Embryotoxic effects of Rovral® for early chicken (Gallus gallus) development.

Rovral® is a fungicide used to control pests that affect various crops and little is known regarding its effects on embryonic development of amniotes. Thus, this study aimed to determine the influence of Rovral® during chicken organogenesis using acute in ovo contamination. Fertilized eggs were inoculated with different concentrations of Rovral® (100, 300, 500 or 750 µl/ml), injected into the egg's air chamber. After 7 days, embryos were examined for possible malformations, staging, weight and mortality. Subsequently, head, trunk, limbs and eyes were measured for morphometry and asymmetry. For blood analysis, eggs were treated with 300 µl/ml Rovral® and glucose, presence of micronuclei and erythrocyte nuclei abnormalities determined. Treatments with Rovral® affected the mortality rate in a concentration-dependent manner. LC50 value was found to be 596 µl/ml which represents 397-fold higher than the recommended concentration for use. Rovral® produced several malformations including hemorrhagic, ocular and cephalic abnormalities. No significant changes were observed in body weight, staging, body measurements, symmetry and glucose levels of live embryos, which indicates this fungicide presents low toxicity under the analyzed conditions. Changes in erythrocyte nuclei were noted; however significant difference was observed only for presence of binucleated erythrocytes. It is important to point out that possibly more significant changes may have occurred at lower concentrations through chronic contamination. Therefore, caution is needed in the use of this fungicide, since it presents teratogenic and mutagenic potential.

Cryptic Diversity in the Terminal Portion of the Chromosomes of the Dogtooth Characins, Family Cynodontidae (Ostariophysi: Characiformes).

The chromosomes of the dogtooth characins, fish species of the family Cynodontidae, have only a relatively small amount of heterochromatin, including the terminal portion. Curiously, in the cynodontid Cynodon gibbus, the terminal portion is rich in repetitive DNAs, including transposable retroelements and microsatellite sequences. Given this, this study investigated the composition of the terminal portion of the chromosomes of two cynodontid species (Rhaphiodon vulpinus and Hydrolycus armatus), to compile a database for the evaluation of all three cynodontid genera, and in particular, verify the possible tendency for the accumulation of repetitive DNAs in the terminal portion of the chromosomes of C. gibbus, H. armatus, and R. vulpinus. The Rex1, Rex3, and Rex6 transposable retroelements and the (CA)15, (GA)15, (GATA)8, (GACA)8, (CAT)10, and (CAC)10 microsatellite motifs are found primarily in the terminal portion of the chromosomes of the species analyzed in this study, except R. vulpinus, which has no evidence of the presence of Rex1 or Rex3 through the fluorescent in situ hybridization technique. The mapping of the repetitive sequences, principally the microsatellite motifs, indicates a marked tendency for the accumulation of these sequences in the terminal portions of the chromosomes, which may have played a fundamental role in the differentiation of the three species.

Looking for genetic effects of polluted anthropized environments on Caiman crocodilus crocodilus (Reptilia, Crocodylia): A comparative genotoxic and chromosomal analysis.

The Amazon aquatic ecosystems have been modified by the human population growth, going through changes in their water bodies and aquatic biota. The spectacled alligator (Caiman crocodilus crocodilus) has a wide distribution and adaptability to several environments, even those polluted ones. This study aimed to investigate if a Caiman species living in urban streams of Manaus city (Amazonas State, Brazil) is affected by environmental pollution. For that, it was used classical and molecular cytogenetic procedures, in addition to micronucleus and comet assays. Although the karyotype macrostructure remains unaltered (2 n = 42 chromosomes; 24 t + 18 m/sm; NF = 60), the genotoxic analysis and the cytogenetic mapping of repetitive DNA sequences demonstrated that polluted environments alter the genome of the specimens, affecting both the chromosomal organization and the genetic material.

Cytogenetic analysis of three Ctenidae species (Araneae) from the Amazon.

Cytogenetic characterization was performed on three wandering spiders: Ctenus amphora Mello-Leitão, 1930, C. crulsi Mello-Leitão, 1930 and C. villasboasi Mello-Leitão, 1949. The three species had similar karyotypes, with 2n = 28 (26 + X1X20) in males, with sex chromosomes exhibiting positive heteropicnosis in meiotic cells. 18S rDNA mapping revealed gene sites at the terminal region of one chromosomal pair for all species, with one C. crulsi individual, showing markings in two pairs. C. villasboasi showed markers only in the pachytene phase. The distribution pattern of constitutive heterochromatin was found to be characteristic for the genus, with markings in the centromeric region of all chromosomes, suggesting an acrocentric morphology for all chromosomes of the three analysed species. The results support the fusion of sex chromosomes as an evolutionary tendency for this spider group.

Transposable DNA Elements in Amazonian Fish: From Genome Enlargement to Genetic Adaptation to Stressful Environments.

Transposable elements have driven genome evolution and plasticity in many ways across a range of organisms. Different types of biotic and abiotic stresses can stimulate the expression or transposition of these mobile elements. Here, we cytogenetically analyzed natural fish populations of the same species living under different environmental conditions to test the influence and organization of transposable elements in their genome. Differential behavior was observed for the markers Rex 1, Rex 3, and Rex 6 in the chromosomes of individuals of the same species but coming from different environments (polluted and unpolluted). An increase in the number of Rex transposable elements in the chromosomes and their influence on the genome of populations living in a polluted environment indicates that they must be under constant adaptive evolution.

First Record on Sex Chromosomes in a Species of the Family Cynodontidae: Cynodon gibbus (Agassiz, 1829).

The fish family Cynodontidae belongs to the superfamily Curimatoidea, together with the Hemiodontidae, Serrasalmidae, Parodontidae, Prochilodontidae, Chilodontidae, Curimatidae, and Anostomidae. The majority of the species of this superfamily that have been analyzed to date have a diploid chromosome number of 2n = 54. Differentiated sex chromosomes (with female heterogamety) have been observed only in the Prochilodontidae, Parodontidae, and Anostomidae. The present study provides the first description of differentiated sex chromosomes in the cynodontid species Cynodon gibbus, which has a ZZ/ZW system, and shows that repetitive DNA has played a fundamental role in the differentiation of these sex chromosomes.

Effects of environmental pollution on the rDNAomics of Amazonian fish.

Pollution is a growing environmental problem throughout the world, and the impact of human activities on biodiversity and the genetic variability of natural populations is increasingly preoccupying, given that adaptive processes depend on this variability, in particular that found in the repetitive DNA. In the present study, the mitochondrial DNA (COI) and the distribution of repetitive DNA sequences (18S and 5S rDNA) in the fish genome were analysed in fish populations inhabiting both polluted and unpolluted waters in the northern Amazon basin. The results indicate highly complex ribosomal sequences in the fish genome from the polluted environment because these sequences are involved primarily in the maintenance of genome integrity, mediated by a systematic increase in the number of copies of the ribosomal DNA in response to changes in environmental conditions.

Comparative Cytogenetics of Omophoita abbreviata and O. aequinoctialis (Coleoptera, Chrysomelidae, Alticini) from the Adolpho Ducke Forest Reserve in Brazilian Amazonia: Intrapopulation Variation in Karyotypes.

The chromosomes of 2 flea beetle species from central Amazonia, Omophoita abbreviata and O. aequinoctialis (Alticini), were investigated through analysis of meiotic and mitotic cells. These species belong to the subtribe Oedionychina, a taxon that has unique cytogenetic features, such as giant sex chromosomes which are aligned at a distance during meiosis I (asynaptic). O. abbreviata and O. aequinoctialis have a meiotic formula of 10II + X + y, which is predominant in this subtribe. While the species of the genus Omophoita possess a relatively stable karyotype, a typical feature for Oedionychina, the present study identified inter- and intrapopulational variation in chromosome morphology, constitutive heterochromatin, and the presence and number of B chromosomes in O. aequinoctialis. In addition, FISH mapping of telomeric sequences revealed signals in the collochores, raising several questions on the chromosomal evolution in this group.

Heterochromatin variation and LINE-1 distribution in Artibeus (Chiroptera, Phyllostomidae) from Central Amazon, Brazil.

Species in the subgenus Artibeus Leach, 1821 are widely distributed in Brazil. Conserved karyotypes characterize the group with identical diploid number and chromosome morphology. Recent studies suggested that the heterochromatin distribution and accumulation patterns can vary among species. In order to assess whether variation can also occur within species, we have analyzed the chromosomal distribution of constitutive heterochromatin in A. planirostris (Spix, 1823) and A. lituratus (Olfers, 1818) from Central Amazon (North Brazil) and contrasted our findings with those reported for other localities in Brazil. In addition, Ag-NOR staining and FISH with 18S rDNA, telomeric, and LINE-1 probes were performed to assess the potential role that these different repetitive markers had in shaping the current architecture of heterochromatic regions. Both species presented interindividual variation of constitutive heterochromatin. In addition, in A. planirostris the centromeres of most chromosomes are enriched with LINE-1, colocated with pericentromeric heterochromatin blocks. Overall, our data indicate that amplification and differential distribution of the investigated repetitive DNAs might have played a significant role in shaping the chromosome architecture of the subgenus Artibeus.

Genomic Organization of Repetitive DNAs and Differentiation of an XX/XY Sex Chromosome System in the Amazonian Puffer Fish, Colomesus asellus (Tetraodontiformes).

The genus Colomesus is the sole representative of the family Tetraodontidae in the Amazon region. Here, Colomesus asellus was analyzed using conventional and molecular cytogenetic protocols. Its diploid chromosome number is 2n = 46 with 12 meta-, 10 submeta-, 16 subtelo-, and 8 acrocentric chromosomes and a fundamental number of FN = 84. An XX/XY sex chromosome system was identified. Mapping of 18S rDNA correlated with the nucleolus organizer regions (Ag-NORs) in the short arms of the 2 X chromosomes in females and in the Y chromosome in males. C-banding revealed heterochromatin in the centromeric regions of all chromosomes, except for pair 3. Prominent sex chromosome-specific heterochromatin amplification was observed, covering the short arms of the Y chromosome almost entirely. FISH with telomeric and tropomyosin (tpm1) sequences, respectively, revealed terminal signals in all chromosomes. The analysis of extended DNA fibers confirmed the colocalization and the interspersed pattern of the telomeric and tpm1 sequences. Thus, this study highlights the remarkable evolutionary dynamism presented by the Amazonian puffer fish regarding the differentiation of a heteromorphic XY sex chromosome system and a particular sex-specific amplification of rDNA sites. This is the first record of such an association in the Tetraodontidae family.

Differential chromosomal organization between Saguinus midas and Saguinus bicolor with accumulation of differences the repetitive sequence DNA.

Saguinus is the largest and most complex genus of the subfamily Callitrichinae, with 23 species distributed from the south of Central America to the north of South America with Saguinus midas having the largest geographical distribution while Saguinus bicolor has a very restricted one, affected by the population expansion in the state of Amazonas. Considering the phylogenetic proximity of the two species along with evidence on the existence of hybrids between them, as well as cytogenetic studies on Saguinus describing a conserved karyotypic macrostructure, we carried out a physical mapping of DNA repeated sequences in the mitotic chromosome of both species, since these sequences are less susceptible to evolutionary pressure and possibly perform an important function in speciation. Both species presented 2n = 46 chromosomes; in S. midas, chromosome Y is the smallest. Multiple ribosomal sites occur in both species, but chromosome pairs three and four may be regarded as markers that differ the species when subjected to G banding and distribution of retroelement LINE 1, suggesting that it may be cytogenetic marker in which it can contribute to identification of first generation hybrids in contact zone. Saguinus bicolor also presented differences in the LINE 1 distribution pattern for sexual chromosome X in individuals from different urban fragments, probably due to geographical isolation. In this context, cytogenetic analyses reveal a differential genomic organization pattern between species S. midas and S. bicolor, in addition to indicating that individuals from different urban fragments have been accumulating differences because of the isolation between them.

Karyotypic Comparison of Hoplias malabaricus (Bloch, 1794) (Characiformes, Erythrinidae) in Central Amazon.

Hoplias malabaricus comprises seven karyomorphs (A-G) and evolutionary units have been described in some of them. In this study, the karyotypic composition and genomic organization of individual H. malabaricus from Central Amazon are described and to verify whether they can be classified according to known karyomorphs. Individuals from the Ducke Reserve have 2n = 42 chromosomes, similar to karyomorph A. Individuals from Catalão Lake and Marchantaria Island exhibit 2n = 40 chromosomes, similar to karyomorph C. Regarding the constitutive heterochromatin, individuals from all locations present centromeric/pericentromeric blocks, in addition to some bitelomeric and interstitial markings. The number of chromosomes with nucleolar organizer region, 5S rDNA and 18S rDNA sites varied among the different locations. The Rex 3 element has a compartmentalized distribution at the terminal and centromeric regions of most chromosomes, with subtle differences among populations. Fluorescence in situ hybridization performed with a telomeric probe allowed the detection of these regions only at the terminal ends of the chromosomes. Thus, only the chromosomal macrostructure (karyomorphs A-G) is not sufficient to establish evolutionary units within the H. malabaricus group, considering differences in the genome organization that are found among their populations. Such differences in the genomic organization could be mainly caused by the sedentary habits of this species.

Intense genomic reorganization in the genus Oecomys (Rodentia, Sigmodontinae): comparison between DNA barcoding and mapping of repetitive elements in three species of the Brazilian Amazon.

Oecomys Thomas, 1906 is one of the most diverse and widely distributed genera within the tribe Oryzomyini. At least sixteen species in this genus have been described to date, but it is believed this genus contains undescribed species. Morphological, molecular and cytogenetic study has revealed an uncertain taxonomic status for several Oecomys species, suggesting the presence of a complex of species. The present work had the goal of contributing to the genetic characterization of the genus Oecomys in the Brazilian Amazon. Thirty specimens were collected from four locations in the Brazilian Amazon and three nominal species recognized: Oecomys auyantepui (Tate, 1939), Oecomys bicolor (Tomes, 1860) and Oecomys rutilus (Anthony, 1921). COI sequence analysis grouped Oecomys auyantepui, Oecomys bicolor and Oecomys rutilus specimens into one, three and two clades, respectively, which is consistent with their geographic distribution. Cytogenetic data for Oecomys auyantepui revealed the sympatric occurrence of two different diploid numbers, 2n=64/NFa=110 and 2n=66/NFa=114, suggesting polymorphism while Oecomys bicolor exhibited 2n=80/NFa=142 and Oecomys rutilus 2n=54/NFa=90. The distribution of constitutive heterochromatin followed a species-specific pattern. Interspecific variation was evident in the chromosomal location and number of 18S rDNA loci. However, not all loci showed signs of activity. All three species displayed a similar pattern for 5S rDNA, with only one pair carrying this locus. Interstitial telomeric sites were found only in Oecomys auyantepui. The data presented in this work reinforce intra- and interspecific variations observed in the diploid number of Oecomys species and indicate that chromosomal rearrangements have led to the appearance of different diploid numbers and karyotypic formulas.

Differential repetitive DNA composition in the centromeric region of chromosomes of Amazonian lizard species in the family Teiidae.

Differences in heterochromatin distribution patterns and its composition were observed in Amazonian teiid species. Studies have shown repetitive DNA harbors heterochromatic blocks which are located in centromeric and telomeric regions in Ameiva ameiva (Linnaeus, 1758), Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868), and Tupinambis teguixin (Linnaeus, 1758). In Cnemidophorus sp.1, repetitive DNA has multiple signals along all chromosomes. The aim of this study was to characterize moderately and highly repetitive DNA sequences by C ot1-DNA from Ameiva ameiva and Cnemidophorus sp.1 genomes through cloning and DNA sequencing, as well as mapping them chromosomally to better understand its organization and genome dynamics. The results of sequencing of DNA libraries obtained by C ot1-DNA showed that different microsatellites, transposons, retrotransposons, and some gene families also comprise the fraction of repetitive DNA in the teiid species. FISH using C ot1-DNA probes isolated from both Ameiva ameiva and Cnemidophorus sp.1 showed these sequences mainly located in heterochromatic centromeric, and telomeric regions in Ameiva ameiva, Kentropyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin chromosomes, indicating they play structural and functional roles in the genome of these species. In Cnemidophorus sp.1, C ot1-DNA probe isolated from Ameiva ameiva had multiple interstitial signals on chromosomes, whereas mapping of C ot1-DNA isolated from the Ameiva ameiva and Cnemidophorus sp.1 highlighted centromeric regions of some chromosomes. Thus, the data obtained showed that many repetitive DNA classes are part of the genome of Ameiva ameiva, Cnemidophorus sp.1, Kentroyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin, and these sequences are shared among the analyzed teiid species, but they were not always allocated at the same chromosome position.

Is the Karyotype of Neotropical Boid Snakes Really Conserved? Cytotaxonomy, Chromosomal Rearrangements and Karyotype Organization in the Boidae Family.

Boids are primitive snakes from a basal lineage that is widely distributed in Neotropical region. Many of these species are both morphologically and biogeographically divergent, and the relationship among some species remains uncertain even with evolutionary and phylogenetic studies being proposed for the group. For a better understanding of the evolutionary relationship between these snakes, we cytogenetically analysed 7 species and 3 subspecies of Neotropical snakes from the Boidae family using different chromosomal markers. The karyotypes of Boa constrictor occidentalis, Corallus hortulanus, Eunectes notaeus, Epicrates cenchria and Epicrates assisi are presented here for the first time with the redescriptions of the karyotypes of Boa constrictor constrictor, B. c. amarali, Eunectes murinus and Epicrates crassus. The three subspecies of Boa, two species of Eunectes and three species of Epicrates exhibit 2n = 36 chromosomes. In contrast, C. hortulanus presented a totally different karyotype composition for the Boidae family, showing 2n = 40 chromosomes with a greater number of macrochromosomes. Furthermore, chromosomal mapping of telomeric sequences revealed the presence of interstitial telomeric sites (ITSs) on many chromosomes in addition to the terminal markings on all chromosomes of all taxa analysed, with the exception of E. notaeus. Thus, we demonstrate that the karyotypes of these snakes are not as highly conserved as previously thought. Moreover, we provide an overview of the current cytotaxonomy of the group.

Comparative Cytotaxonomy of Two Species of Fish from the Genus Satanoperca Reveals the Presence of a B Chromosome.

The taxonomy of Satanoperca spp. is still unresolved, especially because coloring, one of the main diagnostic characters, is variable among species of this genus. Thus, the aim of this study was to elucidate the relationship between the genome and the organization of the chromosome in two Satanoperca species. Our main goal was to develop a method to better differentiate taxa and understand the evolution of Satanoperca jurupari and Satanoperca lilith karyotypes, which we analyzed with classical and molecular cytogenetics. Both species have the same diploid number (2n) of 48 and location of 5S rDNA sites on pair 5. Nonetheless, the distribution of heterochromatin and 18S rDNA sites followed a species-specific pattern. The interstitial telomeric sites were not highlighted in either species. Regardless, a single B chromosome was identified in some metaphases of S. lilith. These data show that Satanoperca species harbor chromosomal features that can be used to identify the two species of Satanoperca studied here, allowing for the use of cytogenetic markers to make taxonomic inferences within the genus.

Genomic Organization Under Different Environmental Conditions: Hoplosternum Littorale as a Model.

The Amazon has abundant rivers, streams, and floodplains in both polluted and nonpolluted environments, which show great adaptability. Thus, the goal of this study was to map repetitive DNA sequences in both mitotic chromosomes and erythrocyte micronuclei of tamoatás from polluted and nonpolluted environments and to assess the possible genotoxic effects of these environments. Individuals were collected in Manaus, Amazonas (AM), and submitted to classical and molecular cytogenetic techniques, as well as to a blood micronucleus test. Diploid number equal to 60 chromosomes are present in all individuals, with 18S ribosomal DNA sites present in one chromosome pair and no interstitial telomeric sites on chromosomes. The micronucleus test showed no significant differences in pairwise comparisons between environments or collection sites, but the Rex3 retroelement was dispersed on the chromosomes of individuals from unpolluted environments and compartmentalized in individuals from polluted environments. Divergent numbers of 5S rDNA sites are present in individuals from unpolluted and polluted environments. The mapping of repetitive sequences revealed that micronuclei have different compositions both intra- and interindividually that suggests different regions are lost in the formation of micronuclei, and no single fragile region undergoes breaks, although repetitive DNA elements are involved in this process.

Karyoevolution in Potamorhina (Cope, 1878) (Ostariophysi, Curimatidae): Using Repetitive DNA for the Elucidation of Genome Organization.

Some families of Characiformes present the tendency toward stability of the karyotypic macrostructure as Curimatidae, which contains species with a 2n = 54 karyotype and metacentric and submetacentric chromosomes, however, some Potamorhina species contradict to this tendency. Some species of the central Amazon exhibit different diploid number and show intraspecific variation in the location of heterochromatin. By performing cytogenetic characterization by localization of heterochromatin and the nucleolus organizer region, as well as physical chromosome mapping using probes targeting 5S and 18S ribosomal DNA (rDNA), retroelement of Xiphophorus 1 (Rex1), Rex3, telomeres, and tropomyosin 1 (TPM1), we attempted to understand the evolutionary mechanisms involved in the differentiation of the Potamorhina species. The analyses showed that the heterochromatic regions of the examined species are distinct and transposable elements are involved in this evolutionary process, considering that the dynamic regions of the genome appear to include the terminal regions and particularly the heterochromatin-rich centromeric regions, which are involved in fission and fusion processes and promote the differentiation of chromosome pairs that bear ribosomal sites; these pairs were similar in the central Amazon species. Thus, we propose a phylogeny for this genus.

Intra-generic and interspecific karyotype patterns of Leptodactylus and Adenomera (Anura, Leptodactylidae) with inclusion of five species from Central Amazonia.

The genera Leptodactylus and Adenomera comprise 92 species distributed throughout the Neotropical region. These species have a modal diploid chromosome number 2n = 22. However, chromosome rearrangements are evident in the differentiation of five intra-generic groups in the genus Leptodactylus (L. fuscus, L. latrans, L. marmoratus (formally composed by the species of the genus Adenomera), L. melanonotus, L. pentadactylus), yet it is not clear if there is a karyotype pattern for each group. Aiming to understand the intra-generic and interspecific karyotype patterns of Leptodactylus and Adenomera, cytogenetic analyses were performed in A. andreae, L. macrosternum, L. pentadactylus, L. petersii, and L. riveroi using conventional staining, C-banding, nucleolus organizer region (NOR) and hybridization in situ fluorescent (FISH). The karyotype of Leptodactylus riveroi was described for the first time. Adenomera andreae had 2n = 26, while the remaining species 2n = 22. The NOR was found on pair No. 8 of A. andreae, L. macrosternum, L. pentadactylus, and L. riveroi, whereas L. petersii had it on pairs Nos. 6 and 10. These locations were confirmed by the FISH with 18S rDNA probe, except for pair No. 10 of L. petersii. The C-banding pattern was evident at the centromeres of chromosomes of all species and some interspecific variations were also observed. 2n = 22 was observed in the species of the L. latrans group, as well as in the intra-generic groups L. fuscus and L. pentadactylus; in the L. melanonotus group there were three diploid chromosome numbers 2n = 20, 22 and 24; and a larger variation in 2n was also evident in the L. marmoratus group.