Comparative cytogenetics of three species of Dichotomius (Coleoptera, Scarabaeidae)

Meiotic and mitotic chromosomes of Dichotomius nisus, D. semisquamosus and D. sericeus were analyzed after conventional staining, C-banding and silver nitrate staining. In addition, Dichotomius nisus and D. semisquamosus chromosomes were also analyzed after fluorescent in situ hybridization (FISH) with an rDNA probe. The species analyzed had an asymmetrical karyotype with 2n = 18 and meta-submetacentric chromosomes. The sex determination mechanism was of the Xyp type in D. nisus and D. semisquamosus and of the Xy r type in D. sericeus. C-banding revealed the presence of pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of the three species. After silver staining, the nucleolar organizer regions (NORs) were located in autosomes of D. semisquamosus and D. sericeus and in the sexual bivalent of D. nisus. FISH with an rDNA probe confirmed NORs location in D. semisquamosus and in D. nisus. Our results suggest that chromosome inversions and fusions occurred during the evolution of the group

Análise citogenética em três espécies do gênero Deltochilum (Coleoptera: Scarabaeidae)

O objetivo deste trabalho foi analisar citogeneticamente três espécies de coleópteros pertencentes ao gênero Deltochilum (Scarabaeidae), D. irroratum, D. morbillosum e D. verruciferum, através da coloração convencional, bandeamentos cromossômicos e hibridização in situ fluorescente (FISH). Deltochilum irroratum e D. morbillosum apresentaram número diplóide 2n=14 e mecanismo sexual neo-XY enquanto D. verruciferum possui cariótipo 2n=20,XYp. As três espécies possuem cromossomos meta-submetacêntricos. O bandeamento C revelou predominantemente cromossomos difásicos, com os braços longos heterocromáticos em D. irroratum e D. morbillosum e curtos heterocromáticos em D. verruciferum. A coloração com nitrato de prata marcou as seqüências correspondentes à heterocromatina constitutiva (HC) em D. morbillosum e D. verruciferum. Nesta última o lúmen do bivalente sexual também foi marcado. Em D. irroratum esta coloração evidenciou a HC dos cromossomos autossômicos difásicos. A coloração com fluorocromos CMA3 e DAPI revelou blocos CMA3 + na HC da espécie D. verruciferum e nos autossomos difásicos e bivalente sexual de D. irroratum. Em D. morbillosum as sequências CMA3 + estão restritas às regiões terminais do braço longo dos pares 1, 2 e do X. Nas três espécies a FISH identificou sítios de DNAr em dois pares autossômicos e no cromossomo X. A utilização destas técnicas permitiu a localização de marcadores citogenéticos e a análise dos possíveis rearranjos envolvidos ao longo da diferenciação cariótipica destas espécie

Microsatellite Organization in the Grasshopper Abracris flavolineata (Orthoptera: Acrididae) Revealed by FISH Mapping: Remarkable Spreading in the A and B Chromosomes

With the aim of acquiring deeper knowledge about repetitive DNAs chromosomal organization in grasshoppers, we used fluorescent in situ hybridization (FISH) to map the distribution of 16 microsatellite repeats, including mono-, di-, tri- and tetra-nucleotides, in the chromosomes of the species Abracris flavolineata (Acrididae), which harbors B chromosome. FISH revealed two main patterns: (i) exclusively scattered signals, and (ii) scattered and specific signals, forming evident blocks. The enrichment was observed in both euchromatic and heterochromatic areas and only the motif (C)(30) was absent in heterochromatin. The A and B chromosomes were enriched with all the elements that were mapped, being observed in the B chromosome more distinctive blocks for (GA)(15) and (GAG)(10). For A complement distinctive blocks were noticed for (A)(30), (CA)(15), (CG)(15), (GA)(15), (CAC)(10), (CAA)(10), (CGG)(10), (GAA)(10), (GAC)(10) and (GATA)(8). These results revealed an intense spreading of microsatellites in the A. flavolineata genome that was independent of the A+T or G+C enrichment in the repeats. The data indicate that the microsatellites compose the B chromosome and could be involved in the evolution of this element in this species, although no specific relationship with any A chromosome was observed to discuss about its origin. The systematic analysis presented here contributes to the knowledge of repetitive DNA chromosomal organization among grasshoppers including the B chromosomes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Organização cromossômica de elementos repetitivos de DNA em representantes da sufamília Scarabaeinae (Coleoptera: Scarabaeidae)

O mapeamento cromossômico de seqüências repetitivas de DNA tem se mostrado uma eficiente ferramenta nos estudos comparativos e evolutivos em diversos organismos. Estudos cromossômicos com besouros da subfamília Scarabaeinae têm revelado ampla variabilidade, entretanto a análise da organização cromossômica de DNAs repetitivos neste grupo é escassa e direcionada unicamente ao mapeamento do DNA ribossomal (DNAr) 18S. O presente trabalho teve como objetivo caracterizar cromossomicamente DNAs repetitivos em espécies de Scarabaeinae, utilizando bandeamentos cromossômicos e mapeamento físico cromossômico de seqüências repetitivas, incluindo famílias multigênicas de RNAr 18S, RNAr 5S e histona H3 e a fração de DNA C0t-1. Ampla variabilidade foi observada relacionada ao número/localização dos sítios de DNAr 18S, aparentemente associada a diversificação da heterocromatina. Por outro lado, os genes de RNAr 5S e histona H3, mostraram-se amplamente conservados e co-localizados em um par cromossômico, com aparente intercalação. Análises em representantes de Dichotomius revelaram conservação dos blocos de heterocromatina, entretanto com aparente compartimentalização dos mesmos. O uso da fração DNA C0t-1 confirmou o enriquecimento em DNAs repetitivos da heterocromatina, que se apresentou diversificada entre as espécies, utilizando como referência D. geminatus. Por outro lado, regiões terminais dos cromossomos apresentaram-se amplamente conservadas entre as seis espécies. Além disso, a análise da fração de DNAs repetitivos em D. geminatus indicou origem intraespecífica do cromossomo B desta espécie que possivelmente pode estar sofrendo homogeneização com seqüências encontradas no complemento A. Os resultados indicam distintos padrões de diversificação para o DNA repetitivo nos representantes de Scarabaeinae, sugerindo extensiva reorganizaçãomicrogenômica ao longoThe chromosomal mapping of repeated DNAs has been used as an efficient tool in comparative and evolutionary studies in some organism. The chromosomal studies in beetles belonging to the subfamily Scarabaeinae have revealed wide variability, although the analysis of chromosomal organization of repeated DNAs in this group is scarce and directed solely for 18S rDNA mapping. The present study aimed in chromosomal characterization of repeated DNAs in Scarabaeinae species using chromosomal banding and physical chromosome mapping of repeated sequences, including the multigene families for 18S and 5S rRNAs and H3 histone genes and the C0t-1 DNA fraction. Wide variability was observed concerning the number and location of 18S rDNA sites, apparently associated to the heterochromatin diversification. On the other hand, the 5S rRNA and H3 histone genes were widely conserved and co-located in one chromosomal pair, showing apparently interspersion. Analysis in Dichotomius representatives revealed conservation for heterochromatic blocks, although an apparent compartmentalization was observed. The use of C0t-1 DNA fraction confirmed the heterochromatin repeated DNAs enrichment, which is diversified among the species, using as reference D. geminatus. On the other hand, the terminal regions of the chromosomes were highly conserved among the six species. Moreover, the analysis of repeated DNA fraction from D. geminatus indicated intraspecific origin of a B chromosome in this species that possibly could be suffering homogenization with A complement sequences. The results indicate distinct diversification patterns for repeated DNAs in Scarabaeinae representatives, suggesting extensive microgenomic reorganization along the cladogenesis of the groupConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

FISH mapping for six microsatellite motifs in embryo mitotic cells of <i>A. flavolineata</i> with scattered distribution.

<p>Each microsatellite is indicated in each image. The B and X chromosomes are pointed in each image and the sex of the embryo can be noticed by the occurrence of one (male X0) or two (female XX) X chromosomes. Note the absence of signals in the heterochromatic areas for (C)₃₀. Bar  = 5 µm.</p

C-banding in embryo female mitotic metaphase of <i>Abracris flavolineata</i>.

<p>The X and B chromosomes are indicated. Note that the C-positive blocks in the centromeric regions extend to the short arms of the A chromosomes and the euchromatic nature of the B element. The inset highlights the euchromatic B chromosome. Bar  = 5 µm.</p

Chromosomal mapping of repetitive DNAs in the beetle Dichotomius geminatus provides the first evidence for an association of 5S rRNA and histone H3 genes in insects, and repetitive DNA similarity between the B chromosome and A complement

Chromosomal banding techniques and repetitive DNA mapping are useful tools in comparative analysis and in the elucidation of genome organization of several groups of eukaryotes. In this study, we contributed to the knowledge of Coleoptera genomes by reporting the chromosomal organization of repetitive DNA sequences, as well as the presence and characteristics of a B chromosome in two natural populations of Dichotomius geminatus (Coleoptera; Scarabaeidae) using classical, chromosomal banding and molecular cytogenetic techniques. As in other coleopteran species, the heterochromatin was mainly concentrated in pericentromeric regions and the B chromosome was composed almost entirely of heterochromatin. Physical mapping using double fluorescent in situ hybridization was performed for the first time in Coleoptera; using DNA probes for 5S and 18S ribosomal RNA (rRNA) and histone H3 genes, we showed that ribosomal 18S rDNAs are located in chromosomes 3 and 4, whereas 5S rRNA and histone H3 genes are colocalized in chromosomal pair 2 and show an apparently interspersed organization. Moreover, these genes are not present in the B chromosome, suggesting that the B chromosome did not originate from chromosomal pairs 2, 3 or 4. on the other hand, mapping of the C(0)t-1 DNA fraction showed that the B chromosome is enriched in repetitive DNA elements, also present in the standard complement, indicating an intraspecific origin of this element in D. geminatus. These results will contribute to our understanding of genome organization and evolution of repetitive elements in Coleoptera and other insects regarding both A and B chromosomes. Heredity (2010) 104, 393-400; doi: 10.1038/hdy.2009.126; published online 16 September 2009Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Heterochromatin and molecular characterization of DsmarMITE transposable element in the beetle Dichotomius schiffleri (Coleoptera: Scarabaeidae)

Cytogenetic studies of the Neotropical beetle genus Dichotomius (Scarabaeinae, Coleoptera) have shown dynamism for centromeric constitutive heterochromatin sequences. In the present work we studied the chromosomes and isolated repetitive sequences of Dichotomius schiffleri aiming to contribute to the understanding of coleopteran genome/chromosomal organization. Dichotomius schiffleri presented a conserved karyotype and heterochromatin distribution in comparison to other species of the genus with 2n = 18, biarmed chromosomes, and pericentromeric C-positive blocks. Similarly to heterochromatin distributional patterns, the highly and moderately repetitive DNA fraction (C (0) t-1 DNA) was detected in pericentromeric areas, contrasting with the euchromatic mapping of an isolated TE (named DsmarMITE). After structural analyses, the DsmarMITE was classified as a non-autonomous element of the type miniature inverted-repeat transposable element (MITE) with terminal inverted repeats similar to Mariner elements of insects from different orders. The euchromatic distribution for DsmarMITE indicates that it does not play a part in the dynamics of constitutive heterochromatin sequences.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq