The synthetic dye malachite green found in food induces cytotoxicity and genotoxicity in four different mammalian cell lines from distinct tissuesw.

Malachite green (MG) is a synthetic dye that uses ranges from its application as a tissue dye to that as an antiparasitic in aquaculture. Several studies have reported the presence of this compound in food dyes and in the meat of fish raised in captivity for human consumption, suggesting risks both for the end user and for as those who handle these products because of MG toxic properties described in the literature. Here we evaluated the cytotoxic and genotoxic profiles of MG in four different cell lines (ACP02, L929, MNP01, and MRC-5). Two of these cell lines are stomach cells (normal and cancer lineages) and the potential ingestion of MG makes this a relevant cell type. Cells were treated with MG at concentrations ranging from 0.1 µM to 100 µM, and tested by MTT assay, a differential apoptosis/necrosis assay (EB/OA), the micronucleus test (MN), and the comet assay. MG exhibits dose-dependent cytotoxicity toward all of the tested cell types; higher concentrations of MG cause cell necrosis, while lower concentrations induce apoptosis. MG has a genotoxic profile increasing the rates of micronuclei, nucleoplasmic bridges, nuclear buds, and DNA fragmentation; L929 and MRC-5 showed more sensibility than ACP02 and MNP01.

Karyotypes of Manatees: New Insights into Hybrid Formation (Trichechus inunguis × Trichechus m. manatus) in the Amazon Estuary.

Great efforts have been made to preserve manatees. Recently, a hybrid zone was described between Trichechus inunguis (TIN) and the Trichechus manatus manatus (TMM) in the Amazon estuary. Cytogenetic data on these sirenians are limited, despite being fundamental to understanding the hybridization/introgression dynamics and genomic organization in Trichechus. We analyzed the karyotype of TMM, TIN, and two hybrid specimens ("Poque" and "Vitor") by classical and molecular cytogenetics. G-band analysis revealed that TMM (2n = 48) and TIN (2n = 56) diverge by at least six Robertsonian translocations and a pericentric inversion. Hybrids had 2n = 50, however, with Autosomal Fundamental Number (FNA) = 88 in "Poque" and FNA = 74 in "Vitor", and chromosomal distinct pairs in heterozygous; additionally, "Vitor" exhibited heteromorphisms and chromosomes whose pairs could not be determined. The U2 snDNA and Histone H3 multi genes are distributed in small clusters along TIN and TMM chromosomes and have transposable Keno and Helitron elements (TEs) in their sequences. The different karyotypes observed among manatee hybrids may indicate that they represent different generations formed by crossing between fertile hybrids and TIN. On the other hand, it is also possible that all hybrids recorded represent F1 and the observed karyotype differences must result from mechanisms of elimination.

Isolation and establishment of skin-derived and mesenchymal cells from south American bat Artibeus planirostris (Chiroptera - Phyllostomidae).

Animal models represent a crucial tool for biological research, so the establishment of new cultures is fundamental for the discovery of new therapies and the understanding of mechanisms of cell development in the most diverse animals. Here, we report the successful establishment of two new primary cell cultures derived from a South American bat (Artibeus planirostris). The establishment of a new bat culture can help in the investigation of new zoonoses since bats have been proposed as carriers of these diseases. We evaluated the chromosomal stability of cells from different passages. Primary cultures were collected from ear tissues and bone marrow of A. planirostris. Cultures were expanded, and osteogenic and adipogenic inductions were conducted for 21 days. For osteogenic differentiation, the medium was supplemented with 0.1 µM dexamethasone, 3 mM ß-glycerophosphate, and 10 µM L-ascorbic acid 2-phosphate. For adipogenic differentiation, the medium was supplemented with 5 µM rosiglitazone, 0.4 µM insulin, 0.1 mM indomethacin, and 0.1 µM dexamethasone. After the induction period, the cells were stained with Alizarin Red to assess osteogenic differentiation and Oil Red O to assess adipogenic differentiation. We observed the appearance of lipid droplets in adipocytes and the extracellular deposition of calcium matrix by osteocytes, indicating that bone marrow-derived cells and skin-derived cells of A. planirostris could successfully differentiate into these lineages. Also, the number of chromosomes remained stable for both primary cultures during passages 2, 4, 6, and 8.

Chromosome evolution in Lophyohylini (Amphibia, Anura, Hylinae).

The hyline tribe Lophyohylini includes 87 species of treefrogs, of which cytogenetics aspects have been studied in less than 20% of them. In order to evaluate the evolution of some of its chromosome characters (NOR position, C-bands, and DAPI/CMA3 bands), we studied the karyotypes of 21 lophyohylines, 16 of them for the first time, and analyzed them in a phylogenetic context. Most species showed similar karyotypes regarding chromosome number (2n = 24) and morphology (FN = 48), excepting Phyllodytes edelmoi and Osteocephalus buckleyi with 2n = 22 (FN = 44) and 2n = 28 (FN = 50), respectively. The NOR location was variable among species and provided valuable phylogenetic information. This marker was located in pair 11 in all species of Trachycephalus, Itapotihyla langsdorffii, and Nyctimantis arapapa, representing the plesiomorphic condition of Lophyohylini. Besides, other apomorphic states were recovered for the clades comprising N. rugiceps and N. siemersi (NOR in pair 5), and Dryaderces pearsoni, Osteocephalus, and Osteopilus (NOR in pair 9). Phyllodytes presented variation for NORs position; they were in pair 2 in P. edelmoi, pair 7 in P. melanomystax, and pair 8 in P. gyrinaethes and P. praeceptor. Polymorphisms in size, number, and activity of this marker were observed for N. siemersi, Osteocephalus fuscifacies, and some species of Trachycephalus. Remarkably, in N. siemersi NORs were detected on a single chromosome in the two specimens studied by this technique, raising the question of how this complex polymorphism is maintained. Interstitial telomeric sequences were found in P. edelmoi, P. melanomystax, and Osteocephalus buckleyi, and their presence seems to be not related to the chromosome reorganization events. Finally, some species showed spontaneous rearrangements, possibly as a consequence of an uncommon phenomenon in anuran cytogenetics: the presence of fragile sites or secondary constrictions not associated with NORs. We propose that this rare feature would have played an important role in the evolution of this group of frogs. From the evidence obtained in this and previous studies, we conclude that Lophyohylini presents a complex chromosome evolution.

Andiroba oil (Carapa guianensis Aubl) shows cytotoxicity but no mutagenicity in the ACPP02 gastric cancer cell line.

Andiroba (Carapa guianensis Aubl) is an Amazonian plant whose oil has been widely used in traditional medicine for various purposes, including anti-inflammation. Research reports indicate that the oil can confer antitumor activity due to the presence of fatty acids, which can directly influence cell death mechanisms. Thus, andiroba oil (AO) has gained interest for its potential to be used in antineoplastic therapies. Here, we report an in vitro analysis of the cytotoxic and mutagenic potential of AO in the gastric cancer cell line, ACP02. Cell survival was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, differential staining with ethidium bromide and acridine orange assessed apoptosis-necrosis, and mutagenesis was assessed by the micronucleus test. The apolar oil was first diluted in 0.1% dimethyl sulfoxide (DMSO) and then further diluted to six concentrations (0.01, 0.1, 1, 10 and 100 µg/mL and 1 mg/mL) in RPMI medium. Controls included RPMI alone (negative control) and 0.1% DMSO diluted in medium (vehicle control). The MTT test showed that AO significantly reduced cell viability (P < .05) only when the highest tested concentration was applied for 48 hours. The apoptosis/necrosis test showed that the highest concentration of AO induced cell death by apoptosis at 24 and 48 hours. There was no statistically significant increase in the frequency of micronuclei. The ability of the AO to decrease the viability of ACP02 cells via apoptosis, without exerting mutagenic effects, suggests that the oil could be useful as an alternative therapeutic agent for primary tumors of stomach cancer.

Vitrification of domestic cat (Felis catus) ovarian tissue: Effects of three different sugars.

We aimed to evaluate the effect of three extracellular cryoprotectants on the morphology of vitrified feline preantral follicles. Feline ovarian fragments (0.5 × 2 × 2 mm) collected from five domestic adult cats subjected to ovariohysterectomy for routine castration were vitrified with ethylene glycol (EG) 40% combined or not with sucrose (0.1 or 0.5 M), trehalose (0.1 or 0.5 M), or raffinose (0.1 M). After vitrification using the solid-surface method and warming of the tissues, cryoprotectants were washed out of the ovarian tissues, which were fixed for histological analysis. The percentages of normal follicles were similar to the control (fresh) (62.9 ± 4.1%) only for tissues exposed and cryopreserved with EG + trehalose at concentrations of 0.1 (35.8 ± 8.3%) and 0.5 M (33.4 ± 5.4%). All the other sugars decreased the percentages of morphologically normal follicles as compared to the control group and the trehalose groups. Based on the results of the present study, we recommend the use of trehalose as the extracellular cryoprotectant for the vitrification of feline ovarian tissue.

Karyotypic Diversity and Evolution in a Sympatric Assemblage of Neotropical Electric Knifefish.

Chromosome changes can perform an important role in speciation by acting as post-zygotic reproductive barriers. The Neotropical electric fish genus Brachyhypopomus (Gymnotiformes, Hypopomidae) has 28 described species, but cytogenetic data are hitherto available only for four of them. To understand karyotype evolution and investigate the possible role of chromosome changes in the diversification of this genus, we describe here the karyotype of eight species of Brachyhypopomus from a sympatric assemblage in the central Amazon basin. We analyzed cytogenetic data in the context of a phylogenetic reconstruction of the genus and known patterns of geographical distribution. We found a strong phylogenetic signal for chromosome number and noted that sympatric species have exclusive karyotypes. Additional insights into the role of chromosome changes in the diversification of Brachyhypopomus are discussed.

Extensive Karyotype Reorganization in the Fish Gymnotus arapaima (Gymnotiformes, Gymnotidae) Highlighted by Zoo-FISH Analysis.

The genus Gymnotus (Gymnotiformes) contains over 40 species of freshwater electric fishes exhibiting a wide distribution throughout Central and South America, and being particularly prevalent in the Amazon basin. Cytogenetics has been an important tool in the cytotaxonomy and elucidation of evolutionary processes in this genus, including the unraveling the variety of diploid chromosome number (2n = from 34 to 54), the high karyotype diversity among species with a shared diploid number, different sex chromosome systems, and variation in the distribution of several Repetitive DNAs and colocation and association between those sequences. Recently whole chromosome painting (WCP) has been used for tracking the chromosomal evolution of the genus, showing highly reorganized karyotypes and the conserved synteny of the NOR bearing par within the clade G. carapo. In this study, painting probes derived from the chromosomes of G. carapo (GCA, 2n = 42, 30 m/sm + 12 st/a) were hybridized to the mitotic metaphases of G. arapaima (GAR, 2n = 44, 24 m/sm + 20 st/a). Our results uncovered chromosomal rearrangements and a high number of repetitive DNA regions. From the 12 chromosome pairs of G. carapo that can be individually differentiated (GCA1-3, 6, 7, 9, 14, 16, and 18-21), six pairs (GCA 1, 9, 14, 18, 20, 21) show conserved homology with GAR, five pairs (GCA 1, 9, 14, 20, 21) are also shared with cryptic species G. carapo 2n = 40 (34 m/sm + 6 st/a) and only the NOR bearing pair (GCA 20) is shared with G. capanema (GCP 2n = 34, 20 m/sm + 14 st/a). The remaining chromosomes are reorganized in the karyotype of GAR. Despite the close phylogenetic relationships of these species, our chromosome painting studies demonstrate an extensive reorganization of their karyotypes.

Chromosome evolution in Cophomantini (Amphibia, Anura, Hylinae).

The hylid tribe Cophomantini is a diverse clade of Neotropical treefrogs composed of the genera Aplastodiscus, Boana, Bokermannohyla, Hyloscirtus, and Myersiohyla. The phylogenetic relationships of Cophomantini have been comprehensively reviewed in the literature, providing a suitable framework for the study of chromosome evolution. Employing different banding techniques, we studied the chromosomes of 25 species of Boana and 3 of Hyloscirtus; thus providing, for the first time, data for Hyloscirtus and for 15 species of Boana. Most species showed karyotypes with 2n = 2x = 24 chromosomes; some species of the B. albopunctata group have 2n = 2x = 22, and H. alytolylax has 2n = 2x = 20. Karyotypes are all bi-armed in most species presented, with the exception of H. larinopygion (FN = 46) and H. alytolylax (FN = 38), with karyotypes that have a single pair of small telocentric chromosomes. In most species of Boana, NORs are observed in a single pair of chromosomes, mostly in the small chromosomes, although in some species of the B. albopunctata, B. pulchella, and B. semilineata groups, this marker occurs on the larger pairs 8, 1, and 7, respectively. In Hyloscirtus, NOR position differs in the three studied species: H. alytolylax (4p), H. palmeri (4q), and H. larinopygion (1p). Heterochromatin is a variable marker that could provide valuable evidence, but it would be necesserary to understand the molecular composition of the C-bands that are observed in different species in order to test its putative homology. In H. alytolylax, a centromeric DAPI+ band was observed on one homologue of chromosome pair 2. The band was present in males but absent in females, providing evidence for an XX/XY sex determining system in this species. We review and discuss the importance of the different chromosome markers (NOR position, C-bands, and DAPI/CMA3 patterns) for their impact on the taxonomy and karyotype evolution in Cophomantini.

Cryptic Species in Proechimys goeldii (Rodentia, Echimyidae)? A Case of Molecular and Chromosomal Differentiation in Allopatric Populations.

The spiny rats of the genus Proechimys have a wide distribution in the Amazon, covering all areas of endemism of this region. We analyzed the karyotype and cytochrome b (Cyt b) sequences in Proechimys goeldii from 6 localities representing 3 interfluves of the eastern Amazon. A clear separation of P. goeldii into 2 monophyletic clades was observed, both chromosomally and based on Cyt b sequences: cytotype A (2n = 26x2640;/27x2642;, NF = 42) for samples from the Tapajos-Xingu interfluve and cytotype B (2n = 24x2640;/25x2642;, NF = 42) for samples from the Xingu-Tocantins interfluve and east of the Tocantins River. The karyotypes differ in a pericentric inversion and a centric fusion/fission and an average nucleotide divergence of 6.1%, suggesting cryptic species. Meiotic analysis confirmed the presence of a XX/XY1Y2 multiple sex chromosome determination system for both karyotypes. The karyotypes also vary from the literature (2n = 24, NF = 42, XX/XY). The autosome translocated to the X chromosome is different both in size and morphology to P. cf. longicaudatus, which also has a multiple sex chromosome determination system (2n = 14x2640;/15x2640;x2642;/16x2640;/17x2642;, NF = 14). The Xingu River is a barrier that separates populations of P. goeldii, thus maintaining their allopatric nature and providing an explanation for the molecular and cytogenetic patterns observed for the Xingu River but not the Tocantins River.

Detrimental Effect of Phenol Red on the Vitrification of Cat (Felis catus) Ovarian Tissue.

The aim of this study was to investigate the effects of different media with or without phenol red or the antioxidant trolox on the successful vitrification of feline ovarian tissue. In a first experiment, ovarian cortical pieces from three cats were vitrified in solutions of Roswell Park Memorial Institute (RPMI)-1640 medium, Minimum Essential Medium, Dulbecco's modified Eagle's medium, or Tissue Culture Medium 199 as basic medium, supplemented or not with 50 µM of trolox, all containing 40% ethylene glycol (EG) and 1 M of sucrose. RPMI-1640 (phenol red-free) without trolox was the only medium that preserved the percentage of morphologically normal preantral follicles similar to control (80%). The main difference between RPMI-1640 and the other media was the absence of phenol red and CaCl2. In a second experiment, ovarian cortical pieces from three cats were vitrified in a solution containing RPMI-1640 as basic medium, 40% EG, 1 M of sucrose, supplemented or not with phenol red or CaCl2 alone, or in combination. It was observed that phenol red supplementation led to follicular degeneration. Finally, to evaluate the interaction between phenol red and the cryoprotectant agent (i.e., EG), ovarian tissue was exposed to RPMI-1640 supplemented with phenol red and EG at different concentrations (10%, 20%, or 40%). There was an inverse relationship between EG concentration and free phenol red in the medium after exposure. It is suggested that vitrification of feline ovarian tissue should be performed in a phenol red-free medium. Medium supplementation with 50 µM of trolox was deleterious for follicular morphology.

Protein markers of synaptic behavior and chromatin remodeling of the neo-XY body in phyllostomid bats.

The XX/XY system is the rule among mammals. However, many exceptions from this general pattern have been discovered since the last decades. One of these non-conventional sex chromosome mechanisms is the multiple sex chromosome system, which is evolutionary fixed among many bat species of the family Phyllostomidae, and has arisen by a translocation between one original gonosome (X or Y chromosome), and an autosome, giving rise to a "neo-XY body." The aim of this work is to study the synaptic behavior and the chromatin remodeling of multiple sex chromosomes in different species of phyllostomid bats using electron microscopy and molecular markers. Testicular tissues from adult males of the species Artibeus lituratus, Artibeus planirostris, Uroderma bilobatum, and Vampyrodes caraccioli from the eastern Amazonia were analyzed by optical/electron microscopy and immunofluorescence of meiotic proteins involved in synapsis (SYCP3 and SYCE3), sister-chromatid cohesion (SMC3), and chromatin silencing (BRCA1, γ-H2AX, and RNApol 2). The presence of asynaptic axes-labeled by BRCA1 and γ-H2AX-at meiotic prophase in testes that have a normal development of spermatogenesis, suggests that the basic mechanism that arrests spreading of transcriptional silencing (meiotic sex chromosome inactivation (MSCI)) to the autosomal segments may be per se the formation of a functional synaptonemal complex between homologous or non-homologous regions, and thus, this SC barrier might be probably related to the preservation of fertility in these systems.

Diversity and Karyotypic Evolution in the Genus Neacomys (Rodentia, Sigmodontinae).

Neacomys (Sigmodontinae) comprises 8 species mainly found in the Amazonian region. We describe 5 new karyotypes from Brazilian Amazonia: 2 cytotypes for N. paracou (2n = 56/FNa = 62-66), 1 for N. dubosti (2n = 64/FNa = 68), and 2 for Neacomys sp. (2n = 58/FNa = 64-70), with differences in the 18S rDNA. Telomeric probes did not show ITS. We provide a phylogeny using Cytb, and the analysis suggests that 2n = 56 with a high FNa is ancestral for the genus, as found in N. paracou, being retained by the ancestral forms of the other species, with an increase in 2n occurring independently in N. spinosus and N. dubosti. Alternatively, an increase in 2n may have occurred in the ancestral taxon of the other species, followed by independent 2n-reduction events in Neacomys sp. and in the ancestral species of N. tenuipes, N. guianae, N. musseri, and N. minutus. Finally, a drastic reduction event in the diploid number occurred in the ancestral species of N. musseri and N. minutus which exhibit the lowest 2n of the genus. The karyotypic variations found in both intra- and interspecific samples, associated with the molecular phylogeny, suggest a chromosomal evolution with amplification/deletion of constitutive heterochromatin and rearrangements including fusions, fissions, and pericentric inversions.

Karyotypic and morphological divergence between two cryptic species of Eigenmannia in the Amazon basin with a new occurrence of XX/XY sex chromosomes (Gymnotiformes: Sternopygidae)

Eigenmannia species are widely distributed in the Neotropics, with eight valid species currently recognized. Populations of Eigenmannia from three locations in the eastern Amazon were investigated using cytogenetic and morphological techniques, revealing two taxa designated here as Eigenmannia sp. "A" and Eigenmannia sp. "B". The species differ in three morphometric characters, two meristic characters, and one osteological character. Eigenmannia sp. "A" presents 2n = 34 (22 m/sm+12 st/a) and Eigenmannia sp. "B" presents 2n = 38 (14 m/sm+24st/a) and simple differentiated sex chromosomes of the type XX/XY. In both species the Constitutive Heterochromatin (CH) rich in A-T bases is distributed in the centromeric region of all chromosomes. Eigenmannia sp. "B" also presents CH blocks in the interstitial region of chromosome pairs 8, 9 and X which are positively stained with CMA3, indicating G-C rich regions. The NOR is located on the short arm of chromosome pair 17 of Eigenmannia sp. "A" and on the short arm of pair 14 of Eigenmannia sp. "B". FISH with rDNA probes hybridized to different-sized regions between homologs, suggesting heteromorphism. The differentiation of the X chromosome in Eigenmannia sp. "B" could be the result of amplification of repetitive DNA sequences.

Espécies de Eigenmannia estão amplamente distribuídas na região Neotropical, com oito espécies válidas atualmente reconhecidas. Populações de Eigenmannia de três localidades do leste da Amazônia foram investigadas usando técnicas citogenéticas e morfológicas, revelando dois táxons designados aqui como Eigenmannia sp. "A" e Eigenmannia sp. "B". As espécies diferem em três caracteres morfométricos, dois merísticos e um osteológico. Eigenmannia sp. "A" apresenta 2n = 34 (22 m/sm+12st/a) e Eigenmannia sp. "B" apresenta 2n = 38 (14 m/sm+24st/a) e cromossomos sexuais de diferenciação simples, do tipo XX/XY. Em ambas espécies a Heterocromatina Constitutiva (HC) rica em bases A-T está distribuída na região centromérica de todos os cromossomos. Eigenmannia sp. "B" também apresenta blocos de HC na região intersticial dos pares cromossômicos 8, 9 e X que coraram positivamente para CMA3, indicando regiões ricas em G-C. A NOR está localizada no braço curto do par 17 em Eigenmannia sp. "A" e no braço curto do par 14 em Eigenmannia sp. "B". FISH com sondas de rDNA hibridizaram em regiões de tamanhos diferentes entre os homólogos, sugerindo heteromorfismo. A diferenciação do cromossomo X em Eigenmannia sp. "B" pode ser o resultado de amplificação de sequências repetitivas de DNA.

FISH in micronucleus test demonstrates aneugenic action of rotenone in a common freshwater fish species, Nile tilapia (Oreochromis niloticus).

Aneuploidies are numerical genetic alterations that lead to changes in the normal number of chromosomes due to abnormal segregation during cell division. This type of alteration can occur spontaneously or as a result of exposure to mutagenic agents. The presence of these agents in the environment has increased concern about potential damage to human health. Rotenone, derived from plants of the genera Derris and Lonchocarpus, is a product that is used all over the world as a pesticide and piscicide. Before establishing its potential and efficiency for these purposes, it is essential to know more about the possible adverse effects that it may cause. The current work aimed to evaluate the mutagenic potential of rotenone using fish from the species Oreochromis niloticus, as well as to help in understanding its action mechanism. Our results showed the mutagenic potential of rotenone evidenced by increased formation of micronuclei and nuclear buds at low doses of exposure. The use of fluorescence in situ hybridisation technique made it possible to measure the aneugenic potential of the substance, probably due to its impairment of mitotic spindle formation.

Two new cytotypes reinforce that Micronycteris hirsuta Peters, 1869 does not represent a monotypic taxon.

BACKGROUND: The genus Micronycteris is a diverse group of phyllostomid bats currently comprising 11 species, with diploid number (2n) ranging from 26 to 40 chromosomes. The karyotypic relationships within Micronycteris and between Micronycteris and other phyllostomids remain poorly understood. The karyotype of Micronycteris hirsuta is of particular interest: three different diploid numbers were reported for this species in South and Central Americas with 2n = 26, 28 and 30 chromosomes. Although current evidence suggests some geographic differentiation among populations of M. hirsuta based on chromosomal, morphological, and nuclear and mitochondrial DNA markers, the recognition of new species or subspecies has been avoided due to the need for additional data, mainly chromosomal data. RESULTS: We describe two new cytotypes for Micronycteris hirsuta (MHI) (2n = 26 and 25, NF = 32), whose differences in diploid number are interpreted as the products of Robertsonian rearrangements. C-banding revealed a small amount of constitutive heterochromatin at the centromere and the NOR was located in the interstitial portion of the short arm of a second pair, confirmed by FISH. Telomeric probes hybridized to the centromeric regions and weakly to telomeric regions of most chromosomes. The G-banding analysis and chromosome painting with whole chromosome probes from Carollia brevicauda (CBR) and Phyllostomus hastatus (PHA) enabled the establishment of genome-wide homologies between MHI, CBR and PHA. CONCLUSIONS: The karyotypes of Brazilian specimens of Micronycteris hirsuta described here are new to Micronycteris and reinforce that M. hirsuta does not represent a monotypic taxon. Our results corroborate the hypothesis of karyotypic megaevolution within Micronycteris, and strong evidence for this is that the entire chromosome complement of M. hirsuta was shown to be derivative with respect to species compared in this study.

Proechimys (Rodentia, Echimyidae): characterization and taxonomic considerations of a form with a very low diploid number and a multiple sex chromosome system.

BACKGROUND: Proechimys is the most diverse genus in family Echimyidae, comprising 25 species (two of which are polytypic) and 39 taxa. Despite the numerous forms of this rodent and their abundance in nature, there are many taxonomic problems due to phenotypic similarities within the genus and high intraspecific variation. Extensive karyotypic variation has been noted, however, with diploid numbers (2n) ranging from 14 to 62 chromosomes. Some heteromorphism can be found, and 57 different karyotypes have been described to date. RESULTS: In the present work, we describe a cytotype with a very low 2n. Specimens of Proechimys cf. longicaudatus were collected from two different places in northern Mato Grosso state, Brazil (12°54″S, 52°22″W and 9°51'17″S, 58°14'53″W). The females and males had 16 and 17 chromosomes, respectively; all chromosomes were acrocentric, with the exception of the X chromosome, which was bi-armed. The sex chromosome system was found to be XY1Y2, originating from a Robertsonian rearrangement involving the X and a large acrocentric autosome. Females had two Neo-X chromosomes, and males had one Neo-X and two Y chromosomes. NOR staining was found in the interstitial region of one autosomal pair. CONCLUSIONS: Comparison of this karyotype with those described in the literature revealed that Proechimys with similar karyotypes had previously been collected from nearby localities. We therefore suggest that this Proechimys belongs to a different taxon, and is either a new species or one that requires reassessment.

Are NORs always located on homeologous chromosomes? A FISH investigation with rDNA and whole chromosome probes in Gymnotus fishes (Gymnotiformes).

Gymnotus (Gymnotiformes, Gymnotidae) is the most diverse known Neotropical electric knife fish genus. Cytogenetic studies in Gymnotus demonstrate a huge karyotypic diversity for this genus, with diploid numbers ranging from 34 to 54. The NOR are also variable in this genus, with both single and multiple NORs described. A common interpretation is that the single NOR pair is a primitive trait while multiple NORs are derivative. However this hypothesis has never been fully tested. In this report we checked if the NOR-bearing chromosome and the rDNA site are homeologous in different species of the genus Gymnotus: G. carapo (2n = 40, 42, 54), G. mamiraua (2n = 54), G. arapaima (2n = 44), G. sylvius (2n = 40), G. inaequilabiatus (2n = 54) and G. capanema (2n = 34), from the monophyletic group G. carapo (Gymnotidae-Gymnotiformes), as well as G. jonasi (2n = 52), belonging to the G1 group. They were analyzed with Fluorescence in situ hybridization (FISH) using 18S rDNA and whole chromosome probes of the NOR-bearing chromosome 20 (GCA20) of G. carapo (cytotype 2n = 42), obtained by Fluorescence Activated Cell Sorting. All species of the monophyletic G. carapo group show the NOR in the same single pair, confirmed by hybridization with CGA20 whole chromosome probe. In G. jonasi the NORs are multiple, and located on pairs 9, 10 and 11. In G. jonasi the GCA20 chromosome probe paints the distal half of the long arm of pair 7, which is not a NOR-bearing chromosome. Thus these rDNA sequences are not always in the homeologous chromosomes in different species thus giving no support to the hypothesis that single NOR pairs are primitive traits while multiple NORs are derived. The separation of groups of species in the genus Gymnotus proposed by phylogenies with morphologic and molecular data is supported by our cytogenetic data.

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.

Chromosome phylogeny of the subfamily Pitheciinae (Platyrrhini, Primates) by classic cytogenetics and chromosome painting.

BACKGROUND: The New World monkey (Platyrrhini) subfamily Pitheciinae is represented by the genera Pithecia, Chiropotes and Cacajao. In this work we studied the karyotypes of Pithecia irrorata (2n = 48) and Cacajao calvus rubicundus (2n = 45 in males and 2n = 46 in females) by G- and C-banding, NOR staining and chromosome painting using human and Saguinus oedipus whole chromosome probes. The karyotypes of both species were compared with each other and with Chiropotes utahicki (2n = 54) from the literature. RESULTS: Our results show that members of the Pitheciinae have conserved several chromosome forms found in the inferred ancestral Platyrrhini karyotype (associations of human homologous segments 3a/21, 5/7a, 2b/16b, 8a/18, 14/15a and 10a/16a). Further, the monophyly of this subfamily is supported by three chromosomal synapomorphies (2a/10b, an acrocentric 15/14 and an acrocentric human 19 homolog). In addition, each species presents several autapomorphies. From this data set we established a chromosomal phylogeny of Pitheciinae, resulting in a single most parsimonious tree. CONCLUSIONS: In our chromosomal phylogeny, the genus Pithecia occurred in a more basal position close to the inferred ancestor of Platyrrhini, while C. c. rubicundus and C. utahicki are closely related and are linked by exclusive synapomorphies.