of its evolution. In this study, we use the oriental fire bellied toad, Bombina orientalis, a basal anuran to investigate the sequence and timing of larval cartilaginous development of the head skeleton from the appearance of mesenchymal Anlagen in post-neurulation stages until the premetamorphic larvae. We use different methodological approaches like classic histology, clearing and staining, and antibody staining to examine the larval skeletal morphology. Our results show that in contrast to other vertebrates, the ceratohyals are the first centers of chondrification. They are followed by the palatoquadrate and the basihyal. The latter later fuses to the ceratohyal and the branchial basket. Anterior elements like Meckel's cartilage and the rostralia are delayed in development and alter the ancestral anterior posterior pattern observed in other vertebrates. The ceratobranchials I–IV, components of the branchial basket, follow this strict anterior–posterior pattern of chondrification as reported in other amphibians. Chondrification of different skeletal elements follows a distinct pattern and the larval skeleton is nearly fully developed at Gosner Stage 28. We provide baseline data on the pattern and timing of early cartilage development in a basal anuran species, which may serve as guidance for further experimental studies in this species as well as an important basis for the understanding of the evolutionary changes in head development among amphibians and vertebrates.     

Bombinin-like peptides with antimicrobial activity from skin secretions of the Asian toad, Bombina orientalis.

The structures and hemolytic and bactericidal activities of three bombinin-like peptides, or BLP-1-3, from the skin of Bombina orientalis are described. The peptides were isolated from the skin of B. orientalis and sequenced by tandem mass spectrometry and are amphipathic, cationic peptides of 25-27 amino acids in length. The sequence of the most abundant member (BLP-1) is: Gly-Ile-Gly-Ala-Ser-Ile-Leu-Ser-Ala-Gly-Lys-Ser-Ala-Leu-Lys-Gly-Leu- Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asn-NH2. All three peptides were found to share considerable, but not complete, homology with bombinin, an antimicrobial, hemolytic peptide first isolated by Michl and Csordas (Csordas, A., and Michl, A. (1970) Monatsh. Chem. 101, 182-189) from the skin of Bombina variegata. The BLPs have been assayed for antibiotic and hemolytic activity and found to be more potent than magainin 2 (a related antimicrobial peptide from Xenopus laevis) in their ability to kill bacteria. However, no significant hemolytic activity was found for these peptides which suggests a selectivity for prokaryotic over eukaryotic membranes. The molecular basis for antibacterial activity is presumed to be due to their predicted amphipathic alpha-helical structures which is supported by circular dichroism measurements that found significant helical content (63-69% alpha-helix) in 40% trifluoroethanol. Last, a cDNA library was constructed from the skin of B. orientalis and screened with an oligonucleotide probe complementary to the COOH terminus of BLP-1. Several clones were isolated and sequenced that encode BLP-1 and BLP-3, as well as an additional peptide (BLP-4) that differs by two amino acid substitutions from BLP-3.     

Ultrastructure of the taste disc in the red-bellied toad Bombina orientalis (Discoglossidae,Salientia)

The taste disc of the red-bellied toad Bombina orientalis (Discoglossidae) has been investigated by light and electron microscopy and compared with that of Rana pipiens (Ranidae). Unlike the frog, B. orientalis possesses a disc-shaped tongue that cannot be ejected for capture of prey. The taste discs are located on the top of fungiform papillae. They are smaller than those in Ranidae, and are not surrounded by a ring of ciliated cells. Ultrastructurally, five types of cells can be identified (mucus cells, wing cells, sensory cells, and both Merkel cell-like basal cells and undifferentiated basal cells). Mucus cells are the main secretory cells of the taste disc and occupy most of the surface area. Their basal processes do not synapse on nerve fibers. Wing cells have sheet-like apical processes and envelop the mucus cells. They contain lysosomes and multivesicular bodies. Two types of sensory cells reach the surface of the taste disc; apically, they are distinguished by either a brush-like arrangement of microvilli or a rod-like protrusion. They are invaginated into lateral folds of mucus cells and wing cells. In contrast to the situation in R. pipiens, sensory cells of B. orientalis do not contain dark secretory granules in the perinuclear region. Synaptic connections occur between sensory cells (presynaptic sites) and nerve fibers. Merkel cell-like basal cells do not synapse onto sensory cells, but synapse-like connections exist between Merkel cell-like basal cells (presynaptic site) and nerve fibers.     

Mitochondrial DNA variation in the hybridizing fire-bellied toads, Bombina bombina and B. variegata

Using five restriction enzymes, geographical variation of mitochondrial DNA (mtDNA) in Bombina bombina and B. variegata was studied in samples from 20 locations. Each restriction enzyme produced a species-specific fragment pattern. B. bombina haplotypes A and B were closely related to each other. In contrast, haplotypes A and B of B. variegata formed two distinct lineages. A very distinctive haplotype (C) was found in the Carpathian Mountains, whereas two other haplotypes, D and E (differing by a single AvaI site), were present in western Europe and the Balkans, respectively. Populations polymorphic for haplotypes D and E occurred in the central Balkans where the haplotypes could replace each other clinally. mtDNA sequence divergence between B. bombina and B. variegata was estimated as 6.0-8.1% and 4.7-5.2% between type C and types D/E of B. variegata. The latter divergence is contrary to allozyme and morphological data that place the western and Carpathian B. v. variegata together (Nei's D = 0.07) and separate them from the Balkan subspecies B. v. scabra (Nei's D = 0.18). Broad interspecific correlation among morphology, allozymes and mtDNA types in European fire-bellied toads argues that, despite continuous hybridization (interrupted perhaps during Pleistocene glacial maxima), little or no mtDNA introgression between the species has occurred outside the narrow hybrid zones that separate these parapatric species.     

Pigment cell pattern formation in Taricha torosa: The role of the extracellular matrix in controlling pigment cell migration and differentiation

The neural crest is a population of highly migratory mesenchymal cells that ultimately localize in specific sites and differentiate into a variety of cell types. This report describes studies on the factors governing the migratory pathways, differentiation, and ultimate localization of the neural crest-derived pigment cells (black melanophores and yellow xanthophores) in the California newt, Taricha torosa. Melanophores first appear scattered in the dorsal portion of the lateral neural crest migratory pathway (between the somites and the ectoderm). These cells are eventually found in two stripes: a dorsal stripe that runs along the apex of the somites, and a midbody stripe near the somite-lateral plate mesoderm border. Melanophores are not seen in the dorsal fin of prehatching embryos. Xanthophores can be identified with the light microscope using NH₄OH-induced autofluorescence of pteridines and in the transmission electron microscope (TEM) by the presence of pterinosomes. Xanthophores first appear scattered among the melanophores over the surface of the somites; these cells eventually are found between the two melanophore stripes and in the dorsal fin. We were interested in determining the roles of the extracellular matrix (ECM) in controlling the formation of pigment cell patterns in T. torosa. Immunocytochemistry, Alcian blue staining of paraffin sections and ruthenium red staining of thin sections (accompanied by Streptomyces hyaluronidase and chondroitinase ABC digestion) were used to identify the composition and distribution of the ECM surrounding the pigment cells at various stages during development. The adhesive glycoprotein fibronectin is found in the dorsal portion of the lateral neural crest migratory pathway as well as in the dorsal fin matrix. Glycosaminoglycans (GAG) are found primarily in the dorsal fin and in the ECM surrounding the notochord. The dorsal fin ECM contains hyaluronate (HA), which was identified in the TEM as Streptomyces hyaluronidase-sensitive 3–5 nm microfibrils, as well as sulfated proteoglycan aggregates. We then confronted T. torosa neural crest cells in vitro with known ECM molecules. When neural folds are explanted onto tissue culture plastic in half-strength L-15 medium containing 10% fetal calf serum (FCS), cells migrate from the explant and differentiate into melanophores after 6 to 9 days. Xanthophores appear in the cultures 2 to 4 days after the appearance of melanophores. When cultured on three-dimensional collagen gels, xanthophores migrate significantly farther (P < 0.01) onto and into the collagen than melanophores (336 ± 183 vs 196 ± 160 μm from the edge of the explant). When 2.5 mg/ml chondroitin sulfate (CS) is present in the collagen gel, the distance that both pigment cell types migrate from the explant is reduced, with the result being that only xanthophores invade the GAG-rich matrix. When 1 mg/ml HA is present in the collagen gel, the differentiation of pigment cells is inhibited. Melanophores appear 48 hr later than in control gels without HA, and the number of melanophores in the explant after 10 days is significantly reduced (P < 0.01; 26.6 vs 1.1 melanophores/explant). When 1 mg/ml of HA is added to the FCS-enriched medium over neural crest cells spreading on tissue culture plastic, there is a similar delay and inhibition of pigment cell differentiation. With 2 mg/ml of CS there is no effect on pigment cell differentiation in vitro. Melanophores eventually appear in the dorsal fin of T. torosa several weeks after hatching. When fragments of dorsal fin that contain no apparent melanophores are transferred onto tissue culture plastic, melanophores appear in the explants after a few days in culture. These results suggest the following model of ECM-cell interactions during pigment cell pattern formation in T. torosa: Pigment cells differentiate in regions of the embryo that contain relatively little GAG. Xanthophores are able to invade the GAG-rich dorsal fin, but melanophores can not. The melanophores that eventually appear in the dorsal fin are derived from the neural crest cells that invaded the fin during early development, and were delayed in differentiating by the presence of HA.     

The cell cycle and transplantation of blastula nuclei in Bombina orientalis

The ability of blastula nuclei in various portions of the cell cycle to support the development of recipient eggs was examined. A non-nutritive saline medium was developed in which isolated blastula cells remained mitotically active. This medium also allowed for ready separation of daughter cells for nuclear transplantation. Donor nuclei were derived from mitotically synchronized populations of blastula cells, obtained by a manual isolation procedure. Nuclei taken from late portions of the cell cycle frequently induced a premature second-cleavage furrow, resulting in abnormal development. However, portions of the cell cycle that preclude the participation of the donor nucleus in normal development were not detected. The donor nuclei need not have initiated or completed the S phase, nor must they be “synchronized” with the recipient egg cytoplasm.     

Evolutionary history of the oriental fire‐bellied toad (Bombina orientalis) in Northeast China

The evolutionary history of a species is generally affected by the combination of geological events and climate fluctuations. By analyzing the population features, genetic structure and the effective population historical dynamics of existing species, the population evolutionary history can be reestablished. In recent years, geological evidence shows that the Yilan–Yitong fault zone located in Northeast Asia experienced strong and frequent geological changes in the late Quaternary period. Species population history has been shaped by the combination of the complex climatic conditions of the Quaternary and Pleistocene glacial interglacial cycles and palaeogeological events in Northeast Asia and it has become a research focus for evolutionary biology researchers. In this study, mitochondrial and microsatellite molecular markers were used to reveal the population features, genetic structure, and the effective population historical dynamics of the Oriental fire‐bellied toad (Bombina orientalis). The results showed that the strong seismic activity of the Yilan–Yitong fault zone in the late Quaternary period was the main reason for the population differentiation of Oriental fire‐bellied toad in northeast China. The Quaternary Pleistocene glacial interglacial cycles led to the significant bottleneck effect of the western population located in the Maoer mountain area. As a result, the western population has low genetic diversity. Recent gene flow between eastern and western populations and historical evidence of population expansion proved that the dispersal behavior of the western populations was the main cause of the low genetic diversity and mitochondrial and nuclear discordance. Human economic activity may be the mainly driving factor. These evidences showed that the comprehensive influence of geology, climate, human activities and other factors should be considered in the process of exploring the evolutionary history of species.     

Mating patterns in a hybrid zone of fire-bellied toads (Bombina): inferences from adult and full-sib genotypes

We present two novel methods to infer mating patterns from genetic data. They differ from existing statistical methods of parentage inference in that they apply to populations that deviate from Hardy-Weinberg and linkage equilibrium, and so are suited for the study of assortative mating in hybrid zones. The core data set consists of genotypes at several loci for a number of full-sib clutches of unknown parentage. Our inference is based throughout on estimates of allelic associations within and across loci, such as heterozygote deficit and pairwise linkage disequilibrium. In the first method, the most likely parents of a given clutch are determined from the genotypic distribution of the associated adult population, given an explicit model of nonrandom mating. This leads to estimates of the strength of assortment. The second approach is based solely on the offspring genotypes and relies on the fact that a linear relation exists between associations among the offspring and those in the population of breeding pairs. We apply both methods to a sample from the hybrid zone between the fire-bellied toads Bombina bombina and B. variegata (Anura: Disco glossidae) in Croatia. Consistently, both approaches provide no evidence for a departure from random mating, despite adequate statistical power. Instead, B. variegata-like individuals among the adults contributed disproportionately to the offspring cohort, consistent with their preference for the type of breeding habitat in which this study was conducted.     

Hybridization between the fire-bellied toads Bombina bombina and Bombina variegata in the karst regions of Slovakia and Hungary: morphological and allozyme evidence

Geographic variability and genetic interactions in the contact zone between the fire-bellied toads, Bombina bombina and B. variegata, were studied using analysis of morphological and genetic variation in sixteen samples from the Slovak Karst and Aggtelek Karst regions. Genotype frequencies at four marker loci (Ldh-1, Mdh-1, Adk, Hem) demonstrate the existence of a hybrid zone with highly variable population structures. While some samples appear to represent panmictic hybrid populations, other samples are very heterogeneous. Pure individuals of both species occurred together with hybrids at one site. Habitat segregation among genetically differentiated demes probably causes this heterogeneity of population structures. Increased frequencies of the allele Ldh-1^M, which is present in low proportion in B. bombina south of the contact zone, were found in some hybrid populations.