Observations on the cytology of Bipes (Amphisbaenia) with special reference to its lampbrush chromosomes

The positions and general anatomical and histological characteristics of the gonads of Bipes biporus and B. canaliculatus are described. The amounts of DNA per haploid chromosome set have been measured in both species, the values being 1.83 and 2.0 pg for biporus and canaliculatus respectively. The karyotypes of both species are described on the basis of data from mitotic and meiotic metaphase chromosome sets and from lampbrush chromosomes. B. biporus has 10 macrochromosomes and 11 microchromosomes. B. canaliculatus has 11 macrochromosomes and 11 microchromosomes. The karyotypes of the two species differ distinctly with regard to the shapes of 3 of the macrochromosomes. Chiasma distribution is described for male meiosis in B. biporus. Studies of the lampbrush chromosomes of both species show the chiasma distribution in the female to be generally similar to that found in the male biporus. In B. canaliculatus, lampbrush chromosomes with maximally extended lateral loops are found in oocytes that are oblate spheroids measuring 0.7×1.0 mm along their short and long axes respectively, these being well before the start of the major phase of vitellogenesis. Smaller oocytes have more distinct chromomeres and shorter loops. Microchromosomes take the form of typical small lampbrush chromosomes in oocytes. There are at the most 1,000 chromomeres per haploid set of lampbrush chromosomes in B. canaliculatus. Chiasmata are described from lampbrush preparations in which the two half-bivalents are firmly attached to one another without evident association of their axes, indicating the possibility of chiasmate association between the DNA axes of lateral loops. There are remarkably few extrachromosomal nucleoli in Bipes oocytes, and its is suggested that this may indicate a level of ribosomal gene amplification that is much lower than that found in fish and Amphibia. The observations are particularly discussed in relation to current ideas concerning the structure and function of lampbrush chromosomes.     

CHROMOSOMAL LOCALIZATION OF REPETITIVE DNA IN THE NEWT, TRITURUS

The repetitive DNA sequences of the newt, Triturus viridescens, have been studied by nucleic acid hybridization procedures. Complementary RNA was synthesized enzymatically from unfractionated newt DNA. This RNA hybridized strongly to the centromeric regions of both somatic and lampbrush chromosomes It also bound to other loci scattered along the lengths of the chromosomes The amplified ribosomal DNA in the multiple oocyte nucleoli was demonstrated by in situ hybridization     

Chromosome banding in amphibia

The distribution of constitutive heterochromatin on the chromosomes of Triturus a. alpestris, T. v. vulgaris and T. h. helveticus (Amphibia, Urodela) was investigated. Sex-specific chromosomes were determined in the karyotypes of T. a. alpestris (chromosomes 4) and T. v. vulgaris (chromosomes 5). The male animals have one heteromorphic chromosome pair, of which only one homologue displays heterochromatic telomeres in the long arms; the telomeres of the other homologue are euchromatic. This chromosome pair is always homomorphic and without telomeric heterochromatin in the female animals. There is a highly reduced crossing-over frequency between the heteromorphic chromosome arms in the male meiosis of T. a. alpestris; in T. v. vulgaris no crossing-over at all occurs between the heteromorphic chromosome arms. No heteromorphisms between the homologues exist on the corresponding lampbrush chromosomes of the female meiosis. In T. h. helveticus no sex-specific heteromorphism of the constitutive heterochromatin could be determined. The male animals of this species, however, already possess a chromosome pair with a greatly reduced frequency of chiasma-formation in the long arms. The C-band patterns and the pairing configurations of the sex-specific chromosomes in the male meiosis indicate an XX/XY-type of sex-determination for the three species. A revision of the literature about experimental interspecies hybridizations, gonadic structure of haploid and polyploid animals, and sex-linked genes yielded further evidence in favor of male heterogamety. The results moreover suggest that the heterochromatinization of the Y-chromosome was the primary step in the evolution of the sex chromosomes.     

Banding patterns on lampbrush chromosomes of Triturus marmoratus (Amphibia Urodela) by the Giemsa stain

Lampbrush chromosome preparations from the newt species Triturus marmoratus have been submitted to a banding procedure by using a Giemsa stain technique (C-banding) as well as variants of the method. Centromeres, most of telomeres, the nucleolus organizing region and some segments along the chromosome axes appear to be differently stained. The centromere positions have been indicated on the maps of the lampbrush complement of the species. The possible relationships between banding and chromosome structure and organization are briefly discussed.     

The introduced fish, rotan (Perccottus glenii), depresses populations of aquatic animals (macroinvertebrates, amphibians, and a fish)

The fish rotan (Perccottus gleniiDybowski) was accidentally introduced into European Russia from the Amur River basin. Rotan is capable of colonising small waterbodies – favourable breeding sites of native amphibians. To reveal its influence on the native aquatic fauna, monitoring of small waterbodies has been carried out since 1994 in the region of Lake Glubokoe Reserve (Moscow Province, Russia). The fish's diet includes a wide range of animal species of all trophic levels. Rotan considerably decreases the species richness of aquatic macroinvertebrates and larval amphibians. As a rule, most amphibian species (Triturus cristatus, T. vulgaris, Rana temporaria, R. arvalis, R. lessonae) and the fish Carassius carassius failed to breed successfully in ponds inhabited by rotan. In contrast, the toad Bufo bufo bred successfully in such sites because its larvae are distasteful to rotan. Rotan–amphibian interactions are discussed.     

An immimohistochemical study of regulatory peptides in lungs of amphibians

Summary The lungs of five species of European Anura and one species of Urodela (Triturus alpestris) have been studied by immunohistochemical methods to determine the occurrence, localization and distribution of serotonin, neuron-specific enolase, and eight regulatory peptides reported in the mammalian respiratory tract.Single and groups of serotonin-immunoreactive cells, corresponding to neuroendocrine cells of the mammalian lung, were identified in lungs of all amphibian species studied. Immunoreactivity for neuron-specific enolase was localized mainly in pulmonary nerves, nerve cell bodies and neuroendocrine cells. The localization and distribution of regulatory peptides varied among species. Bombesin and gastrin-releasing peptide immunoreactivities (predominant peptides in human lung) were localized mostly in submucosal nerves. Single bombesin-immunoreactive cells were found only in lungs of Urodela, i.e., Triturus alpestris. Occasional single cells, immunoreactive for somatostatin and leu-enkephalin were identified in lungs of Bombina variegata and a few cholecystokinin-immunoreactive cells in Hyla arborea. In all anuran species, numerous substance P-immunoreactive nerves were identified in submucosa, pulmonary septa and around blood vessels. No immunoreactive cells or nerves were demonstrated with antibodies against calcitonin and vasoactive intestinal peptide.The term pulmonary neuroendocrine (NE) cells (used here) does not imply neural origin or classical endocrine function for these cells, but rather indicates their potential involvement in neurohormonal regulation of pulmonary function (Cutz 1982)Supported by grant to E.C. from Medical Research Council of Canada (MT-7641)     

Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians

Buccal swabs have recently been used as a minimally invasive sampling method in genetic studies of wild populations, including amphibian species. Yet it is not known to date what is the level of reliability for microsatellite genotypes obtained using such samples. Allelic dropout and false alleles may affect the genotyping derived from buccal samples. Here we quantified the success of microsatellite amplification and the rates of genotyping errors using buccal swabs in two amphibian species, the Alpine newt Triturus alpestris and the Green tree frog Hyla arborea, and we estimated two important parameters for downstream analyses, namely the number of repetitions required to achieve typing reliability and the probability of identity among genotypes. Amplification success was high, and only one locus tested required two to three repetitions to achieve reliable genotypes, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. This sampling method which allows avoiding the controversial toe-clipping will likely prove very useful in the context of amphibian conservation.     

Identification of the lampbrush chromosome loops which transcribe 5S ribosomal RNA in Notophthalmus (Triturus) viridescens

The loops which transcribe 5S ribosomal RNA in lampbrush chromosomes of the newt, Notophthalmus (Triturus) viridescens, were identified by hybridizing purified 5S DNA to nascent 5S RNA in situ. The genes which code for 5S RNA were found near the centromeres of chromosomes 1, 2, 6, and 7 by hybridizing iodinated 5S RNA to denatured lampbrush and mitotic chromosomes in situ. These genes and their intervening spacer DNA were isolated from Xenopus laevis using sequential silver-cesium sulfate equilibrium centrifugations. This purified 5S DNA was iodinated and hybridized to non-denatured lampbrush chromosomes in situ, where it bound to nascent 5S RNA on loops at the base of the centromeres of chromosomes 1, 2, 6, and 7. The number of 5S genes present in the haploid chromosome complement of N. viridescens was determined. — The 5S loops were chosen for study, since (1) the synthesis of 5S RNA has been demonstrated during the lampbrush stage, (2) both 5S RNA and 5S DNA could be isolated in pure form, and (3) the localization of the repetitive 5S genes could be verified by conventional in situ hybridization procedures. These methods may be applicable to the identification of other loops, leading to a better understanding of lampbrush chromosome function.     

Chromosome location of the genes for 28S, 18S and 5S ribosomal RNA in Triturus marmoratus (Amphibia Urodela)

The localization of the 28S, 18S and 5S rRNA genes in the mitotic chromosomes, and of the 5S rRNA genes in the lampbrush chromosomes of Triturus marmoratus has been studied by RNA/DNA in situ hybridization. The 28S and 18S genes are located in a subterminal position, and the 5S genes in an intermediate position, on the long arm of mitotic chromosome X. In situ hybridization on lampbrush chromosomes has shown that the 5S genes are located at or near a dense matrix loop landmark. The cytogenetic implications of these findings are briefly discussed.     

Funktionsstrukturen im Oocytenkern von Locusta migratoria

Examination of living oocyte nuclei of Locusta migratoria has revealed the presence of thread-like struktures. They are paired and are thought to be the uncoiled chromosomes since they are broken into fragments by treatment with DNase. The greater part of the threads carries lateral loops like the lampbrush chromosomes of Amphibia (Fig. 14). A smaller part has no loops hut bears a series of conspicious granules with bright appearance under positive phase contrast optics (pearl-string segments) (Fig. 2). — The visibility of the chromosomes has been investigated in solutions with several ions. In hypertonic media the chromosomes contract, the granules fuse, and the pearl-string segments become lumpy (Fig. 21). In nitrogenous atmosphere and if kept at low temperature the pearl-string structures are likewise transformed into a few lumps (Fig. 19). After return to normal conditions they reconstitute their characteristic beaded appearance. — In autoradiographs obtained by injection of H³-uridine into the body cavity and by incubation of isolated nuclei in vitro, a rather uniformly distributed labelling occurs over the oocyte nuclei up to 30 min incubation time (Fig. 23). With prolonged incubation the activity of the pearl-string segments becomes more intense than the labelling of the lampbrush chromosomes (Fig. 24). After treatment with actinomycin RNA synthesis is stopped, the pearl-string axes and the lampbrush chromosomes contract, and the granules disappear more and more (Figs. 25-28). — The pearlstring segments look very much like the nucleoli in the oocytes of Amphibia, where the nucleolar substance is likewise distributed as a series of beads of rather uniform size on an axis. Therefore, the pearl-string structures may have nucleolar function in Locusta too. If so, the only difference to the Amphibian nucleoli would be the continued attachment to the lampbrush chromosomes.     

Die Ausbildung der Asymmetrie der Nuclei habenulae des Zwischenhirns bei Amphibien in Unabhängigkeit vom Blutkreislauf

Zusammenfassung Zur Prüfung der Fräge, ob der Blutkreislauf bei der Bestimmung der Asymmetrie der Nuclei habenulae des Zwischenhirns eine Rolle spielt, wurden Larven vonTriturus alpestris ohne Blutkreislauf durch Entfernung der Herzanlage hergestellt. Die Nuclei habenulae entwickelten auch ohne Kreislauf ihre normale Asymmetrie.Isolate aus dem Kopfbezirk der Neurula vonTriturus alpestris, die aus cephalem Ekto und Mesoderm bestanden und manchmal auch vorderes Entoderm enthielten, entwickelten in günstigen Fallen an ihrem Gehirn die Nuclei habenulae. Diese besaßen trotz Abwesenheit von Blutkreislauf und der anderen asymmetrischen Organe die normale Asymmetrie.

---

The development of asymmetrical nuclei habenulae in the diencephalon of amphibians, independent of blood circulation

Summary The problem whether the blood circulation influences or determines the asymmetry of Nuclei habenulae was tested by removing the heart-anlage of the embryo ofTriturus alpestris. The resulting larvae without any blood circulation developed their normal asymmetry of habenulae.Isolates from the head region of neurulae ofTriturus alpestris, consisting of cephalic ecto and mesoderm, sometimes also of entoderm, under favorable conditions could develop brains with Nuclei habenulae. These were normal, asymmetric in spite of missing the blood circulation and other asymmetrical organs.

---

---

Ausgefürhrt mit Unterstützung der Deutschen Forschugagemeinschaft.     

Silver staining of the lampbrush chromosomes ofTriturus cristatus carnifex

Lampbrush chromosomes fromTriturus cristatus carnifex were stained using the ammoniacal silver staining (AgAS) technique. Many of the recognized marker structures proved to be silver positive, plus between six and fifteen lateral loop pairs. None of the stained loop pairs corresponded to known sites of the nucleolus organizers, although the extrachromosomal nucleoli were silver positive. The ammoniacal silver staining technique does not demonstrate the specificity for active ribosomal cistrons in lampbrush chromosomes that it does in a wide variety of mammalian mitotic chromosomes.     

Karyotype diversity and interspecific 4C DNA variation inBupleurum

Investigation on karyotype, 4C nuclear DNA amount and interphase nuclear volume (INV) of different HimalayanBupleurum species belonging toUmbelliferae revealed genetic differentiation. Numerical and structural alternation of chromosomes in interspecific level were manifested in their statistically significant altered species specific 4C nuclear DNA content. Somatic chromosome number ranged between 2n = 14 and 2n = 16.B. himalayense was reported for the first time having 2n = 16 chromosomes. Correlation coefficient among the various chromosomal and nuclear parameters showed no significant progressive or regressive interdependence except in between INV and nuclear DNA amount. Critical differences between 4C DNA content showed interspecific variation.