First report on C-banding,fluorochrome staining and NOR location in holocentric chromosomes of Elasmolomus (Aphanus) sordidus Fabricius, 1787 (Heteroptera,Rhyparochromidae)

In spite of various cytogenetic works on suborder Heteroptera, the chromosome organization, function and its evolution in this group is far from being fully understood. Cytologically, the family Rhyparochromidae constitutes a heterogeneous group differing in chromosome numbers. This family possesses XY sex mechanism in the majority of the species with few exceptions. In the present work, multiple banding techniques viz., C-banding, base-specific fluorochromes (DAPI/CMA₃) and silver nitrate staining have been used to cytologically characterize the chromosomes of the seed plant pest Elasmolomus (Aphanus) sordidus Fabricius, 1787 having 2n=12=8A+2m+XY. One pair of the autosomes was large while three others were of almost equal size. At diplotene, C-banding technique revealed, that three autosomal bivalents show terminal constitutive heterochromatic bands while one medium sized bivalent was euchromatic. Microchromosomes (m-chromosomes) were positively heteropycnotic. After DAPI and CMA₃ staining, all the autosomal bivalents showed equal fluorescence, except CMA₃ positive signals, observed at both telomeric heterochromatic regions of one medium sized autosomal bivalent. Silver nitrate staining further revealed that this chromosome pair carries Nucleolar Organizer Regions (NORs) at the location of CMA₃ positive signals. The X chromosome showed a thick C-band, positive to both DAPI /CMA₃ while Y, otherwise C-negative, was weakly positive to DAPI and negative to CMA₃, m-chromosomes were DAPI bright and CMA₃ dull.     

A new approach to the auchenorrhyncha (Hemiptera, Insecta) cytogenetics: chromosomes of the meadow spittlebug Philaenus spumarius (L.) examined using various chromosome banding techniques

The karyotype of the meadow spittlebug Philaenus spumarius (L.) was studied using conventional chromosome staining, C- and AgNOR- banding, and fluorescent CMA3- and DAPI- techniques. This is the first report on differential staining of the holocentric chromosomes of Auchenorrhyncha. The karyotype of Ph. spumarius includes 2n = 22 + XX/X0. The autosomal pair 1 is large and carries a gap in every homologue. After silver staining, NORs were revealed in both this chromosome pair and a middle-sized pair, most likely 6 or 7. In spermatocyte meiosis, the majority of bivalents formed one chiasma each. The bivalent 1 showed from 1 to 4 chiasmata, the value of 1 or 2 being prevalent. Two further bivalents also showed two chiasmata in some cells. After C-banding, terminal and interstitial dot-type C-heterochromatic blocks were revealed in the chromosomes. In 4 of 11 studied males, the autosomal pair 1 was polymorphic for an extra segment attached to one of the homologues. The segment consisted of both heterochromatic and euchromatic portions. No defined signals were observed in any chromosome treated with DAPI. After CMA3- staining, bright fluorescent signals were obtained in the NOR-bearing chromosomes, suggesting GC-rich DNA bound to the NORs.     

Meiotic chromosome pairing in Stethophyma grossum spermatocytes studied by a surface-spreading and silver-staining technique

Spermatocytes of Stethophyma grossum were prepared for light microscope examination by a surface-spreading and silver-staining technique adapted with very little modification from a mammalian procedure. Very clear preparations of a variety of prophase I stages were obtained which revealed many details of chromosome and nuclear organisation. These observations and preliminary observations on two other grasshopper species demonstrate the ready applicability of these techniques to Orthopteran spermatocytes. A detailed study of six pachytene stage spermatocytes gave conclusive confirmation that three bivalents achieve full pairing in male meiosis of Stethophyma grossum, the other eight bivalents being partially paired at their procentric ends only.     

Mapping of pachytene bivalents of female codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae)

Spread pachytene nuclei of codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae) females of a Syrian strain (SY) were used to investigate chromomere patterns of chromosome bivalents and determine their length. The karyotype of female codling moths consists of 28 chromosome bivalents, of which seven are clearly distinguishable using chromosome length and the number and size of the chromomeres in the pachytene stage. One autosome bivalent has two nucleolar organizing regions (NORs) that are located at the opposite ends of the chromosome and appear as distinct structural landmarks. In female codling moths, the WZ sex chromosome bivalent was easily identified in pachytene oocytes according to the heterochromatic thread of the W chromosome. This study contributed to the knowledge and identification of pachytene chromosomes of female codling moths.     

Synapsis with and without recombination in the male meiosis of the leaf-footed bug <Emphasis Type="Italic">Holhymenia rubiginosa</Emphasis> (Coreidae,Heteroptera)

In organisms with chiasmatic meiosis two different relationships have been described between crossing over and synapsis: in one group of organisms synapsis depends on the initiation of meiotic recombination while in the other group it is independent of this initiation. These patterns have been observed mainly in organisms where all meiotic bivalents in the set have similar behaviors. In some heteropteran insects a pair of chromosomes named m chromosomes is known to behave differently from autosomes regarding synapsis and recombination. Here we used immunodetection of a synaptonemal complex component and acid-fixed squashes to investigate the conduct of the small m chromosome pair during the male meiosis in the coreid bug Holhymenia rubiginosa. We found that the m chromosomes form a synaptonemal complex during pachytene, but they are not attached by a chiasma in diakinesis. On the other hand, the autosomal bivalents synapse and recombine regularly. The co-existence of these variant chromosome behaviors during meiosis I add further evidence to the absence of unique patterns regarding the interdependence of synapsis and recombination.     

Pachytene mapping in the female silkworm,Bombyx mori L. (Lepidoptera)

Pachytene preparations of the chromosome complement of female larvae of Bombyx mori were improved to give a distinct chromomere pattern of the bivalents suitable for chromosome mapping. Six of the 28 bivalents are described and can be identified regularly in the bivalent complements, among them the bivalent containing the nucleolus organizer. The relative lengths of these bivalents compared with one another change during development of pachytene. In contrast to other members of the Lepidoptera there is no conspicuous heterochromatic W-chromosome, which corresponds to the female-specific heterochromatin body present in the nuclei of somatic tissues. This tissue-specific heteropycnosis indicates a different functional state of the responsible chromosome or chromosomal segment in germ line and somatic cells.     

The meiotic structure and behavior of the strongly heteromorphic X/Y sex chromosomes of neotropical plethodontid salamanders of the genus Oedipina

Plethodontid salamanders in the genus Oedipina are characterized by a strongly heteromorphic sex-determining pair of X/Y chromosomes. The telocentric X chromosome and the subtelocentric Y chromosome are clearly distinguished from the autosomes and their behavior during meiosis can be sequentially followed in squash preparations of spermatocytes. In Oedipina the sex chromosomes are not obscured by an opaque sex vesicle during early meiotic stages, making it possible to observe details of sex bivalent structure and behavior not directly visible in other vertebrate groups. The sex chromosomes can first be distinguished from autosomal bivalents at the conclusion of zygotene, with X and Y synapsed only along a short segment at their non-centromeric ends, forming a bivalent that contrasts sharply with the completely synapsed autosomes. During pachytene, the XY bivalent becomes progressively shortened and more compact, disappearing as a visible structure when pachytene progresses into the diffuse stage of male meiosis. Diplotene bivalents gradually emerge from the diffuse nuclei, presumably by the return of the loops of chromatin into their respective chromomeres. During early diplotene, the X/Y bivalent is clearly visible with a single chiasma within the synapsed segment. This chiasma is terminalized by first meiotic metaphase with the X and Y appearing either in end-to-end synaptic contact or as univalents separated at opposite poles relative to the equatorially distributed autosomal bivalents. In C-banded preparations, the Y is entirely heterochromatic while the X contains a large centromeric C-band and another block of heterochromatin located at the telomeric end, in the region of synapsis with the Y. We find no cytological evidence of dosage compensation, such as differential staining of the X chromosomes or Barr bodies, in mitotic or interphase cells from female animals.     

淡黄蚊蝎蛉染色体特征及其系统发育意义

【目的】染色体特征在昆虫系统发育研究中起着重要作用。然而,长翅目(Mecoptera)蚊蝎蛉科(Bittacidae)昆虫的染色体研究却比较匮乏。【方法】通过室内饲养获得淡黄蚊蝎Bittacus flavidus Huang & Hua各龄幼虫、蛹和成虫。取淡黄蚊蝎雄性末龄(4龄)幼虫、蛹和新羽化成虫精巢细胞进行染色体制片,通过4′,6-二脒基-2-苯基吲哚(4′,6-diamidino-2-phenylindole, DAPI)荧光染色,研究染色体核型、减数分裂行为和性别决定机制。【结果】结果表明,淡黄蚊蝎蛉的染色体数目为2n=26+X0,染色体均具有中着丝粒,核型高度对称。二价体绝对长度为(3.20±0.07)~(1.53±0.19) μm,相对长度为(5.31±0.29)~(2.73±0.24),均呈梯度变化。淡黄蚊蝎蛉的减数分裂为交叉型,其中核的平均交叉频率为11.5,二价体的平均交叉频率为0.88。性别决定机制为XX/X0型。DAPI荧光带型显示,粗线期二价体一端呈现高亮的AT富集区块。【结论】染色体特征(包括染色体数目、染色体臂基本数和核型公式等)在蚊蝎蛉科中表现出显著变异,表明染色体重排(尤其是融合、断裂和倒位)在蚊蝎蛉科昆虫的谱系分化和染色体演化中起着重要作用。     

Synaptonemal complexes with associated chromatin in a moth,Ephestia kuehniella Z.

Chromosome structure and pairing behaviour of the pachytene bivalents in the wildtype and in W chromosome mutants were studied using a microcentrifugation technique. The spread bivalents display a characteristic lampbrush structure with lateral loops having the typical appearance of nucleosomal fibers, in autosomes as well as in the W and Z chromosomes. While the autosomal loops are always completely dispersed by the spreading forces, the loops of the heterochromatic W chromosome frequently are found to be condensed in tangles. These tangles contain supranucleosomal globular particles of a diameter of 37.7±1.2 nm. — Pairing of the WZ can be complete or partial, probably depending on the stage of the pachytene. Incomplete pairing normally is interpreted as demonstrating non-homology. Pairing was weak, however, even between homologous segments of the W chromosome, which were introduced into the genome in homozygous form by a translocation chromosome.     

Heterochromatin and nucleolus-organizer-region behaviour at male pachytene of Sus scrofa domestica

In the domestic pig (2n=38) two types of constitutive heterochromatin can be differentiated by fluorescence counterstaining techniques. All 24 biarmed autosomes and the X chromosome have chromomycin A₃-positive centromeric C-bands, whereas all 12 acrocentric chromosomes exhibit DA-DAPI-positive centromeric heterochromatin. Fluorescence analysis of male pachytene nuclei revealed that the DA-DAPI-positive C-bands form one or two large chromocentres per cell, while the chromomycin A₃-bright C-material is well scattered. Hence, the bivalents formed by the acrocentric chromosome pairs are centromerically associated, whilst the submetacentric bivalents are not. —Counce-Meyer spreading techniques were used to study the structure of synaptonemal complexes (SCs) both by light and electron microscopy. In general, the SCs of the domestic pig resemble those described for other mammals. The SC formed by the X and the Y may include up to 94.5% of the Y chromosome. In silver-stained microspreads each of the bivalents (nos. 8 and 10) bearing the nucleolus-organizer-regions (NORs) is connected to a pair of nucleoli, indicating that all four NORs are active during early meiotic stages. By contrast, in the majority of mitotic metaphases of phytohaemagglutinin-stimulated lymphocytes only one pair (no. 10) exhibited Ag-NOR staining. — The significance of the chromosome disposition in the pachytene nucleus is discussed with regard to heterochromatin composition and karyotype evolution.This paper is dedicated to Prof. Hans Bauer on the occasion of his 80th birthday     

Differential elongation of autosomal pachytene bivalents related to their DNA content in human spermatocytes

The establishment of the complete karyotype of human pachytene spermatocytes reveals differences in stretching of chromosomes between meiosis and mitosis. Bivalents or specific regions of bivalents which exhibit many R-bands are particularly elongated. In mitotic chromosomes, the DNA contained in such bands is known to be early replicating. The study of variations in the total length and the centromeric index of bivalent 1 suggests that differential elongation of pachytene bivalents is a premeiotic event, taking place during the last DNA replication.     

B Chromosome Polymorphism in the Fish, Astyanax scabripinnis

Four populations of Astyanax scabripinnis (Pisces, Characidae) were analyzed for B chromosome features. This species contains a metacentric macrochromosome (B_M), similar in size to the first chromosome of the karyotype, besides two variant forms, a large submetacentric (B_SM) and a small metacentric (B_m), both showing a reduced frequency. These variant forms were observed only in the populations from the Campos do Jordão region (São Paulo State, Brazil), although not present in all the populations sampled. The three B chromosome forms are heterochromatic and at least the B_M and B_SM are also GC-rich, as evidenced by C-banding and chromomycin A₃ staining. In spite of the morphological similarity between the B_M form and the first chromosome pair, they differ in the G-banding pattern, which does not favor a possible relationship among these chromosomes. FISH with a telomeric DNA probe evidenced signals on the terminal region of all chromosomes, including the Bs. Interstitial telomeric bands, indicative of some chromosomal rearrangements such as translocation or tandem fusion in the origin of the B chromosomes, were not observed. B_SM and B_m are probable derivative B chromosome forms from an ancestral B_M chromosome, showing a restricted occurrence and low frequency in the populations.     

Chromosome organisation in the Australian plague locust,Chortoicetes terminifera

In Chortoicetes terminifera, G-banding, produced by the trypsin treatment of air-dried slides followed by Giemsa staining, leads to light staining gaps at the secondary constrictions on autosomal pair 6 and regions proximal to the centromere on the long arms of pair 4. The variable short arms of two of the three smallest pairs were usually flared and lightly stained after treatment. In contrast to the relatively minor response of the normal chromosome set to G-banding, the large supernumerary chromosomes of C. terminifera show a spectacular series of dark bands alternating with lightly stained gaps. Two G-band variants of the B-chromosome were found in a laboratory stock. These patterns of G-banding are discernable both at mitosis in adults and embryos of both sexes and at all stages of male meiosis. Some regions which are gaps after G-banding appear as dark bands after C-banding. Consequently the supernumerary chromosome is mainly darkly stained with C-banding. In addition the centromeres and some telomeres are C-banded along with narrow interstitial bands and polymorphic heterochromatic blocks. — C-banding was not always successful, the technique often yields a mixture of G- and C-banding. The disparity of banding between the normal complement and the B-chromosome implies that whatever the source of origin of the B it has undergone spectacular changes in organisation since its origin.     

Cytological localization of the genes for the four classes of ribosomal RNA (25S, 18S, 5.8S and 5S) in polytene chromosomes of Phaseolus coccineus

Homologous tritiated 25S, 18S and 5.8S rRNAs were used separately for in situ hybridization to the polytene chromosomes of the embryo suspensor cells of Phaseolus coccineus. Hybridization occurred at the same chromosomal sites which were labeled in previous in situ hybridization experiments with 25+18S rRNAs in the same material (Avanzi et al., 1972), namely: nucleolus organizing system (satellite, nucleolar constriction and organizer) of chromosome pairs I (S₁) and V (S₂), proximal heterochromatic segment of the long arm of chromosome pair I, and terminal heterochromatic segment of chromosome pair II. Competition hybridization experiments confirmed for P. coccineus the high sequence homology between 25S and 18S rRNA already known for other plants.Homologous ¹²⁵I-5S rRNA was found to hybridize to three sites in the polytene chromosomes of P. cocdneus: the proximal heterochromatic segment in the long arm of chromosome pair I (which also bears the sequences complementary to 25S, 18S and 5.8S RNAs), most of the proximal heterochromatic segment plus a small portion of adjoining euchromatin in the long arm of chromosome pair VI and the large intercalary heterochromatic segment in the same chromosome pair. Simultaneous labeling of the two 5S RNA sites in chromosome VI was quite rare (3%), the rule being labelling of one site to the exclusion of the other, with a labeling frequency of 43.7% and 53.3% for sites no. 1 and no. 2 respectively. These results are interpreted as being due to differential hybridizability of chromosomal sites such as described in other materials.     

Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species

Thirty-nine cultivars of cassava and eight related wild species of Manihot were analyzed in this work for number, morphology and size of chromosomes, prophase condensation pattern and the structure of the interphase nucleus. In four accessions, the chromosome size was measured and in some others, the number of secondary constrictions, meiotic behavior, C-band pattern, CMA/DAPI bands, nucleoli number and the location of 5S and 18S-5.8S-28S rDNA sites were also observed. All investigated accessions showed a similar karyotype with 2n = 36, small metacentric to submetacentric chromosomes. Two pairs of terminal secondary constrictions were observed in the chromosome complement of each accession except Manihot sp. 1, which presented two proximal secondary constrictions. The prophase chromosome condensation pattern was proximal and the interphase nuclei structure was areticulate to semi-reticulate. The meiosis, investigated in seven cultivars and four wild species, was regular, displaying 18 bivalents. C-banding revealed heterochromatin in 9 or 10 chromosomes. The analysis with fluorochromes frequently showed four chromosome pairs with a single CMA+ terminal or subterminal band and a few other chromosomes with DAPI+ unstable bands. Six 45S rDNA sites were revealed by FISH, which seemed to colocalize with six CMA+ bands. Only one chromosome pair presented a 5S rDNA site. The maximum nucleoli number observed per nucleus was also six. These data suggest that all Manihot species present a very similar chromosome complement.     

Matthiola Tricuspidata R. BR. (Cruciferae): Analisi Cariologica Ed Embriologica

Abstract

<Matthiola tricuspidata> R. Br.: karyological and embryological studies. — Karyological and embryological observations on specimens of Matthiola tricuspidata R. Br. (Cruciferae) from Siracusa (Sicily) are reported. The chromosome number, 2n = 14, is confirmed, its karyotype having the following features:

z = 2n = 14 = 2M₁ + 2M³ ₂ + 2M₃ + 2M₄ + 2S₁ + 2S₂ + 2S₃

A mutant metaphase is reported. The mutation concerns only one chromosome of pair 2S₁. The development of the megagametophyte follows the normal or Polygonum type. The three antipodal cells disappear very soon. The hypostase is probably connected with the xerotermic habitat in which the species lives. The endosperm is of the nuclear type.     

Parallel polymorphism for supernumerary segments inChorthippus parallelus (zetterstedt)

A complex and parallel pattern of polymorphism for heterochromatic supernumerary segments in the M₇ and S₈ chromosomes has been found in 14 populations of the meadow grasshopperChorthippus parallelus. Nine distinct karyotype classes for these two chromosome pairs occur though they are not equally represented in different populations. Populations differ also with respect to the frequency of supernumerary segments they contain. In all populations the presence of supernumerary segments leads to a significant elevation of mean cell chiasma frequency compared to individuals from the same population lacking such segments. The extent of the effect appears to differ in different populations. The observed frequencies of S₈ karyotypes conform to the expectations of a Hardy-Weinberg distribution. Those of the M₇, however, do not, and in all but one of the 14 populations there is a significant excess of homokaryotypes. In the Ashurst population 26% of the individuals sampled were characterised by germ-line polysomy for the M₄ chromosome, either in the form of entire tetrasomics or more usually as mosaics ranging from tri- to hepta-somics. In all these polysomics the M₄ chromosomes in excess of two were regularly heteropycnotic at first meiotic prophase from zygotene to diakinesis. As a consequence of this multivalents are rare. Extra M₄ chromosomes do not modify the chiasma characteristics of the other chromosomes in the complement. Nor do they modify the action of the supernumerary segments in any way.     

Pseudomultiple production in Ageneotettix deorum deorum

Meiosis in Ageneotettix deorum deorum is characterised by extensive pseudomultiple formation during prophase. The association of non-homologous chromosomes takes place prior to pairing and chiasma formation and occurs to a varying degree in all prophase cells. These pseudomultiples originate during interphase as a consequence of the association of heterochromatin. All autosomes carry procentric heterochromatic segments of variable size and the L₁, L₃ and M₅ chromosomes also possess terminal heterochromatic regions. The association of non-homologous chromosomes during zygotene and pachytene does not appear to impede pairing or the frequency and distribution of chiasmata. — A majority of the pseudomultiples dissociate after diakinesis, during orientation and congression on the spindle. However in 4% of the cells examined, associations, mainly quadrivalents, persist through metaphase. — Heterochromatic associations of non-homologous chromosomes are again evident during second prophase, though here they involve only the centric heterochromatic regions; 9% of these associations persist through second metaphase. — The nature and behaviour of the pseudomultiples in Ageneotettix are pertinent to the interpretation of terminal associations in other Orthoptera and provide evidence that persistent associations can arise following a non-chiasmate association.On educational leave from the Forest Research Laboratory, Fredericton, N.B. Canada.     

Chromosome banding and genome compartmentalization in fishes

The diploid chromosome number of the Chinese raccoon dog varies from 54 (no B chromosomes) to 58 (4 B chromosomes). The B chromosomes are totally heterochromatic. An electron microscopic study was made of the synaptonemal complexes (SC) in spermatocytes of these animals. The SC karyotype consists of 27 regular chromosome pairs (autosomes and the sex chromosomes) plus the B chromosomes. The Bs pair effectively with one another at pachytene, but the SC axes of the B chromosomes are much denser than those of the A chromosomes. Depending on the number of Bs, both bivalents and multivalents have been observed. When three B chromosomes are present in a cell, parallel alignment of all three SCs can be seen. Formation of multivalents indicates high homology among these supernumerary heterochromatic chromosomes. Fusiform bulges are found along unpaired regions of all chromosomes which are particularly pronounced in diplotene.     

Non-relationship between nucleolus and sex chromosome system Xyp in Chelymorpha variabilis boheman (Coleoptera: Chrysomelidae)

Nucleolar-organizer region, nucleolus and mode of association of the sex bivalent were analyzed in spermatecytes of Chelymorpha variabilis Boheman. This species (2n=10II+Xy_p) shows the typical sex chromosome system of the group Polyphaga. The results of silver staining techniques showed the nucleolar organizer region localized in a subterminal position of an autosomal bivalent. During meiotic prophase the nucleolus was distinguished with the silver staining and acridine orange fluorescence technique up to diakinesis. The independence of nucleolus and sex bivalent Xy_p during meiosis is demonstrated. The positively silver staining but negatively orange-red material found within the parachute could be involved in the regular co-orientation of both sex chromosomes. After a longer hypotonic treatment, sex bivalents were observed elongated and paired only at one end during the pachytene stage. Along these sex chromosomes, C-bands showed positive blocks located in the pericentromeric and telomeric regions. Heterochromatic association of both sex chromosomes was suggested.