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.

A step forward in the genome characterization of the sugarcane borer, Diatraea saccharalis: karyotype analysis, sex chromosome system and repetitive DNAs through a cytogenomic approach.

Moths of the family Crambidae include a number of pests that cause economic losses to agricultural crops. Despite their economic importance, little is known about their genome architecture and chromosome evolution. Here, we characterized the chromosomes and repetitive DNA of the sugarcane borer Diatraea saccharalis using a combination of low-pass genome sequencing, bioinformatics, and cytogenetic methods, focusing on the sex chromosomes. Diploid chromosome numbers differed between the sexes, i.e., 2n = 33 in females and 2n = 34 in males. This difference was caused by the occurrence of a WZ1Z2 trivalent in female meiosis, indicating a multiple sex-chromosome system WZ1Z2/Z1Z1Z2Z2. A strong interstitial telomeric signal was observed on the W chromosome, indicating a fusion of the ancestral W chromosome with an autosome. Among repetitive DNAs, transposable elements (TEs) accounted for 39.18% (males) to 41.35% (females), while satDNAs accounted for only 0.214% (males) and 0.215% (females) of the genome. FISH mapping revealed different chromosomal organization of satDNAs, such as single localized clusters, spread repeats, and non-clustered repeats. Two TEs mapped by FISH were scattered. Although we found a slight enrichment of some satDNAs in the female genome, they were not differentially enriched on the W chromosome. However, we found enriched FISH signals for TEs on the W chromosome, suggesting their involvement in W chromosome degeneration and differentiation. These data shed light on karyotype and repetitive DNA dynamics due to multiple chromosome fusions in D. saccharalis, contribute to the understanding of genome structure in Lepidoptera and are important for future genomic studies.

The extensive amplification of heterochromatin in Melipona bees revealed by high throughput genomic and chromosomal analysis.

Satellite DNAs (satDNAs) and transposable elements (TEs) are among the main components of constitutive heterochromatin (c-heterochromatin) and are related to their functionality, dynamics, and evolution. A peculiar case regarding the quantity and distribution of c-heterochromatin is observed in the genus of bees, Melipona, with species having a low amount of heterochromatin and species with high amount occupying almost all chromosomes. By combining low-pass genome sequencing and chromosomal analysis, we characterized the satDNAs and TEs of Melipona quadrifasciata (low c-heterochromatin) and Melipona scutellaris (high low c-heterochromatin) to understand c-heterochromatin composition and evolution. We identified 15 satDNA families and 20 TEs for both species. Significant variations in the repeat landscapes were observed between the species. In M. quadrifasciata, the repetitive fraction corresponded to only 3.78% of the genome library studied, whereas in M. scutellaris, it represented 54.95%. Massive quantitative and qualitative changes contributed to the differential amplification of c-heterochromatin, mainly due to the amplification of exclusive repetitions in M. scutellaris, as the satDNA MscuSat01-195 and the TE LTR/Gypsy_1 that represent 38.20 and 14.4% of its genome, respectively. The amplification of these two repeats is evident at the chromosomal level, with observation of their occurrence on most c-heterochromatin. Moreover, we detected repeats shared between species, revealing that they experienced mainly quantitative variations and varied in the organization on chromosomes and evolutionary patterns. Together, our data allow the discussion of patterns of evolution of repetitive DNAs and c-heterochromatin that occurred in a short period of time, after separation of the Michmelia and Melipona subgenera.

The relevance of chromosome fissions for major ribosomal DNA dispersion in hymenopteran insects.

Ribosomal DNA (rDNA) loci are essential for cellular metabolism due to their participation in ribosome biogenesis. Although these genes have been widely cytogenetically mapped, the evolutionary mechanisms behind their variability in number and chromosomal location remain elusive, even in well-known biological groups, such as ants, bees and wasps (Insecta: Hymenoptera). To address this question in Hymenoptera and therefore advance the understanding of rDNA evolution in insects in general, we integrated molecular cytogenetic data, a phylogenomic framework, model-based predictions and genome sequencing. Hence, we assessed the main evolutionary trends shaping the chromosomal distribution of rDNA loci in Hymenoptera. We noticed the conservation of one site of rDNA per haploid genome, suggesting that a single 45S rDNA locus is the putative ancestral pattern for aculeate Hymenoptera. Moreover, our results highlighted a nonrandom distribution of rDNA in Hymenoptera karyotypes, as well as a lineage-specific preferential location. The proximal location of rDNA is favoured in species with multiple loci and in the two families of Hymenoptera that show the highest range of chromosome numbers: Formicidae and Vespidae. We propose that chromosome fissions have played a crucial role in the distribution pattern of rDNA loci through the evolutionary diversification of Hymenoptera. Moreover, our genomic analysis of two species, one with a single locus of rDNA and one with multiple loci, supported that loci multiplication is followed by sequence divergence. Our results provide detailed information about the number and chromosomal position of rDNA in Hymenoptera and, therefore, broaden our knowledge regarding rDNA evolutionary dynamics in insects.

Spatial Distribution of Heterochromatin Bodies in the Nuclei of Triatoma infestans (Klug).

Constitutive heterochromatin typically exhibits low gene density and is commonly found adjacent or close to the nuclear periphery, in contrast to transcriptionally active genes concentrated in the innermost nuclear region. In Triatoma infestans cells, conspicuous constitutive heterochromatin forms deeply stained structures named chromocenters. However, to the best of our knowledge, no information exists regarding whether these chromocenters acquire a precise topology in the cell nuclei or whether their 18S rDNA, which is important for ribosome function, faces the nuclear center preferentially. In this work, the spatial distribution of fluorescent Feulgen-stained chromocenters and the distribution of their 18S rDNA was analyzed in Malpighian tubule cells of T. infestans using confocal microscopy. The chromocenters were shown to be spatially positioned relatively close to the nuclear periphery, though not adjacent to it. The variable distance between the chromocenters and the nuclear periphery suggests mobility of these bodies within the cell nuclei. The distribution of 18S rDNA at the edge of the chromocenters was not found to face the nuclear interior exclusively. Because the genome regions containing 18S rDNA in the chromocenters also face the nuclear periphery, the proximity of the chromocenters to this nuclear region is not assumed to be associated with overall gene silencing.

Are the TTAGG and TTAGGG telomeric repeats phylogenetically conserved in aculeate Hymenoptera?

Despite the (TTAGG)n telomeric repeat supposed being the ancestral DNA motif of telomeres in insects, it was repeatedly lost within some insect orders. Notably, parasitoid hymenopterans and the social wasp Metapolybia decorata (Gribodo) lack the (TTAGG)n sequence, but in other representatives of Hymenoptera, this motif was noticed, such as different ant species and the honeybee. These findings raise the question of whether the insect telomeric repeat is or not phylogenetically predominant in Hymenoptera. Thus, we evaluated the occurrence of both the (TTAGG)n sequence and the vertebrate telomere sequence (TTAGGG)n using dot-blotting hybridization in 25 aculeate species of Hymenoptera. Our results revealed the absence of (TTAGG)n sequence in all tested species, elevating the number of hymenopteran families lacking this telomeric sequence to 13 out of the 15 tested families so far. The (TTAGGG)n was not observed in any tested species. Based on our data and compiled information, we suggest that the (TTAGG)n sequence was putatively lost in the ancestor of Apocrita with at least two subsequent independent regains (in Formicidae and Apidae).

Cytogenomic analysis unveils mixed molecular evolution and recurrent chromosomal rearrangements shaping the multigene families on Schistocerca grasshopper genomes.

Multigene families are essential components of eukaryotic genomes and play key roles either structurally and functionally. Their modes of evolution remain elusive even in the era of genomics, because multiple multigene family sequences coexist in genomes, particularly in large repetitive genomes. Here, we investigate how the multigene families 18S rDNA, U2 snDNA, and H3 histone evolved in 10 species of Schistocerca grasshoppers with very large and repeat-enriched genomes. Using sequenced genomes and fluorescence in situ hybridization mapping, we find substantial differences between species, including the number of chromosomal clusters, changes in sequence abundance and nucleotide composition, pseudogenization, and association with transposable elements (TEs). The intragenomic analysis of Schistocerca gregaria using long-read sequencing and genome assembly unveils conservation for H3 histone and recurrent pseudogenization for 18S rDNA and U2 snDNA, likely promoted by association with TEs and sequence truncation. Remarkably, TEs were frequently associated with truncated copies, were also among the most abundant in the genome, and revealed signatures of recent activity. Our findings suggest a combined effect of concerted and birth-and-death models driving the evolution of multigene families in Schistocerca over the last 8 million years, and the occurrence of intra- and interchromosomal rearrangements shaping their chromosomal distribution. Despite the conserved karyotype in Schistocerca, our analysis highlights the extensive reorganization of repetitive DNAs in Schistocerca, contributing to the advance of comparative genomics for this important grasshopper genus.

Histone acetylation and methylation marks in chromatin of Panstrongylus megistus (Hemiptera, Reduviidae).

Panstrongylus megistus, a potential vector of Chagas disease, currently occupies a wider geographic distribution in Brazil than Triatoma infestans, another member of the hemipteran Reduviidae family and a vector of the same disease. A small heterochromatic body (chromocenter) formed by the Y chromosome is evident in the somatic cells of P. megistus, differing in size and chromosome type contribution from the well-studied chromocenters present in T. infestans. While the overall distribution of histone epigenetic marks differ when comparing the heterochromatin and euchromatin territories in T. infestans, no similar data have been established for other hemipteran reduviids, including P. megistus. In the present work, histone acetylation and methylation marks were investigated in cells of Malpighian tubules of P. megistus 5th instar nymphs using immunocytochemical assays and compared to previously published data for T. infestans. Although similarities between these species were found regarding absence of acetylated H3K9, H4K8 and H4K16, and H3K9me and H3K9me2 in the chromocenter, presence of these marks in euchromatin, and presence of H3K9me3 in the chromocenter, no intimate association of acetylated H4K8 and 18S rDNA was revealed in the chromocenter of P. megistus. The elevated abundance of H3K9me2 marks at the nuclear periphery in P. megistus cells, differing from data for T. infestans, is suggested to reflect differences in the interaction of lamina-associated chromatin domains with the nuclear lamina, methyl-transferase modulation and/or association with the last DNA endoreplication step in 5th instar nymphs, which is a matter for further investigation.

Satellite DNAs Unveil Clues about the Ancestry and Composition of B Chromosomes in Three Grasshopper Species.

Supernumerary (B) chromosomes are dispensable genomic elements occurring frequently among grasshoppers. Most B chromosomes are enriched with repetitive DNAs, including satellite DNAs (satDNAs) that could be implicated in their evolution. Although studied in some species, the specific ancestry of B chromosomes is difficult to ascertain and it was determined in only a few examples. Here we used bioinformatics and cytogenetics to characterize the composition and putative ancestry of B chromosomes in three grasshopper species, Rhammatocerus brasiliensis, Schistocerca rubiginosa, and Xyleus discoideus angulatus. Using the RepeatExplorer pipeline we searched for the most abundant satDNAs in Illumina sequenced reads, and then we generated probes used in fluorescent in situ hybridization (FISH) to determine chromosomal position. We used this information to infer ancestry and the events that likely occurred at the origin of B chromosomes. We found twelve, nine, and eighteen satDNA families in the genomes of R. brasiliensis, S. rubiginosa, and X. d. angulatus, respectively. Some satDNAs revealed clustered organization on A and B chromosomes varying in number of sites and position along chromosomes. We did not find specific satDNA occurring in the B chromosome. The satDNAs shared among A and B chromosomes support the idea of putative intraspecific ancestry from small autosomes in the three species, i.e., pair S11 in R. brasiliensis, pair S9 in S. rubiginosa, and pair S10 in X. d. angulatus. The possibility of involvement of other chromosomal pairs in B chromosome origin is also hypothesized. Finally, we discussed particular aspects in composition, origin, and evolution of the B chromosome for each species.