Renata da Rosa

Constitutive heterochromatin, 5S and 18S rDNA genes in Apareiodon sp. (Characiformes, parodontidae) with a ZZ/ZW sex chromosome system

Karyotype, sex chromosome system and cytogenetics characteristics of an unidentified species of the genus Apareiodon originating from Piquiri River (Paraná State, Brazil) were investigated using differential staining techniques (C-banding and Ag-staining) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The diploid chromosome number was 2n = 54 with 25 pairs of meta- (m) to submetacentric (sm) and 2 pairs of subtelocentric (st) chromosomes. The major ribosomal rDNA sites as revealed by Ag-staining and FISH with 18S rDNA probe were found in distal region of longer arm of st chromosome pair 26, while minor 5S sites were observed in the interstitial sites on chromosome pairs 2 (smaller cluster) and 7 (larger one). The C-positive heterochromatin had pericentromeric and telomeric distribution. The heteromorphic sex chromosome system consisted of male ZZ (pair 21) and female middle-sized m/st Z/W chromosomes. The pericentric inversion of heterochromatinized short arm of ancestral Z followed by multiplication of heterochromatin segments is hypothesized for origin of W chromosome. The observed karyotype and chromosomal markers corresponded to those found in other species of the genus.

Cytotaxonomy in distinct populations of Hoplias aff. malabaricus (Characiformes, Erythrinidae) from lower Paranapanema River basin

Cytogenetic and morphometric analyses were carried out in Hoplias aff. malabaricus specimens from six distinct populations from the lower Paranapanema River basin, located between the states of Paraná and São Paulo, Brazil. Measurements were also taken from a specimen collected in Surinam. In a population from a fish farm station at Universidade do Norte do Parana (EPUNOPAR), two sympatric cytotypes (2n = 40 and 2n = 42 chromosomes) are found. A population from a fish farm station at Universidade Estadual de Londrina (EPUEL) shows 2n = 42 meta-submetacentric chromosomes for males and females with a simple sex chromosome system of XX/XY type. Populations from the Vermelho and Rancho Alegre Rivers, Três Bocas Stream and Paranapanema River have 2n = 39 chromosomes in males and 2n = 40 chromosomes in females, showing a multiple sex chromosome system of X(1)X(1)X(2)X(2)/X(1)X(2)Y type. Twenty morphological variables were studied. These measurements were used for an analysis of the canonical variables and standard analysis of proportional measurements. The most variable measurements among the specimens are the maxilla length (MXL) and the pre-dorsal distance (PDD). Analysis of canonical variables indicates three distinct groups in the first canonical axis formed by: (1) Três Bocas Stream, (2) Rancho Alegre + Vermelho River + EPUNOPAR and (3) EPUEL + Paranapanema River. This axis retained 79.4% of information from the original matrix. Analysis of morphometrics reveals differences among populations from the Paranapanema River basin and between these and the specimen from Surinam. The morphometric and cytogenetic differences among the studied populations suggest a species complex.

Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method

Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C0t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C0t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish’s genomes.

Conserved Cytogenetic Features in the Amazonian Arapaima, Arapaima gigas (Schinz 1822) from Jamari River, Rondônia-Brazil

Specimens of Arapaima gigas from Jamari River (RO) were cytogenetically analyzed. A diploid number of 2n=56 chromosomes was found (28m-sm + 28st-a). Secondary constrictions were observed on the short arms of chromosome 3. Nucleolar Organizer Regions (NORs) were detected at the subterminal region on short arms of the third chromosomal pair by both silver nitrate staining and FISH with 45S rDNA probe, being equivalent to secondary constrictions. The ribosomal sites were also characterized by size heteromorphism and presence of CMA3 + /DAPI - blocks. The constitutive heterochromatin was located at pericentromeric region of some chromosomes. After sequential C- banding and base-specific fluorochromes staining, most of the heterochromatins proved to be neutral, i.e., with similar amounts of AT and GC bases. Nonetheless, some heterochromatic regions were marked by GC-specific fluorochromes in one chromosomal pair and by AT-specific fluorochrome staining on two pairs. The present data are in agreement with previous reports in populations from Araguaya River, indicating that conserved cytogenetic features are present in this important fish species.

A New Approach to Chromosomal Evolution in the Giant Water Bug (Heteroptera: Belostomatidae)

The genus Belostoma, known colloquially as “giant water bugs,” presents striking cytogenetic diversity and extensive chromosome variability. Notwithstanding, its karyotype evolution is not well understood. We analyzed 8 species of Belostoma (77 samples). The meiotic analysis revealed 2n = 14 + XY for Belostoma horvathi and Belostoma candidulum; 2n = 22 + XY for Belostoma cummings; 2n = 26 + X1X2Y for Belostoma dentatum, Belostoma elongatum, and Belostoma discretum; and 2n = 26 + X1X2X3Y for Belostoma testacopallidum and Belostoma dilatatum. All species showed holokinetic chromosomes. Based on heterochromatin distribution patterns and 18S rDNA, the species of the genus Belostoma were separated into four groups. The analysis of C0t-1 DNA showed that the repetitive DNA, partly composed of microsatellite DNA, was absent on the Y chromosome. Fluorescent in situ hybridization (FISH) using a microdissected X chromosome in species with simple sex system presents uniform hybridization in the nuclear region corresponding to the X chromosome. Species with multiple systems revealed discrete markings. The present data in conjunction with the existing literature led us to propose a new evolutionary hypothesis for the group, with an ancestral karyotype with a low diploid number, simple sex determination system, and nucleolus organizer regions (NORs) on the sex chromosomes. That karyotype would have originated other karyotypes through agmatoploidy, simploidy, heterochromatinization, and movement of the 18S rDNA.

Spreading of heterochromatin and karyotype differentiation in two Tropidacris Scudder, 1869 species (Orthoptera, Romaleidae)

Abstract Tropidacris Scudder, 1869 is a genus widely distributed throughout the Neotropical region where speciation was probably promoted by forest reduction during the glacial and interglacial periods. There are no cytogenetic studies of Tropidacris, and information allowing inference or confirmation of the evolutionary events involved in speciation within the group is insufficient. In this paper, we used cytogenetic markers in two species, Tropidacris collaris (Stoll, 1813) and Tropidacris cristata grandis (Thunberg, 1824), collected in different Brazilian biomes. Both species exhibited 2n=24,XX for females and 2n=23,X0 for males. All chromosomes were acrocentric. There were some differences in the karyotype macrostructure, e.g. in the chromosome size. A wide interspecific variation in the chromosome banding (C-banding and CMA3/DAPI staining) indicated strong differences in the distribution of repetitive DNA sequences. Specifically, Tropidacris cristata grandis had a higher number of bands in relation to Tropidacris collaris. FISH with 18S rDNA revealed two markings coinciding with the NORs in both species. However, two analyzed samples of Tropidacris collaris revealed a heterozygous condition for the rDNA site of S10 pair. In Tropidacris collaris, the histone H3 genes were distributed on three chromosome pairs, whereas in Tropidacris cristata grandis, these genes were observed on 14 autosomes and on the X chromosome, always in terminal regions. Our results demonstrate that, although the chromosome number and morphology are conserved in the genus, Tropidacris cristata grandis substantially differs from Tropidacris collaris in terms of the distribution of repetitive sequences. The devastation and fragmentation of the Brazilian rainforest may have led to isolation between these species, and the spreading of these repetitive sequences could contribute to speciation within the genus.

First report of B chromosomes in three neotropical thorny catfishes (Siluriformes, Doradidae)

Abstract The family Doradidae (Siluriformes) is an important group of fishes endemic to freshwater ecosystems in South America. Some cytogenetic studies have been conducted focused on the group; however, there are no reports on the occurrence of B chromosomes for the family. In this paper the chromosomal characteristics of Platydoras armatulus (Valenciennes, 1840), Pterodoras granulosus (Valenciennes, 1821) and Ossancora punctata (Kner, 1855) were investigated through classical cytogenetics approaches. The conventional staining reveals 2n=58 in Platydoras armatulus and Pterodoras granulosus, however with distinct karyotypic formulae, possibly originated by pericentric inversions. In Ossancora punctata a derivate karyotype was described with 2n=66 and predominance of acrocentric chromosomes. The C banding pattern was resolutive in discriminating the three species, being considered an important cytotaxonomic marker. All species showed B chromosomes totally heterochromatic with non-Mendelian segregation during meiosis and low frequencies in mitotic cells. The probably origin of these additional elements was through fragmentations of chromosomes of the standard complement, which occurred recently and independently in these three species. The diploid number observed in Ossancora punctata is an evidence of centric fusions and up to the moment it is the highest diploid number reported for Doradidae.

Chromosomal similarity between two species of Apteronotus albifrons complex (Apteronotidae–Gymnotiformes) implications in cytotaxonomy and karyotypic evolution

Abstract Apteronotidae is one of the oldest families from the order Gymnotiformes, and is composed of 94 species that can be found from Panama to northern Argentina. Some groups such as Apteronotus present taxonomic problems, mainly related to interspecific morphological similarity. In the present study, two species belonging to Apteronotus albifrons complex from different hydrographic basins were cytogenetically analyzed in order to define chromosomal structure and propose evolutionary trends for the group. Apteronotus albifrons presented 2n = 24 (14 m + 2sm + 8a) FN = 40 and Apteronotus caudimaculosus 2n = 24 (12 m + 2sm + 10a) FN = 38; this difference in karyotypic formulae can be explained by pericentric inversion involving metacentric and acrocentric chromosomes. The karyotypic similarity between these two species indicates a chromosomal diversification, characterized by changes in karyotype formulae, which were considered an excellent cytotaxonomic marker to discriminate A. caudimaculosus of other populations of Apteronotus albifrons complex.

Evolutionary trends in the family Curimatidae (Characiformes): inferences from chromosome banding

Abstract The family Curimatidae is a fish group usually considered chromosomally conserved in their diploid number. However, some studies show small changes in the karyotype microstructure, and the presence of B chromosomes, indicating a chromosomal diversification within the group, even if structural changes in the karyotypes are not visible. Few studies associate this trait with an evolutionary pattern within the family. This study aimed to characterize the karyotype, nucleolus organizer regions (NORs), and heterochromatin distribution of six species of Curimatidae of the genera Cyphocharax Fowler, 1906 and Steindachnerina Fowler, 1906: Cyphocharax voga (Hensel, 1870), Cyphocharax spilotus (Vari, 1987), Cyphocharax saladensis (Meinken, 1933), Cyphocharax modestus (Fernández-Yépez, 1948), Steindachnerina biornata (Braga et Azpelicueta, 1987) and Steindachnerina insculpta (Fernández-Yépez, 1948) and contribute data to a better understanding of the mechanisms involved in the chromosomal evolution of this group of fish. All specimens had 2n=54, m-sm, and B microchromosomes. Five species exhibited single NORs, except for Steindachnerina biornata, which showed a multiple pattern of ribosomal sites. NORs were chromomycin A3 positive (CMA3+) and 4’-6-diamino-2-phenylindole (DAPI-) negative, exhibiting differences in the pair and chromosomal location of each individual of the species. FISH with 5S rDNA probe revealed sites in the pericentrometic position of a pair of chromosomes of five species. However, another site was detected on a metacentric chromosome of Cyphocharax spilotus. Heterochromatin distributed both in the pericentromeric and some terminal regions was revealed to be CMA3+/DAPI-. These data associated with the previously existing ones confirm that, although Curimatidae have a very conservative karyotype macrostructure, NORs and heterochromatin variability are caused by mechanisms of chromosome alterations, such as translocations and/or inversions, leading to the evolution and diversification of this group of fish.

Effects of thiamethoxam and lambda-cyhalothrin on spermatogenesis of Euschistus heros (Heteroptera: Pentatomidae): Insecticide effects on Euschistus heros

Euschistus heros (Fabricius, 1798) is one of the most harmful insect pests damaging Brazilian soybean crops and has become a major problem due to its high population density and resistance to insecticides. Currently, there are no data on whether alterations of testicular morphology and chromosomal behavior are associated with the resistance mechanisms related to the action of insecticides. This study integrated analyses of the testicular morphology, meiocyte cell division, and chromosomal structure and behavior in the process of spermatogenesis in E. heros. We compared these features among wild‐caught individuals, insecticide‐susceptible and ‐resistant strains. The resistant strain was established through a selection experiment exposing the bugs to insecticides (thiamethoxam + lambda‐cyhalothrin) for 15 generations. No differences were detected in the examined features among the three groups of experimental individuals: the testis comprised six lobes, with the fifth lobe thinner than the others; the karyotype was 2n = 14 (12 + XY), with no evident changes in chromosomal breakage, rearrangement or behavior in the meiosis; and abundant spermatozoa were observed in all testicular lobes. Thus, any effects of the long‐term (15 generations) experimental selection by exposure to the insecticides were not detected on the male germinal tissue and chromosomes, suggesting the irrelevancy of the examined features to insecticide‐resistance mechanisms in E. heros.