Cytogenetic analysis of two related Deltochilum (Coleoptera,Scarabaeidae) species: Diploid number reduction,extensive heterochromatin addition and differentiation

Male mitotic and meiotic chromosomes of two species of the genus Deltochilum (Scarabaeidae) were analyzed through conventional staining, C-banding, base-specific fluorochromes, silver nitrate staining (AgNO₃) and FISH (45S rDNA). The two species possessed karyotypes with 2n = 14, neo-XY and meta-submetacentric chromosomes. The analysis of constitutive heterochromatin (CH) revealed mainly diphasic chromosomes in the two species, showing heterochromatic long arms. Silver nitrate staining labeled the blocks corresponding to CH in D. (Deltohyboma) aff morbillosum while in D. (Deltohyboma) calcaratum, AgNO₃ staining revealed only the CH blocks of the diphasic autosomes. The fluorochrome staining revealed in D. (D.) calcaratum the diphasic autosomes and the sex chromosomes with CMA₃⁺ blocks, and in D. (D.) aff morbillosum, the GC-rich sequences were restricted to the terminal regions of the long arms of the pairs 1 and 2 and the X. The FISH revealed 45S rDNA sites in two autosomic pairs and in the X chromosome. The analyses performed allowed for the identification of cytogenetic markers and the discussion of possible chromosome rearrangements that have been involved in the karyotypic differentiation of these species mainly related to the repetitive genome.     

First description of B chromosomes in the family Auchenipteridae,Parauchenipterus galeatus (Siluriformes) of the São Francisco River basin (MG,Brazil)

B chromosomes are considered additional and non-essential; they likely originate from A chromosomes and follow a distinct evolution. In fish, approximately half of the Neotropical species with B chromosomes are Characiformes and 35% are Siluriformes. There has been no report of B chromosomes in Auchenipteridae until this moment. B chromosomes found in a population of Parauchenipterus galeatus from the São Francisco River basin in the state of Minas Gerais (Brazil) were small, metacentric, totally heterochromatic and exhibited intra-individual and inter-individual variation. The diploid number was 58 chromosomes (22 metacentric, 16 submetacentric, 12 subtelocentric and 8 acrocentric). The nucleolar organizing regions were simple and the heterochromatin intercalated in the ribosomal sites, characterized by CMA₃ and DAPI fluorochromes, was of a GC-rich constitution. The 5S rDNA genes were located in an intercalary position in only one chromosome pair. An hypothesis about the origin of the B chromosomes in P. galeatus and a review on B chromosomes in catfish are also presented in this study.     

Chromatin condensation and sensitivity of DNA in situ to denaturation during cell cycle and apoptosis--a confocal microscopy study

The goal of this study was to construct high resolution 3D confocal images of regions of condensed and extended chromatin in cell nuclei and individual chromosomes. It has been shown previously that sensitivity of DNA in situ to denaturation correlates with chromatin condensation and varies during cell cycle and apoptosis. Thus, detection of DNA which was partially denatured in situ provided a means to image areas of condensed chromatin. DNA denaturation was detected using a metachromatic dye acridine orange (AO) which differentially stains single stranded (ss) and double-stranded (ds) DNA sections. Early studies of denaturability of cellular DNA utilized flow cytometry and standard fluorescence microscopy. These techniques could not reveal small local differences in DNA denaturability within cell nucleus or in individual chromosomes. For instance, it was not possible to detect the initial points of chromosome condensation in G2-phase of the division cycle or in apoptosis. In order to achieve this goal we have recently extended these studies by applying confocal microscopy. We investigated DNA denaturability in normal human fibroblasts and HL-60 leukemic cells, at different stages of cell cycle and apoptosis. Following removal of RNA and partial denaturation of DNA with acid cells were stained with AO. Green (530 nm) and red (640 nm) fluorescence (exc. 457 nm) of non-denatured and denatured DNA was imaged by confocal microscopy. Blind deconvolution was used to further improve the quality of 3D images. Photobleaching of AO fluorescence was minimized and a correction for chromatic aberration and register shift was implemented. Nuclei of interphase cells exhibited predominantly green fluorescence representing AO binding to ds DNA. Punctuate areas of red fluorescence representing AO binding to denatured DNA and most likely associated with local regions of condensed chromatin were also present in all interphase nuclei. The proportion of denatured DNA increased in cells entering mitosis. In prophase individual condensing chromosomes exhibited varied proportions of green and red fluorescence indicating different content of denatured chromatin. In some chromosomes bands of denatured and denaturation-resistant chromatin were clearly resolved. In metaphase and anaphase chromosomes exhibited red fluorescence along all length of their arms indicating the highest and uniform susceptibility to denaturation. In telophase chromosomes contained predominantly denaturation-resistant DNA again and denaturated regions were significantly less abundant. At cytokinesis some decondensing chromosomes were still resolved. At this stage almost all regions of denatured DNA were located close to nuclear envelope. These regions may correspond to pockets of heterochromatin reforming at nuclear periphery. In early apoptosis condensation of chromatin appeared to commence in several distinct regions within nucleus. Some apoptotic bodies contained condensed chromatin surrounding central regions of extended chromatin. At late stages of apoptosis the whole volume of apoptotic bodies was occupied by condensed chromatin.     

Heterochromatin and rDNA 5S and 45S sites as reliable cytogenetic markers for castor bean (Ricinus communis,Euphorbiaceae)

The increasing need for renewable energy resources has led to higher demands for biofuel, a scenario where the castor bean (Ricinus communis L.) seed oil represents a promising source of raw material. Despite that, information regarding the genome organization of R. communis is still scarce, impairing the application of modern biotechnological and breeding procedures. The present work brings the first evaluation of the mitotic chromosomes of this species, including 10 potentially interesting accessions for cultivation in semi-arid environments aiming at the biofuel production. The approach included standard staining, fluorochrome staining (CMA/DAPI), fluorescent in situ hybridization (FISH) with rDNA 5S and 45S, as well as silver impregnation. All accessions were diploid with 2n = 2x = 20, displaying mainly metacentric chromosomes, with CMA-positive bands (GC-rich) in all pairs of the complement. After silver impregnation, one to 14 nucleoli were observed, while the FISH with rDNA 45S revealed two large sites and a variety of minor dots, and the DNAr 5S hybridized in a single pair. The observed features were discussed and compared with literature data regarding pachytene bivalents.     

Cytogenetic analysis of two Coprophanaeus species (Scarabaeidae) revealing wide constitutive heterochromatin variability and the largest number of 45S rDNA sites among Coleoptera

The Coleopterans of Scarabaeinae clade presents Coprophanaeus (Megaphanaeus) ensifer and C. (Coprophanaeus) cyanescens (Scarabaeidae) when they are studied cytogenetically by different techniques. The species present symmetric karyotypes, diploid number of 2n = 20, and meta-submetacentric chromosomes. C. (M.) ensifer present an XY sex-determining mechanism and C. (C.) cyanescens an XY_p parachute mechanism. Analysis of constitutive heterochromatin (CH) in the two species revealed the presence of diphasic autosomes, with log arm heterochromatics. Moreover, an additional heterochromatic block in four autosomal bivalents were observed in C. (M.) ensifer. CMA₃/DA/DAPI fluorochrome staining detected CMA₃ positive heterochromatic blocks restricted to the sex chromosomes in C. (C.) cyanescens, whereas in C. (M.) ensifer CMA₃ positive pericentromeric blocks were present in all autosomes, in the Y chromosome and in the four additional heterochromatic blocks. DAPI staining was neutral in both species. Silver nitrate (AgNO₃) staining was inefficient for the detection of the nucleolar organizer region (NORs), but showed affinity for the heterochromatic regions. Fluorescence in situ hybridization (FISH) revealed the presence of 45S rDNA sites in the terminal region of the three autosomal bivalents of C. (C.) cyanescens and in seven bivalents and the Y chromosome of C. (M.) ensifer. These results contribute to a better understanding of chromosome evolution in the genus Coprophanaeus, and demonstrate a wide CH variability and the largest number of ribosomal sites among Coleoptera.     

Molecular cytogenetics of Salminus fish (Characiformes) based on 5S and 18S rRNA genes hybridization, fluorochrome staining and C-banding

Karyotype, mapping of nucleolar and 5S rRNA genes and distribution of constitutive heterochromatin supposedly AT-rich were characterized on two isolate populations of Salminus brasiliensis, the biggest characid fish, and three population of Salminus hilarii. The diploid number 2n=50 and the karyotype formulae (10M+20SM+20ST/A) were the same to Salminus species studied. The position of 18S rDNA cluster identified by FISH coincide with chromomycin A(3) labeling (CMA(+)) in the long arm telomeric portion of sixth pair. Subtle differences for the disposal of the 5S rRNA gene in the chromosome of the Salminus are presented. The distribution of the constitutive heterochromatins and DA/DAPI(+) bands are described.     

Cytogenetics of the land snails Cantareus aspersus and C. mazzullii (Mollusca: Gastropoda: Pulmonata)

A cytogenetic study was carried out on the chromosomes and nuclear DNA contents of the land snails Cantareus aspersus and C. mazzullii (Gastropoda: Pulmonata). Chromosomes were studied using Giemsa staining, banding methods and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [18S rDNA, (GATA)(n) and (TTAGGG)(n)]. Results were very similar in the two species both showing (1) 54 bi-armed chromosomes [submetacentrics (SM) + metacentrics (M) + subtelocentrics (ST)]; (2) 10 terminal NORs after sequential application of rDNA FISH and silver staining; (3) uniform DNA fluorescence with CMA(3) and DAPI staining and (4) genomic composition considerably enriched both in highly- and moderately-repeated DNAs. The telomeric (TTAGGG)(n) sequence hybridized with the termini of all of the chromosomes in the two species. In spite of their apparent karyological uniformity, flow cytometry DNA assays showed that C. aspersus and C. mazzullii are characterized by different nuclear DNA content (C values are 3.58 and 3.08 pg, respectively) and slightly different base composition in their genomes. Present data on GS and AT% in C. mazzullii and C. aspersus confirm the trend toward high GS values and GC percentages among land snails.     

A conserved karyotype of Sternopygus macrurus (Sternopygidae, Gymnotiformes) in the Amazon region: differences from other hydrographic basins suggest cryptic speciation

We studied the karyotypes of 35 Sternopygus macrurus fishes of four localities from rivers of the Eastern Amazon basin. In these four places the karyotypes have 2n = 46 chromosomes, NF = 92, where 30 are metacentric (M) and 16 submetacentric (SM). The constitutive heterochromatin (CH) is found in the centromeric region of most chromosomes and in the pericentromeric region of pairs 5, 17 and 19. Pair 1 has a large and not common heterochromatic block in the short arm, useful as a marker for this species if not found in other Sternopygus taxa. The NOR is located in the distal region of the short arm of pair 1, showing a size heteromorphism in some specimens. The CMA₃ and DAPI fluorochrome bandings and the fluorescent in situ hybridization (FISH), using pantelomeric human probes techniques are described for the first time for this species. DAPI has banding coincident with the C-banded regions, which suggests that the CH is AT base-pair-rich. CMA₃ banding is coincident with the NOR, meaning that this region is GC base-pair-rich. The FISH showed that the probes hybridized only with the telomeric regions, without any sign of interstitial telomeric regions. The karyotype of the samples from different places in the Amazon basin is quite conserved, probably because of the gene flow among the populations. The karyotype differences among the Sternopygus macrurus from the Amazon basin and the São Francisco and Paraná rivers suggest that these taxa may be different species.     

Repetitive DNAs in the slug Milax nigricans: association of ribosomal (18S-28S and 5S rDNA) and (TTAGGG)n telomeric sequences in the slug M. nigricans (Mollusca: Gastropoda: Pulmonata)

Spermatocyte chromosomes of the slug Milax nigricans (Mollusca: Gastropoda: Pulmonata) were studied using silver staining (Ag-NOR) and fluorescent in situ hybridization (FISH) with four repetitive DNA probes [18S rDNA, 5S rDNA, (TTAGGG)n and (GATA)n]. Silver impregnation was inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) since no silver dots occurred on the chromosomes at spermatogonial metaphase and a diffuse silver stainability could be observed on the bivalents at metaphase-I. Unlike silver staining, single-colour rDNA FISH consistently mapped major ribosomal sites (18S-28S rDNA) on two small-sized chromosomes in spermatogonial cells and on the correspondent metaphase-I bivalent in spermatocytes. While telomeric (TTAGGG)n sequence hybridized to all chromosomes, (GATA)n probe localized abundant hybridization sites, dispersed throughout the genome. Simultaneous double-colour FISH demonstrated a close chromosomal association of 18S-28S rDNA, 5S rDNA and (TTAGGG)n.     

Distribution of 5S and 45S rDNA sites in plants with holokinetic chromosomes and the "chromosome field" hypothesis

Secondary constrictions or 45S rDNA sites are commonly reported to be located mainly in the terminal regions of the chromosomes. This distribution has been assumed to be related to the existence of a "chromosome field" lying between the centromere and the telomere, an area in which certain cytogenetic events may predominantly occur. If this hypothesis is true this distribution should not be observed in holokinetic chromosomes, as they do not have a localized centromere. In order to evaluate this hypothesis, a comparative study was made of the distributions of 5S and 45S rDNA sites using fluorescence in situ hybridization in representatives of the genera Eleocharis, Diplacrum, Fimbristylis, Kyllinga and Rhynchospora, all of which belong to the family Cyperaceae. The numbers of sites per diploid chromosome complement varied from 2 to ～10 for 5S rDNA, and from 2 to ～45 for 45S rDNA. All of the 11 species analyzed had terminally located 45S rDNA sites on the chromosomes whereas the 5S rDNA sites also generally had terminal distributions, except for the Rhynchospora species, where their position was almost always interstitial. These results, together with other previously published data, suggest that the variation in the number and position of the rDNA sites in species with holokinetic chromosomes is non-random and similar to that reported for species with monocentric chromosomes. Therefore, the predominant terminal position of the 45S rDNA sites does not appear to be influenced by the centromere-telomere polarization as suggested by the "chromosome field" hypothesis. Additionally, the hybridization of 5S and 45S rDNA sites provides interesting markers to distinguish several chromosomes on the rather symmetrical karyotypes of Cyperaceae.     

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug

Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA(3)/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.     

Dynamic changes of nucleolar DNA configuration and distribution during the cell cycle in Allium sativum cells

To investigate the correlation between subnucleolar structure and function, the precise distribution and configuration of nucleolar DNA during the cell cycle of Allium sativum were determined using the NAMA-Ur DNA-specific staining technique. We showed that nucleolar DNA is present in two forms: compacted chromatin clumps and a decondensed DNA cloud. The form of the DNA within the nucleolus varied greatly as the cell cycle progressed. During telophase, chromosomes extended into the prenucleolar body. In early G1 phase, DNA was only located in the fibrillar centers in the form of the condensed chromatin clump, while in mid-G1, S and G2 phases, the two forms of DNA were distributed in the fibrillar centers (FC) and dense fibrillar component (DFC). In prophase of mitosis, nucleolar DNA, along with FC and DFC, was linked into a network structure and condensed into a large chromatin clump. The area of the DNA cloud in the dense fibrillar component changed during different phases of the cell cycle. Our results demonstrated that the configuration of nucleolar DNA undergoes a series of decondensations and condensations during the cell cycle to fulfill the function of the nucleoli during the different phases.     

Electron microscopy and atomic force microscopy studies of chromatin and metaphase chromosome structure

The folding of the chromatin filament and, in particular, the organization of genomic DNA within metaphase chromosomes has attracted the interest of many laboratories during the last five decades. This review discusses our current understanding of chromatin higher-order structure based on results obtained with transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and different atomic force microscopy (AFM) techniques.Chromatin isolated from different cell types in buffers without cations form extended filaments with nucleosomes visible as separated units. In presence of low concentrations of Mg²⁺, chromatin filaments are folded into fibers having a diameter of ∼30 nm. Highly compact fibers were obtained with isolated chromatin fragments in solutions containing 1–2 mM Mg²⁺. The high density of these fibers suggested that the successive turns of the chromatin filament are interdigitated. Similar results were obtained with reconstituted nucleosome arrays under the same ionic conditions. This led to the proposal of compact interdigitated solenoid models having a helical pitch of 4–5 nm. These findings, together with the observation of columns of stacked nucleosomes in different liquid crystal phases formed by aggregation of nucleosome core particles at high concentration, and different experimental evidences obtained using other approaches, indicate that face-to-face interactions between nucleosomes are very important for the formation of dense chromatin structures.Chromatin fibers were observed in metaphase chromosome preparations in deionized water and in buffers containing EDTA, but chromosomes in presence of the Mg²⁺ concentrations found in metaphase (5–22 mM) are very compact, without visible fibers. Moreover, a recent cryo-electron microscopy analysis of vitreous sections of mitotic cells indicated that chromatin has a disordered organization, which does not support the existence of 30-nm fibers in condensed chromosomes. TEM images of partially denatured chromosomes obtained using different procedures that maintain the ionic conditions of metaphase showed that bulk chromatin in chromosomes is organized forming multilayered plate-like structures. The structure and mechanical properties of these plates were studied using cryo-EM, electron tomography, AFM imaging in aqueous media, and AFM-based nanotribology and force spectroscopy. The results obtained indicated that the chromatin filament forms a flexible two-dimensional network, in which DNA is the main component responsible for the mechanical strength observed in friction force measurements. The discovery of this unexpected structure based on a planar geometry has opened completely new possibilities for the understanding of chromatin folding in metaphase chromosomes. It was proposed that chromatids are formed by many stacked thin chromatin plates oriented perpendicular to the chromatid axis. Different experimental evidences indicated that nucleosomes in the plates are irregularly oriented, and that the successive layers are interdigitated (the apparent layer thickness is 5–6 nm), allowing face-to-face interactions between nucleosomes of adjacent layers. The high density of this structure is in agreement with the high concentration of DNA observed in metaphase chromosomes of different species, and the irregular orientation of nucleosomes within the plates make these results compatible with those obtained with mitotic cell cryo-sections. The multilaminar chromatin structure proposed for chromosomes allows an easy explanation of chromosome banding and of the band splitting observed in stretched chromosomes.     

Cytogenetics of four Omophoita species (Coleoptera,Chrysomelidae, Alticinae): A comparative analysis using mitotic and meiotic cells submitted to the standard staining and C-banding technique

Omophoita belongs to the tribe Oedionychini and is endemic from Neotropical region. The species of the tribe Oedionychini have revealed certain singular chromosomal features, such as sex chromosomes with extremely large size, asynapsis, and synthelic or amphithelic orientation during meiosis. Additionally, some species also showed post-reductional segregation of the gigantic sex chromosomes in meiotic division. The purpose of this work was to characterize cytogenetically four Omophoita species (O. magniguttis, O. octoguttata, O. personata, and O. sexnotata) in relation to their diploid number, chromosomal morphology, type of sex chromosome system, and constitutive heterochromatin pattern in mitotic and meiotic cells, and compare the obtained data with those of related species to establish the mechanism involved in the chromosomal differentiation of these species during the evolutionary process. The diploid number, 2n = 22 = 20 + X + y, and meiotic formulae, 10II + X + y, observed in these species were similar to those of the same genus and other species related. The autosomal morphology was acrocentric in O. magniguttis and O. octoguttata, metacentric in O. personata, and predominantly metacentric in O. sexnotata. In all these species, the sex chromosomes were metacentric. The secondary constriction occurred in pair 6 and X chromosome of O. personata, and in pair 6 and y chromosome of O. sexnotata. The constitutive heterochromatin was pericentromeric in O. magniguttis and centromeric in O. sexnotata, with the exception of the mitotic sex chromosomes of O. sexnotata, in which centromeric C band was lacking. Additional C bands in the sex chromosomes of O. magniguttis and certain autosomes and sex chromosomes of O. sexnotata were observed. Collochores were indirectly identified in the spermatocytes of O. octoguttata, O. personata, and O. sexnotata. The main mechanisms involved in the karyotype evolution of these species were discussed.     

Specific Mg binding to AT-rich regions of chromatin in the evolution of eukaryotes

At SIMS XIV, we reported SIMS evidence of specific Mg²⁺ binding to the AT-rich regions of human metaphase chromosomes represented by G-bands. Subsequent Mg²⁺-depletion experiments supported a direct role for Mg²⁺ in promoting and maintaining the higher order chromatin structure originating G-bands, possibly due to both Mg²⁺-DNA and Mg²⁺-protein interactions. An in-depth study, reported elsewhere, implicated also Ca²⁺ in the maintenance of chromatin ultrastructure in the scaffold of mammalian chromosomes, in association with topoisomerase II.We examine here the association of Mg²⁺ with AT-rich regions of chromatin in the chromosomes of the Indian muntjac deer (IMD), leading to conclusions similar to the above. To answer the question whether the presumed divalent cation role in the chromosomes of advanced eukaryotes had an evolutionary history to be traced back to earlier evolutionary stages, we have SIMS-mapped Ca²⁺ and Mg²⁺ in BrdU-labeled polytene chromosomes from the salivary gland of the Dipteran Drosophila melanogaster. Striking Ca²⁺ and Mg²⁺ SIMS banding patterns correlating with those of the Br label (a thymidine analogue) implicate unequivocally a close association of both these cations with the AT-rich regions of DNA for these primitive eukaryotes.     

Recent advances in rice genome and chromosome structure research by fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) is an effective method for the physical mapping of genes and repetitive DNA sequences on chromosomes. Physical mapping of unique nucleotide sequences on specific rice chromosome regions was performed using a combination of chromosome identification and highly sensitive FISH. Increases in the detection sensitivity of smaller DNA sequences and improvements in spatial resolution have ushered in a new phase in FISH technology. Thus, it is now possible to perform in situ hybridization on somatic chromosomes, pachytene chromosomes, and even on extended DNA fibers (EDFs). Pachytene-FISH allows the integration of genetic linkage maps and quantitative chromosome maps. Visualization methods using FISH can reveal the spatial organization of the centromere, heterochromatin/euchromatin, and the terminal structures of rice chromosomes. Furthermore, EDF-FISH and the DNA combing technique can resolve a spatial distance of 1 kb between adjacent DNA sequences, and the detection of even a 300-bp target is now feasible. The copy numbers of various repetitive sequences and the sizes of various DNA molecules were quantitatively measured using the molecular combing technique. This review describes the significance of these advances in molecular cytology in rice and discusses future applications in plant studies using visualization techniques.     

The absorption spectrum of 12C2H2 between 12800 and 18500cm−1 II. Rotational analysis

The rotational structure of the vibrational bands of ¹²C₂H₂ is investigated in three spectral energy regions not previously systematically explored at high resolution, 12800–13500 cm^?1, 14000–15200 cm^?1 and 16500–18360 cm^?1, on the basis of new spectral data recorded by intracavity laser absorption spectroscopy. The rotational analysis of 17 new absorption bands arising from the ground state is reported (11 Σ_u ⁺ ? Σ_g ⁺ bands and 6Π_u ? Σ_g ⁺ bands). Four bands in the range studied show strong perturbations affecting both the line positions and intensities. Their detailed analysis is performed in order to determine the nature of the coupling schemes, the vibrational species and the rotational constants of the perturber states. Altogether, the vibration-rotation parameters of 21 newly observed vibrational states are derived.