Karyotype analysis in Bignonieae (Bignoniaceae): chromosome numbers and heterochromatin

ABSTRACT Chromosome numbers and heterochromatin banding pattern variability have been shown to be useful for taxonomic and evolutionary studies of different plant taxa. Bignonieae is the largest tribe of Bignoniaceae, composed mostly by woody climber species whose taxonomies are quite complicated. We reviewed and added new data concerning chromosome numbers in Bignonieae and performed the first analyses of heterochromatin banding patterns in that tribe based on the fluorochromes chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI). We confirmed the predominant diploid number 2n = 40, as well as variations reported in the literature (dysploidy in Mansoa [2n = 38] and polyploidy in Dolichandra ungis-cati [2n = 80] and Pyrostegia venusta [2n = 80]). We also found a new cytotype for the genus Anemopaegma (Anemopaegma citrinum, 2n = 60) and provide the first chromosome counts for five species (Adenocalymma divaricatum, Amphilophium scabriusculum, Fridericia limae, F. subverticillata, and Xylophragma myrianthum). Heterochromatin analyses revealed only GC-rich regions, with six different arrangements of those bands. The A-type (one large and distal telomeric band) were the most common, although the presence and combinations of the other types appear to be the most promising for taxonomic studies.

Comparative cytogenetics in astyanax (characiformes: characidae) with focus on the cytotaxonomy of the group

Astyanax is a diverse group of Neotropical fishes, whose different forms occupy different environments. This great diversity is also reflected on cytogenetic aspects and molecular markers, which have repeatedly been demonstrated by cytogenetic studies. In order to characterize the karyotype of species of this genus, six species were studied: Astyanax altiparanae, A.argyrimarginatus, A. elachylepis, A. xavante, and two new species provisionally called Astyanax sp. and A. aff. bimaculatus. A detailed cytogenetic study based on conventional staining with Giemsa, AgNORs, C-banding, base-specific fluorochromes, and FISH using ribosomal genes 18S and 5S was conducted, aiming to understand some of the chromosomal mechanisms associated with the high diversification that characterizes this group and culminated with the establishment of these species. The results showed 2n = 50 chromosomes for five species and a karyotype with 52 chromosomes in Astyanax sp. Small variations in the macrostructure of the karyotypes were identified, which were quite relevant when analyzed by classical banding, fluorochromes, and FISH methods. These differences among Astyanax spp. (2n = 50) are largely due to changes in the amount and types of heterochromatic blocks. Astyanax sp (2n = 52), in addition to variations due to heterochromatic blocks, has its origin possibly by events of centric fission in a pair of chromosomes followed by minor rearrangements.These results show an interesting karyotypic diversity in Astyanax and indicate the need of a review of the group referred as A. aff. bimaculatus and the description of Astyanax sp., including the possibility of inclusion of this unit in another genus.

Astyanax é um grupo bastante diverso de peixes neotropicais cujas diferentes formas ocupam distintos ambientes. Esta grande variabilidade também se reflete em aspectos citogenéticos e moleculares, que têm sido repetidamente demonstrados por meio de estudos citogenéticos. A fim de caracterizar o cariótipo de representantes deste gênero, seis espécies foram estudadas: Astyanax altiparanae, A. elachylepis, A. xavante, A. argyrimarginatus e duas espécies novas provisoriamente citadas como Astyanax sp. e A. aff. bimaculatus. Um estudo citogenético detalhado com base na coloração convencional com Giemsa, AgNORs, banda C, fluorocromos base-específicos, e FISH com sondas para genes ribossomais 18S e 5S foi realizado com o objetivo de compreender alguns dos mecanismos cromossômicos associados com a alta diversificação que caracteriza este grupo de peixes e que culminou com o estabelecimento dessas espécies. Os resultados revelaram 2n = 50 cromossomos para cinco espécies e 2n = 52 cromossomos para Astyanax sp. Pequenas variações na macroestrutura dos cariótipos foram identificadas e se mostraram relevantes quando analisadas com base nos bandamentos clássicos, coloração por fluorocromos base-específicos e FISH com sondas de DNA 18S e 5S. Esssa diversidade cariotípica detectada indica a necessidade de uma revisão taxonômica no grupo de indivíduos aqui referidos como A. aff. bimaculatus, inclusive com a descrição de Astyanax sp., incluindo a possibilidade de inserção dessa unidade em outro gênero distinto de Astyanax.

Aplicaciones e inconvenientes de la técnica Hibridación in situ Fluorescente (FISH) en la identificación de microorganismos / Applications and inconvenient of Fluorescence in situ hybridization technique (FISH) in the identification of microorganism

Durante el transcurso de los últimos años se ha reportado un gran número de aplicaciones de la técnica FISH, la cual es utilizada en la detección de microorganismos en su propio hábitat sin que requieran de su previo aislamiento y purificación. La importancia de FISH radica en la capacidad que tiene la sonda de ADN de detectar una región específica del ácido nucleico de la célula microbiana y ser visualizada por microscopía de epifluorescencia. En esta revisión se describe los diversos usos que tiene FISH, que van desde la identificación de la microbiota en ambientes acuáticos y su empleo en la biorremediación hasta la detección de patógenos en el diagnóstico clínico. Asimismo, se presentan algunas limitaciones, y los posibles correctivos que se deben tener encuenta cuando se aplica esta técnica.

During these recent years, a large number of FISH technique applications have been reported. These techniques have been used in the detection of microorganisms in their own habitat without requiring their previous isolation and purification. The importance of FISH lies in the ability of the DNA probe to detect a specific region of the nucleic acid of microbial cells and to be visualized by epifluorescence microscopy. This review describes the various FISH uses ranging from the identification of the microbiota in aquatic environments and their use in bioremediation, to the detection of pathogens in clinical diagnosis. It also presents some limitations as well as the potential solutions to be applied when the FISH technique is used.

Empleo de la técnica hibridación in situ fluorescente para visualizar microorganismos / Use of fluorescence in situ hybridization technique to visualize microorganisms

La hibridación in situ fluorescente (FISH), es una técnica que emplea sondas de oligonucleótidos marcadas con fluorocromos las cuales van dirigidas hacia secuencias específicas del ácido ribonucleico ribosomal (ARNr), lo que permite la identificación rápida y específica de células microbianas ya sea que estén como células individuales o se encuentren agrupadas en su ambiente natural. El conocimiento de la composición y distribución de los microorganismos en los hábitats naturales, proporciona un soporte sólido para comprender la interacción entre las diversas especies que componen el micro hábitat. El objetivo de la revisión es presentar la forma como ha evolucionado la hibridación, el empleo del ARNr como molécula diana, los tipos de marcaje, los marcadores fluorescentes empleados hoy en día, la metodología, así como las mejoras que se le han hecho a la técnica FISH al emplearse en conjunto con otras técnicas en la identificación microbiana. Salud UIS 2011; 43 (3): 307-316.

Fluorescence in situ hybridization (FISH), is a technique that uses oligonucleotides probes labeled with fluorochromes which are directed to specific sequences of ribosomal ribonucleic acid (rRNA), this allows the rapid and specific identification of microbial cells whether as individual cells or grouped cells in their natural environment. Knowledge of the composition and distribution of microorganisms in natural habitats provides a solid support to understand interaction between different species in the microhabitat. This review shows how hybridization has evolved, the use of rRNA as target molecule, the type of labeling, the labeled uses today in fluorescent and the methodology, as well as the improvements that have been made to the FISH technique when is used in conjunction with other techniques in microbial identification. Salud UIS 2011; 43 (3): 307-316.

Comparative cytogenetic analysis of two grasshopper species of the tribe Abracrini (Ommatolampinae, Acrididae)

The grasshopper species Orthoscapheus rufipes and Eujivarus fusiformis were analyzed using several cytogenetic techniques. The karyotype of O. rufipes, described here for the first time, had a diploid number of 2n = 23, whereas E. fusiformis had a karyotype with 2n = 21. The two species showed the same mechanism of sex determination (XO type) but differed in chromosome morphology. Pericentromeric blocks of constitutive heterochromatin (CH) were detected in the chromosome complement of both species. CMA3/DA/DAPI staining revealed CMA3-positive blocks in CH regions in four autosomal bivalents of O. rufipes and in two of E. fusiformis. The location of active NORs differed between the two species, occurring in bivalents M6 and S9 of O. rufipes and M6 and M7 of E. fusiformsi. The rDNA sites revealed by FISH coincided with the number and position of the active NORs detected by AgNO3 staining. The variability in chromosomal markers accounted for the karyotype differentiation observed in the tribe Abracrini.

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 CMA3/DA/DAPI staining revealed an R-banding-like pattern with CMA3, 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.

Cytogenetic study of two species of the family Pimelodidae (Siluriformes) collected in lago Guaíba, Rio Grande do Sul, Brazil

A cytogenetic study was conducted on specimens of Parapimelodus nigribarbis and Pimelodus maculatus collected in the lago Guaíba drainage, Porto Alegre, Rio Grande do Sul, Brazil. The two species had a diploid number of 56 chromosomes, with P. nigribarbis showing a karyotype of 20m + 20sm + 4st + 12a with FN of 100, and P. maculatus showing a karyotype of 24m + 20sm + 6st + 6a with FN of 106. NORs were demonstrated in both species in only one pair of subtelocentric chromosomes, in the terminal region of the long arm, which was coincident with C- banding and CMA3 staining, while DAPI staining was negative in these regions. Parapimelodus nigribarbis had a greater number of heterochromatic bands than did P. maculatus, which were distributed mainly in the terminal regions, where the latter species showed an interstitial band on the short arm of the first metacentric pair. C-banding plus CMA3 demonstrated heterochromatin that was associated with GC-rich NORs in both P. nigribarbis and P. maculatus, although other fluorescent regions were also observed in the former species. With C-banding plus DAPI, various chromosomal regions were stained in the two species, along with interstitial staining in P. maculatus, indicating that heterochromatin contained a greater quantity of AT-rich regions.

Exemplares de Parapimelodus nigribarbis e Pimelodus maculatus coletados na bacia do rio Guaíba, Porto Alegre, Rio Grande do Sul, foram analisados citogeneticamente. As duas espécies apresentaram um número diplóide de 56 cromossomos, com P. nigribarbis mostrando um cariótipo de 20m + 20sm + 4st + 12a, NF igual a 100; e P. maculatus com 24m + 20sm + 6st + 6a e NF igual 106. As NORs foram evidenciadas em apenas um par de cromossomos subtelocêntricos, na região terminal do braço longo, nas duas espécies estudadas, sendo coincidentes com a banda C e CMA3; o DAPI nestas regiões se mostrou negativo. Parapimelodus nigribarbis apresentou um maior número de bandas heterocromáticas do que P. maculatus, distribuídas principalmente em regiões terminais, sendo observada nesta última espécie uma banda intersticial no braço curto do primeiro par metacêntrico. Banda C + CMA3 evidenciou em P. nigribarbis e P. maculatus a heterocromatina associada à NOR rica em GC, sendo encontrada na primeira espécie outras regiões fluorescentes. Com banda C + DAPI várias regiões cromossômicas foram observadas nas duas espécies, inclusive a porção intersticial encontrada em P. maculatus, revelando que a heterocromatina possui uma maior quantidade de regiões AT ricas em sua composição.

Cytogenetic studies in fishes of the genera Hassar, Platydoras and Opsodoras (Doradidae, Siluriformes) from Jarí and Xingú Rivers, Brazil

We studied the karyotypes of Hassar cf. orestis and an undescribed Hassar species from the Jarí River and Opsodoras ternetzi, H. orestis and Platydoras cf. costatus from the Xingú River, all with 2n = 58. Constitutive heterochromatin is located in the centromere in most metacentric pairs; in some chromosomes this banding is not present, or it is located on the whole chromosome arm or in the distal regions. The NOR is located on a single biarmed pair at a distal region of the short arm in H. cf. orestis, H. orestis and P. cf. costatus at a distal region of the long arm in O. ternetzi and at a proximal region of the long arm in the Hassar species. In all species (except for Hassar sp.) the CMA3 analysis revealed a rich G-C region coincident with the NOR. Probably inversions occurred in the NOR chromosome during the chromosomal differentiation of the Doradidae species here described.

Cytogenetic characterization of Melipona rufiventris Lepeletier 1836 and Melipona mondury Smith 1863 (Hymenoptera, Apidae) by C banding and fluorochromes staining

The stingless bees Melipona rufiventris and M. mondury were analyzed cytogenetically by conventional staining with Giemsa, C-banding and sequential staining with the fluorochromes CMA3/DA/DAPI. Both species presented 2n = 18 and n = 9, except for one colony of M. rufiventris, in which some individuals had 2n = 19 due to the presence of a B chromosome. After Giemsa staining and C-banding the chromosomes appeared very condensed and presented a high heterochromatic content, making it difficult to localize the centromere and therefore to visualize the chromosomes morphology. The constitutive heterochromatin was located in interstitial chromosome regions covering most of the chromosomes extension and consisted mainly of AT, as shown by DAPI staining. The euchromatin was restricted to the chromosome extremities and was GC-rich, as evidenced by CMA3 staining. The B chromosome was CMA3-negative and DAPI-positive, a heterochromatic constitution similar to that of the A genome chromosomes.

Karyotype relationships among Anastrepha bistrigata, A. striata and A. serpentina (Diptera, Tephritidae)

The species of Anastrepha are arranged into 17 intrageneric groups. Recently, it was proposed that two species of the striata group, Anastrepha striata and A. bistrigata, might be realocated to serpentina group. Anastrepha bistrigata and A. serpentina have an X1X2Y/X1X1 X2X2 sex chromosome system while A. striata has a XY/XX system. It was previously proposed that the karyotype of A. bistrigata could be derived from that of A. striata by an Y:A fusion, and that the karyotype of A. serpentina would be derived from another, hypothetical karyotype. In the present report sequential staining with DAPI and chromomycin A3 (CMA3), followed by C-banding, revealed that the C-banded heterochromatic blocks of the sex chromosomes of A. bistrigata have different affinities to fluorochromes in comparison to the chromosomes of A. striata, from which they have hypothetically derived. The chromosomes of A. serpentina show substantial differences in their cytochemical properties compared to their A. bistrigata and A. striata counterparts. The FISH technique showed that the ribosomal gene sequences are located in DAPI- or DAPI/CMA3-positive heterochromatic blocks of the sex chromosomes, one site on the Y chromosome and one site on the X chromosome (X1 in A. bistrigata and A. serpentina). The data suggest that the karyotype of A. striata and A. bistrigata could be derived from a common ancestral karyotype, while the A. serpentina karyotype probably has a distinct origin.