Cytogenetics of the darkling beetles Zophobas aff. confusus and Nyctobates gigas (Coleoptera, Tenebrionidae)

Males of Zophobas aff. confusus and Nyctobates gigas (Tenebrionidae) collected in the State of Pernambuco, Brazil, were studied through conventional staining, C-banding, silver nitrate impregnation (AgNO(3)), and the base specific fluorochromes CMA(3) and DAPI. Z. aff. confusus was found to have 2n = 20 (9+Xyp) while N. gigas exhibited 2n = 18 (8+neoXY). Large pericentromeric blocks of constitutive heterochromatin (CH) were detected throughout the autosomal complement of the two species, except in one autosomal pair of N. gigas in which no heterochromatic block was observed. The sex chromosomes of both species were almost totally heterochromatic. Double staining with CMA(3)/DA (distamycin) and DAPI/DA marked CH in Z. aff. confusus. However, DAPI staining was more intense. N. gigas was found to possess blocks of CH-positive CMA(3) and homogeneous DAPI. AgNO(3) staining also revealed differences between the two species. In Z. confusus an NOR was observed in the sexual bivalent Xyp and N. gigas was found to have an autosomal NOR.     

Cytogenetic characterization of <Emphasis Type="Italic">Eurysternus caribaeus</Emphasis> (Coleoptera: Scarabaeidae): evidence of sex-autosome fusion and diploid number reduction prior to species dispersion

Mitotic and meiotic chromosomes of several populations of Eurysternus caribaeus (Coleoptera: Scarabaeidae) were analysed through conventional staining, C-banding, base-specific fluorochromes, silver nitrate staining and fluorescent in situ hybridization (FISH). All specimens showed 2n = 8 in their karyotypes, with a neo-XY sex system (Y is a submetacentric and X a metacentric) and three pairs of submetacentric autosomes. The analysis of constitutive heterochromatin (CH) revealed small blocks located in the centromeric region of all chromosomes which do not present positive staining under the fluorochromes CMA3 and DAPI. Silver nitrate staining revealed that the nucleolar organizer region (NORs) is associated with the sex chromosomes. The FISH technique revealed that rDNA sites in the X and Y are different in size. Data from different populations indicate that the diploid number reduction (2n = 8) observed in E. caribaeus is established and presumably has preceded the dispersion of this species. Moreover, this reduction occasioned the translocation of rDNA sites to the sex chromosomes, X and Y, an uncommon pattern in Scarabaeidae that was observed for the first time by the FISH in this work.     

Differential characterization of holocentric chromosomes in triatomines (Heteroptera, Triatominae) using different staining techniques and fluorescent in situ hybridization

A comparative study of holocentric chromosomes in the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans was carried out in order to characterize heterochromatin, rDNA active sites and nucleolar proteins. Cytological preparations of seminiferous tubules were stained by silver impregnation, C banding, fluorochromes cma3/da and dapi/da, and fluorescent in situ hybridization (FISH) with Drosophila melanogaster 28S rDNA probe. Our results showed interesting aspects of the organization of chromatin and chromosomes in the meiotic cells of these insects. In R. pallescens, sex chromosomes (X, Y) were distinct from autosomes, when submitted to silver impregnation, C banding, CMA3 staining, and FISH, confirming that these chromosomes bear nucleolar organizer regions (NORs). In P. megistus, two of the three sex chromosomes were CMA3/DAPI-; at early meiotic prophase and at diakinesis, silver impregnation corresponded with FISH signals, indicating that in this species, two chromosomes (probably a sex chromosome and an autosome) bear NORs. In T. infestans, silver nitrate and FISH also stained corresponding areas on meiotic chromosomes. Our data suggest that in triatomines, in general, the number and location of NORs are species-specific. These regions may be considered important chromosome markers for comparative studies to improve the understanding of evolutionary mechanisms in these hematophagous insects.     

Comparative cytogenetic analysis between Lonchorhina aurita and Trachops cirrhosus (Chiroptera, Phyllostomidae)

Phyllostomidae comprises the most diverse family of neotropical bats, its wide range of morphological features leading to uncertainty regarding phylogenetic relationships. Seeing that cytogenetics is one of the fields capable of providing support for currently adopted classifications through the use of several markers, a comparative analysis between two Phyllostomidae species was undertaken in the present study, with a view to supplying datasets for the further establishment of Phyllostomidae evolutionary relationships. Karyotypes of Lonchorhina aurita (2n = 32; FN = 60) and Trachops cirrhosus (2n = 30; FN = 56) were analyzed by G- and C-banding, silver nitrate staining (Ag-NOR) and base-specific fluorochromes. Chromosomal data obtained for both species are in agreement with those previously described, except for X chromosome morphology in T. cirrhosus, hence indicating chromosomal geographical variation in this species. A comparison of G-banding permitted the identification of homeologies in nearly all the chromosomes. Furthermore, C-banding and Ag-NOR patterns were comparable to what has already been observed in the family. In both species CMA(3) /DA/DAPI staining revealed an R-banding-like pattern with CMA (3) , whereas DAPI showed uniform staining in all the chromosomes. Fluorochrome staining patterns for pericentromeric constitutive heterochromatin (CH) regions, as well as for nucleolar organizing regions (NORs), indicated heterogeneity regarding these sequences among Phyllostomidae species.     

Nature and distribution of constitutive heterochromatin and NOR location in the grasshopper Phaeoparia megacephala (Romaleidae: Orthoptera)

The constitutive heterochromatin (CH) of Phaeoparia megacephala was studied using C-banding and fluorochrome staining (CMA3, DAPI and acridine orange). The nucleolar organizer regions (NOR) were identified with silver staining. The chromosome complement of this species was 2n = 23, XO in males, and 2n = 24, XX in females. The CH was pericentromeric in all chromosomes. L1, L2, L3 and X chromosomes showed large blocks of CH, while the medium and small chromosomes had small blocks. The staining procedure with acridine orange revealed the same pattern. All the pericentromeric regions showed small blocks of CMA3-positive constitutive heterochromatin (GC-rich regions), while only part of the large C-band positive chromosome segments (L1, L2, L3 and X) were CMA3 positive. This character demonstrates an uncommon heterogeneity of constitutive heterochromatin in P. megacephala. The fluorochrome DAPI did not reveal DAPI-positive regions (AT-rich regions). Silver staining revealed only one pair of medium chromosomes with NOR.     

A Comparative Cytogenetic Analysis between the Grasshopper Species Chromacris nuptialis and C. speciosa (Romaleidae): Constitutive Heterochromatin Variability and rDNA Sites

The chromosomes of Chromacris nuptialis and C. speciosa were comparatively analyzed using different cytogenetic techniques, in order to determine the level of karyotypic similarities and differences between the species. The results show similarities in chromosome number (2n = 23,X0) and acrocentric morphology. In some C. nuptialis individuals meiotic irregularities were detected involving the L₂ bivalent. This bivalent was delayed and presented anaphasic bridges and other aberrations. Differences in constitutive heterochromatin (CH) patterns and composition were observed through C-banding and fluorochromes staining. Silver nitrate staining revealed a single medium nucleolar organizer regions (NORs) pair, per species. Differences were also observed in NORs location, which was pericentromeric in C. nuptialis and proximal in C. speciosa. FISH using an rDNA probe confirmed the existence of ribosomal sites coinciding with active regions visualized by silver nitrate. The possible implications of the karyotype differences observed between both species are discussed.     

Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species

Thirty-nine cultivars of cassava and eight related wild species of Manihot were analyzed in this work for number, morphology and size of chromosomes, prophase condensation pattern and the structure of the interphase nucleus. In four accessions, the chromosome size was measured and in some others, the number of secondary constrictions, meiotic behavior, C-band pattern, CMA/DAPI bands, nucleoli number and the location of 5S and 18S-5.8S-28S rDNA sites were also observed. All investigated accessions showed a similar karyotype with 2n = 36, small metacentric to submetacentric chromosomes. Two pairs of terminal secondary constrictions were observed in the chromosome complement of each accession except Manihot sp. 1, which presented two proximal secondary constrictions. The prophase chromosome condensation pattern was proximal and the interphase nuclei structure was areticulate to semi-reticulate. The meiosis, investigated in seven cultivars and four wild species, was regular, displaying 18 bivalents. C-banding revealed heterochromatin in 9 or 10 chromosomes. The analysis with fluorochromes frequently showed four chromosome pairs with a single CMA+ terminal or subterminal band and a few other chromosomes with DAPI+ unstable bands. Six 45S rDNA sites were revealed by FISH, which seemed to colocalize with six CMA+ bands. Only one chromosome pair presented a 5S rDNA site. The maximum nucleoli number observed per nucleus was also six. These data suggest that all Manihot species present a very similar chromosome complement.     

Chromosome banding in the genusPinus

The chromosomes (2n=24) ofPinus densiflora Sieb. et Zucc. andP. thunbergii Parl. collected from several localities were analyzed on their fluorescent banding patterns by sequential staining with the base specifically binding fluorochromes, CMA and DAPI. In both species, the CMA-bands were localized at the proximal and/or interstitial regions of most of the chromosomes. The CMA-banding pattern was constant among the cells in a plant and was specific to respective species with a few variations. After the CMA and DAPI stainings each chromosome was identified individually. The fluorescent banding patterns of the two species were somewhat similar, but were diferent with respect to in some characters.Pinus thunbergii had two pairs of metacentric chromosomes without CMA-band and two pairs of metacentric chromosomes with an additional thin CMA-band at the interstitial region. The 10th and 11th pairs of chromosomes of both species, which showed similarity in interstitial CMA and DAPI banding and chromosome shape, had the proximal CMA-bands inP. densiflora and DAPI-band inP. thunbergii. The interspecific F₁ hybrid between the two species could easily be identified by the fluorescent banding method.     

First report on C-banding,fluorochrome staining and NOR location in holocentric chromosomes of Elasmolomus (Aphanus) sordidus Fabricius, 1787 (Heteroptera,Rhyparochromidae)

In spite of various cytogenetic works on suborder Heteroptera, the chromosome organization, function and its evolution in this group is far from being fully understood. Cytologically, the family Rhyparochromidae constitutes a heterogeneous group differing in chromosome numbers. This family possesses XY sex mechanism in the majority of the species with few exceptions. In the present work, multiple banding techniques viz., C-banding, base-specific fluorochromes (DAPI/CMA₃) and silver nitrate staining have been used to cytologically characterize the chromosomes of the seed plant pest Elasmolomus (Aphanus) sordidus Fabricius, 1787 having 2n=12=8A+2m+XY. One pair of the autosomes was large while three others were of almost equal size. At diplotene, C-banding technique revealed, that three autosomal bivalents show terminal constitutive heterochromatic bands while one medium sized bivalent was euchromatic. Microchromosomes (m-chromosomes) were positively heteropycnotic. After DAPI and CMA₃ staining, all the autosomal bivalents showed equal fluorescence, except CMA₃ positive signals, observed at both telomeric heterochromatic regions of one medium sized autosomal bivalent. Silver nitrate staining further revealed that this chromosome pair carries Nucleolar Organizer Regions (NORs) at the location of CMA₃ positive signals. The X chromosome showed a thick C-band, positive to both DAPI /CMA₃ while Y, otherwise C-negative, was weakly positive to DAPI and negative to CMA₃, m-chromosomes were DAPI bright and CMA₃ dull.     

Comparative cytogenetics of giant trahiras Hoplias aimara and H. intermedius (Characiformes, Erythrinidae): chromosomal characteristics of minor and major ribosomal DNA and cross-species repetitive centromeric sequences mapping differ among morphologically identical karyotypes

Karyotype and cytogenetic characteristics of 2 species of giant trahiras, Hopliasintermedius, S?o Francisco river basin, and Hopliasaimara, Arinos river (Amazon basin), were examined by conventional (C-banding, Ag-NOR, DAPI/CMA(3) double-staining) and fluorescent in situ hybridization (FISH) with 5S, 18S rDNA probes and cross-species Cot-1 DNA probing. Both species invariably had diploid chromosome number 2n = 50 and identical karyotypes composed of 10 pairs of metacentric and 15 pairs of submetacentric chromosomes. On the other hand, staining with base-specific fluorochromes (CMA(3), DAPI) and FISH mapping of repetitive DNA sequences showed extensive interspecific differences: while the genome of H. aimara had one submetacentric pair bearing CMA(3)-positive (DAPI-negative) sites, that of H. intermedius had 4 such pairs; while FISH with a 5S rDNA probe showed one (likely homologous) signal-bearing pair, that with 18S rDNA displayed one signal-bearing pair in H. intermedius and 2 such pairs in H. aimara. Cross-species FISH probing with Cot-1 DNA prepared from total DNA of both species showed no signals of Cot-1 DNA from H. aimara on chromosomes of H. intermedius but reciprocally (Cot-1 DNA from H. intermedius on chromosomes of H. aimara) displayed signals on at least 4 chromosome pairs. Present findings indicate (i) different composition of repetitive sequences around centromeres, (ii) different NOR phenotypes and (iii) distinct taxonomic status of both giant trahira species.     

Cytogenetic characterization of Partamona cupira (Hymenoptera, Apidae) by fluorochromes

Four colonies of the stingless bee Partamona cupira (Hymenoptera: Apidae) were cytogenetically analyzed using conventional staining and the fluorochromes CMA(3) e DAPI. The females have 2n = 34 chromosomes (2K = 32 Mˉ+2 Aˉ). Some females, however, presented an additional large B acrocentric chromosome, to a total of 2n = 35. Chromosome B and the chromosomal pairs 2, 9 and 10 showed CMA (3) (+) bands, indicating an excess of CG base-pairs. A clear association was verified between the P. helleri B chromosome SCAR marker and the presence of a B chromosome in P. cupira. The data obtained suggests that B chromosomes in P. helleri and P. cupira share a common origin.     

Comparative cytogenetics of three species of Dichotomius (Coleoptera, Scarabaeidae)

Meiotic and mitotic chromosomes of Dichotomius nisus, D. semisquamosus and D. sericeus were analyzed after conventional staining, C-banding and silver nitrate staining. In addition, Dichotomius nisus and D. semisquamosus chromosomes were also analyzed after fluorescent in situ hybridization (FISH) with an rDNA probe. The species analyzed had an asymmetrical karyotype with 2n = 18 and meta-submetacentric chromosomes. The sex determination mechanism was of the Xy(p) type in D. nisus and D. semisquamosus and of the Xy (r) type in D. sericeus. C-banding revealed the presence of pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of the three species. After silver staining, the nucleolar organizer regions (NORs) were located in autosomes of D. semisquamosus and D. sericeus and in the sexual bivalent of D. nisus. FISH with an rDNA probe confirmed NORs location in D. semisquamosus and in D. nisus. Our results suggest that chromosome inversions and fusions occurred during the evolution of the group.     

Meiosis Aspects and Nucleolar Activity in Triatoma vitticeps (Triatominae, Heteroptera)

Some aspects of both the nucleolar organizer activity and meiosis were studied in the testes of Triatoma vitticeps (Heteroptera, Triatominae). The techniques used included squashing followed by lacto-acetic orcein staining, silver-ion impregnation, fluorescent banding (CMA₃, Quinacrine mustard and DAPI) and fluorescent in situ hybridization (FISH). A close relationship between heterochromatin and nucleolus in testicular cells was observed. During meiosis, the silver-ion impregnation pattern varied. At metaphase plate, a small body appeared apart from the chromosomes. In the spermatids this small body was seen in preparations stained with orcein and silver- ion impregnation but not with fluorochromes or FISH. These characteristics combined suggest that these corpuscles represent a source of ribonucleoproteins (RNP) – RNA and specific nucleolar proteins. Silver-ion impregnation and (FISH) revealed nucleolar organizer activity in two metaphase sex chromosomes (X). These results indicate that, in these species, nucleolar organizer regions (NORs) are located in the sex chromosomes, X chromosomes were CMA₃⁺ and Y chromosome was DAPI⁺.     

Karyotypic characterization and constitutive heterochromatin in the grasshopper Stiphra robusta (Orthoptera proscopiidae)

Conventional analysis, C-banding, silver nitrate and base specific fluorochrome staining with chromomycin A3 (CMA3) were used to analyse the meiotic chromosomes of the grasshopper Stiphra robusta. Diploid numbers of 2n = 19 in the males and 2n = 20 in the females were observed. The chromosome complement comprised a graded series of uniarmed chromosomes, and the X chromosome was medium sized. The nucleolar organizer regions, restricted to the bivalent chromosomes 6, 7 and 8, were CMA3 positive.     

Sympatric occurrence of four cytotypes and one extra chromosome in Bryconamericus ecai (Characidae): 18S rDNA polymorphism and heterochromatin composition

In the present study, specimens of Bryconamericus ecai collected from the Forquetinha River/RS, were cytogenetically analyzed, disclosing a wide karyotypic diversity in this species. All individuals had 2n = 50, with different karyotypic formulae, resulting in four cytotypes and one B macrochromosome observed in cytotype III. Heterochromatin was distributed in the pericentromeric region of most chromosomes on the four cytotypes and also on a chromosome pair with interstitial markings in cytotype IV. Staining with CMA(3) and DAPI fluorochromes revealed a C-band region rich in AT base pairs in cytotypes I, II and III, and a pair with GC-rich heterochromatin in cytotypes II and III. Cytotype IV presented CMA(3) and DAPI positive heterochromatin. Silver nitrate impregnation, in situ hybridization, and fluorochrome staining showed a multiple system of AgNORs, 18S rDNA and CMA(3) sites in cytotypes I, III and IV, with both inter-and intraindividual variability in the number and location of these sites. Cytotype II had only one pair of NORs coincident with the 18S rDNA and CMA(3) sites, indicating a simple system. The chromosomal polymorphism observed among the specimens of B. ecai added to the literature data show that chromosomal rearrangements, especially pericentric inversions, play an important role in the karyotypic evolution of this group of fish. It can also be implied that more than one species of Bryconamericus is probably occurring, living in sympatry in the Forquetinha River/RS.     

Chromosomal similarities and differences among four Neotropical Elateridae (Conoderini and Pyrophorini) and other related species, with comments on the NOR patterns in Coleoptera

This work deals with the comparative cytogenetic analysis of four Neotropical Elateridae species and reviews the nucleolar organizer region (NOR) patterns on Coleoptera chromosomes, for the first time. The cytogenetic characterization of Conoderus malleatus (Conoderini), Pyrearinus candelarius, Pyrophorus divergens and Pyrophorus punctatissimus (Pyrophorini) was accomplished through the study of mitotic and meiotic cells submitted to standard (Giemsa) and differential staining [silver impregnation and GC‐specific chromomycin A₃ (CMA₃) plus AT‐specific 4′‐6‐diamidino‐2‐phenylindole (DAPI) fluorochromes]. The analysis of spermatogonial cells revealed the diploid numbers: 2n = 17 in C. malleatus and 2n = 15 in P. candelarius, P. divergens and P. punctatissimus. In these species, the X0 type sex‐determination system and the acrocentric morphology of almost all chromosomes were observed. The study of meiotic cells of the four species revealed the occurrence of total synapsis between the autosomes, the presence of one terminal or interstitial chiasma in the majority of the bivalents, and the reductional behaviour and regular segregation of all chromosomes. Although the three Pyrophorini species demonstrated many similar karyotypical characteristics, there was one discrepancy, which was noted in the diplotene cells and concerns the number of bivalents with two chiasmata; P. candelarius only presented one bivalent, P. divergens showed two bivalents and P. punctatissimus exhibited up to four bivalents with two chiasmata. Testicular cells impregnated with silver nitrate demonstrated two terminal NORs located on the fourth autosomal pair of the Conoderini species and on the second autosomal pair of the three Pyrophorini representatives. Use of CMA₃/distamycin A (DA)/DAPI staining on the P. candelarius and P. punctatissimus chromosomes revealed that the CMA₃ labelled regions were coincident with the NORs. The main strategies of karyotypical differentiation that have occurred among the four Elateridae species and other related species, and the general trends of the NOR shifts during Coleoptera chromosomal evolution are discussed in this work.     

Chromosome analysis using different staining techniques and fluorescent in situ hybridization in Cerithium vulgatum (Gastropoda: Cerithiidae)

In the present paper one population of the large" subtidal mollusc Cerithium vulgatum Bruguière, 1792 (Gastropoda: Cerithiidae) from the Northwestern coast of Sicily was investigated from a karyological point of view. The chromosome complement was Giemsa stained, conventionally karyotyped in 18 homomorphic chromosome pairs (10 bi-armed and 8 mono-armed), and subsequently analysed using silver, CMA3 and DAPI staining, and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG)n and (GATA)n]. FISH with the rDNA probe consistently mapped major ribosomal sites (18S-28S rDNA) in the terminal region of the short arms of one small sized mono-armed chromosome pair. Ribosomal DNA was transciptionally active as indicated by its preferential impregnation with silver nitrate (Ag-NOR) and did not contain a high amount of GC base pairs as suggested by the lack of a bright CMA3 fluorescence. The (TTAGGG)n telomeric probe was hybridized to the termini of nearly all chromosomes, thus demonstrating that, in C. tulgatum, this sequence has been conserved during the genomic evolution. The finding of the telomeric hexanucleotide in six species belonging to the three high taxa of Gastropoda supports the notion that this sequence is widespread within this class. The (GATA)n probe did not label any chromosome regions except for a minute terminal area of a single bivalent at pachytene stage.     

Karyotype analysis in Hyacinthella dalmatica (Hyacinthaceae) reveals vertebrate-type telomere repeats at the chromosome ends.

Chromosome analysis of three different populations of Hyacinthella dalmatica (Lallem.) Trinajsti?, an endemic species of the coastal region of southeastern Europe, showed a unique chromosome number, 2n = 2x = 20, and bimodal karyotype with one large and nine smaller pairs of chromosomes. Staining with fluorochromes CMA3 (chromomycin A3) and DAPI (4,6-diamidino-2-phenylindole) revealed heterochromatic regions associated with NORs, centromeres, and several interstitial heterochromatic bands on the longest chromosome pair. Double-target FISH with two ribosomal DNA probes revealed one locus of 5S rRNA genes in the pericentromeric region of chromosome pair 3 and one locus of 18S-5.8S-26S rRNA genes on the short arm of chromosome pair 4 in all plants and populations analyzed. Southern hybridization analysis and FISH experiments demonstrated that the distal ends of H. dalmatica chromosomes contain the vertebrate telomere (5'-TTAGGG-3') repeat type rather than the Arabidopsis (5'-TTTAGGG-3') heptamer, and so suggest that this Asparagales species along with Aloe and Othocallis contains the vertebrate-type telomere repeat.     

Multiple Nors in Bryconamericus aff. exodon (Osteichthyes,Characidae, Tetragonopterinae)

Fifteen examples of Bryconamericus aff. exodon from the Três Bocas stream in the basin of the Tibagi river (Parana, Brazil) were analyzed. They presented a diploid number of 52 chromosomes. Multiple NORS were detected by silver nitrate impregnation in the telomeric region on the short or long arm of different chromosomes, showing a variation from 2 to 5 NOR-bearing chromosomes. Treatment with chromomycin A3 (CMA3) showed regions rich in GC in different chromosomes, probably coincident with the AgNORS. In situ hybridization by fluorescence (FISH) with 18S rDNA probe showed 8 hybridization signs in the telomeric regions of the chromosomes, proving that this B. aff. exodon population has a greater number of ribosomal cistrons than were detected by Ag-banding.