Morphology of the kidney in larvae of Bufo viridis (Amphibia, Anura, Bufonidae)

This study deals primarily with the morphology and ultrastructure of the pronephros in the green toad Bufo viridis during prometamorphosis when the pronephros and the developing mesonephros function simultaneously. Furthermore, the mesonephros was studied during pro- and postmetamorphosis with emphasis on the distal segments of the nephron. The paired kidneys consist of two cranial pronephroi immediately behind the gill region and two more caudal elongated mesonephroi. Each pronephros consists of a single convoluted tubule which opens into the coelom via three nephrostomes. This tubule is divided into three ciliated tubules, three proximal tubule branches, a common proximal tubule and a distal tubule, which in turn continues into the nephric duct. No intermediate segment is present. The length of the pronephric tubule is 12 mm, including the three branches of the ciliated tubules and proximal tubules. Primary urine is formed upon filtration from an external glomerulus, which is a convoluted capillary lined by podocytes, a specialization of the coelomic epithelium. From the coelom the filtrate is swept into the ciliated tubules. In the collecting duct system of the developing mesonephric nephron epithelial cells with conspicuous, apical osmiophilic granules appear in larvae of 9-10 mm. Heterocellularity of mixed intercalated (mitochondria rich) cells and principal cells is observed in the collecting duct system and nephric duct from a larval body length of 14 mm. As the proliferation of mitochondria-rich cells proceeds, the osmiophilic granules disappear and are completely absent from the adult amphibian mesonephros.     

Temporal stability in hybridization between Bufo microscaphus and Bufo woodhousii (Anura: Bufonidae): Behavior and morphology

I analyzed temporal variation in hybridization between the southwestern toad (Bufo microscaphus) and Woodhouse's toad (Bufo woodhousii) along the Virgin River in southwestern Utah and northwestern Arizona. Bufo woodhousii is largely restricted to the floodplain of the Virgin River from southern Nevada to the vicinity of St. George, Utah. By contrast, B. microscaphus is confined primarily to the tributaries of the Virgin River, only occupying the Virgin River proper exclusive of B. woodhousii along its upper reaches east of La Verkin Creek. As in all other zones of sympatry for these bufonids, behavior and morphology provide clear evidence of hybridization at a number of sites along the Virgin River. Analysis of morphology and behavior indicates that the distribution of these taxa and their hybrids is largely unchanged relative to that documented forty years ago by Blair (1955). Comparisons of morphological hybrid index scores reveal slight shifts in relative numbers of parental taxa at only one site. Hybridization between these anurans has been relatively stable geographically, and may be related to the nature of the riparian habitat available for breeding.     

Molecular systematics of African 20-chromosome toads (Anura: Bufonidae)

Intrageneric lineages and the historical biogeography of toads, genus Bufo, are poorly resolved due to their conservative morphology, their highly conserved karyotypes (typically 2N = 22), and erratic patterns of interspecific hybridisation. Here, we use mitochondrial and nuclear DNA sequence data to reconstruct relationships of the 20-chromosome toads, a major component of the African bufonid fauna. Mitochondrial 12S and 16S sequences from 29 species revealed two independent transitions between phenotypically distinct savannah and forest adapted forms. Analyses of mitochondrial 12S, 16S, and ND2, along with nuclear ACTC and Rhodopsin sequences from 12 species greatly increased bootstrap, and likelihood support for internal branches including a basal split into two pan-African 20-chromosome clades. Hybridisation is a weak indicator of phylogenetic relationship as it occurs across these deeply divergent clades, between Bufo rangeri and Bufo gutturalis. These analyses suggest a secondary reversion to 22-chromosomes in Bufo pardalis, within the 20-chromosome group, although we could not reject an alternative hypothesis that this lineage forms a sister to all 2N = 20 toads. Other informally recognised 22-chromosome groups form independent phylogenetic lineages outside the 20-chromosome group, such as the Angusticeps and Vertebralis divisions. Bufo lindneri, from the Taitanus division, is closely related to Stephopaedes anotis, and these species should be considered congeners.     

Ultrastructural aspects of gonadal morphogenesis in Bufo bufo (Amphibia Anura) 1. Sex differentiation

The morphogenesis of gonads in Bufo bufo tadpoles was studied, and ultrastructural differences between sexes were identified. All specimens analyzed initially developed gonads made up of a peripheral fertile layer (cortex) surrounding a small primary cavity. Subsequently a central layer of somatic cells (medulla) developed. Both layers were separated by two uninterrupted basal laminae between which a vestige of the primary cavity persisted. During female differentiation, the peripheral layer continued to be the fertile layer. In males, the central layer blended into the peripheral layer and the basal laminae disappeared. The somatic cells of the central layer came into direct contact with the germ cells; this did not occur in females. Testicular differentiation continued with the migration of germ cells towards the center of the gonad. The somatic elements surrounding the germ cells appeared to play an active role in their transfer to the center of the gonad. The peripheral layer shrank and became sterile. Two basal laminae then re-formed to separate the fertile central layer from the peripheral sterile one. Germ cells have always been thought to perform a passive role in sex differentiation in amphibians. Following the generally accepted "symmetric model", the mechanism of gonad development is symmetrical, with cortical somatic cells determining ovarian differentiation and medullary somatic cells determining testicular differentiation. In contrast, we found that sex differentiation follows an "asymmetric" pattern in which germ cells tend primarily toward a female differentiation and male differentiation depends on a secondary interaction between germ cells and medullary somatic cells.     

Molecular phylogeny and biogeography of West Indian toads (Anura: Bufonidae)

Toads of the Bufo peltocephalus Group (Anura: Bufonidae) occur throughout the Greater Antilles (Cuba to the Virgin Islands), a geographic region of relatively high endemicity. Previous morphological and immunological studies suggested that the West Indian toads are a monophyletic lineage derived from Neotropical Bufo but were unable to clarify relationships within the group. We examined the evolutionary relationships and biogeography of this group of frogs by collecting approximately 2 kb of mitochondrial DNA sequence data from eight West Indian species and selected non-West Indian species from the New World and the Old World. Our analyses support the monophyly of native West Indian toads and a New World origin for the group. Relationships among the West Indian species are less certain, but a Cuban lineage is defined in most analyses.     

A new Ansonia from the Isthmus of Kra, Thailand (Amphibia, Anura, Bufonidae)

A new species of torrent-dwelling bufonid frog of the genus Ansonia is described from the Isthmus of Kra, Thailand. Ansonia kraensis is morphologically similar to Malaysian A. malayana, but differs from it in ventral coloration and larval morphology. Occurrence of A. kraensis in this region suggests a heterogeneous nature of the anuran fauna between northern and southern regions of the Malay Peninsula.     

Interspecific patterns for egg and clutch sizes of African Bufonidae (Amphibia: Anura)

Little is known about reproductive trade-offs in African amphibians, but such data, particularly in the form of quantitative measurements, are a key for investigating life history evolution. Here we compile and analyze known data on African bufonids from published material and new data from preserved museum specimens, to investigate interspecific patterns of egg and clutch sizes variation. Our data is a composite of mixed sources, including ova data from dissected females and laid clutches from observations in the field. Our study shows that, as body size increases, clutch size increases but egg size decreases, and when correcting for body size, egg size is inversely correlated with clutch size. These parameter interactions however, are different for different reproductive modes. In free-swimming larval developing species, the same trends are recovered, but for lecithotrophic viviparous species no significant correlations could be recovered for clutch size and body size nor for the trade-off between clutch size and egg size, and egg size is positively related to body size. The egg size of Nimbaphrynoides occidentalis (Angel, 1943) is a clear outlier, which may be due to its matrotrophic viviparous reproduction. In addition, we observed no statistical difference between ova data collected from dissections and laid clutch data from field observations, which suggests that such a mixed dataset has utility in comparative analyses.     

Advertisement calls of the midwife toads Alytes (Amphibia, Anura, Discoglossidae) in continental Spain

The advertisement calls of six Spanish populations of midwife toads (genus Alytes ) are described presenting characteristic audiospectrograms and oscillograms of the calls. Numerical data concerning the spectral and temporal features of the calls in relation to caller size (SVL) and temperature are also compared. The regression temperature vs. call duration groups the different populations according to the extant recognized phylogeny of the genus. Based on this character, the montane population of A. obstetricans from Penalara (Madrid) appears to be more closely related to the Pyrenean populations of A. o. almogavarii than to the more western populations of A. o. boscai in Central Spain, which are closer geographically.

Zusammenfassung

Paarungsrufe der Geburtshelferköten Alytes (Amphibia, Anura, Discoglossidae) aus zentralspanischen Populationen
Die Paarungsrufe von sechs Geburtshelferkröten aus sechs spanischen Populationen werden hinsichtlich ihrer charakteristischen Tonfrequenz-Spektrogramme und Oszillogramme beschrieben.
Die numerischen Daten der spektralen und zeitlichen Eigenheiten der Rufmerkmale wurden mit der Größe der Männchen (SVL) und der Temperatur verglichen. Die Regression zwischen Temperatur und Dauer des Rufes ordnet die verschiedenen Populationen in der gleichen Weise ein, wie sie schon aus der allgemein anerkannten Phylogenetik der Gattung bekannt ist. Aufgrund dieser Untersuchungen, kann davon ausgegangen werden, daß die Gebirgspopulation A. obstetricans von Pealara (Madrid) enger mit der Pyrenäen-Population A. o. almogavarii verwandt ist als mit der westlichen Population A. o. boscai in Zentralspanien, obwohl sie zu jener geographisch näher liegt.     

Testicular myxosporidiasis in the flat-backed toad, Bufo maculatus (Amphibia: Bufonidae), from Cameroon, Africa.

Macroscopic cysts measuring less than or equal to 860 x 500 microns were found in the testes of a flat-backed toad, Bufo maculatus, collected in Cameroon, West Africa. On histologic examination, the cysts contained numerous spores of a Myxobolus sp. (Myxozoa: Myxobolidae). Spores in fixed tissues measured 9.2 microns long, 8.9 microns wide, and 4.0 microns thick; the range of values for length, width, and thickness were 8.8 to 9.6 microns, 8.6 to 9.4 microns, 3.6 to 4.4 microns, respectively (n = 20). The shape index (length/width) was 1.03, and ranged from 1.00 to 1.09. Pathology was limited to a slight constriction of adjacent seminiferous tubules by the cysts. No host inflammatory response was noted. This myxozoan is distinct from all other members of the genus infecting anurans and is assigned the name Myxobolus bufonis sp. n.     

Structural and mechanical aspects of the skin of Bufo marinus (Anura, Amphibia)

Specific biomechanical characters and some structures possibly related to them were investigated in the skin of the toad Bufo marinus using tensile testing techniques (at constant strain till rupture) as well as morphological methods (histological, immunohistochemical and electronmicroscopical). Mechanical parameters of the native skin varied considerably according to sex, individual variability and/or site of specimen collection. In skin strips of males and females excised from different parts of the body thickness ranged from 0.45 to 0.87 mm, strain (epsilonf) from 96.52 to 211.03, tensile strength (sigmam) from 5.72 to 9.38 MPa, and stiffness (E-modulus) from 5.76 to 6.73. The dermis of B. marinus is provided with a collagenous stratum compactum of considerable thickness, a stratum spongiosum with loosely arranged fibres and a marked calcified layer (substantia amorpha). Collagen appears to be the main determinant of skin mechanics. However, the slope of the J-shaped static stress-strain curves indicates elastin to be responsible for the high values of strain. Contrary to van Gieson and orcein staining, immunostaining with a monoclonal antibody against elastin revealed very few elastic fibers between collagen bundles and in the vertical fiber tracts (perforating bundles), but a considerable amount in the tela subcutanea. This was partly confirmed at the ultrastructural level by tannic acid staining.     

Advertisement calls of the midwife toads Alytes (Amphibia, Anura, Discoglossidae) in continental Spain

The advertisement calls of six Spanish populations of midwife toads (genus Alytes) are described presenting characteristic audiospectrograms and oscillograms of the calls. Numerical data concerning the spectral and temporal features of the calls in relation to caller size (SVL) and temperature are also compared. The regression temperature vs. call duration groups the different populations according to the extant recognized phylogeny of the genus. Based on this character, the montane population of A. obstetricans from Penalara (Madrid) appears to be more closely related to the Pyrenean populations of A. o. almogavarü than to the more western populations of A. oboscai in Central Spain, which are closer geographically.     

Acanthocephalus bufonis (Acanthocephala) from Bufo marinus (Bufonidae: Amphibia) in Hawaii.

During a survey of the helminth parasites of the introduced toad, Bufo marinus, on O'ahu, Hawaii, an acanthocephalan corresponding to Acanthocephalus bufonis (Shipley, 1903) was found in the intestinal tract. This is a new host and locality record for A. bufonis which has only previously been recorded from amphibians in the Orient. Possible mechanisms for the introduction of A. bufonis to Hawaii, and its transmission to the toad, are discussed. Almost 98% of toads were infected with a mean intensity of 28.6 acanthocephalans per infected toad. There was a significant negative correlation between host length and intensity of infection with subadult toads having significantly higher infection levels than adult male and female toads. Trunk length of both male and female acanthocephalans was significantly related to host length.     

Yolk proteins and their plasmatic precursor in the tetraploid Odontophrynus americanus (Amphibia, Anura)

The vitellogenin of Odontophrynus americanus is a large (426.5 kDa) plasmatic protein. The vitellogenin is composed of two different phosphoglycopeptides: VTG1 = 207.5 kDa and VTG2 = 202.4 kDa. The vitellins originating from the partial proteolysis of the plasmatic vitellogenin on the ovary cells are composed of lipovitellins and phosphoproteins. Lipovitellin 1 has two glycopeptides with different amino acid sequences as determined by peptide mapping (LV1 alpha, 104.6 kDa; and LV1 beta, 92.6 kDa). Lipovitellin 2 is composed of three kinds of polypeptides (LV2 alpha, 31.7 kDa; LV2 beta, 29.7 kDa; LV2 gamma, 27.8 kDa). There are three phosphopeptides in the yolk: phosvitin (PV, 37.4 kDa) and phosvettes 1 (PVT1, 27.7 kDa) and 2 (PVT2, 26.1 kDa).     

Positive Darwinian selection results in resistance to cardioactive toxins in true toads (Anura: Bufonidae)

Members of the Family Bufonidae, true toads, are famous for their endogenously synthesized cardioactive steroids that serve as defensive toxins. Evolution of resistance to these toxins is not understood. We sequenced a key region of the toxin''s binding site in the Na⁺/K⁺ ATPase for relevant taxa representing Hyloidea (including bufonids), Ranoidea and Archaeobatrachia and tested for positive selection in a phylogenetic context. Bufonidae were distinct from other Hyloidea at 4–6 of 12 sites and, with one exception, had a homologous amino acid sequence. Melanophryniscus stelzneri had a distinct sequence, consistent with other independent evidence for a differentiated toxin. Tests within Bufonidae detected positive selection within the binding region, providing, to our knowledge, the first evidence of this type for positive selection within Amphibia. There was no evidence for positive selection on Bufonidae or M. stelzneri lineages. Sequence change in Leptodactylus ocellatus, a leptodactylid predator of Bufonidae, provides a molecular basis for predator resistance possibly associated with gene duplication.     

The filter apparatus of Rana temporaria and Bufo bufo larvae (Amphibia,Anura)

Summary In larvae of Rana temporaria and Bufo bufo the location of filter apparatus within the larval organization, the arrangement of the morphological parts as branchial food trap, ventral velum, and filter rows, as well as their surface anatomy, are similar to that of other species of Orton's larval type IV. The means by which mucous with its entrapped food particles is transported from the filter rows to the esophagus is finally resolved. The dorsally positioned ciliary cushion extends far ventrally between the filter plates. From their contact with the filter rows, the cilia transport the mucous to Kratochwill's caudally positioned Flimmerrinne and from there to the esophagus. The original chordate principle of mucous entrapment and ciliary transport is thus retained by these anuran larvae. The only modification specific to the latter is the division into a ventral filter apparatus, whose epithelia serve for mucus entrapment, and a dorsal ciliary area.Six different types of cell may be distinguished ultrastructurally: (1) The ubiquitous squamous epithelium with merocrine extrusions; (2) the large supporting cells of the filter rows and of the ventral velum; (3) the ciliary cells of the ciliary cushion; (4) three different types of mucous producing secretory cells: (a) A type of cell similar to the goblet cell is found in the ciliary cushion (merocrine extrusion); (b) The secretory pits of the ventral velum and the secretory ridges have similar bottle-shaped merocrine secretory cells; (c) The merocrine apical cells of the filter rows are the final kind. It is evident that the ciliary cushion epithelium resembles that of both the manicotto glandulare of anuran larvae and the trachea and bronchus of Mammalia.Supported by the Deutsche Forschungsgemeinschaft-DFG     

The calcified amorphous layer of the skin of Bufo marinus (Amphibia: Anura)

The integument of Bufo marinus is surveyed. It is similar to that of other anurans and contains the three chromatophores common to anurans. The skin of these toads contains more than 28% minerals deposited as small crystals in a mucopolysaccharide-positive amorphous layer between the stratum compactum and the stratum spongiosum. These crystals reveal a high content of calcium and phosphorus aggregated in and near membrane-bound vesicles which have an appearance very similar to matrix vesicles. Electron microscopy gives the appearance that these vesicles are associated with flbroblasts located between alternating bundles of collagen. Histochemical studies indicate that the amorphous layer possesses many of the characteristics commonly associated with mineralized cartilage or bone.     

Checklist of helminth parasites of the cane toad Bufo marinus (Anura: Bufonidae) from Mexico

Thirty-four adult cane toads Bufo marinus L. (12 males and 22 females) collected from 2 localities in Mexico (Cerro de Oro and Temascal Dams, Oaxaca) in September 2003 were examined for helminth parasites. In total, 14,749 helminths belonging to 14 taxa were collected. Included were 2 adult digeneans (Choledocystus hepaticus, Mesocoelium monas); 1 larval cestode (an unidentified pseudophyllidean); and 11 nematodes, including 3 species of larvae (Contracaecum sp., Physaloptera sp., Physocephalus sexalatus) and 8 species of adults (Aplectana itzocanensis, Cosmocerca sp., Cruzia morleyi, Ochoterenella digiticauda, Oswaldocruzia sp., Raillietnema sp., Rhabdias americanus, and Rhabdiasfuelleborni). Higher species richness was recorded in B. marinus from Cerro de Oro (12 taxa versus 9 in those from Temascal); hosts from both localities shared 7 taxa. There were 25 new locality records, and 2 taxa were registered in Mexico for the first time. To date, 112 helminth species have been recorded parasitizing B. marinus along its native and introduced range of distribution, with 40.5% of them reported from Mexico.     

Cytoplasmic Influence on Lethality in Nucleocytoplasmic and Natural Hybrids between Bufo Calamita Laur and Bufo Bufo L. (Amphibia, Anura)

Nucleocytoplasmic interactions have been studied through nuclear and cytoplasmic grafts. Bufo bufo nuclei transplanted in Bufo calamita eggs provoked 95% lethality during gastrulation. Influence of the cytoplasm in such a nucleocytoplasmic incompatibility has been studied through cytoplasmic fractionation and grafting of the fractions obtained. Fractionation of egg cytoplasm gave three fundamental fractions: lipid, hyaline cytoplasm and vitellus. After injection of B. calamita fractions into B. bufo fertilized eggs, the hyaline cytoplasmic fraction provoked a gastrulation block in the same ratio (95%) as in the interspecific nuclear graft experiment. Influence of the hyaline cytoplasmic fractions of both species has been tested on nucleocytoplasmic and natural hybrid developments. The B. calamita hyaline cytoplasmic fraction provoked in each case a developmental block at the beginning of gastrulation. The B. bufo hyaline cytoplasmic fraction acts later at the yolk plug stage and in some cases partially corrects the developmental bolck or exogastrulation of young hybrid gastrula. It can be generalized that in both kinds of hybrids, the hyaline cytoplasmic fraction of B. calamita exerted a lethal effect on B. bufo nucleus.