Amount Of Heterochromatin Research Articles

Satellitome analysis on the pale-breasted thrush Turdus leucomelas (Passeriformes; Turdidae) uncovers the putative co-evolution of sex chromosomes and satellite DNAs

Do all birds' sex chromosomes follow the same canonical one-way direction of evolution? We combined cytogenetic and genomic approaches to analyze the process of the W chromosomal differentiation in two selected Passeriform species, named the Pale-breasted Thrush Turdus leucomelas and the Rufous-bellied thrush T. rufiventris. We characterized the full catalog of satellite DNAs (satellitome) of T. leucomelas, and the 10 TleSatDNA classes obtained together with 16 microsatellite motifs were in situ mapped in both species. Additionally, using Comparative Genomic Hybridization (CGH) assays, we investigated their intragenomic variations. The W chromosomes of both species did not accumulate higher amounts of both heterochromatin and repetitive sequences. However, while T. leucomelas showed a heterochromatin-poor W chromosome with a very complex evolutionary history, T. rufiventris showed a small and partially heterochromatic W chromosome that represents a differentiated version of its original autosomal complement (Z chromosome). The combined approach of CGH and sequential satDNA mapping suggest the occurrence of a former W-autosomal translocation event in T. leucomelas, which had an impact on the W chromosome in terms of sequence gains and losses. At the same time, an autosome, which is present in both males and females in a polymorphic state, lost sequences and integrated previously W-specific ones. This putative W-autosomal translocation, however, did not result in the emergence of a multiple-sex chromosome system. Instead, the generation of a neo-W chromosome suggests an unexpected evolutionary trajectory that deviates from the standard canonical model of sex chromosome evolution.

Gonadal Morphology of Stillborn Hawksbill Sea Turtle (Eretmochelys imbricata) Hatchlings.

This study aimed to investigate sexual dimorphism in stillborn hawksbill sea turtles (Eretmochelys imbricata) through gonadal morphological characterizations. Macroscopic, light microscopy, and transmission electron analyses were performed for 30 gonad-mesonephros complexes. Female gonads were spindle-shaped and present a translucent whitish appearance with a grainy texture. Male gonads were approximately ovoid with a smooth opaque white surface. A primary sexual difference concerns different marrow structures, with females presenting organized cellularity featuring oocytes, lacunae, and blood vessels, while males presented a distinct organizational medulla pattern marked by testicular cords extending throughout the gonad length. Ultrastructurally, female's stroma presented interstitial cells and an abundant cytoplasm rich in electrodense droplets and large oval germline cells, with a conspicuous and noncentral nucleus. Males, on the other hand, presented testicular cord cells containing small amounts of heterochromatin and approximately triangular apical and basal cytoplasms with an evident nucleolus characteristic of support cells. Additionally, there were cells with a large spherical nucleus compared with the cell size and a relatively scarce cytoplasm, identified as gonocytes. These findings indicate that macroscopic, microscopic, and ultrastructural evaluations are effective and reliable techniques for the sexual identification of stillborn E. imbricata hatchlings.

Increased genome size is caused by heterochromatin addition in two non-related bat species, Hesperoptenus doriae and Philetor brachypterus (Vespertilionidae, Chiroptera, Mammalia).

The average genome size (GS) of bats, which are the only mammals capable of powered flight, is approximately 18% smaller than that of closely related mammalian orders. The low nuclear DNA content of Chiroptera is comparable to that of birds, which are also characterized by a high metabolic rate. Only a few chiropteran taxa possess notable amounts of constitutive heterochromatin. Here, we studied the karyotypes of two non-related vesper bat species with unusually high amounts of constitutive heterochromatin: Hesperoptenus doriae and Philetor brachypterus. Conventional staining methods and whole-chromosome painting with probes derived from Myotismyotis (2n = 44), showing a karyotype close to that of the presumed ancestor of Vespertilionidae, revealed Robertsonian fusions as the main type of rearrangement leading to the exceptionally reduced diploid chromosome number of 2n = 26 in both species. Moreover, both karyotypes are characterized by large blocks of pericentromeric heterochromatin composed of CMA-positive and DA-DAPI-positive segments. In H. doriae, the heterochromatin accumulation has resulted in a genome size of 3.22pg (1C), which is 40% greater than the mean genome size for the family. For P. brachypterus, a genome size of 2.94pg was determined, representing an increase of about 28%. Most notably, in H. doriae, the presence of additional constitutive heterochromatin correlates with an extended mitotic cell cycle duration in vitro. A reduction in diploid chromosome number to 30 or lower is discussed as a possible cause of the accumulation of pericentromeric heterochromatin in Vespertilionidae.

Dicentric chromosome breakage in Drosophila melanogaster is influenced by pericentric heterochromatin and occurs in nonconserved hotspots.

Chromosome breakage plays an important role in the evolution of karyotypes and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster, we find that breakage under tension also tends to occur in specific hotspots. Our experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. We analyzed the breakage patterns of 3 different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all 3 chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, we found that the hotspot locations are not conserved between the 3 chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment vary significantly between the 3 chromosomes and are correlated with the origin of the centromere and the amount of pericentric heterochromatin. We suggest that different centromere strengths could account for this.

Analysis in Proceratophrys boiei genome illuminates the satellite DNA content in a frog from the Brazilian Atlantic forest.

Satellite DNAs (satDNAs) are one of the most abundant elements in genomes. Characterized as tandemly organized sequences that can be amplified into multiple copies, mainly in heterochromatic regions. The frog P. boiei (2n = 22, ZZ♂/ZW♀) is found in the Brazilian Atlantic forest and has an atypical pattern of heterochromatin distribution when compared to other anuran amphibians, with large pericentromeric blocks on all chromosomes. In addition, females of Proceratophrys boiei have a metacentric sex chromosome W showing heterochromatin in all chromosomal extension. In this work, we performed high-throughput genomic, bioinformatic, and cytogenetic analyses to characterize the satellite DNA content (satellitome) in P. boiei, mainly due to high amount of C-positive heterochromatin and the highly heterochromatic W sex chromosome. After all the analyses, it is remarkable that the satellitome of P. boiei is composed of a high number of satDNA families (226), making P. boiei the frog species with the highest number of satellites described so far. Consistent with the observation of large centromeric C-positive heterochromatin blocks, the genome of P. boiei is enriched with high copy number of repetitive DNAs, with total satDNA abundance comprising 16.87% of the genome. We successfully mapped via Fluorescence in situ hybridization the two most abundant repeats in the genome, PboSat01-176 and PboSat02-192, highlighting the presence of certain satDNAs sequences in strategic chromosomal regions (e.g., centromere and pericentromeric region), which leads to their participation in crucial processes for genomic organization and maintenance. Our study reveals a great diversity of satellite repeats that are driving genomic organization in this frog species. The characterization and approaches regarding satDNAs in this species of frog allowed the confirmation of some insights from satellite biology and a possible relationship with the evolution of sex chromosomes, especially in anuran amphibians, including P. boiei, for which data were not available.

Chromosome diversity in Buthidae and Chactidae scorpions from Brazilian fauna: Diploid number and distribution of repetitive DNA sequences.

In this work, we analyzed cytogenetically eight Chactidae and Buthidae, including the localization of repetitive DNA sequences. The chactids possess monocentric chromosomes and the highest diploid numbers (2n=50 in Brotheas amazonicus, 2n=36 in Chactopsis amazonica, 2n=30 in Neochactas sp.) when compared with buthids (2n=10 in Tityus bahiensis, 2n=14 in Tityus apiacas and Tityus metuendus, 2n=18 in Tityus aba, 2n=26 in Ischnotelson peruassu). The localization of rDNA genes and (TTAGG)n sequences exhibited a conserved pattern of two terminal/subterminal ribosomal cistrons and terminal telomere signals. However, the comparison between the data of C-banding, DAPI after FISH and Cot-DNA fraction indicated a variable quantity and distribution of these regions, as follow: (i) positive heterochromatin and Cot-DNA signals (B. amazonicus and I. peruassu), (ii) small blocks of heterochromatin with large Cot-DNA signals (T. metuendus), (iii) positive heterochromatic regions and absence of Cot-DNA signals (T. aba and T. apiacas), and (iv) negative heterochromatin and Cot-DNA signals (T. bahiensis). Therefore, our results revealed that there still is not a clear relation between quantity of heterochromatin and presence of monocentric or holocentric chromosomes and occurrence of chromosomal rearrangements, indicating that repetitive regions in scorpions must be analyzed using different cytogenetic approaches.

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L.).

Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the "Branco Mineiro Piauí" accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Chromosome analysis and the occurrence of B chromosomes in fish parasite Acanthocephalus anguillae (Palaeacanthocephala: Echinorhynchida).

The cytogenetics of Acanthocephala is a neglected area in the study of this group of endoparasites. Chromosome number and/or karyotypes are known for only 12 of the 1,270 described species, and molecular cytogenetic data are limited to rDNA mapping in two species. The standard karyological technique and mapping of 18S rRNA and H3 histone genes on the chromosomes of Acanthocephalus anguillae individuals from three populations, one of which originated from the unfavorable environmental conditions of the Zemplínska Šírava reservoir in eastern Slovakia, were applied for the first time. All specimens had 2n=7/8 (male/female); n=1m+1m-sm+1a+1a (X). Fluorescence in situ hybridization (FISH) revealed three loci of 18S rDNA on two autosomes and dispersion of H3 histone genes on all autosomes and the X chromosome. In addition to the standard A chromosome set, 34% of specimens from Zemplínska Šírava possessed a small acrocentric B chromosome, which was always found to be univalent, with no pairing observed between the B chromosome and the A complement. The B chromosome had a small amount of heterochromatin in the centromeric and telomeric regions of the chromosomal arms and showed two clusters of H3 genes. It is well known that an environment permanently polluted with chemicals leads to an increased incidence of chromosomal rearrangements. As a possible scenario for the B chromosome origin, we propose chromosomal breaks due to the mutagenic effect of pollutants in the aquatic environment. The results are discussed in comparison with previous chromosome data from Echinorhynchida species.

Revision and analysis of the chromosome variability in the speciose genus Akodon (Rodentia, Sigmodontinae), including new data from Argentina.

Rodentia has a high species number and chromosomal variability. The South American genus Akodon is one of the most speciose muroids, with more than 40 species included in several species groups. Here, we characterize cytogenetically specimens of Akodon from central-western Argentina. Subsequently, we reviewed and analyzed the cytogenetic data for this genus, build a phylogeny and mapped chromosome changes to interpret its evolution. Specimens of A. dolores from central-western Argentina have 2n=42-44/FNa=44 (46, 48) due to a Robertsonian rearrangement. Our data expand the distribution range known for this polymorphism and confirm its geographic structure. Other specimens had 2n=40/FNa=40, representing populations of A. oenos, A. polopi, and A. spegazzinii. All karyotypes have a low amount of heterochromatin, concentrated in centromeres and sex chromosomes, as in other rodents. The complement with 2n=40/FNa=40 is the most frequent in Akodon and is shared by most species in some groups. Chromosome numbers are very diverse. The FNa shows less variability; FNa=42 was recovered as ancestral, excluding A. mimus, which was connected at the base of the Akodon tree and has FNa=44. This indicates a complex chromosome evolution in Akodon, and suggests that reductions and increases in the 2n and FNa evolved independently in some lineages.

Chromosomal rearrangements and the first indication of an ♀X1 X1 X2 X2 /♂X1 X2 Y sex chromosome system in Rineloricaria fishes (Teleostei: Siluriformes).

Rineloricaria is the most diverse genus within the freshwater fish subfamily Loricariinae, and it is widely distributed in the Neotropical region. Despite limited cytogenetic data, records from southern and south-eastern Brazil suggest a high rate of chromosomal rearrangements in this genus, mirrored in remarkable inter- and intraspecific karyotype variability. In the present work, we investigated the karyotype features of Rineloricaria teffeana, an endemic representative from northern Brazil, using both conventional and molecular cytogenetic techniques. We revealed different diploid chromosome numbers (2n) between sexes (33♂/34♀), which suggests the presence of an ♀X1 X1 X2 X2 /♂X1 X2 Y multiple sex chromosome system. The male-limited Y chromosome was the largest and the only biarmed element in the karyotype, implying Y-autosome fusion as the most probable mechanism behind its origination. C-banding revealed low amounts of constitutive heterochromatin, mostly confined to the (peri)centromeric regions of most chromosomes (including the X2 and the Y) but also occupying the distal regions of a few chromosomal pairs. The chromosomal localization of the 18S ribosomal DNA (rDNA) clusters revealed a single site on chromosome pair 4, which was adjacent to the 5S rDNA cluster. Additional 5S rDNA loci were present on the autosome pair 8, X1 chromosome, and in the presumed fusion point on the Y chromosome. The probe for telomeric repeat motif (TTAGGG)n revealed signals of variable intensities at the ends of all chromosomes except for the Y chromosome, where no detectable signals were evidenced. Male-to-female comparative genomic hybridization revealed no sex-specific or sex-biased repetitive DNA accumulations, suggesting a presumably low level of neo-Y chromosome differentiation. We provide evidence that rDNA sites might have played a role in the formation of this putative multiple sex chromosome system and that chromosome fusions originate through different mechanisms among different Rineloricaria species.

Meiotic Analysis of Gomphidae Species Sheds Light on the Large X Chromosome of the Family (Anisoptera, Odonata)

In most Anisoptera families, the modal diploid number is 25 in males (24 autosomes + X), and the X chromosome is one of the smallest elements of the complement. The family Gomphidae is an exception, as it has a modal diploid number of 23 (22 + X), and the X chromosome is the largest of the complement and of medium-to-large size in many species. We studied the meiosis of three gomphid species from Argentina: Aphylla cf. distinguenda (Campion, 1920), Phyllocycla propinqua Belle, 1972 and Phyllocycla sp. Chromosome number is 2n = 23, n = 11 + X, except for Phyllocycla propinqua, showing n = 10 + X. The X chromosome of these species is medium-sized and presents heteropyknotic blocks of different sizes. Despite the small number of gomphid species analysed, there is a clear trend of increasing size of the X chromosome with the increasing amount of heterochromatin. Our results, together with those from the literature, suggest that its large size might have been due to a progressive accumulation of repetitive DNA and heterochromatinisation and not to fusion, as previously suggested. This led us to propose that the ancestral number coincided with the modal number of Gomphidae. A revision of the derived sex-determining systems in Odonata is also provided.

Comparative repeatome analysis reveals new evidence on genome evolution in wild diploid Arachis (Fabaceae) species.

Opposing changes in the abundance of satellite DNA and long terminal repeat (LTR) retroelements are the main contributors to the variation in genome size and heterochromatin amount in Arachis diploids. The South American genus Arachis (Fabaceae) comprises 83 species organized in nine taxonomic sections. Among them, section Arachis is characterized by species with a wide genome and karyotype diversity. Such diversity is determined mainly by the amount and composition of repetitive DNA. Here we performed computational analysis on low coverage genome sequencing to infer the dynamics of changes in major repeat families that led to the differentiation of genomes in diploid species (x = 10) of genus Arachis, focusing on section Arachis. Estimated repeat content ranged from 62.50 to 71.68% of the genomes. Species with different genome composition tended to have different landscapes of repeated sequences. Athila family retrotransposons were the most abundant and variable lineage among Arachis repeatomes, with peaks of transpositional activity inferred at different times in the evolution of the species. Satellite DNAs (satDNAs) were less abundant, but differentially represented among species. High rates of evolution of an AT-rich superfamily of satDNAs led to the differential accumulation of heterochromatin in Arachis genomes. The relationship between genome size variation and the repetitive content is complex. However, largest genomes presented a higher accumulation of LTR elements and lower contents of satDNAs. In contrast, species with lowest genome sizes tended to accumulate satDNAs in detriment of LTR elements. Phylogenetic analysis based on repetitive DNA supported the genome arrangement of section Arachis. Altogether, our results provide the most comprehensive picture on the repeatome dynamics that led to the genome differentiation of Arachis species.

Sequencing of laser captured Z and W chromosomes of the tocantins paradoxical frog (Pseudis tocantins) provides insights on repeatome and chromosomal homology.

Pseudis tocantins is the only frog species of the hylid genus Pseudis that possesses highly heteromorphic sex chromosomes. Z and W chromosomes of Ps. tocantins differ in size, morphology, position of the nucleolar organizer region (NOR) and the amount and distribution of heterochromatin. A chromosomal inversion and heterochromatin amplification on the W chromosome were previously inferred to be involved in the evolution of this sex chromosome pair. Despite these findings, knowledge related to the molecular composition of the large heterochromatic band of this W chromosome is restricted to the PcP190 satellite DNA, and no data are available regarding the gene content of either the W or the Z chromosome of Ps. tocantins. Here, we sequenced microdissected Z and W chromosomes of this species to further resolve their molecular composition. Comparative genomic analysis suggests that Ps. tocantins sex chromosomes are likely homologous to chromosomes 4 and 10 of Xenopus tropicalis. Analyses of the repetitive DNA landscape in the Z and W assemblies allowed for the identification of several transposable elements and putative satellite DNA sequences. Finally, some transposable elements from the W assembly were found to be highly diverse and divergent from elements found elsewhere in the genome, suggesting a rapid amplification of these elements on the W chromosome.

Y chromosome-related genetic diseases

As a male-specific chromosome, the structure of Y chromosome is complex and lacks of recombination, with numerous repeating, amplifying and palindromic sequences. The research of Y chromosome is difficult and slow since there are few protein coding genes and a large amount of heterochromatin which has caused extreme difficulty for sequencing. In recent years, an increasing number of studies have been focused on the Y chromosome. With the completion of the sequencing of human Y chromosome, the rapid development of sequencing technology, and the composition of DNA sequences in human Y chromosomes and the determination of gene content. This paper has summarized the structural composition and genes function of human Y chromosome, as well as the related hereditary diseases, with an aim to provide reference for Y chromosome-related genetic research.

Chromosome Evolution in the Genus Partamona (Apidae: Meliponini), with Comments on B Chromosome Origin

The genus Partamona includes 33 species of stingless bees, of which 11 were studied cytogenetically. The main goal of this study was to propose a hypothesis about chromosomal evolution in Partamona by combining molecular and cytogenetic data. Cytogenetic analyses were performed on 3 Partamona species. In addition, the molecular phylogeny included mitochondrial sequences of 11 species. Although the diploid number was constant within the genus, 2n = 34, B chromosomes were reported in 7 species. Cytogenetic data showed karyotypic variations related to chromosome morphology and the amount and distribution of heterochromatin and repetitive DNA. The molecular phylogenetic reconstruction corroborated the monophyly of the genus and separated the 2 clades (A and B). This separation was also observed in the cytogenetic data, in which species within each clade shared most of the cytogenetic characteristics. Furthermore, our data suggested that the B chromosome in the genus Partamona likely originated from a common ancestor of the species that have it in clade B and, through interspecific hybridization, it appeared only in Partamona rustica from clade A. Based on the above, Partamona is an interesting genus for further investigations using molecular mapping of B chromosomes as well as for broadening phylogenetic data.

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.

Genetic implications of a biological invasion: Chromosomal and DNA barcode monomorphism in Old World populations of Colorado potato beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Once introduced into new area, invasive species can be expected to have low genetic diversity due to the founder effect. Here we tested this prediction using cytogenetic and molecular analysis of Armenian and Belarusian populations of Colorado potato beetle Leptinotarsa decemlineata (Say, 1824) and by comparing the results with those of native (North America) and those introduced into Europe. This revealed that the karyotype of males from Armenia and Belarus is remarkably conserved with 2n = 35 (34 + X0), n = 17AA + X0; and includes a pair of large acrocentric chromosomes. Thus, these populations belong to the so-called acrocentric chromosome race of the Colorado potato beetle. At diakinesis there are clearly visible argentophilic signals, probably NORs (the nucleolus organizer regions) present on some autosomal bivalents, while the X chromosome was homogenously argentophilic during different stages of meiosis. C-banding revealed a small amount of constitutive heterochromatin weakly visible in the pericentromeric regions of some chromosomes. Analysis of the DNA-barcode fragment of the gene cytochrome c oxidase subunit I (COI) revealed a single haplotype (we call it "the European haplotype") and lack of inter-population variability in all the samples collected from different locations in Armenia and Belarus. The comparison of our karyological and molecular data with that available in the literature and GenBank shows that all the populations studied from the Old World are monomorphic with respect to karyotype and the mitochondrial DNA-barcode. We assume that (1) the presence of acrocentric chromosomes in the karyotype and (2) the European haplotype of mitochondrial genome are the ancestral states for all populations in the Old World and inherited from the New World invaders who colonized Europe 100 years ago. New World populations are polymorphic with respect to karyotype and mitochondrial genes; however, the European haplotype has not yet been found in America. We believe that in the future it will be found in North America, which will shed light on the origin of populations of this dangerous pest in Eurasia.

Dynamics of pathomorphological changes in the structural organization of the intervertebral disc at the end of the seventh and fourteenth day of experimental opioid exposure at the ultrastructural level

In general, the modern literature pays attention to the issues of spine pathology and intervertebral discs. A significant percentage of vertebral disorders - scoliosis, osteochondrosis, spinal disc herniation, etc., occur as a result of exposure to various factors and manifest in changes of the intervertebral discs. The aim of our work was to study at the ultrastructural level the features of pathomorphological manifestations in the structural components of the intervertebral disc at the end of the seventh and fourteenth days of experimental opioid exposure. Materials and methods of research. The objects of the study were 32 sexually mature, white, male rats, weighing 92 - 103 g, aged 4.5 months. Animals were injected with nalbupine intramuscularly once daily (at 10-11 a.m.) for 14 days. The initial dose of nalbuphine was 8 mg/kg during the first week, 15 mg/kg during the second week. It created the conditions of chronic opioid exposure. Before sampling, the animals were withdrawn from the experiment using dibutyl ether. Intervertebral discs of rats were used as a material for ultrastructural study. Ultrastructural specimens were prepared according to the accepted methods. The results of the study. As a result of the sampling after 7 days of opioid exposure we found inhomogeneous osmiophilicity and compaction of the nucleus pulposus matrix in which intensively accumulated osmiophilic grains of glycogen proteoglycans, increased the number of collagen fibers, some of them were heterogeneous. It was also noted the development of moderate degenerative changes in some notochondral cells, which was accompanied by increased vacuolization of the cytoplasm by inhomogeneous compaction of the nucleus and an increase of heterochromatin there. After 14 days necrotic changes in the cells of the nucleus pulposus, as well as the destruction of collagen fibers of the annulus fibrosus were found. In particular, an increase in the amount of heterochromatin in the nucleui of notochondral cells, which was accompanied by a decrease in the volume of the nucleui and inhomogeneous swelling of the cytoplasm. Active fibroblasts were often visualized in the annulus fibrosus. Intense osmiophilicity and thickening of collagen fibers of the annulus fibrosus were observed in some areas of the fibrous ring. Focal destruction of collagen fibers was also noted. In the areas of destruction the fibrils of collagen fibers disintegrated into an inhomogeneous fine-grained stratified mass and were located loosely. Conclusions. At the end of the first week we found that the cytoplasmic processes of chondrocytes decreased in volume, shortened, underwent fragmentation and destruction, some of them detached from the surface of the plasmolemma. At the end of the second week signs of opioid exposure progressed and manifested by an increase in the destruction of cytoplasmic processes in chondrocytes. Also focal destruction of collagen fibers was noted.

Incipient speciation in Calceolaria polyrhiza complex (Calceolariaceae): cytogenetic analysis supports molecular and morphological data

ABSTRACT Calceolaria polyrhiza is a perennial herb distributed in southern South America. The species is highly polymorphic and includes four described morphotypes that were previously considered different species. We performed a detailed study of the karyotype of each morphotype considering at least two localities for each one, with the aim of contributing to the taxonomic delimitation of the Calceolaria polyrhiza complex. In addition, a principal component analysis and a mapping of chromosome traits onto a phylogeny of nuclear DNA sequences were performed. This detailed karyotypic study has revealed specific markers, which have allowed to characterize each morphotype. ‘Prichardii’ morphotype was consistently polyploid. ‘Mendocina’ and ‘Lanceolata’ morphotypes had the highest total heterochromatin amount, with two pairs of chromosomes carrying NORs. ‘Mendocina’ samples showed the most asymmetrical karyotypes. ‘Polyrhiza’ morphotype had the most symmetrical karyotypes of the four morphotypes and the simplest fluorescent chromosome banding pattern. Thus, the four morphotypes could be characterized by chromosomal data and, together with the phylogenetic analysis of molecular data, could be considered taxonomic units in the process of diversification.

Satellitome Analysis of the Pacific Oyster Crassostrea gigas Reveals New Pattern of Satellite DNA Organization, Highly Scattered across the Genome.

Several features already qualified the invasive bivalve species Crassostrea gigas as a valuable non-standard model organism in genome research. C. gigas is characterized by the low contribution of satellite DNAs (satDNAs) vs. mobile elements and has an extremely low amount of heterochromatin, predominantly built of DNA transposons. In this work, we have identified 52 satDNAs composing the satellitome of C. gigas and constituting about 6.33% of the genome. Satellitome analysis reveals unusual, highly scattered organization of relatively short satDNA arrays across the whole genome. However, peculiar chromosomal distribution and densities are specific for each satDNA. The inspection of the organizational forms of the 11 most abundant satDNAs shows association with constitutive parts of Helitron mobile elements. Nine of the inspected satDNAs are dominantly found in mobile element-associated form, two mostly appear standalone, and only one is present exclusively as Helitron-associated sequence. The Helitron-related satDNAs appear in more chromosomes than other satDNAs, indicating that these mobile elements could be leading satDNA propagation in C. gigas. No significant accumulation of satDNAs on certain chromosomal positions was detected in C. gigas, thus establishing a novel pattern of satDNA organization on the genome level.