What is a B chromosome? Early definitions revisited.

Since the discovery of B chromosomes, multiple different definitions of these selfish genetic elements have been put forth. We reconsidered early definitions in light of recently published studies. While there are many characteristics that vary among different B chromosomes, such as their evolutionary origins, size, segregation behaviors, gene content, and function, there is one defining trait of all B chromosomes: they are nonessential for the organism. The points raised here may be useful for framing future B chromosome studies and help guide the categorization of new chromosomal elements that are uncovered in genomic studies.

The new highest number of B chromosomes (Bs) in Leisler's bat Nyctalusleisleri (Kuhl, 1817).

B chromosomes (Bs) are supernumerary to the standard chromosome set, from which they prevalently derive. Variation in numbers both among individuals or populations and among cells within individuals is their constant feature. Leisler's bat Nyctalusleisleri (Kuhl, 1817) is one of only four species of Chiroptera with detected Bs. Four males of N.leisleri were collected from two localities on the territory of Serbia and cytogenetically analysed. All animals had Bs with interindividual variability ranging from two to five heterochromatic micro Bs. The highest number of Bs was detected in this species. Among mammals, Rodentia and Chiroptera are orders with the largest number of species, but Bs frequently appear in rodents and rarely in chiropterans. Possible explanations for this difference are offered.

Canalization and developmental stability of the yellow-necked mouse (Apodemus flavicollis) mandible and cranium related to age and nematode parasitism.

BACKGROUND: Mammalian mandible and cranium are well-established model systems for studying canalization and developmental stability (DS) as two elements of developmental homeostasis. Nematode infections are usually acquired in early life and increase in intensity with age, while canalization and DS of rodent skulls could vary through late postnatal ontogeny. We aimed to estimate magnitudes and describe patterns of mandibular and cranial canalization and DS related to age and parasite intensity (diversity) in adult yellow-necked mice (Apodemus flavicollis). RESULTS: We found the absence of age-related changes in the levels of canalization for mandibular and cranial size and DS for mandibular size. However, individual measures of mandibular and cranial shape variance increased, while individual measures of mandibular shape fluctuating asymmetry (FA) decreased with age. We detected mandibular and cranial shape changes during postnatal ontogeny, but revealed no age-related dynamics of their covariance structure among and within individuals. Categories regarding parasitism differed in the level of canalization for cranial size and the level of DS for cranial shape. We observed differences in age-related dynamics of the level of canalization between non-parasitized and parasitized animals, as well as between yellow-necked mice parasitized by different number of nematode species. Likewise, individual measures of mandibular and cranial shape FA decreased with age for the mandible in the less parasitized category and increased for the cranium in the most parasitized category. CONCLUSIONS: Our age-related results partly agree with previous findings. However, no rodent study so far has explored age-related changes in the magnitude of FA for mandibular size or mandibular and cranial FA covariance structure. This is the first study dealing with the nematode parasitism-related canalization and DS in rodents. We showed that nematode parasitism does not affect mandibular and cranial shape variation and covariance structure among and within individuals. However, parasite intensity (diversity) is related to ontogenetic dynamics of the levels of canalization and DS. Overall, additional studies on animals from natural populations are required before drawing some general conclusions.

New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis.

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

B Chromosomes' Sequences in Yellow-Necked Mice Apodemus flavicollis-Exploring the Transcription.

B chromosomes (Bs) are highly polymorphic additional chromosomes in the genomes of many species. Due to the dispensability of Bs and the lack of noticeable phenotypic effects in their carriers, they were considered genetically inert for a long time. Recent studies on Bs in Apodemus flavicollis revealed their genetic composition, potential origin, and spatial organization in the interphase nucleus. Surprisingly, the genetic content of Bs in this species is preserved in all studied samples, even in geographically distinct populations, indicating its biological importance. Using RT-PCR we studied the transcription activity of three genes (Rraga, Haus6, and Cenpe) previously identified on Bs in A. flavicollis. We analysed mRNA isolated from spleen tissues of 34 animals harboring different numbers of Bs (0-3).The products of transcriptional activity of the analysed sequences differ in individuals with and without Bs. We recorded B-genes and/or genes from the standard genome in the presence of Bs, showing sex-dependent higher levels of transcriptional activity. Furthermore, the transcriptional activity of Cenpe varied with the age of the animals differently in the group with and without Bs. With aging, the amount of product was only found to significantly decrease in B carriers. The potential biological significance of all these differences is discussed in the paper.

The prevalence of pathogenic forms of Leptospira in natural populations of small wild mammals in Serbia.

The greatest epidemiological significance of leptospirosis in Europe comes from the fact that it is the most widespread zoonosis in the world. However, epizootiological data, especially information on maintenance hosts such as small wild mammals, are largely missing. To fill this gap in data in Serbia, we used RT-PCR for the detection of pathogenic Leptospira species and analysed 107 animals belonging to six species of small wild mammals (Apodemus agrarius, Apodemus flavicollis, Microtus arvalis, Myodes glareolus, Microtus subterraneus and Sorex araneus) collected from two localities. The animals from the first locality that was situated in a tourist area, were collected for four consecutive years (2014-2017). We found persistent incidence of infection from year to year ranging from 6.67% to 78.57%. The average frequency of infected animals was 33.3% with the highest frequency in 2014, the year characterised by a very high number of flooding days. All animals proved to be infected with pathogenic Leptospira species that were collected from the second locality situated in an agricultural area in a single year, 2014. The findings show a variable but constant presence of pathogenic Leptospira species in populations of small wild mammals in the studied areas, which indicates the need for constant monitoring.

Population genetic structure of the Mediterranean horseshoe bat Rhinolophus euryale in the central Balkans.

Migratory behaviour, sociality and roost selection have a great impact on the population structure of one species. Many bat species live in groups, and movements between summer and hibernation sites are common in temperate bats. The Mediterranean horseshoe bat Rhinolophus euryale is a cave-dwelling species that exhibits roost philopatry and undertakes seasonal movements which are usually shorter than 50 km. Its distribution in Serbia is restricted to karstic areas in western and eastern parts of the country, with a lack of known roosts between them. In this study, microsatellite markers were used to evaluate genetic variation in this species in the Central Balkans. Specifically, spatial genetic structuring between geographic regions and relatedness within different colony types were assessed. All analysed loci were polymorphic, and there was no significant inbreeding coefficient recorded. A moderate degree of genetic differentiation among the sampled colonies was found, and significant isolation by distance was recorded. Our results revealed that populations show a tendency to segregate into three clusters. Unexpectedly, populations from Montenegro and Eastern Serbia tended to group into one cluster, while populations from Western Serbia and Slovenia represented second and third cluster, respectively. The majority of variance was partitioned within colonies, and only a small but significant portion among clusters. Average relatedness within colony members was close to zero, did not differ significantly between the different colony types, and kinship is unlikely to be a major grouping mechanism in this species.

B Chromosomes in Populations of Mammals Revisited.

The study of B chromosomes (Bs) started more than a century ago, while their presence in mammals dates since 1965. As the past two decades have seen huge progress in application of molecular techniques, we decided to throw a glance on new data on Bs in mammals and to review them. We listed 85 mammals with Bs that make 1.94% of karyotypically studied species. Contrary to general view, a typical B chromosome in mammals appears both as sub- or metacentric that is the same size as small chromosomes of standard complement. Both karyotypically stable and unstable species possess Bs. The presence of Bs in certain species influences the cell division, the degree of recombination, the development, a number of quantitative characteristics, the host-parasite interactions and their behaviour. There is at least some data on molecular structure of Bs recorded in nearly a quarter of species. Nevertheless, a more detailed molecular composition of Bs presently known for six mammalian species, confirms the presence of protein coding genes, and the transcriptional activity for some of them. Therefore, the idea that Bs are inert is outdated, but the role of Bs is yet to be determined. The maintenance of Bs is obviously not the same for all species, so the current models must be adapted while bearing in mind that Bs are not inactive as it was once thought.

Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and Apodemus B chromosomes.

Supernumerary chromosomes sporadically arise in many eukaryotic species as a result of genomic rearrangements. If present in a substantial part of species population, those are called B chromosomes, or Bs. This is the case for 70 mammalian species, most of which are rodents. In humans, the most common types of extra chromosomes, sSMCs (small supernumerary marker chromosomes), are diagnosed in approximately 1 of 2000 postnatal cases. Due to low frequency in population, human sSMCs are not considered B chromosomes. Genetic content of both B-chromosomes and sSMCs in most cases remains understudied. Here, we apply microdissection of single chromosomes with subsequent low-pass sequencing on Ion Torrent PGM and Illumina MiSeq to identify unique and repetitive DNA sequences present in a single human sSMC and several B chromosomes in mice Apodemus flavicollis and Apodemus peninsulae. The pipeline for sequencing data analysis was made available in Galaxy interface as an addition to previously published command-line version. Human sSMC was attributed to the proximal part of chromosome 15 long arm, and breakpoints leading to its formation were located into satellite DNA arrays. Genetic content of Apodemus B chromosomes was species-specific, and minor alterations were observed in both species. Common features of Bs in these Apodemus species were satellite DNA and ERV enrichment, as well as the presence of the vaccinia-related kinase gene Vrk1. Understanding of the non-essential genome elements content provides important insights into genome evolution in general.

Exploring Supernumeraries - A New Marker for Screening of B-Chromosomes Presence in the Yellow Necked Mouse Apodemus flavicollis.

Since the density of simple sequence repeats (SSRs) may vary between different chromosomes of the same species in eukaryotic genomes, we screened SSRs of the whole genome of the yellow necked mouse, Apodemus flavicollis, in order to reveal SSR profiles specific for animals carrying B chromosomes. We found that the 2200 bp band was amplified by primer (CAG)4AC to a highly increased level in samples with B chromosomes. This quantitative difference (B-marker) between animals with (+B) and without (0B) B chromosomes was used to screen 20 populations (387 animals). The presence/absence of Bs was confirmed in 96.5% of 342 non mosaic individuals, which recommends this method for noninvasive B-presence detection. A group of 45 animals with mosaic and micro B (µB) karyotypes was considered separately and showed 55.6% of overall congruence between karyotyping and molecular screening results. Relative quantification by qPCR of two different targeted sequences from B-marker indicated that these B-specific fragments are multiplied on B chromosomes. It also confirms our assumption that different types of Bs with variable molecular composition may exist in the same individual and between individuals of this species. Our results substantiate the origin of Bs from the standard chromosomal complement. The B-marker showed 98% sequence identity with the serine/threonine protein kinase VRK1 gene, similarly to findings reported for Bs from phylogenetically highly distant mammalian species. Evolutionarily conserved protein-coding genes found in Bs, including this one in A. flavicollis, could suggest a common evolutionary pathway.

Possible influence of B chromosomes on genes included in immune response and parasite burden in Apodemus flavicollis.

Genetic background underlying wild populations immune response to different parasites is still not well understood. We studied immune response to multiple infections and to competition between different parasite species at different developmental stages in population of yellow-necked mouse, Apodemus flavicollis. Quantitative real-time PCR was used to investigate associations of MHC II-DRB, IL-10 and Tgf-ß genes expressions with presence of intestinal parasites at different developmental stages. Furthermore, we were interested whether the host related characteristics (sex, age, body condition, presence of B chromosomes or expression of other genes) or characteristics of present parasites (number of adult parasites of each identified species, egg count of each parasite genus, total number of nematode individuals) affect differential expression of the studied genes. A significant invert association between the expression of MHC II-DRB and Tgf-ß gene was found, which together with absence of IL-10 association confirmed modified Th2 as the main type of immune response to nematode infections. Effect of recorded parasites and parasite life-cycle stage on expression levels of MHC II-DRB gene was detected only through interactions with host-related characteristics such as sex, age, and the presence of B chromosomes. The presence of B chromosomes is associated with lower expression level of Tgf-ß gene. Although the influence of host genetic background on parasite infection has already been well documented, this is the first study in mammals that gave presence of B chromosomes on immune response full consideration.

Potential genotoxic effects of melted snow from an urban area revealed by the Allium cepa test.

The presence of well-known atmospheric pollutants is regularly screened for in large towns but knowledge about the effects of mixtures of different pollutants and especially their genotoxic potential is largely missing. Since falling snow collects pollutants from the air, melted snow samples could be suitable for evaluating potential genotoxicity. For this purpose the Allium cepa anaphase-telophase test was used to analyse melted snow samples from Belgrade, the capital city of Serbia. Samples of snow were taken at two sites, characterized by differences in pollution intensity, in three successive years. At the more polluted site the analyses showed a very high degree of both toxicity and genotoxicity in the first year of the study corresponding to the effects of the known mutagen used as the positive control. At the other site the situation was much better but not without warning signals. The results showed that standard analyses for the presence of certain contaminants in the air do not give an accurate picture of the possible consequences of urban air pollution because the genotoxic potential remains hidden. The A. cepa test has been demonstrated to be very convenient for evaluation of air pollution through analyses of melted snow samples.

Genotoxicity screening of the river Rasina in Serbia using the Allium anaphase-telophase test.

Evaluation of the presence of genotoxic substances is especially important in rivers that serve as a source of drinking water. Nine water samples collected along the river Rasina in Serbia were analyzed for potential toxic and genotoxic effects using the Allium anaphase-telophase test. Inhibition of root growth relative to the negative control (synthetic water) was observed in all samples. Analysis of the genotoxic potential, through scoring anaphase and telophase aberrations, showed that in seven of the nine samples the level of aberrations was significantly increased relative to the negative control but was lower than that obtained for the positive control (methyl methanesulfonate). Changes in the relation between spindle and chromosome types of aberrations were found in some samples, indicating differences in the potential genotoxic substances present at the analyzed sites. The data, which were obtained from samples collected at the highest level of river water, warn that during periods of low flow the values could reach genotoxic activity. The Allium anaphase-telophase test can be recommended as an monitoring system, that can serve as the first alert for the presence of genotoxic environmental pollutants.

Intra individual variation in the number of B chromosomes in the yellow-necked mouse, Apodemus flavicollis (Mammalia, Rodentia).

The yellow-necked mouse, Apodemus flavicollis, is characterized by a frequent occurrence of B chromosomes. The frequency of intra individual mosaicism of Bs was studied in 995 animals collected at six localities in Serbia. It was found that 329 (33.06%) possessed B chromosomes. Among these, 87 animals (26.44%) were mosaics. A total of 32 mosaic animals with more than one B chromosome were analyzed for distribution of Bs which was found to be quite different between groups of animals with different numbers of Bs and increases with their number. The frequency of mosaics differs between localities and ranges from 0.22 to 0.55.

Differential gene expression in yellow-necked mice Apodemus flavicollis (Rodentia, Mammalia) with and without B chromosomes.

Most B chromosomes are heavily heterochromatic, promoting the general idea that they are genetically inert. The B chromosomes of Apodemus flavicollis are euchromatic and show a high degree of homology with the A chromosomes. The euchromatic nature of B chromosomes in A. flavicollis suggests that they may carry active genes and have transcriptional activity. We applied the differential display reverse transcription-polymerase chain reaction (DD RT-PCR) in order to analyze and compare gene expression in animals possessing B chromosomes and animals without B chromosomes. After a second and third round of amplification, three cDNA fragments were differentially expressed in +B mice compared with 0B animals. These cDNAs were Chaperonin containing TCP-1, subunit 6b (zeta) (CCT6B), Fragile histidine triad gene (FHIT) and hypothetical gene XP transcript. The differential expression pattern was confirmed by Real Time RT-PCR. We suggest that altered expression of these important genes is due to the presence of B chromosomes. In elevating the expression of these genes, B chromosomes of A. flavicollis affect some of the crucial processes in the cell. The significance of these effects and the nature of B chromosomes of A. flavicollis are discussed in the context of the data presented.