Cytogenetic markers reveal a reinforcement of variation in the tension zone between chromosome races in the brachypterous grasshopper Podisma sapporensis Shir. on Hokkaido Island.

The cytogenetic characteristics of the grasshopper Podisma sapporensis (two races 2n = 23♂ X0/XX and 2n = 22♂ neo-XY/neo-XX) were analysed through fluorescence in situ hybridization with rDNA and telomeric DNA probes, C-banding, fluorochrome and silver staining. For the first time, samples from the neighbourhood of a hybrid population (i.e., Mikuni Pass population) were studied. Our results indicated a significant degree of chromosomal differentiation between P. sapporensis races when comparing the number and position of the rDNA sites, as well as the heterochromatin composition and distribution obtained by C-banding and DAPI/CMA3 staining. Telomeric signals were usually detected at the distal and/or subdistal position of the autosomes; however, some chromosome ends lacked signals, probably due to a low number of telomeric repeats. On the other hand, telomeric DNA sequences were found as interstitial telomeric repeats in some autosomes, which can trigger a variety of genome instability. B chromosomes were found in specimens belonging to both main races from nine out of 22 localities. Four types of X chromosomes in the X0/XX race were identified. It was concluded that the physical mapping of rDNA sequences and heterochromatin are useful as additional markers for understanding the phylogeographic patterns of cytogenetic differentiation in P. sapporensis populations.

Comparative analysis of C-heterochromatin, ribosomal and telomeric DNA markers in chromosomes of Pamphagidae grasshoppers from Morocco.

The karyotypes and the localization of C-bands, clusters of ribosomal DNA and telomeric repeats of 10 species of the family Pamphagidae from Morocco are described for the first time. The species studied belong to the subfamilies Pamphaginae and Thrinchinae. All species have karyotypes consisting of 19 and 20 acrocentric chromosomes and X0/XX sex chromosome system in males and females, respectively (2n♂=19, NF=19; 2n♀=20, NF=20). Despite the karyotype conservatism, we revealed differences in the location and size of C-heterochromatin blocks and ribosomal DNA clusters. A comparative analysis of these differences shows that karyotype divergences in this group is connected not to structural chromosome rearrangements, but to the evolution of repetitive DNA.

Comparative analysis of chromosomal localization of ribosomal and telomeric DNA markers in three species of Pyrgomorphidae grasshoppers.

The karyotypes of three species of Pyrgomorphidae grasshoppers were studied: Zonocerus elegans (Thunberg, 1815), Pyrgomorpha guentheri (Burr, 1899) and Atractomorpha lata (Mochulsky, 1866). Data on karyotypes of P. guentheri and Z. elegans are reported here for the first time. All species have karyotypes consisting of 19 acrocentric chromosomes in males and 20 acrocentric chromosomes in females (2n♂=19, NF=19; 2n♀=20, NF=20) and X0/XX sex determination system. A comparative analysis of the localization of C-heterochromatin, clusters of ribosomal DNA, and telomere repeats revealed inter-species diversity in these cytogenetic markers. These differences indicate that the karyotype divergence in the species studied is not associated with structural chromosome rearrangements, but with the evolution of repeated DNA sequences.

Sex chromosome diversity in Armenian toad grasshoppers (Orthoptera, Acridoidea, Pamphagidae).

Although previous cytogenetic analysis of Pamphagidae grasshoppers pointed to considerable karyotype uniformity among most of the species in the family, our study of species from Armenia has discovered other, previously unknown karyotypes, differing from the standard for Pamphagidae mainly in having unusual sets of sex chromosomes. Asiotmethis turritus (Fischer von Waldheim, 1833), Paranocaracris rubripes (Fischer von Waldheim, 1846), and Nocaracris cyanipes (Fischer von Waldheim, 1846) were found to have the karyotype 2n♂=16+neo-XY and 2n♀=16+neo-XX, the neo-X chromosome being the result of centromeric fusion of an ancient acrocentric X chromosome and a large acrocentric autosome. The karyotype of Paranothrotes opacus (Brunner von Wattenwyl, 1882) was found to be 2n♂=14+X1X2Y and 2n♀=14+X1X1X2X2., the result of an additional chromosome rearrangement involving translocation of the neo-Y and another large autosome. Furthermore, evolution of the sex chromosomes in these species has involved different variants of heterochromatinization and miniaturization of the neo-Y. The karyotype of Eremopeza festiva (Saussure, 1884), in turn, appeared to have the standard sex determination system described earlier for Pamphagidae grasshoppers, 2n♂=18+X0 and 2n♀=18+XX, but all the chromosomes of this species were found to have small second C-positive arms. Using fluorescent in situ hybridization (FISH) with 18S rDNA and telomeric (TTAGG)n DNA repeats to yield new data on the structural organization of chromosomes in the species studied, we found that for most of them, clusters of repeats homologous to 18S rDNA localize on two, three or four pairs of autosomes and on the X. In Eremopeza festiva, however, FISH with labelled 18S rDNA painted C-positive regions of all autosomes and the X chromosome; clusters of telomeric repeats localized primarily on the ends of the chromosome arms. Overall, we conclude that the different stages of neo-Y degradation revealed in the Pamphagidae species studied make the family a very promising and useful model for studying sex chromosome evolution.

DNA content of the B chromosomes in grasshopper Podisma kanoi Storozh. (Orthoptera, Acrididae).

A DNA library derived from the B chromosome of Podisma kanoi was obtained by chromosome microdissection. A total of 153 DNA clones were isolated from the microdissected DNA library. Twenty of them were sequenced. A comparison of B chromosome DNA sequences with sequences of other species from the DDBJ/GenBank/EMBL database ( http://www.ddbj.nig.ac.jp/ ) was performed. Different patterns of signals were observed after FISH with labeled cloned DNA fragments. FISH signals with cloned DNA fragments painted either whole Bs or their different regions. Some clones also gave signals in pericentromeric regions of A chromosomes. Other cloned DNA fragments gave only background-like signals on A and B chromosomes. Comparative FISH analysis of B chromosomes in Podisma kanoi and P. sapporensis with DNA probes derived from the Bs of these species revealed homologous DNA that was confined within pericentromeric and telemetric regions of the B chromosome in P. kanoi. In contrast to the B chromosomes in P. sapporensis containing large regions enriched with rDNA, only a small cluster of rDNA was detected in one of the examined B chromosomes in P. kanoi. The data strongly suggest an independent origin of B chromosomes in two closely related Podisma species.

Experimental hybridisation between X0 and XY chromosome races in the grasshopper Podisma sapporensis Shir. (Orthoptera, Acrididae). I. Cytological analysis of embryos and F1 hybrids.

The results of experimental hybridisation between some chromosome subraces belonging to the X0 and XY chromosome races of the brachypterous grasshopper P. sapporensis are presented. Pre-zygotic reproductive isolation mechanisms in experimental pairs were not confirmed. In crossings of XY-standard x X0-standard and XY-standard x X0-Naganuma chromosome subraces, a zygotic barrier has been found. All embryos of XY-standard x X0-standard crosses and the vast majority of embryos of XY-standard x X0-Naganuma crosses were obtained from female diploid or haploid/diploid cells as a result of parthenogenesis. In very rare cases, when the zygotic barriers had been surmounted, normal embryo heterozygotes and a F1 hybrid generation were obtained in XY-standard x X0-Naganuma crosses. On the contrary, crosses between the XY-Tanno and X0-standard subraces gave viable offspring in spite of many chromosome differences such as a X-A translocation and fixed pericentric inversions in four pairs of autosomes. The results obtained do not support the hypothesis that chromosomal differences play a key role in restricting gene flow between X0 and XY races of P. sapporensis. The presence of crossing barriers explains the phenomena of the purity of the X0 and XY chromosomes races.

B chromosomes of the Podisma sapporensis Shir. (Orthoptera, Acrididae) analysed by chromosome microdissection and FISH.

The analysis of the distribution of repetitive DNA of the B chromosomes of Podisma sapporensis in the A and B chromosomes of the natural populations and in A chromosomes of three other species of the Podismini grasshoppers were made. DNA-libraries of the B chromosome and the euchromatic segment of the A chromosome of P. sapporensis were generated by meiotic chromosome microdissection followed by degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR). Paints based on these DNA-libraries were used for FISH analysis to detect localization of homologous sequences in A and B chromosomes of P. sapporensis from different natural populations. On the basis of the FISH analysis the authors suggest that evolution of the B chromosomes in Podisma sapporensis was associated mainly with the insertions of "alien DNA sequences" into ancestral A chromosome and their further amplification. The number of initial sites of amplifications differed in the different Bs, the distance between these sites also varying. Karyotype evolution in P. sapporensis was associated partly with the insertion of "alien DNA sequences" into pericentromeric chromosomal regions. Insertion into the small short arms of the acrocentric chromosomes followed, with the DNA amplification leading to the formation of the additional C-heterochromatic arms or euchromatic-like regions of different size.

Chromosome C-banding patterns in isolated population of the grasshopper Podisma pedesis (L.) (Orthoptera, Acrididae) from the Altai Mts.

The C-banding patterns in the embryo chromosomes of the grasshopper Podisma pedestris (L.) from the Altai Mts are reported. The additional second C-heterochromatic arms in at least five pairs of autosomes and in the X-chromosome were revealed. The paracentromeric, interstitial, and telomeric C-bands were observed. The studied population of P. pedestris shows some differences in the distribution and amount of the heterochromatin in comparison with European populations.

Constitutive heterochromatin in karyotypes of two Cicadidae species from Japan (Cicadoidea, Hemiptera).

Karyotypes of males of cicadas Tibicen bihamatus (Motschulski) and Platypleura kuroiwae Matsumura were studied using C-banding technique. In Tibicen bihamatus two types of C-band distribution were observed. Two chromosome pairs have C-bands at one of the chromosome ends, while in the other, including the sex chromosome, C-heterochromatin blocks occurred at both ends. Platypleura kuroiwae has a smaller amount of C-heterochromatin located as small subterminal blocks. The intercalar C-bands were seen in the early spermatogonial metaphase chromosomes.