Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

Mitotic and polytene chromosome analysis in Dacus oleae (Diptera: Tephritidae).

The present study constitutes the first attempt to construct a photographic map of the polytene chromosomes of Dacus oleae, a pest of the olive tree that causes serious financial damage in all olive oil producing countries. The map was constructed by using the larval fat body cells, the chromosomes of which are representative of the polytene chromosomes of other polytene tissues. In addition, the mitotic chromosomes of brain ganglia were examined, permitting tentative correlations between mitotic and polytene elements. This investigation shows that D. oleae is suitable for cytogenetic analysis in both mitotic and polytene chromosomes, a fact that may prove very useful for obtaining more detailed genetic information on the pest's natural populations.     

Genetic and cytogenetic analysis of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae)

The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactrocera oleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomes including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubules of this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b) the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter as an accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence of toroid tips of the chromosome arms. The in situhybridization of genes or DNA sequences to the salivary gland polytene chromosomes of B. oleaeprovided molecular markers for all five autosomes and permitted the establishment of chromosomal homologies among B. olea, B. tryoniand Ceratitis capitata. The heat shock response of B. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophila melanogaster.     

Polytene chromosome maps of the melon fly Bactrocera cucurbitae (Diptera: Tephritidae).

Standard photographic maps of the polytene chromosomes are presented for the melon fly Bactrocera cucurbitae, a serious pest of fleshy fruits and vegetables. Five larval salivary gland polytene chromosomes (10 polytene arms) were isolated, and their characteristic features and landmarks have been recognized. Banding patterns of each of the polytene arms are presented, where variation in band intensity and puffs appear to reflect fundamental differences in chromosomes. The whole polytene genome has been typically mapped by dividing it into 100 sections and the subsections were lettered. The mitotic chromosomes of larval brain ganglia are also examined, five pairs of autosomes and an XX/XY sex chromosome pair. In addition, a heterochromatic mass corresponding to the sex chromosomes are observed in the polytene nuclei of salivary gland tissue. This investigation showed that B. cucurbitae has excellent cytological material for polytene chromosome analysis and proved to be very useful for obtaining more detailed genetic information on the pest's natural populations.     

Conservation of nucleolar structure in polytene tissues of Ceratitis capitata (Diptera: Tephritidae)

Nucleolar structure was studied in mitotic and three polytene tissues of the Mediterranean fruit fly, Ceratitis capitata using in situ hybridization with a tritium-labelled rDNA probe and silver staining. In mitotic metaphase chromosomes nucleolar organiser regions were localised in the short arms of both sex chromosomes. In polytene nuclei of trichogen cells, salivary glands and fat body rDNA was detected within nucleoli. Nucleoli in these tissues have a similar structure with rDNA labelling concentrated in a central core. Silver staining resulted in very heavy staining of polytene nucleoli and interphase nucleoli in diploid cells. Silver staining of nucleolar organisers in metaphase chromosomes is weak or absent although the X chromosome has numerous dark silver bands in other locations. The results suggest that nucleolar structure is conserved in polytene tissues contrasting with the variability of autosomal banding patterns and sex chromosome structure. They also indicate that silver staining is not necessarily specific for nucleolar regions.     

The interrelation of the polytene chromosomes of generative tissues in Drosophila melanogaster]

The principles of three dimensional organization of primary and secondary orders polytene chromosomes in ovarian nurse cells of Drosophila melanogaster were elucidated. Contrary to somatic tissues, no joining of chromosome arms into local chromocentre was discovered. The chromosomes are separated in the nuclear space and are attached to the nuclear envelope by the centromeric (and the XL arm--by the telomeric) sites, the arms of autosomes (especially primary polytene chromosomes) being separated in the area of attachment. Polytenized XR arm of the X chromosomes were discovered. The architecture of chromosomes discovered in ovarian nurse cells is tissue-specific and differs considerably from the organization of polytene chromosomes of somatic tissues.     

Cytogenetic analysis of the Ethiopian fruit fly <Emphasis Type="Italic">Dacus ciliatus</Emphasis> (Diptera: Tephritidae)

The Ethiopian fruit fly, Dacus ciliatus, is an important pest of cucurbits, which recently invaded the Middle East. The genetics and cytogenetics of D. ciliatus have been scarcely studied. Such information is, however, an essential basis for understanding the biology of insect pests, as well as for the design of modern control strategies. We report here the mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this species. The mitotic metaphase complement consists of six pairs of chromosomes, including one pair of heteromorphic sex (XX/XY) chromosomes. The heterogametic sex is ascribed to the male. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei, and a heterochromatic mass corresponding to the sex chromosomes. Banding patterns, as well as the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between D. ciliatus and Bactrocera oleae are proposed by comparing chromosome banding patterns and by in situ hybridization of the hsp70 gene.     

Genetic and cytogenetic analysis of the fruit fly Rhagoletis cerasi (Diptera: Tephritidae).

The European cherry fruit fly, Rhagoletis cerasi, is a major agricultural pest for which biological, genetic, and cytogenetic information is limited. We report here a cytogenetic analysis of 4 natural Greek populations of R. cerasi, all of them infected with the endosymbiotic bacterium Wolbachia pipientis. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this pest species are presented here. The mitotic metaphase complement consists of 6 pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement has shown a total of 5 long chromosomes (10 polytene arms) that correspond to the 5 autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes. The most prominent landmarks of each polytene chromosome, the "weak points", and the unusual asynapsis of homologous pairs of polytene chromosomes at certain regions of the polytene elements are also presented and discussed.     

Similarities in the chromosomal distribution of AG and AC repeats within and between Drosophila, human and barley chromosomes

Two simple sequence repeats (SSRs), AG and AC, were mapped directly in the metaphase chromosomes of man and barley (Hordeum vulgare L.), and in the metaphase and polytene chromosomes of Drosophila melanogaster. To this end, synthetic oligonucleotides corresponding to (AG)(12) and (AC)(8) were labelled by the random primer technique and used as probes in fluorescent in situ hybridisation (FISH) under high stringency and strict washing conditions. The distribution and intensity of the signals for the repeat sequences were found to be characteristic of the chromosomes and genomes of the three species analysed. The AC repeat sites were uniformly dispersed along the euchromatic segments of all three genomes; in fact, they were largely excluded from the heterochromatin. The Drosophila genome showed a high density of AC sequences on the X chromosome in both mitotic and polytene nuclei. In contrast, the AG repeats were associated with the euchromatic regions of the polytene chromosomes (and in high density on the X chromosome), but were only seen in specific heterochromatic regions in the mitotic chromosomes of all three species. In Drosophila, the AG repeats were exclusively distributed on the tips of the Y chromosome and near the centromere on both arms of chromosome 2. In barley and man, AG repeats were associated with the centromeres (of all chromosomes) and nucleolar organizer regions, respectively. The conserved chromosome distribution of AC within and between these three phylogenetically distant species, and the association of AG in specific chromosome regions with structural or functional properties, suggests that long clusters of these repeats may have some, as yet unknown, role.     

兴义维蚋多线染色体研究（英文）

首次在国内对兴义维蚋Simulium (Wilhelmia) xingyiense的多线染色体进行研究, 并提供其多线染色体标准图。选取兴义维蚋的成熟幼虫, 用改良苯酚品红染色法进行唾腺多线染色体制备, 并进行测量、 描述及分析。结果表明： 兴义维蚋多线染色体数目为3对（2n=6）。Ⅰ号染色体具中央着丝粒, Ⅱ和Ⅲ号染色体均为亚中央着丝粒染色体。核仁组织者区位于Ⅰ号染色体短臂近着丝粒端。巴尔比尼氏环和双泡位于Ⅱ号染色体短臂近中央位置。3对染色体的着丝粒区可形成明显的染色中心。兴义维蚋多线染色体具有多态性的倒位, 倒位频率为0.64。兴义维蚋多线染色体的着丝粒、 核仁组织区、 巴氏环、 双泡等主要特征性结构的位置及形态恒定一致,可作为该种的重要鉴别特征。其多态性的倒位可为该蚋种在细胞水平上进行蚋类分类鉴别和系统发育等研究提供基础资料。     

Homologous banding patterns in the polytene chromosomes from the larval salivary glands and ovarian nurse cells of Anopheles stephensi liston (Culicidae)

A comparison of the banding patterns of two homologous polytene chromosome arms from the larval salivary gland and ovarian nurse cell complement of Anopheles stephensi is presented. The homologous chromosomes from the somatic larval salivary glands and germ-line derived ovarian nurse cells have essentially the same band-interband organisation. An analysis of the ³H-uridine labelling patterns of a small chromosome segment from the two tissues indicates that germ-line polytene chromosomes are not radically different from somatic polytene chromosomes in their patterns of gene expression.     

Chromosome organization in wheat endosperm and embryo

We have analysed the chromosome organization in endosperm and embryo of bread wheat (Triticum aestivum L.), in order to compare these tissues with developing anthers, in which the centromeres associate, and the developing root xylem vessel cells, in which the chromosomes endoreduplicate to become polytene and associate via their centromeres. Both endosperm and embryo showed a typical Rabl configuration and a degree of non-homologous centromere association and the endosperm also showed extensive telomere association. Wheat endosperm is initially triploid and during its development a percentage of the nuclei increase their DNA content to 6C and 12C. 6C nuclei showed twice as many centromeres as 3C nuclei and the centromere number increased further in 12C nuclei. The higher the C-content of a nucleus the more the telomeres associated in endosperm. The vast majority of 12C nuclei showed six rye chromosome arms, although a few showed three associated groups of rye chromosome arms. This means that during endosperm development wheat nuclei show both polyploidization and polytenization.     

Genetic and cytogenetic analysis of the American cherry fruit fly, Rhagoletis cingulata (Diptera: Tephritidae)

The American eastern cherry fruit fly, Rhagoletis cingulata, a pest of cherries in the western hemisphere, invaded Europe in 1983, and since then dispersed to several European countries. Information on the genetics and cytogenetics of this pest is very scarce. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of R. cingulata are presented here. The mitotic metaphase complement consists of six pairs of chromosomes with the sex chromosomes being very small and similar in size. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei and an extrachromosomal heterochromatic mass, which corresponds to the sex chromosomes. The banding patterns and the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between R. cingulata, R. completa and R. cerasi are also proposed, based on the comparison of chromosome banding patterns. Furthermore, the detection and characterization of Wolbachia pipientis in the R. cingulata population studied is presented and the potential correlation with the asynaptic phenomena found in its polytene complement is discussed. In addition, 10 out of 24 microsatellite markers developed for other Rhagoletis species are cross-amplified, evaluated and proposed as useful markers for population and genetic studies in R. cingulata.     

Karyotype structure in species of Propsilocerus genus (Diptera, Chironomidae)

Karyotype structure and polytene chromosome banding patterns were studied in two Orthocladiinae siblings--Propsilocerus akamusi (China) and Propsilocerus jacuticus (Russia). Both species have haploid number of chromosome typical for Orthocladiinae (n = 3). An unusual structure of centromeric regions was observed in all three chromosomes of karyotypes in both species. Photomaps of polytene chromosomes are presented. A comparison of karyotypes of P. akamusi and Propsilocerus jacuticus revealed a high level of homology in their banding sequences, however, the presence of fixed paracentric inversions in chromosomal arms IR, IIR, IIIR of Propsilocerus jacuticus has shown a clear-cut phylogenetic divergence. No chromosomal polymorphism was found in both species.     

Structural evolution of the germ line-limited chromosomes in Acricotopus

The elimination of chromatin or whole chromosomes from the future somatic nuclei during germ line-soma differentiation in early embryogenesis is a genetic phenomenon found in a wide variety of animal species. Less is known about the origin, structure, and function of the germ line-limited chromosomes. In the chironomid Acricotopus lucidus fluorescence in situ hybridization (FISH) with labeled soma DNA to "Keimbahn" chromosomes (Ks) and soma chromosomes (Ss) of spermatogonial mitoses revealed that each of the nine different K types possesses large S-homologous sections, mostly in the distal parts of both chromosome arms. Painting probes of the three Ss and of each of their chromosome arms were generated by microdissection of polytene salivary gland chromosomes and subsequent amplification by the degenerate oligonucleotide-primed polymerase chain reaction. Multicolor FISH demonstrated that each of the Ks, with the exception of one K type, was painted by only one of the three S probes. Furthermore, in seven Ks, one chromosome arm was painted by the long-arm probe and the other by the short-arm probe of the S concerned. The hybridization pattern strongly suggests that each of these K types is derived from a specific S. One function of the S-homologous K sections is thought to be determination of the regular occurrence of crossover events, with the resulting chiasmata in these sections ensuring correct segregation of the K homologs during meiosis. Reverse chromosome painting on polytene S sets with a probe generated from metaphase Ks corroborates the above results and produces conclusive evidence for the hypothesis that during evolution the Ks have developed from the Ss by endopolyploidization and rearrangements followed by the accumulation of germ line-specific repetitive DNA sequences in the centromeric regions.     

Interspecies differences of co-orientation of primary polytene chromosomes in ovarian nurse cells in Drosophila melanogaster, D. simulans and D. mauritiana]

Essential differences in the architecture of primary polytene chromosomes in ovarian nurse cells between Drosophila melanogaster, D. simulans and D. mauritiana were discovered. The individual chromosomes of D. melanogaster (as well as their arms) are separated significantly from each other and are attached to the nuclear envelope by the centromeric (and the XL arm--by the telomeric) sites. D. simulans has the combination of the centromeric parts of the chromosomes X, 2, 3 into the "elongated" chromocentre, and in addition, the arms of chromosomes are also separated and attached to the nuclear envelope. Unlike the rest of the chromosomes, D. mauritiana has the chromosome 3 with firm combinated arms and the large heterochromatic block in the centromere, without being attached to the nuclear envelope.     

Polyteny and endomitosis in supergiant trophoblast cells of the gray vole Microtus subarvalis

A cytomorphological study was made of peculiarly structured polytene chromosomes in supergiant trophoblast cells of Microtus subarvalis. The polyteny level was extremely high (over 1024C). The polytene chromosomes are characterized by a rather high degree of condensation of single chromosomes, and, as a consequence, close chromosome junctions and the typical disk pattern are lacking. The presence of complex nucleoli in the nuclei of these cells also testifies to a great detachment of chromonemes in polytene chromosomes of the studied supergiant trophoblast cells. Compared to other rodent species, a lower degree of chromoneme junction in the vole polytene chromosomes may cause their easy dissociation into single chromonemata, whose further condensation results in endomitotic chromosome formation. The chromosome depolytenization, earlier suggested from the analysis of interphase nucleus markers, has been traced here in detail. The process of polytene chromosome splitting was most obvious in the nucleolus-organizing chromosomes. A hony-combed nucleolus splits into numerous micronucleoli. The nucleus pattern becomes altered. Once in the polytene nucleus, chromosome bundles were located below the nuclear membrane and the central zone of the karyoplasm was not completely filled up. However, after dissociation of polytene chromosomes the whole karyoplasm was filled up with small nucleoli, and a thin layer of endomitotic chromosomes was seen beneath the nuclear membrane. The correlation between endomitosis and polyteny is discussed in terms of the dissociation of polytene chromosomes and formation of endomitotic chromosomes.     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.     

Cytofluorometry and visualisation of DNA base composition in the chromosomes of Drosophila melanogaster

The DNA base composition determined cytofluorometrically with the dyes CMA and DAPI in individual mitotic chromosomes of Drosophila melanogaster agrees very well with reference data obtained by hybridisation. Measurements in polytene chromosomes showed: (1) The base composition in the chromocenter, in chromosome 4 and bands X 1 and 3R 81 is lower than would be expected if they consisted of satellite DNAs only. (2) In the chromosome arms, bands with deviating base composition were found also where no satellite DNAs have been localized. With two visualisation methods — a photographic technique and image analysis — a complex pattern of base composition heterogeneity in the arms of the polytene chromosomes was established. In part this pattern may reflect the intercalary heterochromatin shown by weak point behaviour, ectopic pairing, and late replication.