Vilma Loreto

Chromosomal mapping of rDNAs and H3 histone sequences in the grasshopper rhammatocerus brasiliensis (acrididae, gomphocerinae): extensive chromosomal dispersion and co-localization of 5S rDNA/H3 histone clusters in the A complement and B chromosome.

BackgroundSupernumerary B chromosomes occur in addition to standard karyotype and have been described in about 15% of eukaryotes, being the repetitive DNAs the major component of these chromosomes, including in some cases the presence of multigene families. To advance in the understanding of chromosomal organization of multigene families and B chromosome structure and evolution, the distribution of rRNA and H3 histone genes were analyzed in the standard karyotype and B chromosome of three populations of the grasshopper Rhammatocerus brasiliensis.ResultsThe location of major rDNA was coincident with the previous analysis for this species. On the other hand, the 5S rDNA mapped in almost all chromosomes of the standard complement (except in the pair 11) and in the B chromosome, showing a distinct result from other populations previously analyzed. Besides the spreading of 5S rDNA in the genome of R. brasiliensis it was also observed multiple sites for H3 histone genes, being located in the same chromosomal regions of 5S rDNAs, including the presence of the H3 gene in the B chromosome.ConclusionsDue to the intense spreading of 5S rRNA and H3 histone genes in the genome of R. brasiliensis, their chromosomal distribution was not informative in the clarification of the origin of B elements. Our results indicate a linked organization for the 5S rRNA and H3 histone multigene families investigated in R. brasiliensis, reinforcing previous data concerning the association of both genes in some insect groups. The present findings contribute to understanding the organization/evolution of multigene families in the insect genomes.

Possible autosomal origin of macro B chromosomes in two grasshopper species.

The acrocentric macro B chromosomes of Rhammatocerus brasiliensis (Acrididae, Gomphocerinae) and Xyleus discoideus angulatus (Romaleidae, Romaleinae) are highly similar to the X chromosome in each species in terms of morphology, size, and pycnosis. However, the results of FISH experiments using 45S and 5S rDNA probes suggest that in both species the B chromosomes are most likely of autosomal origin. In R. brasiliensis, the B chromosome presented 5S rDNA but not 45S rDNA, in resemblance to the L2, L3, M5 and S11 autosomes, but the X chromosome lacks both rDNA families. In X. d. angulatus, 45S rDNAs is absent from the B chromosome, whereas the X chromosome contains one of the two 45S rDNA clusters in the genome. The occurrence of B chromosomes in all nine R. brasiliensis populations analyzed indicates that they are widely distributed in Northeastern Brazil, and the small amount of interpopulation variation found for B chromosome prevalence suggests the existence of high gene flow, presumably due to the abundance of this grasshopper species on several types of vegetation and its relatively high flight capability.

Karyotype, constitutive heterochromatin and nucleolar organizer regions (NORs) in Belosacris coccineipes (Acrididae-Leptysminae)

Several techniques including C-banding, fluorochromes and silver staining were used to obtain information about heterochromatin patterns in the grasshopper B. coccineipes. Conventional staining showed a karyotype with 2n = 23 chromosomes in males and 2n = 24 in females, as well as XO:XX sex determination and acrotelocentric chromosomes. The medium-sized X chromosome was heteropycnotic positive at the beginning of prophase I and negative in metaphase I. C-banding revealed heterochromatic blocks in the pericentromeric regions of all chromosomes. Silver nitrate staining in this species showed three small bivalents (S9-S11) as nucleolar organizers with NORs located in the pericentromeric regions. CMA3-positive blocks were seen in pericentromeric regions of pairs M6, S9, S10 and S11. Sequential staining with CMA3/AgNO3 revealed homology between the CMA3-positive bands and NORs of the bivalents S9, S10 and S11. The CMA3-positive block of the bivalent M6 could represent a latent secondary NOR. The results obtained permit us to distinguish two categories of the constitutive heterochromatin in B. coccineipes.

U1 snDNA clusters in grasshoppers: chromosomal dynamics and genomic organization

The spliceosome, constituted by a protein set associated with small nuclear RNA (snRNA), is responsible for mRNA maturation through intron removal. Among snRNA genes, U1 is generally a conserved repetitive sequence. To unveil the chromosomal/genomic dynamics of this multigene family in grasshoppers, we mapped U1 genes by fluorescence in situ hybridization in 70 species belonging to the families Proscopiidae, Pyrgomorphidae, Ommexechidae, Romaleidae and Acrididae. Evident clusters were observed in all species, indicating that, at least, some U1 repeats are tandemly arrayed. High conservation was observed in the first four families, with most species carrying a single U1 cluster, frequently located in the third or fourth longest autosome. By contrast, extensive variation was observed among Acrididae, from a single chromosome pair carrying U1 to all chromosome pairs carrying it, with occasional occurrence of two or more clusters in the same chromosome. DNA sequence analysis in Eyprepocnemis plorans (species carrying U1 clusters on seven different chromosome pairs) and Locusta migratoria (carrying U1 in a single chromosome pair) supported the coexistence of functional and pseudogenic lineages. One of these pseudogenic lineages was truncated in the same nucleotide position in both species, suggesting that it was present in a common ancestor to both species. At least in E. plorans, this U1 snDNA pseudogenic lineage was associated with 5S rDNA and short interspersed elements (SINE)-like mobile elements. Given that we conclude in grasshoppers that the U1 snDNA had evolved under the birth-and-death model and that its intragenomic spread might be related with mobile elements.

Chromosomal evolution of rDNA and H3 histone genes in representative Romaleidae grasshoppers from northeast Brazil.

BackgroundGrasshoppers from the Romaleidae family are well distributed in the Neotropical Region and represent a diversified and multicolored group in which the karyotype is conserved. Few studies have been conducted to understand the evolutionary dynamics of multigene families. Here, we report the chromosomal locations of the 18S and 5S rDNA and H3 histone multigene families in four grasshopper species from the Romaleidae family, revealed by fluorescent in situ hybridization (FISH).ResultsThe 5S rDNA gene was located in one or two chromosome pairs, depending on the species, and was found in a basal distribution pattern. Its chromosomal location was highly conserved among these species. The 18S rDNA was located in a single medium-sized chromosomal pair in all species analyzed. Its chromosomal location was near the centromere in the proximal or pericentromeric regions. The location of the H3 histone gene was highly conserved, with slight chromosomal location differences among some species. To our knowledge, this is the first report of a megameric chromosome carrying both the chromosomal markers 18S rDNA and the H3 histone genes, thereby expanding our understanding of such chromosomes.ConclusionsThe 5S and 18S rDNA genes and the H3 histone genes showed a conservative pattern in the species that we analyzed. A basal distribution pattern for 5S rDNA was observed with a location on the fourth chromosomal pair, and it was identified as the possible ancestral bearer. The 18S rDNA and H3 histone genes were restricted to a single pair of chromosomes, representing an ancestral pattern. Our results reinforce the known taxonomic relationships between Chromacris and Xestotrachelus, which are two close genera.

Ommexecha virens (Thunberg, 1824) and Descampsacris serrulatum (Serville, 1831) (Orthoptera, Ommexechidae): karyotypes, constitutive heterochromatin and nucleolar organizing regions

Abstract Chromosomes of Ommexecha virens and Descampsacris serrulatum (Ommexechidae) were analyzed through conventional staining, C-banding, base specific fluorochromes, silver nitrate impregnation (AgNO3), and fluorescent in situ hybridization (FISH) with probe for 45S rDNA. The two species presented diploid number 2n= 23,X0 in males and acrocentric autosomes, except the pair one that presented submetacentric morphology. The X chromosome has distinct morphology in the two analyzed species, being a medium acrocentric in Ommexecha virens and large submetacentric in Descampsacris serrulatum. The C-banding revealed pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of Descampsacris serrulatum. For Ommexecha virens it was evidenced that the blocks of CH are preferentially located in the pericentromeric area (however some bivalents presents additional blocks) or in different positions. The staining with CMA3/DA/DAPI showed GC rich CH blocks (CMA3+) in some chromosomes of the two species. The nucleolar organizer regions (NORs) were located in the bivalents L2, S9, S10 of Ommexecha virens and M5, M6, M7, S11 of Descampsacris serrulatum. The FISH for rDNA showed coincident results with the pattern of active NORs revealed by AgNO3. This work presents the first chromosomal data, obtained through differential cytogenetics techniques in Ommexechidae, contributing to a better characterization of karyotypic evolution for this grasshopper family.

Patterns of rDNA and telomeric sequences diversification: contribution to repetitive DNA organization in Phyllostomidae bats

Chromosomal organization and the evolution of genome architecture can be investigated by physical mapping of the genes for 45S and 5S ribosomal DNAs (rDNAs) and by the analysis of telomeric sequences. We studied 12 species of bats belonging to four subfamilies of the family Phyllostomidae in order to correlate patterns of distribution of heterochromatin and the multigene families for rDNA. The number of clusters for 45S gene ranged from one to three pairs, with exclusively location in autosomes, except for Carollia perspicillata that had in X chromosome. The 5S gene all the species studied had only one site located on an autosomal pair. In no species the 45S and 5S genes collocated. The fluorescence in situ hybridization (FISH) probe for telomeric sequences revealed fluorescence on all telomeres in all species, except in Carollia perspicillata. Non-telomeric sites in the pericentromeric region of the chromosomes were observed in most species, ranged from one to 12 pairs. Most interstitial telomeric sequences were coincident with heterochromatic regions. The results obtained in the present work indicate that different evolutionary mechanisms are acting in Phyllostomidae genome architecture, as well as the occurrence of Robertsonian fusion during the chromosomal evolution of bats without a loss of telomeric sequences. These data contribute to understanding the organization of multigene families and telomeric sequences on bat genome as well as the chromosomal evolutionary history of Phyllostomidae bats.

Chromosome mapping of ribosomal genes and histone H4 in the genus Radacridium (Romaleidae)

In this study, two species of Romaleidae grasshoppers, Radacridium mariajoseae and R.nordestinum, were analyzed after CMA3/DA/DAPI sequential staining and fluorescence in situ hybridization (FISH) to determine the location of the 18S and 5S rDNA and histone H4 genes. Both species presented karyotypes composed of 2n = 23, X0 with exclusively acrocentric chromosomes. CMA3+ blocks were detected after CMA3/DA/DAPI staining in only one medium size autosome bivalent and in the X chromosome in R. mariajoseae. On the other hand, all chromosomes, except the L1 bivalent, of R. nordestinum presented CMA3+ blocks. FISH analysis showed that the 18S genes are restricted to the X chromosome in R. mariajoseae, whereas these genes were located in the L2, S9 and S10 autosomes in R. nordestinum. In R. mariajoseae, the 5S rDNA sites were localized in the in L1 and L2 bivalents and in the X chromosome. In R. nordestinum, the 5S genes were located in the L2, L3, M4 and M5 pairs. In both species the histone H4 genes were present in a medium size bivalent. Together, these data evidence a great variability of chromosome markers and show that the 18S and 5S ribosomal genes are dispersed in the Radacridium genome without a significant correlation.

Spreading of heterochromatin and karyotype differentiation in two Tropidacris Scudder, 1869 species (Orthoptera, Romaleidae)

Abstract Tropidacris Scudder, 1869 is a genus widely distributed throughout the Neotropical region where speciation was probably promoted by forest reduction during the glacial and interglacial periods. There are no cytogenetic studies of Tropidacris, and information allowing inference or confirmation of the evolutionary events involved in speciation within the group is insufficient. In this paper, we used cytogenetic markers in two species, Tropidacris collaris (Stoll, 1813) and Tropidacris cristata grandis (Thunberg, 1824), collected in different Brazilian biomes. Both species exhibited 2n=24,XX for females and 2n=23,X0 for males. All chromosomes were acrocentric. There were some differences in the karyotype macrostructure, e.g. in the chromosome size. A wide interspecific variation in the chromosome banding (C-banding and CMA3/DAPI staining) indicated strong differences in the distribution of repetitive DNA sequences. Specifically, Tropidacris cristata grandis had a higher number of bands in relation to Tropidacris collaris. FISH with 18S rDNA revealed two markings coinciding with the NORs in both species. However, two analyzed samples of Tropidacris collaris revealed a heterozygous condition for the rDNA site of S10 pair. In Tropidacris collaris, the histone H3 genes were distributed on three chromosome pairs, whereas in Tropidacris cristata grandis, these genes were observed on 14 autosomes and on the X chromosome, always in terminal regions. Our results demonstrate that, although the chromosome number and morphology are conserved in the genus, Tropidacris cristata grandis substantially differs from Tropidacris collaris in terms of the distribution of repetitive sequences. The devastation and fragmentation of the Brazilian rainforest may have led to isolation between these species, and the spreading of these repetitive sequences could contribute to speciation within the genus.

B Chromosome Variants of the Grasshopper Xyleus discoideus angulatus Are Potentially Derived from Pericentromeric DNA.

B chromosomes, extra elements present in the karyotypes of some eukaryote species, have been described in the grasshopper Xyleus discoideus angulatus. Although some studies have proposed an autosomal origin of the B chromosome in X. d. angulatus, little is known about its repetitive DNA composition and evolutionary dynamics. The aim of the present work was to shed light on the B chromosome evolution in X. d. angulatus by cytogenetic analysis of 27 populations from Pernambuco and Ceará states (Brazil). The frequency of B chromosomes in the different populations was determined, and chromosome measurements and fluorescence in situ hybridization (FISH) with C0t-DNA and telomeric and B chromosome sequences were performed in cells from B-carrying individuals. The results revealed variations in B chromosome prevalence among the populations and showed that some B chromosomes were smaller in certain populations. FISH produced similar patterns for the C0t-DNA probe in all hybridized individuals, whereas telomeric and B chromosome probes, obtained by microdissection, exhibited variations in their distribution. These results indicate the presence of 3 morphotypes of B chromosomes in X. d. angulatus, with variation in repetitive DNA composition during their evolution. In this species, B chromosomes have an intraspecific origin and probably arose from the pericentromeric region of A chromosomes.