Evolution of satDNAs on holocentric chromosomes: insights from hemipteran insects of the genus Mahanarva.

Satellite DNAs (satDNAs) constitute one of the main components of eukaryote genomes and are involved in chromosomal organization and diversification. Although largely studied, little information was gathered about their evolution on holocentric species, i.e., diffuse centromeres, which, due to differences in repeat organization, could result in different evolutionary patterns. Here, we combined bioinformatics and cytogenetic approaches to evaluate the evolution of the satellitomes in Mahanarva holocentric insects. In two species, de novo identification revealed a high number of satDNAs, 110 and 113, with an extreme monomer length range of 18-4228 bp. The overall abundance of satDNAs was observed to be 6.67% in M. quadripunctata and 1.98% in M. spectabilis, with different abundances for the shared satDNAs. Chromosomal mapping of the most abundant repeats of M. quadripunctata and M. spectabilis on other Mahanarva reinforced the dynamic nature of satDNAs. Variable patterns of chromosomal distribution for the satDNAs were noticed, with the occurrence of clusters on distinct numbers of chromosomes and at different positions and the occurrence of scattered signals or nonclustered satDNAs. Altogether, our data demonstrated the high dynamism of satDNAs in Mahanarva with the involvement of this genomic fraction in chromosome diversification of the genus. The general characteristics and patterns of evolution of satDNAs are similar to those observed on monocentric chromosomes, suggesting that the differential organization of genome compartments observed on holocentric chromosomes compared with monocentric chromosomes does not have a large impact on the evolution of satDNAs. Analysis of the satellitomes of other holocentric species in a comparative manner will shed light on this issue.

U1 snDNA chromosomal mapping in ten spittlebug species (Cercopidade, Auchenorrhyncha, Hemiptera).

Spittlebugs, which belong to the family Cercopidae (Auchenorrhyncha, Hemiptera), form a large group of xylem-feeding insects that are best known for causing damage to plantations and pasture grasses. The holocentric chromosomes of these insects remain poorly studied in regards to the organization of different classes of repetitive DNA. To improve chromosomal maps based on repetitive DNAs and to better understand the chromosomal organization and evolutionary dynamics of multigene families in spittlebugs, we physically mapped the U1 snRNA gene with fluorescence in situ hybridization (FISH) in 10 species of Cercopidae belonging to three different genera. All the U1 snDNA clusters were autosomal and located in interstitial position. In seven species, they were restricted to one autosome per haploid genome, while three species of the genus Mahanarva showed two clusters in two different autosomes. Although it was not possible to precisely define the ancestral location of this gene, it was possible to observe the presence of at least one cluster located in a small bivalent in all karyotypes. The karyotype stability observed in Cercopidae is also observed in respect to the distribution of U1 snDNA. Our data are discussed in light of possible mechanisms for U1 snDNA conservation and compared with the available data from other species.

Karyotypes and Repetitive DNA Evolution in Six Species of the Genus Mahanarva (Auchenorrhyncha: Cercopidae).

Insects of the Cercopidae family are widely distributed and comprise 59 genera and 431 species in the New World. They are xylemophagous, causing losses in agricultural and pasture grasses, and are considered as emerging pests. Chromosomally, these insects have been studied by standard techniques, revealing variable diploid numbers and primarily X0 sex chromosome systems (males). We performed chromosome studies in 6 Mahanarva (Cercopidae) species using standard and differential chromosome staining as well as mapping of repetitive DNAs. Moreover, the relationship between the repetitive DNAs was analyzed at the interspecific level. A diploid chromosome number of 2n = 19,X0 was documented, with chromosomes gradually decreasing in size. Neutral or GC-rich regions were detected which varied depending on the species. Fluorescence in situ hybridization with a (TTAGG)n telomeric motif probe revealed terminal signals, matching those of the Cot DNAs obtained from each species, that were also restricted to the terminal regions of all chromosomes. Dot blot analysis with the Cot fraction from M. quadripunctata showed that at least part of the repetitive genome is shared among the 6 species. Our data highlight the conservation of chromosomal features and organization of repetitive DNAs in the genus Mahanarva, suggesting a low differentiation for chromosomes and repetitive DNAs in most of the 6 species studied.

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.

Organization of some repetitive DNAs and B chromosomes in the grasshopper Eumastusia koebelei koebelei (Rehn, 1909) (Orthoptera, Acrididae, Leptysminae).

B chromosomes occur in approximately 15% of eukaryotes and are usually heterochromatic and rich in repetitive DNAs. Here we describe characteristics of a B chromosome in the grasshopper Eumastusia koebelei koebelei (Rehn, 1909) through classical cytogenetic methods and mapping of some repetitive DNAs, including multigene families, telomeric repeats and a DNA fraction enriched with repetitive DNAs obtained from DOP-PCR. Eumastusia koebelei koebelei presented 2n=23, X0 and, in one individual, two copies of the same variant of a B chromosome were noticed, which are associated during meiosis. The C-positive blocks were located in the pericentromeric regions of the standard complement and along the entire length of the B chromosomes. Some G+C-rich heterochromatic blocks were noticed, including conspicuous blocks in the B chromosomes. The mapping of 18S rDNA and U2 snDNA revealed only autosomal clusters, and the telomeric probe hybridized in terminal regions. Finally, the DOP-PCR probe obtained from an individual without a B chromosome revealed signals in the heterochromatic regions, including the entire length of the B chromosome. The possible intraspecific origin of the B chromosomes, due to the shared pool of repetitive DNAs between the A and B chromosomes and the possible consequences of their association are discussed.