Longitudinal differentiation in Melipona mandacaia (Hymenoptera,Meliponini) chromosomes

Melipona mandacaia is a stingless bee endemic to northeast Brasil. We describe the M. mandacaia karyotype using C-banding technique. fluorochrome staining and treatment with restriction enzymes and discuss the position of this species in the context of the phylogeny of the genus. Melipona mandacaia has 2n = 18 (14 SM + 2 M + 2 A). Heterochromatin was detected in the pericentromeric region of pairs 1, 2 and 8 and in the form of small blocks in the remaining pairs. Staining with base-specific fluorochromes showed that this heterochromatin was rich AT (QM and DAPI), except in the region corresponding to the NOR which was rich GC (CMA3) and was cleaved by the HaeIII enzyme. Melipona mandacaia is a member of Group I Melipona. Treatment with DraI/Giemsa discloses a larger number of bands than treatment with DraI/QM. Pre-cleavage with DraI gave rise to a larger number of bands following QM staining; a circumstance evidently due to a removal of the DNA-protein complex that prevented the association of the fluorochrome with AT-rich DNA. The results highlight the complex nature of heterochromatin.     

DNA methylation in stingless bees with low and high heterochromatin contents as assessed by restriction enzyme digestion and image analysis.

BACKGROUND: The stingless bee genus Melipona has been divided into two groups, based on their heterochromatin content. Melipona quadrifasciata and Melipona rufiventris have low and high levels of heterochromatin, respectively. Since condensed chromatin may be rich in methylated DNA sequences, M. quadrifasciata and M. rufiventris nuclei may contain different amounts of methylated CpG. These differences could be assessed by comparing Feulgen-DNA values obtained by image analysis of cells treated with the restriction enzymes Msp I and Hpa II that distinguish between methylated and unmethylated DNA. Msp I and Hpa II cleave the sequence -CCGG-, but there is no cleavage by Hpa II if the cytosine of the central CG dinucleotide is methylated. METHODS: Malpighian tubules of M. quadrifasciata and M. rufiventris were treated with Msp I and Hpa II prior to the Feulgen reaction, and analyzed by automatic scanning microspectrophotometry. RESULTS: The Feulgen-DNA values for the heterochromatin of M. rufiventris and for the small heterochromatin and some euchromatin domains of M. quadrifasciata mostly decreased after treatment with Msp I, but were unchanged after treatment with Hpa II. CONCLUSION: CpG methylation, although detected in diverse chromatin compartments in different bee species, may induce silencing effects required for the same cell physiology.     

Cytogenetic study in Corydoras britskii (Siluriformes: Callichthyidae), using different chromosomal banding and fluorescence hybridization in situ with rDNA probes

Cytogenetic analyses were performed on Corydoras britskii from the Miranda River basin, an important river located in the Pantanal, Mato Grosso do Sul State, Brazil. The karyotype of this species comprises 90 chromosomes and a karyotype formula of 4m + 10 sm + 22 st + 54a. The nucleolus organizer regions were detected by impregnation with silver nitrate and FISH with an 18S rDNA probe on the short arm of three acrocentric chromosomes. The constitutive heterochromatin is distributed in pericentromeric and interstitial positions, and also associated with the NORs. The HinfI restriction endonuclease was used and showed homology with practically all types of heterochromatin observed in C. britskii, except for two interstitial heterochromatic blocks present in a subtelocentric pair. The fluorochrome staining evidenced six chromosomes with chromomycin-positive signals indicating that both the heterochromatin interspersed with NORs and some heterochromatic blocks were rich in GC base pairs. FISH using a 5S rDNA probe revealed the presence of these regions in only one subtelocentric pair in the interstitial position. The obtained data substantiate the karyotype diversity of the genus Corydoras and provide novel information about the composition of heterochromatin and location of 5S and 18S rDNA sites.     

Differential sensitivity of constitutive and facultative heterochromatin in orthopteran chromosomes to digestion by DNaseI

In situ pancreatic DNaseI digestions were used as probes to study the structural organization of facultative and constitutive heterochromatin during both mitotic and meiotic divisions. Three different types of heterochromatic regions from three insect species were chosen for this study. These regions had been previously characterized by in situ treatments with restriction endonucleases (AT and GC rich DNA sequences). Progressive increase in DNaseI concentration (from 10 to 200 ng/ml) or in incubation time (from 5 to 30 min) revealed a specific pattern of sequential digestion of the constitutive heterochromatic regions, the centromeric ones (AT-rich DNA) being the most resistant to DNaseI action. The interstitial C-bands (with AT or GC-rich DNA) were more sensitive to DNaseI, and the band 4.4 from Baetica ustalata was the most resistant of the non-centromeric bands. Similar results were obtained during meiosis, but increased accessibility to DNAseI was observed compared to mitosis. DNA methylation in the non-centromeric band 4.4 of B. ustulata could be responsible for its differential digestion with respect to the remaining intercalar heterochromatin. Facultatively heterochromatic regions (X chromosomes) were found to exhibit a differential response to DNaseI attack from mitosis to meiosis. While they behaved as cuchromatin during mitosis, they were the most resistant together with centromeric heterochromatin regions, during metaphase I and II. The different responses to digestion of the X chromosome and X-derived regions between somatic and meiotic divisions are probably a consequence of the changes in the organization of this chromosome during the facultative heterochromatinization process.     

Restriction enzyme and fluorochrome banding analysis of the constitutive heterochromatin of Saguinus species (Callitrichidae, Primates)

Metaphases of Saguinas fuscicollis fuscicollis and Saguinas mystax were subjected to restriction enzyme banding (Alu I, Hae III, Hin fI, Rsa I, Dde I, Mbo I and Msp I) and sequenced C-banding, together with fluorochrome staining (CMA3 and DAPI). Both species showed large C-bands in the pericentromeric regions. S. f. fuscicollis also manifested distal C-bands in both arms of pair 5 and in the short arms of pairs 8-15. In each species the heterochromatin revealed different reactions to the restriction enzymes and fluorochromes. This was related to its location in the genome (centromeric, pericentromeric, distal), making possible the identification of distinct categories of constitutive heterochromatin. In S. f. fuscicollis there were at least five types, namely centromeric in bi-armed chromosomes, centromeric in acrocentrics, pericentromeric, distal, and cryptic bands, detected only with the Alu I. There were three types in S. mystax, viz centromeric in bi-armed chromosomes, centromeric in acrocentric, and pericentromeric chromosomes. Several aspects of their constitution and origin are discussed.     

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.     

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.     

Homolog pairing and sister chromatid cohesion in heterochromatin in <Emphasis Type="Italic">Drosophila</Emphasis> male meiosis I

Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X–Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.     

Molecular cytogenetics and characterization of a ZZ/ZW sex chromosome system in Triportheus nematurus (Characiformes, Characidae)

Chromosomes of Triportheus nematurus, a fish species from family Characidae, were analyzed in order to establish the conventional karyotype, location of C-band positive heterochromatin, Ag-NORs, GC- and AT-rich sites, and mapping of 18S and 5S rDNA with fluorescence in situ hybridization (FISH). The diploid number found was 2n = 52 chromosomes in both males and females. However, the females presented a pair of differentiated heteromorphic chromosomes, characterizing a ZZ/ZW sex chromosome system. The Z chromosome was metacentric and the largest one in the karyotype, bearing C-positive heterochromatin at pericentromeric and telomeric regions. The W chromosome was middle-sized submetacentric, appearing mostly heterochromatic after C-banding and presenting heterogeneous heterochromatin composed of GC- and AT-rich regions revealed by fluorochrome staining. Ag-NORs were also GC-rich and surrounded by heterochromatic regions, being located at the secondary constriction on the short arms of the second chromosome pair, in agreement with 18S rDNA sites detected with FISH. The 18S and 5S rDNA were aligned in tandem, representing an uncommon situation in fishes. The results obtained reinforce the basal condition of the ZZ/ZW sex system in the genus Triportheus, probably arisen prior to speciation in the group.     

Cytogenetic analysis of three species of Pseudacteon (Diptera, Phoridae) parasitoids of the fire ants using standard and molecular techniques

Pseudacteon flies, parasitoids of worker ants, are being intensively studied as potentially effective agents in the biological control of the invasive pest fire ant genus Solenopsis (Hymenoptera: Formicidae). This is the first attempt to describe the karyotype of P. curvatus Borgmeier, P. nocens Borgmeier and P. tricuspis Borgmeier. The three species possess 2n = 6; chromosomes I and II were metacentric in the three species, but chromosome pair III was subtelocentric in P. curvatus and P. tricuspis, and telocentric in P. nocens. All three species possess a C positive band in chromosome II, lack C positive heterochromatin on chromosome I, and are mostly differentiated with respect to chromosome III. P. curvatus and P. tricuspis possess a C positive band, but at different locations, whereas this band is absent in P. nocens. Heterochromatic bands are neither AT nor GC rich as revealed by fluorescent banding. In situ hybridization with an 18S rDNA probe revealed a signal on chromosome II in a similar location to the C positive band in the three species. The apparent lack of morphologically distinct sex chromosomes is consistent with proposals of environmental sex determination in the genus. Small differences detected in chromosome length and morphology suggests that chromosomes have been highly conserved during the evolutionary radiation of Pseudacteon. Possible mechanisms of karyotype evolution in the three species are suggested.     

Composition,Antibiofilm, and Antibacterial Potential of Volatile Oils from Geopropolis of Different Stingless Bees’ Species**

We aimed to characterize and investigate the antibacterial potential of the native stingless bees geopropolis volatile oils (VO) for the search of potentially new bioactive compounds. Geopropolis samples from Melipona bicolor schencki, M. compressipes manaosensis, M. fasciculata, M. quadrifasciata, M. marginata and M. seminigra merrillae were collected from hives in South Brazil. VO were obtained by hydrodistillation and characterised by gas chromatography coupled to mass spectrometry (GC/MS). Antimicrobial activity was assessed by microplate dilution method. The lowest MIC against cell walled bacteria was 219±0 μg mL⁻¹ from M. quadrifasciata geopropolis VO with Staphylococcus aureus. The M. b. schencki geopropolis VO minimal inhibition concentration (MIC) was 424±0 μg mL⁻¹ against all the mycoplasma strains evaluated. Fractionation resulted in the reduction of 50 % of the MIC value from the original oil. However, its compounds’ synergism seems to be essential to this activity. Antibiofilm assays demonstrated 15.25 % eradication activity and 13.20 % inhibition of biofilm formation after 24 h for one subfraction at 2× its MIC as the best results found. This may be one of the essential mechanisms by which geopropolis VOs perform their antimicrobial activity.     

Counterstained enhancement of TaqI resistant sites after distamycin A/diamidinophenylindole treatment

Numerous selective and differential staining techniques have been used to investigate the hierarchical organisation of the human genome. This investigation demonstrates the unique characteristics that are produced on fixed human chromosomes when sequential procedures involving restriction endonuclease TaqI, distamycin A (DA) and 4,6-diamidino-2-phenylindole (DAPI) are employed. TaqI produces extensive gaps in the heterochromatic regions associated with satellite II and III DNAs of human chromosomes 1, 9, 15, 16 and Y. DA/DAPI selectively highlights, as brightly fluorescent C-bands, the heterochromatin associated with the alpha, beta, satellite II and III DNAs of these chromosomes. When DA and DAPI are used on chromosomes before TaqI digestion, and then stained with Giemsa, the centromeric regions appear to be more resistant, producing a distinct C-banding pattern and gaps in the heterochromatin regions. Sequential use of the DA/DAPI technique after TaqI treatment produces a bright fluorescence on the remaining pericentromeric regions of chromosomes 1, 9, 16 and Y, which also displayed a cytochemically unique banding pattern. This approach has produced specific enhanced chromosomal bands, which may serve as tools to characterize genomic heterochromatin at a fundamental level.     

Human (Homo sapiens) and chimpanzee (Pan troglodytes) share similar ancestral centromeric alpha satellite DNA sequences but other fractions of heterochromatin differ considerably

The euchromatic regions of chimpanzee (Pan troglodytes) genome share approximately 98% sequence similarity with the human (Homo sapiens), while the heterochromatic regions display considerable divergence. Positive heterochromatic regions revealed by the CBG-technique are confined to pericentromeric areas in humans, while in chimpanzees, these regions are pericentromeric, telomeric, and intercalary. When human chromosomes are digested with restriction endonuclease AluI and stained by Giemsa (AluI/Giemsa), positive heterochromatin is detected only in the pericentromeric regions, while in chimpanzee, telomeric, pericentromeric, and in some chromosomes both telomeric and centromeric, regions are positive. The DA/DAPI technique further revealed extensive cytochemical heterogeneity of heterochromatin in both species. Nevertheless, the fluorescence in situ hybridization technique (FISH) using a centromeric alpha satellite cocktail probe revealed that both primates share similar pericentromeric alpha satellite DNA sequences. Furthermore, cross-hybridization experiments using chromosomes of gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) suggest that the alphoid repeats of human and great apes are highly conserved, implying that these repeat families were present in their common ancestor. Nevertheless, the orangutan's chromosome 9 did not cross-hybridize with human probe. The euchromatic regions of chimpanzee (Pan troglodytes) genome share approximately 98% sequence similarity with the human (Homo sapiens), while the heterochromatic regions display considerable divergence. Positive heterochromatic regions revealed by the CBG-technique are confined to pericentromeric areas in humans, while in chimpanzees, these regions are pericentromeric, telomeric, and intercalary. When human chromosomes are digested with restriction endonuclease AluI and stained by Giemsa (AluI/Giemsa), positive heterochromatin is detected only in the pericentromeric regions, while in chimpanzee, telomeric, pericentromeric, and in some chromosomes both telomeric and centromeric, regions are positive. The DA/DAPI technique further revealed extensive cytochemical heterogeneity of heterochromatin in both species. Nevertheless, the fluorescence in situ hybridization technique (FISH) using a centromeric alpha satellite cocktail probe revealed that both primates share similar pericentromeric alpha satellite DNA sequences. Furthermore, cross-hybridization experiments using chromosomes of gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) suggest that the alphoid repeats of human and great apes are highly conserved, implying that these repeat families were present in their common ancestor. Nevertheless, the orangutan's chromosome 9 did not cross-hybridize with human probe. © 1995 Wiley-Liss, Inc.     

Reduced meiotic fitness in hybrids with heterozygosity for heterochromatin in the speciating<Emphasis Type="Italic">Mus terricolor</Emphasis> complex

Mus terricolor I, II and III are the three chromosomal species which differ in stable autosomal short-arm heterochromatin variations established in homozygous condition. Analysis of meiosis in the laboratory-generated F₁ male hybrids from crosses (both ways) betweenM. terricolor I and II and betweenM. terricolor I and III shows high frequencies of pairing abnormalities at pachytene. The backcross (N3 generation) male hybrids betweenM. terricolor I and II have meiotic abnormalities as in the F₁male hybrids, though to a lesser extent. They show difference in pairing abnormalities in the different karyotypic forms; the backcross hybrids heterozygous for the heterochromatic short arms have more anomalies compared to the homokaryotypic hybrids. This suggests a negative influence of the heterochromatin heterozygosity in meiotic pairing. The results indicate a role for heterochromatin variations in the development of a reproductive barrier in the speciatingM. terricolor complex.     

Cytogenetic analysis of three species of the genus <Emphasis Type="Italic">Haemulon</Emphasis> (Teleostei: Haemulinae) from Margarita Island,Venezuela

This paper describes the karyotype analysis of Haemulon aurolineatum, Haemulon bonariensis and Haemulon plumierii, by Giemsa staining, C-banding, Ag-staining and fluorescent in situ hybridization (FISH), to locate the 18S and 5S rRNA genes. Diploid modal count in the three species was 2n = 48 acrocentric elements. Except for pair 24, which exhibited an unmistakable secondary constriction in all three species, it was not possible to classify them as homologous to each other because differences in chromosome size were too slight between adjacent pairs within a size-graded series. Ag-NOR clusters were located in pair 24 in the three species with signal located on the secondary constriction of these chromosomes. C-banding demonstrated that the three species share the same distribution pattern of the constitutive heterochromatin with centromeric heterochromatic blocks in the 23 chromosome pairs and a pericentromeric block in pair 24 which is coincident with the NORs. FISH experiments showed that 18S rDNA sequences were located coincident with the Ag-NOR site in the three species; however, differences in both the number and chromosome distribution of 5S-rDNA cluster were detected among them. Our data suggest that chromosome evolution of Haemulon has been preserved from major changes in the karyotypic macrostructure, whereas microstructural changes have occurred.     

Epigenetic profiling of heterochromatic satellite DNA

Sugar beet (Beta vulgaris) chromosomes consist of large heterochromatic blocks in pericentromeric, centromeric, and intercalary regions comprised of two different highly abundant DNA satellite families. To investigate DNA methylation at single base resolution at heterochromatic regions, we applied a method for strand-specific bisulfite sequencing of more than 1,000 satellite monomers followed by statistical analyses. As a result, we uncovered diversity in the distribution of different methylation patterns in both satellite families. Heavily methylated CG and CHG (H=A, T, or C) sites occur more frequently in intercalary heterochromatin, while CHH sites, with the exception of CAA, are only sparsely methylated, in both intercalary and pericentromeric/centromeric heterochromatin. We show that the difference in DNA methylation intensity is correlated to unequal distribution of heterochromatic histone H3 methylation marks. While clusters of H3K9me2 were absent from pericentromeric heterochromatin and restricted only to intercalary heterochromatic regions, H3K9me1 and H3K27me1 were observed in all types of heterochromatin. By sequencing of a small RNA library consisting of 6.76 million small RNAs, we identified small interfering RNAs (siRNAs) of 24 nucleotides in size which originated from both strands of the satellite DNAs. We hypothesize an involvement of these siRNAs in the regulation of DNA and histone methylation for maintaining heterochromatin.     

Localization of repetitive DNA sequences in the pericentromeric heterochromatin of malarial mosquitoes of the "Anopheles maculipennis" complex

Distribution of eight fragments of conserved repetitive DNA from pericentromeric heterochromatin of chromosome 2 of Anopheles atroparvus has been investigated by in situ hybridization on polytene chromosomes of An. atroparvus and An. messeae. We have shown that heterochromatic regions of all chromosomes both in An. atroparvus and An. messeae vary in combinations of, at least, conserved repeats. Some repeats have been found only in pericentromeric heterochromatic regions of chromosomes 2 (clones Atr2R-46a, Atr2R-73, Atr2R-85a in An. atroparvus and Atr2R-25 in An. messeae). Others have been found in two (clones Atr2R-25a and Atr2R-90 in An. atroparvus, Atr2R-25a in An. messeae) and more (clones Atr2R-118, Atr2R-136 in An. atroparvus, Atr2R-73 in An. messeae) pericentromeric heterochromatic regions of chromosomes. DNA comparison of pericentromeric heterochromatic regions of chromosomes in species of the "Anopheles maculipennis" complex is species- and chromosome-specific, due, in particular, to different maintenance of conserved repeates.