Exploring the Relationship between Spontaneous Sister Chromatid Exchange and Genome Instability in Two Cryptic Species of Non-Human Primates.

There are extensive studies on chromosome morphology and karyotype diversity in primates, yet we still lack insight into genomic instability as a key factor underlying the enormous interspecies chromosomal variability and its potential contribution to evolutionary dynamics. In this sense, the assessment of spontaneous sister chromatid exchange (SCE) frequencies represents a powerful tool for evaluating genome stability. Here, we employed G-banding, fluorescence plus Giemsa (FPG), and chromosome orientation fluorescence in situ hybridization (CO-FISH) methodologies to characterize both chromosome-specific frequencies of spontaneously occurring SCE throughout the genome (G-SCE) and telomere-specific SCE (T-SCE). We analyzed primary fibroblast cultures from two male species of Ateles living in captivity: Ateles paniscus (APA) and Ateles chamek (ACH). High frequencies of G-SCEs were observed in both species. Interestingly, G-SCEs clustered on evolutionary relevant chromosome pairs: ACH chromosomes 1, 2, 3, 4, and 7, and APA chromosomes 1, 2, 3, 4/12, 7, and 10. Furthermore, a statistically significant difference between the observed and expected G-SCE frequencies, not correlated with chromosome size, was also detected. CO-FISH analyses revealed the presence of telomere-specific recombination events in both species, which included T-SCE, as well as interstitial telomere signals and telomere duplications, with APA chromosomes displaying higher frequencies, compared to ACH. Our analyses support the hypothesis that regions of Ateles chromosomes susceptible to recombination events are fragile sites and evolutionary hot spots. Thus, we propose SCE analyses as a valuable indicator of genome instability in non-human primates.

Genotoxic effects induced for sub-cytotoxic concentrations of tebuconazole fungicide in HEp-2 cell line.

Tebuconazole (TB) is an active ingredient in formulations applied to control of fungal diseases in plants and classified as a possible human carcinogen. In the present study, the sub-cytotoxic concentrations of TB were evaluated for genotoxicity response in HEp-2 cell line. The HEp-2 cell line was exposed to 20, 40, and 50 µg/mL of TB in the Cytokinesis-block micronucleus (CBMN) assay and 40, 60, and 80 µg/mL in the comet assay (CA). Both negative centromere micronuclei (MNC-) as DNA damage were induced by the concentrations of TB assayed, suggesting a mostly clastogenic effect. The results obtained from the comet assay suggest that part of the endogenous DNA damage in the HEp-2 cell line could be repaired in presence of low TB concentrations by promoting the damage response. In conclusion, exposition to sub-cytotoxic concentrations of TB induces genotoxicity in the HEp-2 cell line. Therefore, the safety of their application should be evaluated.

Assessment of genetic variability in captive capuchin monkeys (Primates: Cebidae).

Capuchin monkeys (genera Cebus and Sapajus) show a wide range distribution, from Honduras to Argentina. The aim of this work was to evaluate the genetic and phenotypic variability of captive specimens putatively belonging to S. cay (SCY) and S. nigritus (SNI) at their southernmost distribution limit. Forty-four individuals held in five captive centers from Argentina were analyzed based on external morphology, karyology and DNA sequences of mitochondrial control region (mtDNA-CR). Three morphotypes associated with their probable geographical origin in SCY and a single morphotype in SNI were found. For SCY we could associate each morphotype with the most frequent karyotype. SNI showed a single phenotype and a homogenous karyotype. Heterochromatin showed geographical patterns within species. A 515-bp mtDNA-CR fragment was sequenced, defining fourteen haplotypes at 59 polymorphic sites. A network constructed with our 14 haplotypes and other 77 from S. apella, S. macrocephalus, S. cay and S. nigritus from bibliography revealed some phylogeographic signals. Our SCY and SNI samples rendered four groups that differed in multiple mutational steps, with SCY being more similar to S. apella than to S. macrocephalus. Also, we identified two genetic divergent SCY groups: samples from NOA and from NEA with high mitochondrial diversity. Our results highlight the relevance of using complementary genetic tools throughout the distribution ranges of SCY and SNI for a better assessment of their diversity.

Genetic analysis of an insular population of Sapajus nigritus (Primates: Cebidae) in Rio de Janeiro state, Brazil.

The black-horned capuchin (Sapajus nigritus) is a neotropical primate with wide distribution from southeastern Brazil to northeastern Argentina. Although this species has been described with coat pattern variation, even with intrapopulational differences, and characterized as having the greatest genetic diversity among Sapajus species, there are still few studies on natural populations that contribute to the knowledge of this intraspecific variability. We examined individuals from an as yet unstudied population of Ilha da Marambaia, Rio de Janeiro (RJ) state, Brazil, compared with published data for S. nigritus. We sought to confirm the species through phenotypic and genetic characterization using C-banding and fluorescence in situ hybridization with #11qHe+/21WCP probes for chromosomal constitutive heterochromatin (He+) patterns, and cytochrome c oxidase I and II gene sequences for phylogenetic analysis. The coat presented two color patterns, varying from brown to blackish on the body, yellow to brown on the chest, and white to yellow on the face, besides the presence and shape of the tufts on the head, corresponding to S. nigritus. He+ was identified in pairs 4, 12, 13 and 17, and less consistently in pairs 6, 19 and 21, already described for this species. While most Sapajus species have a large He+ block, here pair 11 was identified without extracentromeric He+, the same as reported for S. nigritus from Argentina. Molecular analysis showed divergence of this population from other S. nigritus sequences, reinforcing a trend already demonstrated when samples from RJ are compared with the rest of the distribution, which may represent an evolutionary deviation.

Geographic distribution of sex chromosome polymorphism in Anastrepha fraterculus sp. 1 from Argentina.

BACKGROUND: Anastrepha fraterculus is recognized as a quarantine pest in several American countries. This fruit fly species is native to the American continent and distributed throughout tropical and subtropical regions. It has been reported as a complex of cryptic species, and at least eight morphotypes have been described. Only one entity of this complex, formerly named Anastrepha fraterculus sp. 1, is present in Argentina. Previous cytogenetic studies on this morphotype described the presence of sex chromosome variation identified by chromosomal size and staining patterns. In this work, we expanded the cytological study of this morphotype by analyzing laboratory strains and wild populations to provide information about the frequency and geographic distribution of these sex chromosome variants. We analyzed the mitotic metaphases of individuals from four laboratory strains and five wild populations from the main fruit-producing areas of Argentina, including the northwest (Tucumán and La Rioja), northeast (Entre Ríos and Misiones), and center (Buenos Aires) of the country. RESULTS: In wild samples, we observed a high frequency of X1X1 (0.94) and X1Y5 (0.93) karyomorphs, whereas X1X2 and X1Y6 were exclusively found at a low frequency in Buenos Aires (0.07 and 0.13, respectively), Entre Ríos (0.16 and 0.14, respectively) and Tucumán (0.03 and 0.04, respectively). X2X2 and X2Y5 karyomorphs were not found in wild populations but were detected at a low frequency in laboratory strains. In fact, karyomorph frequencies differed between wild populations and laboratory strains. No significant differences among A. fraterculus wild populations were evidenced in either karyotypic or chromosomal frequencies. However, a significant correlation was observed between Y5 chromosomal frequency and latitude. CONCLUSIONS: We discuss the importance of cytogenetics to understand the possible route of invasion and dispersion of this pest in Argentina and the evolutionary forces acting under laboratory conditions, possibly driving changes in the chromosomal frequencies. Our findings provide deep and integral genetic knowledge of this species, which has become of relevance to the characterization and selection of valuable A. fraterculus sp. 1 strains for mass rearing production and SIT implementation.

Oxidative Response and Micronucleus Centromere Assay in HEp-2 Cells Exposed to Fungicide Iprodione.

The fungicide agents are a key component in the fruits and vegetables production. The Iprodione residues are one of the pesticide more frequently found in food products. The available data about the cytotoxicity of iprodione and its metabolites are scarce and do not allow characterization of its genotoxic potential and define the risk assessment.The human larynx epidermoid carcinoma cell line (HEp-2) has been shown to be sensitive to the toxic effects of xenobiotics of different origin and have been often used in citotoxicity and genotoxicity studies. The purpose of this paper is to evaluate the induction of genotoxicity and the role of oxidative stress in HEp-2cell line by exposure to the IP. The MTT test for viability resulted in CL50 85.86 (77.05-95.68) µg/mL of Iprodione. On the basis of this result, we proceeded to expose the cells to the sublethal concentrations (below the CL50) during 24 h to analyze the mitotic index and nuclear division index in order to determine the subcytotoxic concentrations of IP which the genotoxicity was evaluated. The subcytotoxic concentrations of 7, 17, and 25 µg/mL IP induced aneugenic effects as micronuclei centromere positive whereas 17 µg/mL was a threshold for centromere negative micronuclei induction in HEp-2 cells. The abnormal mitosis was induced for exposition of Hep-2 cells to the three concentrations. According to the result obtained, citotoxicity and genotoxicity oxidative stress studies were performed in 1.5, 7.0, and 25 µg/mL of IP. The results showed that the GSH intracellular content, the SOD activity and the levels of oxidative damage of the proteins were affected lead to redox imbalance. The decreased in the SOD activity and protein oxidation were in according to the result obtained to genotoxicity, suggesting that different biological targets could be affected.

Association between Genomic Instability and Evolutionary Chromosomal Rearrangements in Neotropical Primates.

During the last decades, the mammalian genome has been proposed to have regions prone to breakage and reorganization concentrated in certain chromosomal bands that seem to correspond to evolutionary breakpoints. These bands are likely to be involved in chromosome fragility or instability. In Primates, some biomarkers of genetic damage may be associated with various degrees of genomic instability. Here, we investigated the usefulness of Sister Chromatid Exchange as a biomarker of potential sites of frequent chromosome breakage and rearrangement in Alouatta caraya, Ateles chamek, Ateles paniscus, and Cebus cay. These Neotropical species have particular genomic and chromosomal features allowing the analysis of genomic instability for comparative purposes. We determined the frequency of spontaneous induction of Sister Chromatid Exchanges and assessed the relationship between these and structural rearrangements implicated in the evolution of the primates of interest. Overall, A. caraya and C. cay presented a low proportion of statistically significant unstable bands, suggesting fairly stable genomes and the existence of some kind of protection against endogenous damage. In contrast, Ateles showed a highly significant proportion of unstable bands; these were mainly found in the rearranged regions, which is consistent with the numerous genomic reorganizations that might have occurred during the evolution of this genus.

Sex chromosomes in mitotic and polytene tissues of Anastrepha fraterculus (Diptera, Tephritidae) from Argentina: a review.

Cytogenetics, which is considered a fundamental tool to understand basic genetic and genomic issues of species, has greatly contributed to the description of polymorphisms both at inter- and intra-specific level. In fact, cytogenetics was one of the first approaches used to propose Anastrepha fraterculus (Diptera: Tephritidae) as a complex of cryptic species. Different morphological variants of sex chromosomes have been reported among Argentinean populations of Anastrepha fraterculus. However, since this high structural variability in sex chromosomes does not pose a reproductive barrier, their role in speciation is yet to be unveiled. This review provides an update on general aspects of cytogenetics in Argentinean Anastrepha fraterculus populations, focused on the prevalence of X-Y arrangements.

Primates karyological diagnosis and management programs applications.

BACKGROUND: Captive primates are often maintained in groups without geographic origin or genetic heritage information. This could lead to an incorrect assignment of species, which could result in an inadequate management of the colonies. METHODS: We present a cytogenetic protocol adapted to be successfully used in an accurate taxonomic diagnosis of non-human primates (Platyrrhini), including lymphocyte culture, G- and C-banding, meiosis, and fluorescent in situ hybridization technique (FISH). RESULTS: Using classical cytogenetic diagnosis, the species status was determined in 541 Platyrrhini individuals. Of these, 99 were previously erroneously sexed or assigned to a different species using only morphological characteristics. CONCLUSIONS: The cytogenetic results highlight the relevance of the genetic characterization of primates both in captivity and in the wild. These techniques had been used in our research group for more than 30 years in different research projects, not only for characterizing hundreds of primates, but also different for topics regarding primates genomes and evolution.

Multiple sex chromosome systems in howler monkeys (Platyrrhini, Alouatta).

In light of the multiple sex chromosome systems observed in howler monkeys (Alouatta Lacépède, 1799) a combined cladistic analysis using chromosomal and molecular characters was applied to discuss the possible origin of these systems. Mesoamerican and South American howlers were karyologically compared. FISH analysis using the chromosome painting probes for the #3 and #15 human chromosomes was applied to corroborate the homeology of the sexual systems. We found that the HSA3/15 syntenic association, present in the sex chromosome systems of South American Howlers, is not present in those of Mesoamerican ones. The autosomes involved in the translocation that formed the sexual systems in the Mesoamerican and South American species are different, thus suggesting an independent origin. Parsimony analysis resolved the phylogenetic relationships among howler species, demonstrating utility of the combined approach. A hypothesis for the origin of the multiple sex chromosome systems for the genus is proposed.

Algunas certeza entre las tantas preguntas de os últimos diez años acerca dell genoma de los primaess / Some Certainties Among the Manyquestions of the Last 10 Years about the Primate Genome

En el año 2000 se presentó lo que se dio en llamar nuestro libro de la vida, el primer borrador del genoma humano. Aquello generó grandes expectativas por sus potenciales en beneficio de las ciencias biológicas. ¿Qué ha sucedido diez años después? Se conoce el número de genes que forman parte de nuestro genoma y se determinó la función de algunos de ellos. Se conocen las secuencias de tres genomas completos de mamíferos, Mus musculus, Pan troglodytes y Sus scrofa y genomas completos o borradores de otros numerosos eucariota (otros animales, plantas, hongos y protistas) y procariota (Archea y Bacterias), ver: http://ncbi.nlm.nih.gov/genomes. Sin embargo, el estudio del genoma no se limita a la mera descripción de las secuencias que lo componen. Las respuestas que se elaboren tendrán enfoques muy diversos, desde evolución y conservación de la biodiversidad hasta terapia génica y transformación maligna, donde el estudio de las particularidades individuales y poblacionales requiere fuentes de información tanto pasadas como actuales sobre estos genomas en estudio. Así, los avances en ciencia siempre son provisorios y por tanto, plausible de continuarse, completarse e incluso reinterpretarse ya que, conforme avanzamos en el conocimiento van surgiendo nuevos interrogantes.

In 2000 the first draft of the human genome, what became known as our book of life, was presented. It generated high expectations for its potential applications to the benefit of the biological sciences. What happened 10 years later? We know how many genes we have in our genome and analyzed the function of some of them. Nowadays, we know the sequences of 3 mammalians genomes: M. musculus, P. troglodytes y S. scrofa and the genomes or borradores from other eucaryotes (other animals, plants, fungi and protists) and procaryotes (Archea and Bacterias). However, the study of the genome is not merely a description of the sequences that compose it. The answers provided will have very different approaches from evolution and conservation of biodiversity to gene therapy and malignant transformation, where the study of individual and population particularities requires sources of information both past and present on these genomes under survey. Thus, advances in science are always provisional and therefore liable to be continued, completed and even reinterpreted as we advance in knowledge, new questions arise.

Analysis of the heterochromatin of Cebus (Primates, Platyrrhini) by micro-FISH and banding pattern comparisons.

The karyotype of the neotropical primate genus Cebus (Platyrrhini: Cebidae), considered the most ancestral one, shows the greatest amount of heterochromatin described among Platyrrhini genera. Banding techniques and restriction enzyme digestion have previously revealed great variability of quantity and composition of heterochromatin in this genus. In this context, we use fluorescence in situ hybridization (FISH) to analyse this genomic region and discuss its possible role in the diversification of Cebus.We used a heterochromatin probe for chromosome 11 of Cebus libidinosus (11qHe+ CLI probe), obtained by chromosome microdissection. Twenty-six specimens belonging to the families Atelidae, Cebidae, Callitrichidae and Pithecidae (Platyrrhini) were studied. Fourteen out of 26 specimens were Cebus (Cebidae) individuals of C. libidinosus, C. xanthosternos, C. apella, C. nigritus, C. albifrons, C. kaapori and C. olivaceus. In Cebus specimens, we found 6 to 22 positive signals located in interstitial and telomeric positions along the different species. No hybridization signal was observed among the remaining Ceboidea species, thus reinforcing the idea of a Cebus-specific heterochromatin composed of a complex system of repetitive sequences.

Biomarkers of genotoxicity and genomic instability in a non-human primate, Cebus libidinosus (Cebidae, Platyrrhini), exposed to nitroimidazole derivatives.

The genotoxicity of two nitroimidazole derivatives, ornidazole (ONZ) and metronidazole (MTZ) in the peripheral blood lymphocytes of Cebus libidinosus (CLI) (Primates, Cebidae) was assessed. Endpoints measured included sister chromatid exchange (SCE) frequency, cell proliferation kinetics (CPK), replication index (RI), mitotic index (MI), and damage incidence in or near CLI heterochromatin regions. MI and SCE values following ONZ or MTZ treatments were significantly different (p<0.001) from control. SCE frequency per chromosome was not proportional to chromosome length. The chromosomes most affected for SCE were 1, 2, 4, 6, 11-13, 17, and 18, many of which possess interstitial or terminal heterochromatin. In the CLI genome, chromosomes 11 and 17 showed higher susceptibility to damage RI was the only biomarker that did not show statistically significant differences between control and treated cultures. C. libidinosus bands 11q1.4 and 11q1.5 may be hot-spots in the context of nitroimidazole exposure.

Meiotic characterization and sex determination system of neotropical primates: Bolivian squirrel monkey Saimiri boliviensis (primates: Cebidae).

The chromosomal sex determination system differs among platyrrhine monkeys more than any other group of primates. Although a number of studies have investigated mitotic chromosomes across platyrrhine species, the meiotic chromosomes of many genera have not yet been described. The goal of this study was to characterize the sex determination system of Saimiri boliviensis. We described for the first time the meiotic cycle, confirming the sexual system in germ cells from testicular biopsies of four adult male S. boliviensis. All specimens were weighed and testicular volume was measured. We observed 22 bivalents corresponding to 2N = 44, and a "human-like" XY bivalent was found in diakinesis/metaphase I. In addition, mitotic studies from blood samples of both sexes were performed and G- and C-banding patterns agreed with previously reported karylogy of S. boliviensis boliviensis. Further meiotic studies should be performed in New World primates based on the great value of those studies for systematic evolutionary biology and conservation programs.

Phylogenetic relationships among some Ateles species: the use of chromosomic and molecular characters.

As with most platyrrhines, the systematics of Ateles is under discussion. In order to help clarify its systematic, we employed chromosomic and molecular characters to analyze the phylogenetic relationship among some species of the genus Ateles. Chromosomic studies were conducted on 14 atelid specimens: eight Ateles from A. paniscus, A. chamek, A. belzebuth and A. geoffroyi, and six Alouatta caraya. Ateles paniscus showed 2N=32, whereas A. chamek, A. belzebuth and A. geoffroyi presented 2N=34, XX/XY (with a submetacentric X and a variable Y) corroborated by male meiosis. Nucleotide sequence variation at the mitochondrial cytochrome c oxidase subunit II gene (COII) was analyzed in ten New World monkey specimens. Parsimony trees showed consistent phylogenetic relationships using both chromosomic forms and mitochondrial COII gene sequences as characters. Particularly, chromosomic phylogenies showed A. hybridus as a divergent taxon from the remaining group, whereas A. chamek, A. belzebuth and A. marginatus form an unresolved clade with A. geoffroyi as sister group.

Heterochromatin and chromosome evolution: a FISH probe of Cebus apella paraguayanus (Primate: Platyrrhini) developed by chromosome microdissection.

Neotropical Primate karyotypes are highly variable, particularly in the heterochromatic regions, not only regarding the amount of heterochromatin, but also the composition. G and C banding and FISH techniques provide useful information to characterize interspecific relationships. We used chromosome microdissection to develop a FISH probe of the chromosome 11 heterochromatic block (11qHe+) of Cebus apella paraguayanus (CAPp). Fragments of the 11qHe+ microdissected from fibroblast cell culture were collected in a PCR tube, amplified by degenerate oligonucleotide primer-PCR and subsequently labeled. The specificity of the FISH probe was confirmed in metaphases of some Ceboidea species. Signals were located in the He+ of chromosomes 4, 11, 12, 13, and 19 of CAPp and in the He+ of chromosomes 4, 12 and 13 of C. a. nigritus (CAPn); no signals were observed when other Ceboidea species were analyzed. We propose that the heterochromatin observed in CAPp and CAPn is specific for these species. We consider this C. apella heterochromatin identity as a possible key for the interpretation of chromosomal evolution in these Ceboidea.