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.     

Analyses of Nucleolus Organizer Regions and Heterochromatin of Pimelodus Maculatus (Pisces, Pimelodidae)

Eighteen specimens of Pimelodus maculatus collected from Tibagi River (Sertaneja, PR, Brazil) were analyzed cytogenetically. The diploid number of 56 chromosomes was observed and karyotype was 20 M + 20 SM + 10 ST + 6 A with fundamental number (FN) of 106. Results of analyses from the nucleolus organizer regions (NORs), obtained by AgNO₃, CMA₃ and C-band staining showed marking in a terminal position on the long arm of a pair of subtelocentric chromosomes. The restriction enzyme AluI produced a linear differentiation similar to C-banding. This revised version was published online in July 2006 with corrections to the Cover Date.     

B chromosomes in two fish species, genus Rhamdia (Siluriformes, Pimelodidae)

Specimens belonging to two fish species genus Rhamdia were cytogenetically analysed from seven localities in Brazil and Argentina. In addition to the 58 chromosomes of the basic karyotype, one to five metacentric B chromosomes were observed carrying conspicuous heterochromatic blocks on the distal regions of both chromosome arms. These B chromosomes are mitotically stable and, in the two best-sampled populations in R. hilarii (Lobo and 29 reservoirs), they showed frequency distributions fitting a binomial distribution, though Bs were more frequent in the latter. The presence of B chromosomes with the same appearance in R. quelen suggests an ancient origin for these B chromosomes, presumably prior to speciation from a common ancestor.     

A Population Analysis of the Structure and Variability of NOR in Salmo Trutta by Ag, CMA3 and ISH

An analysis of the variation in the number and location of rDNA genes has been carried out in two populations of brown trout (Salmo trutta) from Poland by using Ag and CMA₃-staining, and rDNA in situ hybridisation. We observed an interindividual variation in arm number with NF = 100, 101, and 102. This variation was connected with the size polymorphism of the short (NOR-bearing) arm of the chromosome pair 11. The population studied showed a multichromosomal distribution of active NORs. Atypical Ag-NORs consisted of rDNA genes, as evidenced by rDNA-ISH. In addition to individuals with standard NORs, specimens with extra NORs as well as others with only one active NOR and single interphase nucleolus were observed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nature and distribution of constitutive heterochromatin in fishes,genus Hypostomus (Loricariidae)

Some Hypostomus species were studied concerning the features of the karyotype structure and the constitutive heterochromatin. The karyotype of Hypostomus sp. F from the S?o Francisco river (Minas Gerais state, Brazil) is now described for the first time. A diversity in the diploid number, ranging from 2n = 68 to 2n = 80, as well as in the karyotype formulae, is evident in this fish group. Two types of heterochromatin, GC- and AT-rich, could be identified with the use of base-specific fluorochromes. In some species heterochromatic bands are mainly located on the centromeric and telomeric chromosomal regions, while in other species they are also observed at interstitial locations. Hypotheses concerning this heterochromatic distribution in Hypostomus karyotypes are discussed. A case of supernumerary heterochromatic segment and a centric fusion appear to be related with two variant karyotypic formulae observed among specimens from the Mogi-Gua?u and S?o Francisco rivers, respectively. The available data permit us to characterize a divergent karyotypic evolution among the Hypostomus species already analyzed, both at the macro- and microstructural levels, that is, their general karyotype organization and particular features related to chromosomal banding or staining, respectively.     

Karyotype analysis and heterochromatin differentiation with Giemsa C-banding and fluorescent counterstaining inCephalanthera (Orchidaceae)

Detailed studies of the chromosomes of the three Austrian species of the genusCephalanthera showed them all to have basically similar karyotypes. BothC. damasonium (2n = 36) andC. longifolia (2n = 32) have three large and several classes of smaller chromosome pairs. The karyotype ofC. rubra (2n = 44) is composed of four large and several groups of smaller pairs. The heterochromatin in these species amounts to about 10% of total karyotype length. All the chromosomes have Giemsa-positive centromeres, but only a few have intercalary or terminal bands. Using differential fluorescent staining with DAPI/actinomycin D, quinacrine/actinomycin D (both A-T specific), and chromomycin A₃/distamycin A (G-C specific) three different types of major heterochromatic bands can be characterized in respect of their satellite DNA composition: highly A-T rich, slightly A-T rich, and very G-C rich. The chromosomes ofC. longifolia contain more A-T rich C-bands than those ofC. damasonium, while the latter's have more G-C rich heterochromatin. In both species several C-bands appear as secondary constrictions or gaps in the Feulgen-stained chromosomes, but most likely, in each species there is only one pair of chromosomes where the secondary constrictions function as nucleolus organizing regions. No major intraspecific variation could be observed except on one small chromosome pair ofC. longifolia which had a heteromorphic C-band in most individuals. Possible pathways of karyotype evolution involving polyploidy and Robertsonian events are discussed.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

Evolutionary chromosomal differentiation among four species of Conoderus Eschscholtz, 1829 (Coleoptera, Elateridae, Agrypninae, Conoderini) detected by standard staining, C-banding, silver nitrate impregnation, and CMA3/DA/DAPI staining

The speciose Brazilian Elateridae fauna is characterized by high karyotypic diversity, including one species (Chalcolepidius zonatus Eschscholtz, 1829) with the lowest diploid number within any Coleoptera order. Cytogenetic analysis of Conoderus dimidiatus Germar, 1839, C. scalaris (Germar, 1824,) C. ternarius Germar, 1839, and C. stigmosus Germar, 1839 by standard and differential staining was performed with the aim of establishing mechanisms of karyotypic differentiation in these species. Conoderus dimidiatus, C. scalaris, and C. ternarius have diploid numbers of 2n(♂) = 17 and 2n(♀) = 18, and a X0/XX sex determination system, similar to that encountered in the majority of Conoderini species. The karyotype of C. stigmosus was characterized by a diploid number of 2n=16 and a neoXY/neoXX sex determination system that was highly differentiated from other species of the genus. Some features of the mitotic and meiotic chromosomes suggest an autosome/ancestral X chromosome fusion as the cause of the neoXY system origin in C. stigmosus. C-banding and silver impregnation techniques showed that the four Conoderus species possess similar chromosomal characteristics to those registered in most Polyphaga species, including pericentromeric C band and autosomal NORs. Triple staining techniques including CMA₃/DA/DAPI also provided useful information for differentiating these Conoderus species. These techniques revealed unique GC-rich heterochromatin associated with NORs in C. scalaris and C. stigmosus and CMA₃-heteromorphism in C. scalaris and C. ternarius.     

Comparative karyotype analysis ofCeratozamia mexicana andMicrocycas calocoma (Zamiaceae) using fluorochrome banding (CMA/DAPI) and fluorescence in situ hybridization of ribosomal DNA

Orcein staining, differential staining with CMA and DAPI, and FISH with an rDNA probe were used to compare somatic chromosomes ofCeratozamia mexicana andMicrocycas calocoma. CMA-positive dots and hybridization signals appeared on chromosomes at early interphase and mitotic prophase, but in significantly different number in the two species. InCeratozamia mexicana, the CMA-positive and DAPI-negative bands and the hybridization signal were located at the terminal region of the long arm of three median-centromeric chromosomes, the terminal region of the short arm of two median-centromeric chromosomes and the terminal region of the long arm of two subterminal-centromeric chromosomes. InMicrocycas calocoma, they were located at the pericentric region of two median-centromeric chromosomes. These chromosome data suggested thatMicrocycas has no simple Robertsonian relationship toCeratozamia.     

Hidden heterochromatin: Characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae)

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes.     

First report on C-banding and fluorescent banding in species of Dieuches (Rhyparochrominae: Lygaeidae: Heteroptera)

Although the karyotypes of twelve species of Dieuches Dohrn, 1860 belonging to Rhyparochrominae have been described so far, there is no information about heterochromatin and its characterization in terms of base composition for any of the species. In the present paper, C-banding and fluorescent banding have been applied for the first time to three species of Dieuches : D. uniguttatus, D. insignis (2n = 12 = 8A + 2m + XY) and D. coloratus (2n = 14 = 10A + 2m + XY). Dieuches uniguttatus and D. insignis show distinct terminal C-bands along with a few interstitial bands in all the autosomal bivalents, whereas in D. coloratus , one autosomal pair is almost completely heterochromatic, three show C-positive bands while one is totally euchromatic. The sex chromosomes too show heterogeneity in distribution of C-heterochromatin among three Dieuches species. Characterization of heterochromatin in D. uniguttatus and D. insignis using DAPI/CMA₃ staining reveals that in D. uniguttatus , C- heterochromatin blocks of all the autosomal bivalents, which are predominantly A–T rich, whereas in D. insignis , these are rich both in A–T and G–C. In D. uniguttatus, sex chromosomes X and Y have localized G–C rich regions whereas in D. insignis , these are scattered in X and absent in Y. As variations in the heterochromatin represent the main source of karyological differentiation among and within species, it seems that there occurred extensive redistribution of heterochromatin within the complement as the three species evolved. There is need for cytological details of more species to understand evolutionary aspects in the genus Dieuches .     

Cytogenetics of bisexual/unisexual species of Poecilia. II. Analysis of heterochromatin and nucleolar organizer regions in Poecilia mexicana mexicana by C-banding and DAPI, quinacrine, chromomycin A3, and silver staining.

Chromosomes of Poecilia mexicana mexicana, one of the bisexual species involved in the hybrid origin of the unisexual teleost fish species P. formosa, were analyzed by several staining techniques. Sex-specific, differential heterochromatin, found in other congeneric species, was not observed in P. m. mexicana. Nucleolar organizer regions were polymorphic among individual specimens within a given population sample. A single specimen exhibiting intraindividual variability of chromosome pair 1 and a specimen with a triploid karyotype are also described.     

The ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermatogenesis: premeiotic and meiotic periods

The ultrastructure of Sorubim lima spermatogenesis during the premeiotic and meiotic periods was studied. Our observations showed that the germ cells in the cysts are connected by cytoplasmic bridges and the mitotic and meiotic divisions are slightly asynchronous. The first and the last spermatogonial generations differ in the cellular and nuclear volume, nucleolus, chromatin condensation, distribution, size, density, and shape of the mitochondria, presence of 'lamellae anulata', amount and dimension of the 'nuages', and movement of the centrioles. In addition to the nuclear prophase structures, the spermatocyte I shows changes in all other cellular organelles and elongated vesicles appear in the cytoplasm. The accentuated cytoplasmic density and thickened walled vesicles are morphological characteristics that differentiate spermatocytes II from the other germ cells in the cysts of Sorubim lima testis.     

Cytogenetic characterization of F1, F2 and backcross hybrids of the Neotropical catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum (Pimelodidae, Siluriformes)

The cytogenetic characteristics of Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum and their F1, F2 and backcross hybrids were assessed by using chromosome banding techniques. The diploid number of 56 chromosomes was constant in all species and lineages, with a karyotypic formula containing 20 metacentric, 12 submetacentric, 12 subtelocentric and 12 acrocentric chromosomes. Nucleolar organizer regions (NORs) were identified in two subtelocentric chromosomes in the parents and hybrids, with partial nucleolar dominance in F1 and F2 specimens. Heterochromatic blocks were detected in the terminal and centromeric regions of some chromosomes in all individuals. For parental and hybrid lineages, 18S ribosomal clusters corresponding to NORs and 5S ribosomal genes were identified in distinct pairs of chromosomes. The striking conservation in the chromosomal macrostructure of the parental species may account for the fertility of their F1 hybrids. Similarly, the lack of marked alterations in the chromosomal structure of the F1 hybrids could account for the maintenance of these features in post-F1 lineages.     

DAPI staining and DNA content estimation of nuclei in uncultivable microbial eukaryotes (Arcellinida and Ciliates)

Though representing a major component of eukaryotic biodiversity, many microbial eukaryotes remain poorly studied, including the focus of the present work, testate amoebae of the order Arcellinida (Amoebozoa) and non-model lineages of ciliates (Alveolata). In particular, knowledge of genome structures and changes in genome content over the often-complex life cycles of these lineages remains enigmatic. However, the limited available knowledge suggests that microbial eukaryotes have the potential to challenge our textbook views on eukaryotic genomes and genome evolution. In this study, we developed protocols for DAPI (4′,6-diamidino-2-phenylindole) staining of Arcellinida nuclei and adapted protocols for ciliates. In addition, image analysis software was used to estimate the DNA content in the nuclei of Arcellinida and ciliates, and the measurements of target organisms were compared to those  of well-known model organisms. The results demonstrate that the methods we have developed for nuclear staining in these lineages are effective and can be applied to other microbial eukaryotic groups by adjusting certain stages in the protocols.     

Two new species of Pseudancistrus (Siluriformes,Loricariidae) from the Amazon basin,northern Brazil

Two new species of Pseudancistrus, a genus diagnosed by non-evertible cheek plates and hypertrophied odontodes along the snout margin, are described from two drainages of the Brazilian Shield: Pseudancistrus kayabi from the rio Teles Pires (rio Tapajós basin) and Pseudancistrus asurini from the rio Xingu. The new species are distinguished from congeners (Pseudancistrus barbatus, Pseudancistrus corantijniensis, Pseudancistrus depressus, Pseudancistrus nigrescens, Pseudancistrus reus, and Pseudancistrus zawadzkii) by the coloration pattern. Pseudancistrus kayabi has dark bars on the dorsal and caudal fins which are similar to that of Pseudancistrus reus from the Caroní River, Venezuela. Pseudancistrus asurini is unique among Pseudancistrus in having whitish tips of the dorsal and caudal fins in juveniles to medium-sized adults.     

Observations of mitochondria and mitochondrial nuclei by double staining with rhodamine-123 and DAPI in synchronous cultures ofCatharanthus roseus

Mitochondria in cells ofCatharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle ofC. roseus.     

Cytogenetic characterization of two species of Frieseomelitta Ihering, 1912 (Hymenoptera, Apidae, Meliponini)

The cytogenetic analysis of Frieseomelitta dispar and F. francoi revealed the chromosome numbers 2n = 30 and n = 15 and a karyotypic formula 2K = 4M+2M(t)+4A+20A(M). The number of chromosomes observed was consistent with those reported for other Frieseomelitta species. The occurrence of the M(t) chromosome and other features of the karyotype formulae suggest a close relationship between F. dispar, F. francoi and F. varia. Nevertheless, it was possible to differentiate the karyotypes of the species by DAPI/CMA(3) staining, which revealed GC-rich regions on two chromosome pairs of F. dispar: one acrocentric and one pseudoacrocentric. In F. francoi, the same kinds of regions were observed on a pair of metacentrics and on a pair of acrocentrics. Our analysis also confirmed the chromosome number conservation in Frieseomelitta and suggests that infrequent pericentric inversion could constitute a synapomorphy for the group including F. dispar, F. francoi, and F. varia.     

Karyotype and genome size of Iberochondrostoma almacai (Teleostei, Cyprinidae) and comparison with the sister-species I.lusitanicum

This study aimed to define the karyotype of the recently described Iberian endemic Iberochondrostoma almacai, to revisit the previously documented chromosome polymorphisms of its sister species I.lusitanicum using C-, Ag-/CMA(3) and RE-banding, and to compare the two species genome sizes. A 2n = 50 karyotype (with the exception of a triploid I.lusitanicum specimen) and a corresponding haploid chromosome formula of 7M:15SM:3A (FN = 94) were found. Multiple NORs were observed in both species (in two submetacentric chromosome pairs, one of them clearly homologous) and a higher intra and interpopulational variability was evidenced in I.lusitanicum. Flow cytometry measurements of nuclear DNA content showed some significant differences in genome size both between and within species: the genome of I. almacai was smaller than that of I.lusitanicum (mean values 2.61 and 2.93 pg, respectively), which presented a clear interpopulational variability (mean values ranging from 2.72 to 3.00 pg). These data allowed the distinction of both taxa and confirmed the existence of two well differentiated groups within I. lusitanicum: one that includes the populations from the right bank of the Tejo and Samarra drainages, and another that reunites the southern populations. The peculiar differences between the two species, presently listed as "Critically Endangered", reinforced the importance of this study for future conservation plans.