[Intensity of respiration and oxidative phosphorylation in the ontogeny of tailless amphibia (Anura, Amphibia)].
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Amphibia Kupffer cells (i.e., liver resident macrophages) show many common characteristics when compared with Mammalia Kupffer cells: filopodia, microvillous-like structures, lamellipodia, fuzzy coat, coated vesicles, bristled vacuoles, nonspecific esterase activity, and pinocytotic and phagocytic activity are present both in Amphibia and Mammalia Kupffer cells. On the other hand, some differences are present between Kupffer cells of both zoological classes: phagocytosed red cells and their derivatives, iron-protein complexes, and lipofuscin bodies are normally present in Amphibia Kupffer cells, but absent in the same cells of healthy mammals. Worm-like structures are not seen in Amphibia and endogenous peroxidase activity is very weak in these animals compared with Mammalia. The most important difference lies in the ability of Amphibia Kupffer cells to produce melanins: in fact the tyrosinase gene is expressed, "melanosome centers" are present, and dopa oxidase activity is demonstrable.
It has been suggested that the amino acid sequence of pancreatic polypeptide (PP) may provide a useful molecular marker with which to study evolutionary relationships between tetrapods but few PP sequences from amphibia are available to test this hypothesis. PPs have been purified from the pancreata of five species belonging to the different orders of amphibians. Their amino acid sequences were established as: APSEPEHPGD10 NASPDELAKY20 YSDLWQYITF30 VGRPRY for the lesser siren, Siren intermedia (Caudata); GPTEPIHPGK10 DATPEELTKY20 YSDLYDYITL30 VGRSRW for the caecilian, Typhlonectes natans (Gymnophiona); and TPSEPQHPGD10 QASPEQLAQY20 YSDLWQYITF30 VTRPRF for the cane toad, Bufo marinus (Anura). The structure of Rana sylvatica PP is the same as that of Rana catesbeiana PP whereas PP from the green frog Rana ridibunda contains one substitution (His6 --> Gln). The data provide further support for the conclusion that the amino acid sequence of PP has been poorly conserved during evolution with only 17 residues invariant among the eight species of amphibia yet studied and only 8 residues (Pro5, Pro8, Gly9, Ala12, Leu24, Tyr27, Arg33, and Arg35) invariant among all tetrapods. A maximum parsimony analysis based upon the amino acid sequence of PP and using the sequence of frog PYY as outgroup to polarize the in-group taxa generates a consensus phylogenetic tree in which the Amniota and Amphibia form two distinct clades. However, such a tree does not permit valid conclusions to be drawn regarding branching order within the Amphibia.
This survey suggests that about 115 000 amphibia, of which more than half were Rana temporaria, were supplied for use in teaching and research in the UK during 1977. As only 27 000 were recorded as being used by universities, polytechnics and research institutes, probably mostly for teaching, it must be assumed that the majority are used for teaching purposes in schools. There has been an increase in the proportion of amphibia supplied by Recognised Suppliers, and also in that being 'purpose-bred' although breeding in the laboratory is still restricted to Xenopus laevis and Ambystoma mexicanum. Probable cost appears to be a deterent to the use of purpose-bred amphibia. A large number of respondents had encountered problems with using amphibia, mainly losses due to 'red leg' and arrival from the supplier in poor condition, probably interrelated.
In the lungs of 12 species of Amphibia investigated so far, solitary neuroendocrine (NE) cells, as well as groups of these cells called "neuroepithelial bodies" (NEB), are observed. They occur in the position strategic to monitoring gas composition, mainly in the ciliated epithelium of the apical part of the septa. A great diversity in the structure of NEB is observed. The NE cells and NEB in Amphibia are predominantly of the "closed type," separated from the air space by a thin cytoplasmic layer of ciliated cells, goblet cells or pneumocytes. In two species, Bufo marinus and Ambystoma tigrinum, the "open type" of NEB occur, where NEB communicate with the air space, by apical cells in Bufo and type II NE cells in Ambystoma. Both types of cells possess single atypical cilia with an 8+1 microtubule arrangement and microvilli on the free surface. Single and grouped NE cells are characterized by small dense core vesicles (DCV) dispersed in the cytoplasm. In Salamandra and Ambystoma the second type of NE cells with large DCV are observed. The DCV represent sites of storage of serotonin and several neuropeptides. The basal parts of NEB and of some solitary cells are invested by the intraepithelial sensory nerve terminals, both afferent and efferent morphologically. In the lungs of Amphibia, similarly to other vertebrates, NE cells, which act as endocrine/paracrine receptors, form epithelial endocrine systems.
A modified BrdU-Hoechst-Giemsa technique permitted the demonstration of easily reproducible replication patterns in the somatic chromosomes of Amphibia. These banding patterns allow for the first time a precise identification of all chromosomes and the analysis of the patterns of replication in the various stages of S-phase in Amphibia. Several possibilities for the use of this technique were demonstrated on three frog species of the family Ranidae, all differing greatly in their DNA-content. With this method, the homomorphic chromosome pair No. 4 in Rana esculenta could be identified as sex-specific chromosomes of the XX/XY-type. All male animals exhibit an extremely late replicating region in the Y-chromosome, which is lacking in the X-chromosome in the female animals, both X-chromosomes replicate synchronously. These sex-specific chromosomes cannot be distinguished by other banding techniques. In the highly heteromorphic ZZ/ZW-sex chromosome system of Pyxicephalus adspersus a synchronous replication of the two Z-chromosomes of male animals and a very late replication of the short arm of the W-chromosomes of male animals was demonstrated. These results support the assumption that there is no dosage compensation for Z-linked or X-linked genes by the sex chromosome inactivation mechanism in the sex chromosomes of Amphibia.
The jelly coat surrounding the eggs of amphibia is composed of oviducal mucins and plays an important role in the fertilization process. From a structural and chemical point of view, these jellies are very different from one species to another. Bufo viridis is the 13th amphibia species studied in term of carbohydrate structural analysis. The oligosaccharides have been released from the oviducal mucins by reductive beta elimination, purified by various chromatography procedures and analyzed by (1)H and (13)C 1D-2D NMR spectroscopy. Among the 15 compounds, ten have novel structures, although they possess some well-known structural patterns as blood group epitopes (Le(x), Le(y)) or other sequences already observed in other amphibia species. These results reinforce our hypothesis about the strict species-specificity of these carbohydrate chains. It must be noted that such species-specificity does not depend on one particular monosaccharide but it is rather due to a set of particular tri- or tetrasaccharide sequences. Hence, B. viridis species could be characterized by the simultaneous presence of a 2,3,6-trisubstituted galactosyl residue, the GlcNAc(beta 1-3)[Fuc(alpha 1-4)]GlcNAc beta sequence and the Le(x), Le(y) or Cad determinants. The anionic charge of the oligosaccharides is carried only by sialic acid alpha-(2-->6)-linked to GalNAc-ol residue as in Bufo bufo or in Bufo arenarum.
The phylogenetic relationship among toad, frogs and newt from Japan was investigated by means of immunoelectrophoresis. Homologous, heterologous and non-identity reactions between rabbit anti-Bufo bufo antiserum and antigen from each of 13 species and 2 subspecies of Japanese amphibia were examined. The number and positions of arcs appeared in these reactions were compared. The relationship observed in the comparison well coincided with the classification of the upper taxa of amphibia; viz 1) Hyla arborea is more intimately related to B. bufa, 2) members of Ranidae and Rhacophoridae have equal intimacy for B. bufo, and 3) Cynops pyrrhogaster is most distantly related to B. bufo. By contrasting arc obtained in non-identity reaction and those obtained in homologous reaction, it was found that toad, B. bufo carried 22 antigens, and among these antigens, one was observed through all amphibians tested, 12 were carried solely by toad, and some of the remainders were common to antigens carried by various frogs. Newts carried only one antigen common to toad. Based on these results, the phylogeny of Japanese amphibia was discussed.
A three-year field study was conducted during 1993 to 1995 to determine the importance of mosquitoes in the diet of anurans. The study was aimed to assess the impact of biological mosquito control on the populations of amphibia in the Rhine Valley, Germany. Sampling took place in two areas with stands of different deciduous trees at the western bank of the Rhine (north of Karlsruhe, Germany) from early May to late October. The frequency and species composition of the terrestrial stages of Amphibia was monitored by hand catches and by live pitfall trapping. A total of 2,419 Amphibia were caught in the three years. Of these, 95.8% were anurans, consisting of 77% Ranidae, with 25.5% Rana arvalis, and 4.2% were Urodela, Salamandridae. All anura caught were subjected to "stomach flushing" to yield their stomach contents before they were released again. The stomachs of 2,163 anuran specimens contained an average of 7.7 prey items, of which only 0.16% were Culicidae. In R. arvalis the total diet consisted of 33% Collembola, spiders and beetles; 0.1% of the specimens in the diet were Culicidae. The most common culicid species in the study area, Aedes vexans, was also most often found in the anuran stomachs. However, no correlation existed between the number of mosquitoes and their number as prey of Anura. It is concluded that anurans will not be negatively affected by biological mosquito control in the Rhine Valley. Furthermore, the impact of anurans on Culicidae seems to be negligible.
Immunoreactive insulin-like growth factors I and II (IGF-I, IGF-II) were sought in the endocrine pancreas of representative birds, reptiles, and amphibia using antisera specific for mammalian IGF-I and IGF-II and the classical islet hormones insulin (INS), glucagon (GLUC), somatostatin (SOM), and pancreatic polypeptide (PP) in double immunofluorescence. Both IGF-I and IGF-II immunoreactivities were present in the endocrine pancreas of all species. IGF-II immunoreactivity was exclusively found in INS-immunoreactive (-IR) cells, indicating evolutionary conservation of the islet IGF-II system. In contrast, IGF-I immunoreactivity was distributed differently among the species and never occurred in INS-IR cells. In the anuran Xenopus laevis, IGF-I immunoreactivity was present in islet cells showing coexistence of GLUC and PP immunoreactivities. In reptiles, the lizards (Lacerta viridis, Scincus officinalis) exhibited IGF-I immunoreactivity in PP-IR and SOM-IR cells and the snakes (Psamophis leniolatum, Coluber ravergieri) in SOM-IR and GLUC-IR cells. In birds, IGF-I immunoreactivity was located either in SOM-IR cells only (Gallus g. domesticus, Streptopelia roseogrisea) or in PP-IR and SOM-IR cells (Coturnix c. japonica). Thus, the distribution patterns of islet IGF-I immunoreactivities in birds, reptiles, and amphibia are equivalent to those in mammals and most bony fish. They differ, however, from those found in cartilaginous fish, cyclostomes, and protochordates, where a total or partial coexistence of IGF-I and INS immunoreactivities has been obtained. Therefore, the divergence of IGF-I and INS seems to have occurred early in vertebrate phylogeny. Furthermore, the existence of IGF-I immunoreactivity likely is common in the islets of all vertebrates. Finally, no phylogenetic trend to concentrate IGF-I immunoreactivity in a particular islet cell type is apparent.
In this chapter the different categories of homomorphic and heteromorphic sex chromosomes, types of sex-determining mechanisms, known sex-linked genes, and data about sex-determining genes in the Amphibia have been compiled. Thorough cytogenetic analyses have shown that both XY/XX and ZW/ZZ sex chromosomes exist in the order Anura and Urodela. In some species quite unusual systems of sex determination have evolved (e.g. 0W-females/00-males or the co-existence of XY/XX and ZW/ZZ sex chromosomes within the same species). In the third order of the Amphibia, the Gymnophiona (or Apoda) there is still no information regarding any aspect of sex determination. Whereas most species of Anura and Urodela present undifferentiated, homomorphic sex chromosomes, there is also a considerable number of species in which an increasing structural complexity of the Y and W chromosomes exists. In various cases, the morphological differentiation of the sex chromosomes occurred as a result of quantitative and/or qualitative changes to the repetitive DNA sequences in the constitutive heterochromatin of the Y and W chromosomes. The greater the structural differences between the sex chromosomes, the lesser the extent of pairing in meiosis. No dosage compensation of the sex-linked genes in the somatic cells of the homogametic (XX or ZZ) individuals have been detected. The genes located to date on the amphibian sex chromosomes lead to the conclusion that there is no common ancestral or conserved sex-linkage group. In all amphibians, genetic sex determination (GSD) seems to operate, although environmental factors may influence sex determination and differentiation. Despite the accumulated evidence that GSD is operating in Anura and Urodela, there is little substantial information about how it functions. Although several DNA sequences homologous to the mammalian ZFY, SRY and SOX genes have been detected in the Anura or Urodela, none of these genes is an appropriate candidate to explain sex determination in these vertebrates.
The oral, percutaneous and subcutaneous routes of infection of Oswaldocruzia filiformis were investigated in amphibia. Tadpoles of Bufo bufo and Rana temporaria can be infected with O. filiformis when kept temporarily in a suspension of infective larvae in water. Larval stages and subadults were found in tadpoles. All stages of the parasite, including egg-producing females, were found after metamorphosis of the host. However, under natural circumstances infection of tadpoles seems unlikely. Oral infections in metamorphosed hosts of both species were successful in 97.5% of the host animals used. The first eggs appeared 29 days after infection in the faeces. The oral route seems to be normal for O. filiformis in amphibia. Experiments on percutaneous infections did not reveal actual penetration of larvae in or through the skin nor a subsequent migration through host tissues. Sometimes a few larvae were found in the stomach and intestine, but in these particular cases the experimental conditions did not totally exclude the possibility of oral infections. Consequently, the percutaneous route of infection is not plausible for O. filiformis. Subcutaneous inoculation of infective larvae seems to be a possible way of establishing experimental infections. Erratic localisation of the parasite in the enlarged gall bladder of the host was observed.
Despite the important position of amphibia in phylogeny, efforts at the structural characterization of amphibian neurohormonal peptides have largely been confined to the Anurans (frogs and toads). Insulin was purified from an extract of the pancreas of the caecilian, Typhlonectes natans. The primary structure of the peptide was established as: [formula: see text] This amino acid sequence contains several unusual substitutions (Gln-->Lys at A5, His-->Leu at A8, Gln-->Glu at A15, and Gly -->Ala at B20) that are not present in other amphibian insulins. The structure of insulin appears to be less well conserved among the different orders of amphibia, compared with reptiles and birds.
Fishes, amphibia and reptiles, the ectothermic vertebrates, are hosts for a variety of intraerythrocytic parasites including protists, prokaryotes, viruses and structures of uncertain status. These parasites may experience host temperature fluctuations, host reproductive strategies, population genetics, host habitat and migratory behaviour quite unlike those of endothermic hosts. Few blood infections of fishes, amphibia and reptiles have proven pathogenicity, in contrast to the many intraerythrocytic parasites of mammals and some birds which harm their hosts. Although not given the attention afforded to intraerythrocytic parasites of endotherms, those of ectotherms have been studied for more than a century. This review reports on the diversity, general biology and phylogeny of intraerythrocytic parasites of ectotherms. The existence of taxonomic confusion is emphasized and the main taxonomic features of most of the 23 better characterized genera, particularly the kinetoplastid and apicomplexan protists, are summarized. Transmission of protistan infections of aquatic ectotherms is also discussed. Leeches can transfer sporozoties or merozoites to the vertebrate host during feeding. Dormant sporozoites of Lankesterella may permit transmission of species of this genus between vertebrates by predation. The fish haemogregarine, Haemogregarina bigemina, probably has gnathiid isopods, rather than leeches, as its definitive hosts. Hepatozoon spp. in aquatic hosts, and Progarnia of caiman, may also use invertebrate hosts other than leeches. Protistan infections of terrestrial or semi-terrestrial hosts are transmitted by a variety of arthropods, or, in some cases, leeches, contaminated paratenic hosts, or sporocysts free in water. Transfer of protists between vertebrates by predation and congenitally may also occur. The biology of the host cells of these infections, the red blood cells of ectotherm vertebrates, is summarized and compared with that of mammalian erythrocytes. Erythropoiesis, the nature of the surface molecules (especially the possible existence of a major histocompatibility complex), the haemoglobins, and the shape and size of erythrocytes are discussed. The exoerythrocytic sites in which protists, prokaryotes, viruses and structures of uncertain status exist before erythrocyte entry are described. Tissue merogony, tissue cysts and invasion of the white cell series occur in a variety of protistan infections. Intraerythrocytic stages of protistan infections are also discussed, including modes of entry to erythrocytes, survival mechanisms, and multiplication. The impact of infection on host populations is difficult to assess, in part because there is no agreement in the literature on the criteria used to evaluate parasite-induced cost to the host. Almost all studies have been on haemogregarine and Plasmodium infections in, mainly, lizards, but also fishes and snakes. Some infections may be responsible for mortality in their hosts, but hosts themselves may be short-lived, or have a limited ability to recover from infection.
Frogs and toads treated with high doses of anthracene-9-carboxylic acid (A-9-C) develop prolonged muscular contractions and 'divebomber' electromyograms characteristic of myotonia. Hitherto, myotonia has been considered peculiar to homeotherms where it is associated with several hereditable diseases and can be induced by specific treatments, most of which seem to act by decreasing membrane chloride conductance. Our work indicates that myotonia can be induced in amphibia by similar means. We offer possible reasons why others have missed seeing myotonia in amphibia.
Studies have been made of the distribution of mercury and its occurrence as methylmercury in the organs of amphibia collected from different sites mainly in Slovenia, Yugoslavia, including the area around the mercury mine at Idrija. Liver accumulates the highest amounts of mercury, up to 2 ppm in apparent background areas, with values up to 0.5 ppm in muscle, where virtually all mercury is present in the methyl form. Results are reported for some other trace elements in liver. Amphibia may provide useful monitors of the occurrence and spread of mercury contamination.