Differential amplification and contraction of satellite DNAs in the distinct lineages of the beetle Euchroma gigantea.

Satellite DNA (satDNA) consists of tandem repeat sequences that typically evolve rapidly through evolutionary mechanisms, including unequal crossover, transposition events, and others. The evolutionary history of Euchroma gigantea is marked by complex chromosomal evolution between lineages, making this species an interesting model for understanding satDNA evolution at intraspecies level. Therefore, our aim was to comprehend the potential contribution of satDNAs to the greater chromosomal differentiation of evolutionary lineages in E. gigantea by investigating the differential patterns of amplification and contraction of the repeats. To achieve this, we employed de novo identification of satDNA using RepeatExplorer and TAREAN, allowing the satellitome characterization between lineages. A total of 26 satDNA families were identified, ranging from 18 to 1101 nucleotides in length, with most families being shared between individuals/lineages, as predicted by the library hypothesis, except for the satDNA EgiSat21-168 that was absent for Northeast Lineage. The total satDNA content of the individuals was less than 11.2%, and it appeared to increase in two directions following the chromosomal evolution model. Thirteen satDNAs exhibited different patterns of amplification, and nine ones were contracted among individuals. Additionally, most repeats showed a divergence of about 10% for these satDNAs, indicating satellitome differentiation for each lineage/individual. This scenario suggests that the expansion of the satellitome occurred differentially among individuals/lineages of E. gigantea, with the contribution of various DNA turnover mechanisms after geographical isolation, and that they could be involved with karyotype evolution.

Comparative analysis of transposable elements dynamics in fish with different sex chromosome systems.

Transposable elements (TEs) are widespread genomic components with substantial roles in genome evolution and sex chromosome differentiation. In this study, we compared the TE composition of three closely related fish with different sex chromosome systems: Megaleporinus elongatus (Z1Z1Z2Z2/Z1W1Z2W2), Megaleporinus macrocephalus (ZZ/ZW) (both with highly differentiated W sex chromosomes), and Leporinus friderici (without heteromorphic sex chromosomes). We created custom TE libraries for each species using clustering methods and manual annotation and prediction, and we predicted TE temporal dynamics through divergence-based analysis. The TE abundance ranged from 16% to 21% in the three mobilomes, with L. friderici having the lowest overall. Despite the recent amplification of TEs in all three species, we observed differing expansion activities, particularly between the two genera. Both Megaleporinus recently experienced high retrotransposon activity, with a reduction in DNA TEs, which could have implications in sex chromosome composition. In contrast, L. friderici showed the opposite pattern. Therefore, despite having similar TE compositions, Megaleporinus and Leporinus exhibit distinct TE histories that likely evolved after their separation, highlighting a rapid TE expansion over short evolutionary periods.

Telomere organization and the interstitial telomeric sites involvement in insects and vertebrates chromosome evolution.

Telomere has a central role in chromosomal stability events. Chromosome ends organized in telomere-loop prevent activation of DNA damage response (DDR) mechanisms, thus keeping the chromosome structure organized. On the other hand, free chromosome ends, dysfunctional telomeres, and interstitial telomeric sequences (ITS) can trigger chromosome rearrangements. Here, the telomere organization, function, and maintenance mechanisms, in addition to ITS types and their involvement in chromosome changes, were revisited. Despite a general (TTAGGG)n sequence being present in vertebrate telomeres, insects show more diversification of their telomere motif. The relation between ITS and chromosome rearrangements was observed in insects and vertebrates, demonstrating different types of genome organization and distribution. Some ITS cannot be considered relicts of chromosome rearrangements because probable they were inserted during a double-strand break repair mechanism. On the other hand, the involvement of telomere sequences participating or triggering chromosome rearrangements or organizing satellite DNA components in several species groups is evident. The genomic assembling advances and applying other methodologies over ITS, and their flanking regions, can help to understand the telomere participation in the chromosomal evolution in species groups with highly diversified karyotypes.

Establishment and multiapproach characterization of Amblyomma sculptum (Acari: Ixodidae) cell line (ASE-14) from Brazil.

The establishment and characterization of the ASE-14 cell line derived from embryos of Amblyomma sculptum is described here. Primary cultures were started, and after 60 days of culturing a confluent monolayer was formed and the first subculture was then carried out. After this, new subcultures were carried out every 4 weeks. Cryopreservation of cells was successful only after the 14th subculture. We compared the chromosomes of the ASE-14 cell line with those of parental ticks. Cytogenetic analysis revealed occurrences of variable and increased diploid numbers in the ASE-14 cell line in comparison with adult ticks, probably through polyploidization events, chromosome fusions and translocations, which allowed generation of cells with distinct diploid numbers. Confirmation of the origin of the A. sculptum cell line was obtained through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In addition, no DNA from Anaplasma marginale, Anaplasma spp., Babesia/Theileria spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Mycoplasma spp. or Rickettsia spp. was detected in the cells through PCR assays. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect polysaccharides and protein, respectively. In conclusion, a new cell line derived from embryos of A. sculptum was generated and characterized in this study. The ASE-14 cell line was deposited in the Tick Cell Biobank at the University of Liverpool, and in the Tick Cell Biobank South America Outpost at the Oswaldo Cruz Foundation (FIOCRUZ). The ASE-14 cell line is an important addition to the existing panel of tick cell lines and can be used as a tool for advancing research in various areas of the virology, bacteriology, biology and control of this tick.

Establishment and characterization of a cell line (RBME-6) of Rhipicephalus (Boophilus) microplus from Brazil.

Tick cell lines have already proved to be a useful tool for obtaining more information about possible vector species and the factors governing their ability to transmit a pathogen. Here, we established and characterized a cell line (RBME-6) derived from embryos of Rhipicephalus microplus from Brazil. Primary tick cell cultures were prepared in L-15B medium supplemented with 20% fetal bovine serum and 10% tryptose phosphate broth. The cell monolayers were subcultured when they reached a density of approximately 8 × 10 5 cells/mL (95% viability). Only after the sixth subculture were cells thawed from storage in liquid nitrogen successfully. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect total polysaccharides and total protein, respectively . No DNA from Anaplasma spp., Anaplasma marginale, Babesia spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Rickettsia spp. or Mycoplasma spp. was detected in the cells through PCR assays. In addition, we performed chromosomal characterization of the tick cell line and confirmed the R. microplus origin of the cell line through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In conclusion, we established and characterized a new cell line from a Brazilian population of R. microplus, which may form a useful tool for studying several aspects of ticks and tick-borne pathogens.

Universal fluorescence in situ hybridization (FISH) protocol for mapping repetitive DNAs in insects and other arthropods.

Repetitive DNAs comprise large portion of eukaryote genomes. In genome projects, the assembly of repetitive DNAs is challenging due to the similarity between repeats, which generate ambiguities for alignment. Fluorescence in situ hybridization (FISH) is a powerful technique for the physical mapping of various sequences on chromosomes. This technique is thus very helpful in chromosome-based genome assemblies, providing information on the fine architecture of genomes and their evolution. However, various protocols are currently used for FISH mapping, most of which are relatively laborious and expensive, or work properly only with a specific type of probes or sequences, and there is a need for a universal and affordable FISH protocol. Here we tested a FISH protocol for mapping of different DNA repeats, such as multigene families (rDNAs, U snDNAs, histone genes), satellite DNAs, microsatellites, transposable elements, DOP-PCR products, and telomeric motif (TTAGG)n, on the chromosomes of various insects and other arthropods. Different cell types and stages obtained from diverse tissues were used. The FISH procedure proved high quality and reliable results in all experiments performed. We obtained data on the chromosomal distribution of DNA repeats in representatives of insects and other arthropods. Thus, our results allow us to conclude that the protocol is universal and requires only time adjustment for chromosome/DNA denaturation. The use of this FISH protocol will facilitate studies focused on understanding the evolution and role of repetitive DNA in arthropod genomes.

Analysis of Holhymenia histrio genome provides insight into the satDNA evolution in an insect with holocentric chromosomes.

Satellite DNAs (satDNA) are fast-evolving repetitive sequences organized in large tandem arrays, with characteristic enrichment in heterochromatin. Knowledge about evolutionary dynamics of this genome fraction is mostly restricted to its characterization in species with monocentric chromosomes, i.e., localized centromeres. In holocentric species, with non-localized centromeres, satDNAs have been largely ignored. Here we advance the understanding of satDNA evolution among holocentric species by characterization of the most abundant satDNAs in the hemipteran Holhymenia histrio, integrating genomic and chromosomal analyses. High plasticity at chromosomal and molecular levels was noticed for 34 satDNAs populating H. histrio genome. One satDNA family in particular (HhiSat01-184) was highly amplified on multiple chromosomes and also highly polymorphic. Our data support the emergence of a new satDNA family from this abundant satDNA, confined to a single chromosome. Moreover, we present new information about composition of a peculiar chromosome in Coreidae, the m-chromosome, and of the X chromosome. Overall, the molecular and chromosomal patterns for satDNAs in the holocentric species H. histrio seem to be similar to those observed in monocentric species.

Insights into the karyotype evolution and speciation of the beetle Euchroma gigantea (Coleoptera: Buprestidae).

Euchroma Dejean, 1833 (Buprestidae: Coleoptera) is a monotypic genus comprising the species Euchroma gigantea, with populations presenting a degree of karyotypic variation/polymorphism rarely found within a single taxonomic (specific) unit, as well as drastically incompatible meiotic configurations in populations from extremes of the species range. To better understand the complex karyotypic evolution of E. gigantea, the karyotypes of specimens from five populations in Brazil were investigated using molecular cytogenetics and phylogenetic approaches. Herein, we used FISH with histone genes as well as sequencing of the COI to determine differential distribution of markers and relationships among populations. The analyses revealed new karyotypes, with variability for chromosome number and morphology of multiple sex chromosome mechanisms, occurrence of B chromosome variants (punctiform and large ones), and high dispersion of histone genes in different karyotypes. These data indicate that chromosomal polymorphism in E. gigantea is greater than previously reported, and that the species can be a valuable model for cytogenetic studies. The COI phylogenetic and haplotype analyses highlighted the formation of three groups with chromosomally polymorphic individuals. Finally, we compared the different karyotypes and proposed a model for the chromosomal evolution of this species. The species E. gigantea includes at least three cytogenetically polymorphic lineages. Moreover, in each of these lineages, different chromosomal rearrangements have been fixed. Dispersion of repetitive sequences may have favored the high frequency of these rearrangements, which could be related to both adaptation of the species to different habitats and the speciation process.

Satellite DNAs are conserved and differentially transcribed among Gryllus cricket species.

Satellite DNA (satDNA) is an abundant class of non-coding repetitive DNA that is preferentially found as tandemly repeated arrays in gene-poor heterochromatin but is also present in gene-rich euchromatin. Here, we used DNA- and RNA-seq from Gryllus assimilis to address the content and transcriptional patterns of satDNAs. We also mapped RNA-seq libraries for other Gryllus species against the satDNAs found in G. assimilis and G. bimaculatus genomes to investigate their evolutionary conservation and transcriptional profiles in Gryllus. Through DNA-seq read clustering analysis using RepeatExplorer, dotplots analysis and fluorescence in situ hybridization mapping, we found that ∼4% of the G. assimilis genome is represented by 11 well-defined A + T-rich satDNA families. These are mainly located in heterochromatic areas, with some repeats able to form high-order repeat structures. By in silico transcriptional analysis we identified satDNAs that are conserved in Gryllus but differentially transcribed. The data regarding satDNA presence in G. assimilis genome were discussed in an evolutionary context, with transcriptional data enabling comparisons between sexes and across tissues when possible. We discuss hypotheses for the conservation and transcription of satDNAs in Gryllus, which might result from their role in sexual differentiation at the chromatin level, heterochromatin formation and centromeric function.

Uncovering the molecular organization of unusual highly scattered 5S rDNA: The case of Chariesterus armatus (Heteroptera).

One cluster of 5S rDNA per haploid genome is the most common pattern among Heteroptera. However, in Chariesterus armatus, highly scattered signals were noticed. We isolated and characterized the entire 5S rDNA unit of C. armatus aiming to a deeper knowledge of molecular organization of the 5S rDNA among Heteroptera and to understand possible causes and consequences of 5S rDNA chromosomal spreading. For a comparative analysis, we performed the same approach in Holymenia histrio with 5S rDNA restricted to one bivalent. Multiple 5S rDNA variants were observed in both species, though they were more variable in C. armatus, with some of variants corresponding to pseudogenes. These pseudogenes suggest birth-and-death mechanism, though homogenization was also observed (concerted evolution), indicating evolution through mixed model. Association between transposable elements and 5S rDNA was not observed, suggesting spreading of 5S rDNA through other mechanisms, like ectopic recombination. Scattered organization is a rare example for 5S rDNA, and such organization in C. armatus genome could have led to the high diversification of sequences favoring their pseudogenization.

Centromeric enrichment of LINE-1 retrotransposons and its significance for the chromosome evolution of Phyllostomid bats.

Despite their ubiquitous incidence, little is known about the chromosomal distribution of long interspersed elements (LINEs) in mammalian genomes. Phyllostomid bats, characterized by lineages with distinct trends of chromosomal evolution coupled with remarkable ecological and taxonomic diversity, represent good models to understand how these repetitive sequences contribute to the evolution of genome architecture and its link to lineage diversification. To test the hypothesis that LINE-1 sequences were important modifiers of bat genome architecture, we characterized the distribution of LINE-1-derived sequences on genomes of 13 phyllostomid species within a phylogenetic framework. We found massive accumulation of LINE-1 elements in the centromeres of most species: a rare phenomenon on mammalian genomes. We hypothesize that expansion of these elements has occurred early in the radiation of phyllostomids and recurred episodically. LINE-1 expansions on centromeric heterochromatin probably spurred chromosomal change before the radiation of phyllostomids into the extant 11 subfamilies and contributed to the high degree of karyotypic variation observed among different lineages. Understanding centromere architecture in a variety of taxa promises to explain how lineage-specific changes on centromere structure can contribute to karyotypic diversity while not disrupting functional constraints for proper cell division.

The satellite DNA AflaSAT-1 in the A and B chromosomes of the grasshopper Abracris flavolineata.

BACKGROUND: Satellite DNAs (satDNAs) are organized in repetitions directly contiguous to one another, forming long arrays and composing a large portion of eukaryote genomes. These sequences evolve according to the concerted evolution model, and homogenization of repeats is observed at the intragenomic level. Satellite DNAs are the primary component of heterochromatin, located primarily in centromeres and telomeres. Moreover, satDNA enrichment in specific chromosomes has been observed, such as in B chromosomes, that can provide clues about composition, origin and evolution of this chromosome. In this study, we isolated and characterized a satDNA in A and B chromosomes of Abracris flavolineata by integrating cytogenetic, molecular and genomics approaches at intra- and inter-population levels, with the aim to understand the evolution of satDNA and composition of B chromosomes. RESULTS: AflaSAT-1 satDNA was shared with other species and in A. flavolineata, was associated with another satDNA, AflaSAT-2. Chromosomal mapping revealed centromeric blocks variable in size in almost all chromosomes (except pair 11) of A complement for both satDNAs, whereas for B chromosome, only a small centromeric signal occurred. In distinct populations, variable number of AflaSAT-1 chromosomal sites correlated with variability in copy number. Instead of such variability, low sequence diversity was observed in A complement, but monomers from B chromosome were more variable, presenting also exclusive mutations. AflaSAT-1 was transcribed in five tissues of adults in distinct life cycle phases. CONCLUSIONS: The sharing of AflaSAT-1 with other species is consistent with the library hypothesis and indicates common origin in a common ancestor; however, AflaSAT-1 was highly amplified in the genome of A. flavolineata. At the population level, homogenization of repeats in distinct populations was documented, but dynamic expansion or elimination of repeats was also observed. Concerning the B chromosome, our data provided new information on the composition in A. flavolineata. Together with previous results, the sequences of heterochromatic nature were not likely highly amplified in the entire B chromosome. Finally, the constitutive transcriptional activity suggests a possible unknown functional role, which should be further investigated.

High-throughput analysis of the satellitome revealed enormous diversity of satellite DNAs in the neo-Y chromosome of the cricket Eneoptera surinamensis.

Satellite DNAs (satDNAs) constitute large portion of eukaryote genomes, comprising non-protein-coding sequences tandemly repeated. They are mostly found in heterochromatic regions of chromosomes such as around centromere or near telomeres, in intercalary heterochromatin, and often in non-recombining segments of sex chromosomes. We examined the satellitome in the cricket Eneoptera surinamensis (2n = 9, neo-X1X2Y, males) to characterize the molecular evolution of its neo-sex chromosomes. To achieve this, we analyzed illumina reads using graph-based clustering and complementary analyses. We found an unusually high number of 45 families of satDNAs, ranging from 4 bp to 517 bp, accounting for about 14% of the genome and showing different modular structures and high diversity of arrays. FISH mapping revealed that satDNAs are located mostly in C-positive pericentromeric regions of the chromosomes. SatDNAs enrichment was also observed in the neo-sex chromosomes in comparison to autosomes. Especially astonishing accumulation of satDNAs loci was found in the highly differentiated neo-Y, including 39 satDNAs over-represented in this chromosome, which is the greatest satDNAs diversity yet reported for sex chromosomes. Our results suggest possible involvement of satDNAs in genome increasing and in molecular differentiation of the neo-sex chromosomes in this species, contributing to the understanding of sex chromosome composition and evolution in Orthoptera.

Highest Diploid Number Among Gymnotiformes: First Cytogenetic Insights into Rhabdolichops (Sternopygidae).

We report the first comparative cytogenetic analysis of two species from electrogenic fish of genus Rhabdolichops (Sternopygidae, Gymnotiformes): Rhabdolichops troscheli and Rhabdolichops cf eastwardi. R. troscheli has 2n = 54 (fundamental number [FN] = 66), whereas R. cf. eastwardi has 2n = 74 (FN = 78). C-banding revealed centromeric constitutive heterochromatin in both species. Ag-NORs mapped on pair 6 in R. troscheli and pair 30 in R. cf eastwardi. Fluorescense in situ hybridization with 18S rDNA probes confirmed the Ag-NOR staining results and revealed additional (presumably silent) ribosomal genes on pairs 12, 13, 21, 23, 26, and 27 in R. cf eastwardi. 5S rDNA was found on the centromeres of pair 7 in both species. Telomeric probes showed only distal locations. Dispersed signal patterns were obtained using probes for retrotransposons Rex1 and Rex3. Histone H1 and H3 genes were found together on pair 6 in R. cf eastwardi. The high diploid number found in Rhabdolichops suggests that chromosome fission may have contributed to its chromosomal evolution, phylogenetic relationship of the Sternopygidae suggests that this increase in diploid number could be a synapomorphic characteristic of genus Rhabdolichops. Although both species are phylogenetically close related, their karyotype structure has undergone divergent evolutionary directions. All in all, our results strongly suggest that R. cf eastwardi experencied recent intense genome reorganization.

Possible origin of B chromosome in Dichotomius sericeus (Coleoptera).

B chromosomes have so far been described in about 80 species of Coleoptera, mainly using conventional staining analysis. In this study, 152 individuals of the dung beetle Dichotomius sericeus (Coleoptera), collected from three isolated geographical areas in the State of Pernambuco, Brazil, were analyzed to determine the frequency, prevalence, distribution, meiotic behavior, and possible B chromosome origin. The cytogenetic analysis consisted of conventional staining, C-banding, triple fluorochrome staining (CMA3/DA/DAPI), and fluorescent in situ hybridization using ribosomal DNAs (rDNAs) and H3 histone gene as probes, as well as microdissection and chromosome painting of the B chromosome. The B chromosomes were detected in all populations analyzed. Analysis revealed the heterochromatic nature and the presence of G+C-rich blocks and 18S rDNA on the B chromosome. FISH with DNA from microdissected B chromosome painted the entire extension of the B chromosome for all populations, besides the pericentromeric regions of all the autosomes, as well as the X chromosome. Finally, cross-hybridization in nine related species of Dichotomius using the microdissected B chromosome as probe did not reveal any hybridization signal. The results suggest an intraspecific and monophyletic origin for B chromosomes in D. sericeus, probably from the second or third autosomal pair.

Chromosomal evolutionary dynamics of four multigene families in Coreidae and Pentatomidae (Heteroptera) true bugs.

Previous chromosome mapping of multigene families in Pentatomomorpha (Heteroptera) insects, which was restricted to the major rDNA, revealed remarkable conservation of number of clusters and chromosomal positions. Aiming to understand the chromosomal organization and evolutionary patterns of multigene families in karyotypes of Heteroptera, we performed a chromosomal mapping using four distinct multigene families in representatives of Coreidae (ten species) and Pentatomidae (five species). A single pair of the major rDNA cluster (18S rDNA probe) and a single pair of the minor rDNA cluster (5S rDNA probe), both terminally located were primarily observed, being, in most species, located in distinct chromosomes. However, some alternative patterns were also observed. In species in which the U2 snDNA and H4 gene clusters were mapped, they were mainly located in one autosomal pair each, wherein the H4 gene cluster was located in different positions. Our data suggest that the karyotype diversity reported in Coreidae is not reflected in the distribution diversity of multigene families. This contrasts with the data for Pentatomidae, with a conserved gross karyotype but a discrete diversity in the location of the clusters of multigene families, indicating genome dynamics for these markers. The findings are discussed to shed light on the possible causes for the conservation or variation observed and to assist in understanding the chromosomal evolutionary trends in the group.

The 5S rDNA in two Abracris grasshoppers (Ommatolampidinae: Acrididae): molecular and chromosomal organization.

The 5S ribosomal DNA (rDNA) sequences are subject of dynamic evolution at chromosomal and molecular levels, evolving through concerted and/or birth-and-death fashion. Among grasshoppers, the chromosomal location for this sequence was established for some species, but little molecular information was obtained to infer evolutionary patterns. Here, we integrated data from chromosomal and nucleotide sequence analysis for 5S rDNA in two Abracris species aiming to identify evolutionary dynamics. For both species, two arrays were identified, a larger sequence (named type-I) that consisted of the entire 5S rDNA gene plus NTS (non-transcribed spacer) and a smaller (named type-II) with truncated 5S rDNA gene plus short NTS that was considered a pseudogene. For type-I sequences, the gene corresponding region contained the internal control region and poly-T motif and the NTS presented partial transposable elements. Between the species, nucleotide differences for type-I were noticed, while type-II was identical, suggesting pseudogenization in a common ancestor. At chromosomal point to view, the type-II was placed in one bivalent, while type-I occurred in multiple copies in distinct chromosomes. In Abracris, the evolution of 5S rDNA was apparently influenced by the chromosomal distribution of clusters (single or multiple location), resulting in a mixed mechanism integrating concerted and birth-and-death evolution depending on the unit.

Toxicogenetic effects of low concentrations of the pesticides imidacloprid and sulfentrazone individually and in combination in in vitro tests with HepG2 cells and Salmonella typhimurium.

The insecticide imidacloprid and the herbicide sulfentrazone are two different classes of pesticides that are used for pest control in sugarcane agriculture. To evaluate the genotoxic potential of low concentrations of these two pesticides alone and in mixture, the comet assay and the micronucleus (MN) test employing fluorescence in situ hybridization (FISH) with a centromeric probe were applied in human hepatoma cell lines (HepG2), in a 24-h assay. Mutagenicity was assessed by Salmonella/microsome assay with TA98 and TA100 strains in the absence and presence of an exogenous metabolizing system (S9). The results showed significant inductions of MN in HepG2 cells by both pesticides, for all the tested concentrations. As evidenced in the comet assay, only the imidacloprid presented significant responses. When the two pesticides were associated, a significant induction of damage was observed in the HepG2 cells by the comet assay, but not by the MN test. Moreover, the MN induced by the mixtures of the pesticides appeared at lower levels than those induced by sulfentrazone and imidacloprid when tested alone. According to the FISH results, the damage induced by imidacloprid in the HepG2 cells resulted from a clastogenic action of this insecticide (76.6% of the MN did not present a centromeric signal). For the herbicide sulfentrazone and for the mixture of the pesticides, a similar frequency of MN with and without the presence of the centromeric signal (herbicide: 52.45% of the MN without centromeric signal and 47.54% of the MN with centromeric signal; mixture: 48.71% of the MN without centromeric signal and 51.42% of the MN with centromeric signal) was verified. Based on these results, it was concluded that each one of the pesticides evaluated interacts with the DNA of HepG2 cells and causes irreparable alterations in the cells. However, the combination of the pesticides showed an antagonistic effect on the cells and the damage induced was milder and not persistent in HepG2 cells. The results obtained by the Ames test did not point out significant results.

High similarity of U2 snDNA sequence between A and B chromosomes in the grasshopper Abracris flavolineata.

B chromosomes are frequently enriched for a wide variety of repetitive DNAs. Among grasshoppers in the species Abracris flavolineata (Ommatolampidinae) the B chromosomes are submetacentric, C-negative and harbor repetitive DNAs such as, U2 snDNA, C 0 t-1 DNA, two Mariner-like elements and some microsatellites. Here, we provide evidence showing the intragenome similarity between the B chromosome and the A complement in A. flavolineata, combining analysis of microdissection and chromosome painting and B chromosome-specific amplification through polymerase chain reaction (PCR) of U2 snDNA. Chromosome painting revealed signals spread through the C-negative regions, including the A and B chromosomes. Moreover, significant clustered signals forming bands were observed in some A chromosomes, and for the B chromosome, significant signals were located on both arms, which could be caused by accumulation of repetitive DNA sequences. The C-positive regions did not reveal any signals. Sequence comparison of U2 snDNA between that obtained from a genome without the B chromosome and that from µB-DNA revealed high similarity with the occurrence of four shared haplotypes, one of them (i.e., Hap1) being highly prevalent and putatively ancestral. The highest divergence from Hap1 was observed for Hap3, which was caused by only six mutational steps. These data support an intraspecific origin of the B chromosome in A. flavolineata that is highly similar with the A complement, and the low U2 snDNA sequence diversity observed in the B chromosome could be related to its recent origin, besides intrachromosomal concerted evolution for U2 snDNA repeats in the B chromosome.

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.