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.

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.

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.

Heterochromatin base pair composition and diversification in holocentric chromosomes of kissing bugs (Hemiptera, Reduviidae).

The subfamily Triatominae (Hemiptera, Reduviidae) includes 150 species of blood-sucking insects, vectors of Chagas disease or American trypanosomiasis. Karyotypic information reveals a striking stability in the number of autosomes. However, this group shows substantial variability in genome size, the amount and distribution of C-heterochromatin, and the chromosome positions of 45S rDNA clusters. Here, we analysed the karyotypes of 41 species from six different genera with C-fluorescence banding in order to evaluate the base-pair richness of heterochromatic regions. Our results show a high heterogeneity in the fluorescent staining of the heterochromatin in both autosomes and sex chromosomes, never reported before within an insect subfamily with holocentric chromosomes. This technique allows a clear discrimination of the heterochromatic regions classified as similar by C-banding, constituting a new chromosome marker with taxonomic and evolutionary significance. The diverse fluorescent patterns are likely due to the amplification of different repeated sequences, reflecting an unusual dynamic rearrangement in the genomes of this subfamily. Further, we discuss the evolution of these repeated sequences in both autosomes and sex chromosomes in species of Triatominae.

Heterochromatin base pair composition and diversification in holocentric chromosomes of kissing bugs (Hemiptera, Reduviidae)

The subfamily Triatominae (Hemiptera, Reduviidae) includes 150 species of blood-sucking insects, vectors of Chagas disease or American trypanosomiasis. Karyotypic information reveals a striking stability in the number of autosomes. However, this group shows substantial variability in genome size, the amount and distribution of C-heterochromatin, and the chromosome positions of 45S rDNA clusters. Here, we analysed the karyotypes of 41 species from six different genera with C-fluorescence banding in order to evaluate the base-pair richness of heterochromatic regions. Our results show a high heterogeneity in the fluorescent staining of the heterochromatin in both autosomes and sex chromosomes, never reported before within an insect subfamily with holocentric chromosomes. This technique allows a clear discrimination of the heterochromatic regions classified as similar by C-banding, constituting a new chromosome marker with taxonomic and evolutionary significance. The diverse fluorescent patterns are likely due to the amplification of different repeated sequences, reflecting an unusual dynamic rearrangement in the genomes of this subfamily. Further, we discuss the evolution of these repeated sequences in both autosomes and sex chromosomes in species of Triatominae.

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.

Origin and distribution of AT-rich repetitive DNA families in Triatoma infestans (Heteroptera).

Triatoma infestans, one of the most important vectors of Trypanosoma cruzi, is very interesting model, because it shows large interpopulation variation in the amount and distribution of heterochromatin. This polymorphism involved the three large pairs up to almost all autosomal pairs, including the sex chromosomes. To understand the dynamics of heterochromatin variation in T. infestans, we isolated the AT-rich satDNA portion of this insect using reassociation kinetics (C0t), followed by cloning, sequencing and FISH. After chromosome localization, immunolabeling with anti-5-methylcytosine, anti-H4K5ac and anti-H3K9me2 antibodies was performed to determine the functional characteristics of heterochromatin. The results allowed us to reorganize the karyotype of T. infestans in accordance with the distribution of the families of repetitive DNA using seven different markers. We found that two arrays with lengths of 79 and 33bp have a strong relationship with transposable element sequences, suggesting that these two families of satDNA probably originated from Polintons. The results also allowed us to identify at least four chromosome rearrangements involved in the amplification/dispersion of AT-rich satDNA of T. infestans. These data should be very useful in new studies including those examining the cytogenomic and population aspects of this very important species of insect.

Karyotype diversity among predatory Reduviidae (Heteroptera).

Species of infraorder Cimicomorpha of Heteroptera exhibit holokinetic chromosomes with inverted meiosis for sex chromosomes and high variation in chromosome number. The family Reduviidae, which belongs to this infraorder, is also recognized by high variability of heterochromatic bands and chromosome location of 18S rDNA loci. We studied here five species of Reduviidae (Harpactorinae) with predator habit, which are especially interesting because individuals are found solitary and dispersed in nature. These species showed striking variation in chromosome number (including sex chromosome systems), inter-chromosomal asymmetry, different number and chromosome location of 18S rDNA loci, dissimilar location and quantity of autosomal C-heterochromatin, and different types of repetitive DNA by fluorochrome banding, probably associated with occurrence of different chromosome rearrangements. Terminal chromosome location of C-heterochromatin seems to reinforce the model of equilocal dispersion of repetitive DNA families based in the "bouquet configuration".

The conservation of number and location of 18S sites indicates the relative stability of rDNA in species of Pentatomomorpha (Heteroptera).

Fluorescent in situ hybridization (FISH) with rDNA probes has been used for comparative cytogenetics studies in different groups of organisms. Although heteropterans are a large suborder within Hemiptera, studies using rDNA are limited to the infraorder Cimicomorpha, in which rDNA sites are present in the autosomes or sex chromosomes. We isolated and sequenced a conserved 18S rDNA region of Antiteuchus tripterus (Pentatomidae) and used it as a probe against chromosomes of 25 species belonging to five different families of Pentatomomorpha. The clone pAt05, with a length of 736 bp, exhibited a conserved stretch of 590 bp. FISH analysis with the probe pAt05 always demonstrated hybridization signals in sub-terminal positions, except for Euschistus heros. Apparently, there is a tendency for 18S rDNA sites to locate in autosomes, except for Leptoglossus gonagra and Euryophthalmus rufipennis, which showed signals in the m- and sex chromosomes, respectively. Although FISH has produced evidence that rearrangements are involved in rDNA repositioning, whether in different autosomes or between sex and m-chromosomes, we have no conclusive evidence of what were the pathways of these rearrangements based on the evolutionary history of the species studied here. Nevertheless, the diversity in the number of species analyzed here showed a tendency of 18S rDNA to remain among the autosomes.

Cytogenetic characterization reveals differences in nuclear organization of cystic cells among Brazilian species of Triatoma (Heteroptera, Reduviidae).

Cytogenetic studies in triatomines have described the occurrence of holokinetic chromosomes, heterochromatin distribution and the location of rDNA (ribosomal DNA) sites, but few aspects of nuclear organization in this group have been discussed. We have focused on ultrastructural and cytogenetic features and differences in cystic cells of seminiferous tubules between five species of Triatoma. Cystic cells showed evidence of polyploidy events and heterochromatic blocks appeared predominantly in the central region of the nuclei. Cytogenetic analyses showed that there was variation in chromocenter number between species, and that the central regions were AT-rich [DAPI+ (4',6-diamidino-2-phenylindole+)], whereas the periphery was CG-rich (CMA+). Another characteristic was the distribution of 45S rDNA, which differed according to the chromosomal location of this sequence. In all we have compared aspects of nuclear organization, polyploidy, heterochromatin, rDNA site distribution and methylation levels, as well as the relationships between five species of Triatoma from a cystic cell perspective.