Processos carioevolutivos na ordem tetraodontiformes: uma visão através de suas diferentes linhagens

Given the great diversity of fishes, the Order Tetraodontiformes stands to show genetic and morphological characteristics enough singular. The fishes of this order have a compact DNA which favors molecular studies, as well as comparisons with more basal species. Model of genome evolution, there are still many gaps in knowledge about their chromosomal patterns and how evolutionary rearrangements influence the marked variation in DNA content of this order. In view of this, we present cytogenetic analyzes of the species Acanthostracion quadricornis (Ostraciidae), A. polygonius (Ostraciidae) Melichthys niger (Balistidae) Cantherhines macrocerus (Monacanthidae) and C. pullus (Monacanthidae), Lagocephalus laevigatus, Colomesus psittacus and Canthigaster figueiredoi (Tetraodontidae), to contribute with cytogenetic data for this group. The analysis was performed by C-banding, Ag-RONs, coloring with base-specific fluorochromes DAPI-CMA3, restriction enzymes AluI, EcoRI, TaqI, PstI and HinfI and in situ hybridization with probes for ribosomal DNA 18S and 5S. The heterochromatic ultrastructure of A. quadricornis and A. polygonius revealed a outstanding heterochromatin content, which may indicate that the accumulation or loss of extensive heterochromatin content could be responsible for large variations in genomic content displayed in different Tetraodontiformes families. The species Cantherhines macrocerus, C. pullus (Monacanthidae) and Melichthys niger (Balistidae) shows a huge karyotypic similarity both numerically and structural. L. laevigatus showed similar cytogenetic features (2n = 44 and single RONs) to the species of the genus Takifugu, which reinforces the idea of their phylogenetic relationships. C. psittacus presented the highest diploid number described for the family (2n = 56) and large amount of HC, features that related with its sister family Diodontidae. Cytogenetic analysis in C. figueiredoi revealed heterochromatic polymorphisms, RONs multiple and Bs chromosomes. These events are rare in marine fishes, and are possibly associated with the strong restructuring and genomic reduction that this family has been suffered. These features, plus the morphological and molecular data suggests that these species share the same ancestral branch, with a possible monophyletic origin. In this study, new contributions to the knowledge of evolutionary patterns facing by Tetraodontiformes are provided and discussed under cytotaxonomyc, genomic and evolutionary perspectives.

Contribuição à citogenética de testudines e análise de assimetria flutuante em Eretmochelys imbricata, Cheloniidae

This work has contributed to knowledge of the order Testudines from cytogenetic and morphological point of view. With regard to the aspects proposed cytogenetic characterization of the species Mesoclemmys tuberculata (n = 5), endemic to the Caatinga biomes, through conventional techniques of cytogenetics and molecular levels. This species presented 2n = 58, NF = 64, the first submetacentric pair, the second metacentric and third subtelocentric, and the other microchromosome telocentric. This species showed a nucleolar bearing pair, coincident with the 18S ribosomal rDNA and that proved to be heterochromatic. Small heterochromatic blocks were also found in the centromeres of the largest chromosomes, as well as terminal regions in most other chromosomes of the complement, that were GC +. Telomeric sequences showed variable patterns of signal intensity, with some repeats more intense in microchromosomes and subtly in the larger ones. When compared with other species of the genus, the G-banding patterns showed a marked similarity between them. The first karyotypic description of the species will aid in future studies and the understanding of evolutionary aspects of this family. From the morphological point of view, we carried out studies of fluctuating asymmetry in sea turtle Eretmochelys imbricata, using methods of benchmarking between hatchlings and adults and their implications for natural selection. Data were collected at two different times: first during the spawning female and the second during the outbreak and birth of the nest. The analyzed characteristics consisted of measurements of length and width of front and rear flippers (CANT, LANT, CPOS and LPOS) also collected data on the number of hull plates, side plates (NPL), the surrounding plates (NPCIRC), and plastron; plates power plants (NPP), inframarginais plates (NPIM). With the values of asymmetry we calculated the value of strict heritability for these traits, the calculation was based on only one parent. A nonparametric analysis Mann-Whitneywas performed to compare the groups (females X hatchlings, newborn hatchlings X dead hatchlings). Adult females showed no bilateral fluctuating asymmetry (FA = 0) on the number plates of the hull and plastron, while offspring, living and dead, showed a greater level of variation in these meristic parameters. In the analysis of females x hatchlings we found a significant difference between the levels of asymmetry in hoof plates (p=0.006) an the width of hindlimbs (p=0.001). Levels of FA suggest an accurate indicator as to the viability or maintenance of the individual to the reproductive phase. The coefficient of heritability (h2) of FA , obtained from the regression analysis, showed that both have low and not statistically significant values(p> 0.1). In the case of exclusion of the effective role of genetics in the generation of FA, reproductive strategies based on high number of subsidiaries products, such as those observed in E. imbricata seems to implicate the production of individuals with high level of developmental instability

Padrões cromossômicos e mapeamento de genes ribossomais 18S e 5S em peixes pelágicos Atlânticos

Cytogenetic studies in fish have been contributed significantly to a better understanding of the marine biodiversity, presenting information related to characterization, evolution and conservation of species e fisheries stocks. Among the marine species which cytogenetic data are less well known pelagic forms are detached, that despite the economic importance and conservation efforts have been suffering great pressure from the artisanal and industrial fisheries. The present work characterized cytogenetically six species of large pelagic fish in the Atlantic, belonging to the Order Perciformes, among them, four species of Scombridae, Thunnus albacares, T. obesus, Scomberomorus brasiliensis and Acanthocybium solandri and two Coryphaenidae, Coryphaena equiselis and C. hippurus using Classical cytogenetic methods as conventional staining, C-banding and Ag-NORs and molecular through staining fluorochromes AT and GC-specific and mapping of ribosomal multigene families, 18S and 5S. The identification of phylogenetic patterns and cytotaxonomic markers between the species and the presence of sex chromosomes in at least one species of Coryphaenidae, are particularly useful in the formulating of phylogenetic hypotheses, as well as comparisons between groups and populations

Análise citogenética de células-tronco derivadas do subendotélio da veia umbilical humana

Mesenchymal stem cells (MSCs) are known as a population of multi-potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. Several applications of the study of EC can be emphasized the therapeutic techniques such as guided bone regeneration by implantation of EC in the affected site, without the need for bone grafts, using titanium as a vehicle. The process of cryopreservation is essential for the maintenance of cell cultures, since the cell line is frozen, it can be maintained in liquid nitrogen for an indefinite period and then thawed for therapeutic or experimental purposes. The aim of this study was to isolate a population of MSCs derived from the subendothelium of the umbilical vein human (MSCs-SUVH) to assess cytogenetic analysis by the possibility of appearance of chromosomal changes in two different situations: MSCs-SUVH regarding the process of cryopreservation and MSCs-SUVH grown on the surface of titanium. Flow cytometry analysis revealed that, this cell population was positive for the markers CD29, CD73 and CD90, but there was no expression of hematopoietic lineage markers, such as CD14, CD34 and CD45 and demonstrated capacity for osteogenic differentiation. The chromosomes obtained from the primary culture of MSCs-SUVH were analyzed by GTW banding technique, and results are described as guidelines to ISCN 2005. There was not the emergence of clonal chromosomal changes in the MSCs-SUVH in different situations analyzed. However one of the strings presented a balanced paracentric inversion, probably a cytogenetic constitutional alterations, which was present before and after the experimental situations that the MSCs-SUVH was submitted

Levantamento cariotípico em espécies de peixes marinhos costeiros de fundo arenoso (Osteichthypes, Perciformes)

Cytogenetics analyses in fish are important because they compose a private group among the vertebrates, occupying a central position in the animal evolution. The Perciforms Order, dominant in the marine and freshwater environment, it constitutes a model potentially useful in the genetic evaluation of populations, as well as in the understanding of its evolutionary processes. In spite of this, cytogenetics studies in this great group is scarce, above all for the inhabitants of sandy bottom and pelagics habits. The present work proposed to contribute for the cytogenetic characterization of nine species of fish marine of sandy bottom of the coast of Rio Grande do Norte (Brazil), identifying the evolutionary patterns related to the karyotype in these species and the existence of filogenetics affinities between them and other Perciformes. The animals were collected in the beaches of the Redinha, Ponta Negra and Búzios (Coast of Rio Grande do Norte) and in Saint Peter and Saint Paul Archipelago. Later on they were submitted to the cytogenetics technical that consist of mitotic estimulation, obtaining of mitotics chromosomes, proceeded by techniques of conventional coloration (Giemsa) and chromosomic bands (Ag-RONs and C band). Diploid number and fundamental number equal to 48 were observed in most of the species: Menticirrhus americanus, Ophioscion punctatissimus, Pareques acuminatus (Sciaenidae); Chloroscombrus chrysurus (Carangidae); Echeneis sp. 2 (Echeneidae); Archosargus probatocephalus (Sparidae) and Orthopristis ruber (Haemulidae). Trachinotus goodei (NF=52) (Carangidae) and Echeneis sp. 1 (Echeneidae) (NF=54) presented variation in NF, staying constant a diploid number equal to 48. RONs was situated in pericentromeric position in whole the scianids, and in the species Echeneis sp. 2 (22° pair), O. ruber and A. probatocephalus (1° pair), coinciding with great heterocromatics blocks in M. americanus (1° pair), P. acuminatus (2° pairl) and O. ruber (1° pair). RONs was also located in the telomeric area of the short arm of the 5° and 11° acrocentrics pairs in T. goodei, 4° and 19° pairs of C. chrysurus, 1° pair (sm) of Echeneis sp. 1. The C band detected centromeric blocks in most of the chromosomes of the species of Sciaenidae, Carangidae and Echeneidae, with great blocks in A. probatocephalus (4° pair). Heterocromatic blocks in telomeric areas in submetacentrics of Echeneis sp. 1, and pericentromerics in M. americanus (1° and 8° pairs), O. punctatissimus (1° pair) and P. acuminatus (2° pair) were also observed. It is noticed a marked conservatism cromossomic in the species of the family Scianidae and Haemulidae in what says respect to the number of acrocentrics chromosomes and the location of RONs. Even so it is outstanding the presence of heterocromatinization events during the karyotypic evolution of this family. Already in the families Sparidae and Carangidae, the obtained results reaffirm examples of small variations structural resultants of inversion and translocation Robertsonian, as important mechanisms of diversification karyotipical, as well as a pattern numerical evolutionary conserved, also observed in representatives of Echeneidae of Atlantic in relation to Pacific. The presence of RONs multiple, observed in the species T. goodei and C. chrysurus seems to represent a character derived in the family Carangidae. The results for the species O. ruber and A. probatocephalus suggest the presence of possible geographical or climatic barriers among populations of NE of Brazil in relationship the one of the SE

Padrões cariotípicos em tetraodontiformes (Osteichthypes) :redução genômica e mecanismos de diversificação

The Tetraodontiformes order is composed for about 400 species of fish, distributed in ten families, with circuntropical distribution. The morphologic diversity of each family reflects in, part, the different levels of specialization. This group represents an ancestry after-Perciformes and constitutes the last branch of the diffusion of the Teleósteos, occupying a position of prominence. The phylogenetics relationships of the Tetraodontiformes exist diverse works examining and, in all, these families are recognized as groups brothers, being Diodontidae next to Tetraodontidae and Balistidae next to Monacanthidae. Although it possesss a representative number of species, the works involving of the families Balistidae and Monacanthidae are few exemplary, especially species of oceanic islands. In this work cytogenetic studies in five species had been analyzed Cantherhines macrocerus, Cantherhines pullus (Monacanthidae), Melichthys niger (Balistidae), Sphoeroides testudíneus (Tetraodontidae) and Chilomycterus antennatus (Diodontidae); through conventional coloration, Ag-NORs and C banding. Ahead of the different karyological trends of evolution presented by the Tetraodontiformes, the present work also searched to verify the relation existence enters the total size of the chromosomes with the amount of DNA in these groups of Tetraodontiformes. For such, they had been correlated the total size of the chromosomes of these species, with values of content of available DNA in literature. The cytogenetics analyses for the species C.macrocerusJ C.pullus (Monacanthidae) and M.niger (Balistidae), had disclosed 40 chromosomes, all acrocentrics. All possess only one pair of NORs and pericentromeric heterochromatin. For S.testudíneus the found dyploid number was equal 2n=46, with NF=78 (16m+18sm+8st+4a), while that for C.antennatus it possesss 2n=50, with NF=76 (4m+22st+24a). Both species possess simple NORs and pericentromeric heterochromatin blocks. In M.niger, the presence of positive marking (heterochromatin and NOR) in the secondary constriction in the second chromosomic pair suggesting the occurrence of a rearrangement, possibly a fusing involving these homologous ones, indicating that these events had been important for the establishment of the karyological history of this group. A maintenance of the chromosomic constancy found in the populations of C.macrocerus (Monacanthidae) and S.testudineus (Tetraodontidae) perhaps if must for the aiding of the gene flow through oceanic chains. These data contrast with the differentiated kinds of chromosomes of C.antennatus between the Northeast coast and Southeastern, suggesting that the ecological standards of each species, added to the conditions of the marine environment, can be responsible for the karyological delineation of each species. The found characteristics for the species C.macrocerus, C.pullus, M.niger, S.testudineus and C.antennatus add it the available data for other species of Tetraodontiformes. From the data gotten in the present study, it can be inferred that the DNA content possesss direct relation with the total length of the chromosomes

Diversificação cariotípica em cinco espécies de moréias do Atlântico Ocidental (Anguilliformes)

The Anguiliformes is constituted by 15 families, 141 sorts and 737 species. In this group eight families possess at least one karyotyped species, where a prevalence of karyotypes with 2n=38 is evidenced chromosomes and high NF, apparently basal for the Anguiliformes. The only family who shows a different karyotypic pattern from the others is the Muraenidae family. In this, of the eight species already described, all of them present 2n=42 chromosomes. Despite the dimension of this Order, few species present karyotypics descriptions. In the present work, a species of Ophichthidae, Myrichthys ocellatus (2n=38, 8m+14sm+10st+6a, NF=70) and three species of Muraenidae, Enchelycore nigricans (2n=42, 6m+8sm+12st+16a, NF=68), Gymnothorax miliaris (2n=42, 14m+18sm+10st, NF=84), Gymnothorax vicinus (2n=42, 8m+6sm+28a, NF=56) and Muraena pavonina (2n=42, 6m+4sm+32a, NF=52), collected in the coast of the Rio Grande do Norte state, Saint Peter and Paul Rocks and in the coast of Bahia state were analyzed. Mitotics chromosomes had been gotten through mitotic stimulation with yeasts. Among the analyzed species, it is observed the presence of characteristic large metacentric chromosomic pairs (≅10µm). As for the structural standard, heterochromatics regions in these species in centromeric position of the majority of the chromosomic pairs and simple ribosomal sites had been evidenced. For the Ophichthidae family, the gotten data corroborate the hypothesis of karyotypic diversification mediated by the occurrence of pericentrics inversions and robertsonians rearrangements, while in the Muraenidae, the identification of larger chromosomic values (2n=42), suggests derived karyotypes, possibly caused by possible chromosomic fissions

Determinação de haplótipos do gene βs em portadores de anemia falcifome

Haplotypes linked to the βS gene represent patterns of DNA polymorphisms along chromosome 11 of individuals bearing the βS gene. Analysis of haplotypes, in addition to serving as an important source for anthropological studies about the ethnic origin of a population, contributes to a better understanding of the variations in clinical severity of sickle cell anemia. The aim of the present study was to determine βS gene haplotypes in a group of patients with sickle cell anemia treated at the Dalton Barbosa Cunha Hematology Center (Hemonorte) in Natal, Brazil and the Oncology and Hematology Center in Mossoró, Brazil. Blood samples were obtained from 53 non-related patients (27 males and 26 females), aged between 3 months and 61 years (mean age: 16.9 ± 12.1 years). Laboratory analyses consisted of the following: erythrogram, reticulocyte count, hemoglobin electrophoresis at alkaline pH, measurement of hemoglobin A2 and Fetal hemoglobin, solubility test and molecular analysis to determine βS gene haplotypes. DNA samples were extracted by illustra blood genomicPrep Mini Spin kit and βS gene haplotypes were determined by PCR-RFLP, using Xmn I, Hind III, Hinc II and Hinf I restriction enzymes for analysis of six polymorphic restriction sites in the beta cluster. Of 106 βS chromosomes studied, 75.5% were Central African Republic (CAR) haplotype, 11.3% Benin (BEN) and 6.6% Cameroon (CAM). The atypical haplotypes had a frequency of 6.6%. More than half the patients (58.5%) were identified as CAR/CAR genotype carriers, 16.9% heterozygous CAR/BEN, 13.2% CAR/CAM and 1.9% BEN/BEN. Patients with atypical haplotype in one or two chromosomes accounted for 9.5% (CAR/Atp, BEN/Atp and Atp/Atp). The genotype groups showed no statistically significant difference (p < 0.05) in their laboratory parameters. This is the first study related to βS haplotypes conducted in state of Rio Grande do Norte and the higher frequency of Cameroon halotype found, compared to other Brazilian states, suggests the existence of a peculiarity of African origin