Heterochromatin analysis in the fish species Liposarcus anisitsi (siluriformes) and Leporinus elongatus (characiformes)

Heterochromatin analysis in the fish species Liposarcus anisitsi (siluriformes) and Leporinus elongatus (characiformes)

Roberto Ferreira Artoni¹, Wagner Franco Molina², Luis Antonio Carlos Bertollo³ and Pedro Manoel Galetti Junior³

¹Departamento de Biologia Geral, Universidade Estadual de Ponta Grossa, Campus Uvaranas, 84031-510 Ponta Grossa, PR, Brasil.

²Departamento de Genética e Biologia Celular, Universidade Federal do Rio Grande do Norte, 59078-970 Natal, RN, Brasil.

³Departamento de Genética e Evolução, Universidade Federal de São Carlos, Caixa Postal 676, 13565-905 São Carlos, SP, Brasil. Send correspondence to L.A.C.B.

ABSTRACT

The chromosomes of two neotropical freshwater fish species, namely Liposarcus anisitsi (Siluriformes, Loricariidae) and Leporinus elongatus (Characiformes, Anostomidae), were investigated by means of C-banding, Ag-NORs, fluorochrome staining and banding by hot saline solution (HSS) treatment, to reveal patterns of heterochromatin differentiation. The karyotype of L. anisitsi is described for the first time. Staining with the GC-specific fluorescent antibiotic mithramycin (MM) revealed bright signals in some C-banded blocks in both species, suggesting that these MM⁺ heterochromatin contains GC-rich DNA. Banding by denaturation employing HSS, followed by Giemsa staining, yielded corresponding results documenting the thermal stability of GC-rich DNA part of heterochromatin positive after C-banding. In L. elongatus the Ag-NOR also followed the above banding patterns. However, in L. anisitsi the Ag-NOR was MM⁺ but negatively stained after C-banding and HSS treatment. L. elongatus also showed C-banded segments that were negative for mithramycin staining and HSS treatment. The results obtained evidence the heterochromatin heterogeneity in these fish species.

INTRODUCTION

Several chromosome banding techniques have been increasingly used to better understand the structure and organization of fish chromosomes, although in some cases, such as in G-banding, they have showed a poor reproducibility with some exceptions (Medrano et al., 1988; Gold and Li, 1991; Bertollo et al., 1997). The classical C-banding technique (Sumner, 1972) has been the most utilized method for studying heterochromatin in fish chromosomes. Nevertheless, fluorochrome dyes and restriction endonucleases have also proved to be useful in detecting heterochromatin differentiation in this group (Mayr et al., 1988, Sánchez et al., 1991, Sola et al., 1992, among others).

In fish species the nucleolar organizer regions (NORs) have been showed as GC-rich DNA sites, positive for mithramycin A (MM) and/or chromomycin A₃ (CMA₃) staining, both fluorochromes with affinity to GC-base pairs (Mayr et al., 1985; Amemiya and Gold, 1986; Phillips et al., 1988; Sola et al., 1992, 1997; Galetti Jr. and Rasch, 1993 a,b; Galetti Jr. et al., 1995; Souza et al., 1996). On the other hand, AT-rich DNA regions were also observed in fish chromosomes, as in some salmonids and zebrafish, where positive segments for DAPI, an AT-specific fluorochrome, occur together with GC-rich regions (Mayr et al., 1988; Gornung et al., 1997).

Changes related with heterochromatin amount and its distribution on the chromosomes have been reported as an evolutionary karyotypic mechanism in some fish groups. The Anostomidae, e.g., is a freshwater fish family with a relatively stable karyotypic macrostructure, although significant differences concerning the constitutive heterochromatin occur among genera and species (Galetti Jr. et al., 1991). Largely C-banded sex chromosomes were also detected among Leporinus (Galetti Jr. and Foresti, 1986), Triportheus (Bertollo and Cavallaro, 1992) and Parodon (Moreira-Filho et al., 1993) fish species bearing a ZZ/ZW sex chromosome system, appearing as a good material for heterochromatin studies and chromosomal differentiation.

In the present study, heterochromatin heterogeneity was investigated in the chromosomes of two neotropical freshwater fishes, Leporinus elongatus and Liposarcus anisitsi, by means of different banding techniques. While the karyotypic macrostructure of L. elongatus (cited as L. obtusidens) was previously described (Galetti Jr. et al., 1981), the karyotype of L. anisitsi is now being reported for the first time.

MATERIAL AND METHODS

Eight Liposarcus anisitsi (Siluriformes, Loricariidae) specimens - five males and three females - from Preto River (Mirassolândia, São Paulo State, Brazil) and six Leporinus elongatus (Characiformes, Anostomidae) specimens - three males and three females - from Mogi Guaçu River (Pirassununga, São Paulo State, Brazil) were studied.

Both direct preparations (Bertollo et al., 1978) and short-term culture cells (Fenocchio et al., 1991) were used to obtain metaphase chromosomes, using stimulation of mitotic activity (Lee and Elder, 1980).

Chromosome banding

Ag-NOR-banding

Nucleolar organizing regions were detected by silver nitrate staining (Ag-NORs), according to Howell and Black (1980). Briefly, the slides were treated with a gelatin-silver nitrate solution, covered with a coverslip, incubated at 60^oC for 3-5 min, washed in deionized water and air-dried.

C-banding

Constitutive heterochromatin (C-band) was detected according to Sumner (1972), with the following modifications: two-day-old slides were immersed in 0.2 N HCl for 15 min, incubated in 2 x SSC at 60^oC for 15 min and immersed in 5% barium hydroxide at 42^oC for 1 min and 30 s. The slides were then quickly immersed in 0.2 HCl and again incubated in 2 x SSC at 60^oC for 30 min, followed by Giemsa staining (5%) for 5 min. After each of these six steps, slides were washed in deionized water and air-dried.

Fluorochrome staining

Fluorescent signals on the chromosomes were obtained using MM, following the protocol described by Schmid (1980): 150 ml distamycin counterstaining solution (0.1 mg/ml in McIlvaine buffer) was applied on the slides and then covered with a coverslip. After 15 min, the slides were washed in McIlvaine buffer and air-dried. Then, 120 ml mithramycin solution (0.1 mg/ml in McIlvaine buffer) was added, and slides with coverslips were kept staining in the dark for 1 h. After this time, they were vigorously washed three times in McIlvaine buffer, air-dried and mounted under saturated sucrose solution. About 15-day-old slides were analyzed under epifluorescent microscope.

Banding by hot saline solution (HSS) treatment

Some chromosomal bands were also obtained employing the RHG technique (R-bands by heat using Giemsa), described in Verma and Babu (1989). Briefly, the slides were aged for 6 days in an oven at 37°C. Then, they were immersed in Sörensen phosphate buffer, pH 6.8, at 85^oC for 5 min, washed in deionized water and stained with 5% Giemsa for 5 min.

RESULTS

Standard karyotype and Ag-NORs

Liposarcus anisitsi

A diploid chromosome number equal to 2n = 52 was found. The karyotype consisted of 8 metacentric (m), 12 submetacentric (sm), 4 subtelocentric (st) and 2 acrocentric (a) chromosome pairs in both sexes (Figure 1A). No morphologically differentiated sex chromosomes were detected. The sm chromosome pair 14 showed a conspicuous terminal secondary constriction on its long arm, corresponding to a nucleolar organizer region location detected by silver nitrate staining-Ag-NOR (Figure 1C).

Figure 1
- Karyotype and details of some Liposarcus anisitsi chromosome pairs. A, Standard karyotype stained with Giemsa. Pair 14 shows a conspicuous terminal secondary constriction on its long arm, corresponding to the nucleolus organizer region (Ag-NOR). B, Chromosomes 12, 25, and C, chromosome 14 showing the correspondence between C-banded (C) regions, MM⁺ signals and positive segments after hot saline solution (HSS) treatment plus Giemsa staining; the Ag-NOR appears as MM⁺ but negative after C-banding and HSS treatment. D, Metaphase treated with mithramycin showing six big GC-rich (MM⁺) blocks, correspondent to chromosome pairs 12, 14 and 25, besides some small telomeric/centromeric positive blocks on other chromosomes. M, Metacentric; SM, submetacentric; ST, subtelocentric; A, acrocentric.

Leporinus elongatus

A previous description of the karyotype (Galetti Jr. et al., 1981) showed a diploid number equal to 2n = 54 biarmed chromosomes in both sexes and a well-differentiated ZZ/ZW sex chromosome system. While Z was a middle-sized submetacentric chromosome, W was a large subtelocentric one, the biggest in size in the female karyotype (Figure 2B). Ag-NORs, also previously reported (Galetti Jr. et al., 1984), were terminally located on the long arm of the submetacentric pair 5 (Figure 2C).

Figure 2
- Details of some Leporinus elongatus chromosomes. A, Selected first C-banded autosomal pairs showing the negative response of the constitutive heterochromatin after hot saline solution (HSS) treatment. B, ZW sex chromosomes showing the great amount of constitutive heterochromatin on their long arm, as well as the correspondence between some heterochromatic segments and positive regions for HSS treatment and mithramycin (MM) staining in the W chromosome. C, Pair 5 bearing a terminal Ag-NOR on its long arm; this region also appears positive after C-banding, mythramycin staining and HSS treatment.

C-bands

Liposarcus anisitsi

Moderate heterochromatic blocks (C⁺ regions) can be seen on the centromeric and/or telomeric regions of some chromosomes but conspicuous telomeric blocks occur on the chromosomes 12 and 25 (Figure 1B). A subterminal heterochromatic segment can also be observed on the long arm of the chromosome pair 14 just above its terminal secondary constriction, demonstrating that this NOR site is negative after C-banding (Figure 1C).

Leporinus elongatus

Autosomes showed C⁺ regions on the telomeres, besides some positive pericentromeric segments, as illustrated in Figures 2A-C for the first six chromosome pairs of the karyotype. Remarkable results refer to the heteromorphic sex chromosomes, where a large amount of heterochromatin was observed on the big long arm of the W chromosome, as well as on some extent of the Z chromosome (Figure 2B). Chromosome 5 also showed a C⁺ segment corresponding to the NOR site location (Figure 2C).

Fluorescent mithramycin (MM⁺) signals

Liposarcus anisitsi

The large C-banded segments on the chromosomes 12 and 25 were also MM⁺ (Figure 1B). A large MM⁺ signal was equally observed on the NOR bearing chromosome, including the entire NOR site and its flanking heterochromatic block (Figure 1C). A few other chromosomes showed slight MM⁺ signals in their centromeric and/or telomeric regions (Figure 1D), which were also positive after C-banding.

Leporinus elongatus

Except for the NOR-bearing pair 5 and the sex chromosomes, all the remaining ones were devoid of specific labeling with mithramycin staining. In chromosome 5 a slightly MM⁺ region coincided with the C⁺ positive NOR (Figure 2C). The W chromosome showed some distinct MM⁺ signals on its largely C-banded long arm (Figure 2B).

Bands by hot saline solution (HSS) treatment

In both species under study, all chromosome regions that were found to be C-banded and MM⁺ were also positively banded after the HSS treatment plus Giemsa staining (Figures 1B,C and 2B,C). An exception is made for the MM⁺ nucleolar organizer region of Liposarcus anisitsi (pair 14), which appeared negative after this treatment (Figure 1C), although it was not a C⁺ region.

DISCUSSION

Multiple R-bands, opposite to those produced by Q- and G-banding, have been routinely evidenced on human and higher vertebrate chromosomes employing the RHG method (e.g. Dutrillaux and Lejeune, 1971; Drouin et al., 1994). This technique, however, yielded very different results in the fish species under study, evidencing only some specific GC-rich heterochromatic segments. By this reason, we preferred to identify these regions as bands produced by HSS treatment, instead of R-bands commonly referred to the multiple reserve chromosomal bands. It has been reported that some chromosome bands that occur in higher vertebrates may be absent in the lower ones, as amphibians and fish (e.g. Medrano et al., 1988). Apparently, a lack of differentiation in base composition occurs in the chromosomes of the latter group, in contrast with what is observed in other vertebrates, like mammals (Holmquist, 1987, 1988, 1989, Medrano et al., op. cit.). This proposition and/or differences related to chromatin organization in the chromosome core (Saitoh and Laemmli, 1994) might explain the different banding results after various banding procedures between these two major vertebrate groups.

In Liposarcus anisitsi, the HSS treatment revealed most of the heterochromatin blocks visualized by C-banding. Similar results were also observed after mithramycin staining, suggesting that these constitutive heterochromatins are compositionally GC-rich. This congruence was also true for Leporinus elongatus although, in this case, only some heterochromatic segments present in the ZW sex chromosomes and that associated with the NORs were also detected after MM staining and HSS treatment. It has been claimed that a heat treatment, such as that applied here in HSS method, could denature and partially extract AT-rich DNA, whereas GC-rich DNA might remain on the chromosomes (Comings, l978). This might account for the correspondence between banding by HSS treatment and MM⁺ signals, thus evidencing the thermal stability of GC-rich DNA part of the C-banded heterochromatin.

The positive NOR staining with a GC-base pair binding fluorochrome, observed in Leporinus elongatus and Liposarcus anisitsi, agrees with several other results in different fish species, as well as in amphibians (Schmid, 1982, among others). Besides silver staining and GC-specific fluorochrome dyes, the fluorescent in situ hybridization (FISH), with rDNA probes, has been more recently used to localize rDNA cistrons in fish chromosomes. In Salvelinus namaycush, e.g., Reed and Phyllips (1995) showed that all chromomycin bands in the karyotype contain rDNA. Interesting results have also been obtained by Pendás and coworkers (1993a,b) in other salmonids. Thus, in Salmo trutta, the rDNA hybridization signal is interspersed over the whole GC-rich heterochromatic short arm of a subtelocentric chromosome pair, in accordance with a major Ag-NOR location. Only with FISH it was possible to identify several other additional rDNA sites, representing inactive minor NORs (Pendás et al., 1993b). On the other hand, in Salmo salar, while the rDNA also appears over the whole heterochromatic arm of a metacentric chromosome, the active NOR (Ag-NOR) is restricted to the secondary constriction of this arm (Pendás et al., 1993a). Pendás et al. (1993b) and Martínez et al. (1996) consider that chromomycin (or mithramycin) and silver staining are not the best methods to localize rDNA cistrons in fish species such as some salmonids, where a large amount of GC-rich heterochromatin is associated with the Ag-NORs. On the other hand, silver staining is able to detect only the active NORs during the previous interphase and so would be better applied to investigate NOR expression rather than NOR localization (Pendás et al., 1993b). Mayr et al. (1988) and Phillips and Hartley (1988) have also signalized that the heterochromatin adjacent to NORs can be stained by CMA₃ in salmonids, and Gold et al. (1990) emphasize that some caution is required before considering a chromomycin or mithramycin bright chromosomal region as a NOR, since these fluorochromes can also selectively stain heterochromatin.

In Leporinus elongatus the results clearly show that the Ag-NOR is MM⁺, corresponding to a C-banded region also positive for HSS treatment (Figure 2C). Thus, there is an apparent agreement concerning the chromosomal location of a GC-rich DNA segment, the Ag-NOR site and constitutive heterochromatin. In Liposarcus anisitsi the data are particularly interesting since Ag-NOR is also MM⁺, but not a positive region after C-banding and HSS treatment. The MM⁺ signal appeared over a great extent of the long arm of the submetacentric pair 14, including the terminal Ag-NOR and its adjacent constitutive heterochromatin (Figure 1A,C). So, in this species the Ag-NOR was a GC-rich region, not heterochromatic under C-banding. Several amphibian species also show Ag-NORs that are bright after mithramycin or chromomycin staining and negative after C-banding (Schmid, 1982). A high GC-base pair content of the rDNA spacer sequences may be a possible factor related with the positive NOR staining by these fluorochromes in Anura (Schmid, op. cit.). This appear an attractive hypothesis to be considered in the case of Liposarcus anisitsi. On the other hand, in some fish species showing association between rDNA and heterochromatin and where NORs have been intensively studied, there is evidence that the major rDNA genes are interspersed with GC-rich heterochromatin sequences (Martínez et al., 1996). Such situation could be also a suitable hypothesis for Leporinus elongatus.

A heterochromatinization process has been associated with the evolution of a ZZ/ZW sex chromosome system in Leporinus (Galetti Jr. and Foresti, 1986), as well as in other fish species such as Triportheus guentheri (Bertollo and Cavallaro, 1992) and Parodon hilarii (Moreira-Filho et al., 1993), leading to highly differentiated sex chromosomes. In these cases, the W chromosome has been shown largely heterochromatic after C-banding, with a greater amount of constitutive heterochromatin than the Z chromosome. In Leporinus elongatus some positive segments for mithramycin staining and HSS treatment can be seen on the heterochromatic long arm of the W chromosome (Figure 2B), evidencing GC-rich heterochromatin sites. Molina (1995) also found this kind of heterochromatin in the W chromosome of other Leporinus species and suggests that repetitive DNA amplifications are related with heterochromatic differentiations in this chromosome. Fluorescent segments related to sex appear even to represent the initial stage of a sex chromosome differentiation in some fish species, as observed in Poecilia sphenops var. melanistica (Haaf and Schmid, 1984), Salvelinus namaycush (Phillips and Ihssen, 1985) and Eigenmannia virescens (Almeida-Toledo et al., 1988). At this time, the heterochromatin nature in other fish sex chromosomes is under investigation in our laboratory.

Besides the data already discussed, it can also be signalized that several C-banded regions were not positive after mithramycin staining and HSS treatment. This is the case for telomeric and pericentromeric segments in Leporinus elongatus (Figure 2A), which evidence again a heterochromatin heterogeneity probably due to compositional differences. So, taken together, the banding methods here utilized were informative, contributing for a better knowledge of heterochromatin differentiation and fish chromosome organization.

ACKNOWLEDGMENTS

The authors thank Drs. R. Drouin for suggestions on the manuscript and F. Langeani Neto for supplying Liporsarcus anisitsi specimens. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Publication supported by FAPESP.

RESUMO

Cromossomos mitóticos de duas especies de peixes neotropicais, Leporinus elongatus (Characiformes) e Liposarcus anisitsi (Siluriformes), foram estudados por diferentes métodos de bandamentos, com o intuito de investigar a diferenciação da heterocromatina. Enquanto que a macroestrutura cariotípica de L. elongatus já foi objeto de estudos anteriores, o cariótipo de L. anisitsi está sendo apresentado pela primeira vez. Em ambas as espécies, a coloração dos cromossomos com a mitramicina (MM), fluorocromo GC específico, evidenciou sinais brilhantes em alguns segmentos heterocromáticos também positivos ao bandamento C, sugerindo ser esta fração da heterocromatina rica em seqüências de bases GC. O tratamento dos cromossomos com solução salina aquecida e posterior coloração com Giemsa demonstrou resultados similares, documentando a estabilidade térmica do DNA rico em bases GC, presente na heterocromatina constitutiva. Em L. elongatus a Ag-NOR também seguiu os padrões de bandamentos acima. Em L. anisitsi, contudo, a Ag-NOR apresentou-se MM⁺ mas negativamente corada após o bandamento C e o tratamento com solução salina aquecida. Por outro lado, em L. elongatus, alguns segmentos que foram positivos ao bandamento C mostraram-se negativos à coloração com mitramicina, assim como à coloração com Giemsa após o tratamento térmico com solução salina. Os resultados obtidos evidenciam a heterogeneidade da heterocromatina no complemento cariotípico das espécies estudadas.

(Received August 27, 1997)

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Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999

History

• Received
  27 Aug 1997