Karyotypic analysis of Baryancistrus aff. niveatus (ancistrinae, loricariidae) by C-banding, Ag-NOR, CMA 3, DAPI and FISH

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1 CARYOLOGIA Vol. 57, no. 3: , 2004 Karyotypic analysis of Baryancistrus aff. niveatus (ancistrinae, loricariidae) by C-banding, Ag-NOR, CMA 3, DAPI and FISH Augusto C. Paes de Souza 2* ; Aline L. Nascimento 3 ; Jaime R. Carvalho Jr 4.; Regina M.S. Barros 1 ; Eliana Feldberg 5 ; Cleusa Y. Nagamachi 1 and Júlio C. Pieczarka 1 1 Departamento de Genética CCB/UFPA, Belém, Brasil. 2 Bolsista de Mestrado em Zoologia MPEG/UFPA, Belém, Brasil. 3 Bolsista de Mestrado em Genética e Biologia Molecular UFPA, Belém, Brasil. 4 Centro Jovem de Aquarismo (CENJA), Belém, Brasil. 5 Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, Brasil. Abstract Three specimes of Baryancistrus aff. niveatus were studied cytogenetically by conventional staining, C banding, Ag-NORs, CMA 3, DAPI and FISH with human telomeric probes. The results revealed a complement consisting of 52 metacentric, submetacentric and subtelocentric type chromosomes. C banding showed that heterochromatin is located at pairs 1, 3, 6, 10, 11 and 22 in different positions. NORs were located at the short arm of pair 3, coinciding with a positive C-band. This region was also marked with CMA 3, which also marked heterochromatic blocks in chromosomes 11 and 22. These regions were negatively stained by DAPI. These results revealed the presence of two different types of constitutive heterochromatin, probably due to their base composition. Chromosome bearing rdna sites presented a difference in size in the NOR regions, probably due to heterochromatin and ribossomal cistrons amplification. The localization of telomeric sequences by FISH revealed the conservative nature of this region. Key words: Ag-NORs, Baryancistrus aff. niveatus,cbanding, CMA 3, DAPI, FISH INTRODUCTION The Loricariidae family is one of the most diversified among the Siluriformes with about 600 species, distributed in five subfamilies: Ancistrinae, Hypostominae, Hypoptopomatinae, Loricariinae and Neoplecostominae. They are found exclusively in the Neotropical region, from Panamá to Uruguay (Isbrücker, 1980). The front part of the body of these fish is covered by three or more series of bone plates, the mouth is always low with large lips. They feed on algae and microorganisms adhering to the hard substrate or mud on river beds. They generally make nests to lay eggs on the river bottom or banks (Silvan et al, 2001; Burgess 1989). The Ancistrinae subfamily includes about 26 genera and 200 described species (Isbrücker 1980). Corresponding author: Departamento de Genética, UFPA, CCB, 3 Andar. Av. Perimetral SN, CEP , Bairro- Guamá, Belém PA, Brasil. phone-fax: acpaes@ufpa.br In the Xingu river, the fishing of ornamental fish is concentrated on some loricariideum species of the Anicistrinae subfamily: Hypancistrus sp., Hypancistrus zebra, Baryancistrus spp, Hypancistrus sp., Panaque nigrolineatus, Peckoltia vittata, Ancistrus spp., Scobiancistrus spp., Scobiancistrus auratus, Pseudacanthicus leopardus, Pseudancistrus aff. barbatus and Oligancistrus spp. These species represent approximately 90% of the total number of ornamental fish caught in the region of the municipality of Altamira and are considered the most valuable (Torres and Carvalho Jr. 1995; Carvalho Jr. et al, 2002). Loricariideum fishing is one of the most important commercial activities in the region (Zuanon 1999). In spite of their importance for the Amazon region, there are few scientific studies on loricariideums. Cytogenetic studies on this family have shown variation in the diploid number ranging from 48 to 80 chromosomes and a high fundamental number, and the main karyotypic diversification events were the Robertsonian rearrangements (fissions and fusions), pericentric inversions and polyploidy (Michele et al, 1976; Andreate 1991, 1993; Artoni and Bertollo, 1996).

2 220 paes de souza, nascimento, carvalho jr., barros, feldberg, nagamachi and pieczarka MATERIALS AND METHODS One male and two females of Baryancistrus aff. niveatus were analyzed (Figure 1). The were caught in the Xingu River (03 o 1248 S and 52 o W) municipality of Altamira PA The specimens were injected with biological yeast according to the technique described by Cole and Leavens (1971) for amphibians and reptiles, modified by Bertollo (1986) for fish. The metaphases were prepared using the technique described by Bertollo et al. (1978) for fish chromosome studies. The chromosomes were classified morphologically in metacentric (M), submetacentric (SM) subtelocentric (ST) and acrocentric (A) according to Levan et al. (1964). The C-banding method described by Summer (1972) was used to observe the position and size of heterochromatic blocks, while the technique by Howell and Black (1980) was used for NORs staining. The fluorescent bands using CMA 3 and DAPI fluorochromes followed Schweizer (1980) with modifications. In situ hybridization was done using human telomeric probes (Oncor, All Human Telomere Probe, code P5091) following the protocol suggested by the manufacturer. To detect the probe and counterstaining the chromosomes, the FITC and propidium iodide were used. The metaphases in conventional staining and those submitted to C banding and Ag-NORs staining were photographed in a Zeiss Axioscop microscope with Imagelink HQ 33mm perforated film. The metaphases submitted to CMA 3, DAPI and FISH were analyzed under an Zeiss Axioplan II Fluorescence microscope. The best metaphases were shot by an Axiocam MRM camera coupled to the microscope. The images were digitalized by the Axiovision software installed in a PC Pentium IV computer. RESULTS The analysis of a total of 104 cells showed that the males and females presented 2n=52 and NF=104, with 16 metacentric chromosomes, 32 submetacentric and 4 subtelocentric chromosomes (Figure 2). No karyotypic differences or mitotic sexual Fig. 1 Baryancistrus aff. niveatus species analyzed. Fig. 2 Conventional karyotype of the Baryancistrus aff. niveatus species stained by Giemsa. Bar = 10µm.

3 cytogenetic analysis in baryancistrus aff. niveatus 221 Fig. 3 Sequenced C banding, showing the heterochromatin blocks that have heterochromatin rich in G-C on pairs 3, 11 and 22 (see figure 5). Bar = 10µm. chromosome heteromorphism were detected among the individuals The constitutive heterochromatin was detected in the form of conspicuous blocks in the pericentromeric position of pair 6, in the interstitial region on the short arm of pair 3, and were coincident with a secondary constriction, occupying almost completly the long arm of pairs 1 and 10; on the distal portion of the short arm of pair 11 and the long arm of pair 22, and were most evident in one of the homologues. Some of the remaining chromosomes of the complement presented less evident centromeric C-bandings and some did not present C-bandings (Figure 3). The NORs were located in the interstitial position on the short arm of the 3 rd metacentric pair (Figure 4) and were coincident with a posite C-band. CMA 3 stained the region of pair 3 corresponding to the NOR, and the constitutive heterochromatin of pairs 11 and 22, indicating that these regions were rich in G-C base pairs (Figure 5). DAPI marked negatively the constitutive heterochromatin in pairs 3, 11 and 22, confirming that these regions are rich in G-C base pairs. The rest of the karyotype was uniformly stained (Figure 6). Staining by silver nitrate, C-banding and CMA 3 revealed differences in the size of the NOR region between the homologous chromosomes. The telomeric probes marked exclusively the terminal regions of the chromosomes. DISCUSSION The modal number and chromosome formula corroborate the hypothesis suggested by Artoni Fig. 4 Baryancistrus aff. niveatus metaphase showing the NORs stained by silver nitrate. (1996) who considered the karyotypic macrostructure of Ancistrinae preserved when compared to the species in the Hypostominae subfamily. The Ag-NORs study revealed one metacentric pair bearing the rdna sites, with interstitial location on the short arm and positive for CMA 3 and C banding. Pendás et al. (1993) analyzed salmon chromosomes and observed that two probes hybridized with rdna spacer that did not transcribe, suggesting that these spacers, probably rich in G-C, may be respon-

4 222 paes de souza, nascimento, carvalho jr., barros, feldberg, nagamachi and pieczarka Fig. 5 Baryancistrus aff. niveatus metaphase stained with by CMA3. The white arrows indicates the heterochromatin regions of pairs 11 and 22 rich in G-C base pairs and the yellow arrows indicate the NOR regions. 11 th chromosome pair, in the heterochromatin coinciding with the NOR and in the heterochromatin found on pair 22; b) heterochromatin regions negative for CMA 3, correspondiing to the large heterochromatin blocks located on the long arm of the 1 st chromosome pair, in the pericentromeric region of the 6 th pair, on the long arm of the 10 th chromosome pair and in some chromosomes with centromeric and interstitial bandings. Pieczarka et al. (1996) analyzed the constitutive heterochromatin of three New World monkey species belonging to the Callithrix argentata group by restriction enzyme and fluorochromes. They observed that large distal heterochromatin blocks found on some chromosome pairs of these species have the same structure and composition, suggesting a common origin. Margarido and Galetti Jr. (2000) analyzed constitutive heterochromatin in Leporinus desmotes, and suggested that a primordial heterochromatin rich in G-C sequences that is probably associated with the NOR, may have spread along some chromosome in the karyotype by an amplification process. The same hypothesis of a common origin for constitutive heterochromatin as suggested by these authors could be applied to the heterocromatic blocks positively marked with CMA 3 in Baryancistrus aff. niveatus. Another observation found in the C banding pattern is a size heteromorphism on pairs 11 and 22. This size difference is related to an amplification process of the sequences in clusters rich in G-C bases pairs which is confirmed by the banding with CMA 3 fluorochrome, where on one of the homo- Fig. 6. Baryancistrus aff. niveatus metaphase stained with by DAPI. The comparison to figure 5 shows that CMA 3 positivestained regions are negative stained by DAPI (arrows). sible for the coincidence of C banding/nors/ CMA 3. The NORs were heteromorphic in the specimens analyzed, that may have been due to the amplification of the heterochromatin and/or the ribosomal cistron in one of the homologues. Swarca et al. (2001) studied heteromorphism in the rdna size by the FISH technique and observed that these differences are related to an rrna gene accumulation process in one of the homologues. Regarding the nature of the constitutive heterochromatin, two situations were found: a) heterochromatin regions positive for CMA 3 which were found in large distal heterochromatic blocks located on the Fig. 7 Baryancistrus aff. niveatus metaphase hybridized with the human telomeric probes, indicating that these regions are well preserved between humans and fish.

5 cytogenetic analysis in baryancistrus aff. niveatus 223 logues the band produced by this fluorochrome was larger. Staining by DAPI was negative for the heterochromatin regions positive for CMA 3, reinforcing the hypothesis of a common origin for these regions. The rest of the karyotype stained uniformly, showing that those heterochromatin blocks which were negative for CMA 3 had the same behavior with DAPI, showing heterogeneity in the base composition. Regarding in situ hybridization using telomeric probes, they were only observed in the terminal regions showing that, in spite of the large phylogenetic distance among fish and man, the sequences of the chromosome regions are quite preserved. The absence of interstitial signals indicates that if the species suffered fusion-type rearrangement, the telomeric sequences were excluded from the karyotype (Figure 7) REFERENCES Andreata A.A Estudos citogenéticos na subfamília Hypoptopomatine (Pices, Siluriforme, Loricariidae). Mastership Dissertation. Instituto de Biociências da Universidade de São Paulo. 171p. Andreata A.A.; Almeida-Toledo L. F.; Oliveira C. and Toledo-Filho S.A Chromosome studies in Hypoptopomatine (Pices, Siluriforme, Loricariidae). II. ZZ/ZW sex-chromosome system, B chromosome, and constitutive heterochromatin differentiation in Microlepidogaster leucofrenatus. Cytogenet and Cell Genetics., Vol. 63: p. Artoni R.F. and Bertollo L.A.C Cytogenetic studies on Hypostominae (Pisces, Siluriformes, Loricariidae). Considerations on Karyotype evolution in the genus Hypostomus. Caryologia, Vol. 49: 81-90p. Bertollo L.A.C.; Takahashi C. S. and Moreira- Filho O Citotaxonomic considerations on Hoplias lacerdae (Pisces, erythrynidae). Revista Brasileira Genética., Vol. 1: p. Bertollo L.A.C Estimulação de mitoses em peixes.isimpósio de Citogenética e Evolução Aplicada para Peixes Neotropicais. Abstract, 30p. Burgess W.E An atlas of freshwater and marine catfish. T.F.H. Publications, 784p. Carvalho Jr., J.R.; Giarrizzo T.; Camargo M.; Zuanon J. and Isaac V Diversidade e distribuição geográfica dos acaris (Loricariidae) do médio rio Xingu-Pa, estado do conhecimento. XXIV Congresso Brasileiro de Zoologia. Abstract. Cole C.J. and Leavens C.R Chromosome preparations of amphibians and reptiles: improved techique. Herpetol., Vol. 3:102p. Howell W.M. and Black D.A Controlled silver staining nucleolus organizer regions with protective colloidal developer: a 1 step method. Experientia, Vol. 36, p. Isbrücker I.J.H Classification and catalogue of the mailed Loricariidae (Pisces, Siluriformes) Verslagem en Technische Gegevenes, Inst. Taxon. Zool. (Zool. Mus.), Univ. Amesterdam, Vol. 22: 1-181p. Levan A.; Fredga K. and Sandberg A.A Nomeclature for centromeric position of chromosomes. Heredity, Vol. 52: p. Margarido V.P. and Galetti Jr., P.M Amplification of a GC-rich heterochromatin in the freshwater fish Leporinus desmostes (Characiformes, Anostomidae). Genet. and Mol. Biol., Vol. 23: p. Michele J.L.; Takahashi C.S and Ferrari I Karyotypic study of some of the Family. Loricariidae. Cytologia, Vol p. Pendás A.M.; Morán P. and García-Vazquez E Ribossomal RNA genes ar interspersed throughout a heterochromatic chromosome arm in Atlantic salmon. Cytogenetic and Cell Genetics, Vol. 63: p. Pieczarka J.C.; Nagamachi C.Y.; Barros R.M.S. and Mattevi M.S Analysis of constitutive heterochromatin by fuorochromes and in situ digestion with restriction enzymes in species of the group Callithrix argentata (Callitrichidae, Primates). Cytogenet and Cell Genetics, Vol. 72: p. Schweizer D Simultaneous fluorescent staining of R bands and specific heterochromatic regions (DA/DAPI bands) in human chromosomes Cytogenet and Cell Genetics., Vol. 27: p. Silvano R.A.M.; Oyakawa O.T.; do Amaral B.D.; and Begossi A Peixes do alto rio Juruá (Amazônia, Brasil) São Paulo: Editora da Universidade de São Paulo: Imprensa Oficial do Estado, 298p. Sumner A.T A simple technic for demonstrating centromere heterocromatin. Exptl. Cell. Res., Vol. 75: p. Swarca A.C; Giuliano-Caetano L. and Dias A.L Analyses of nucleolus organizer regions and heterochromatin of Pimelodus maculatus (Pisces, Pimelodidae). Genetica, Vol. 110: p. Torres M. F. and Carvalho Jr., J.R A pesca artesanal de peixes ornamentais da família Loricariidae (Siluriformes) no município de Ourém, Pará.XI Encontro Brasileiro de Ictiologia. Resumo. Zuanon J.A.S História natural da ictiofauna de corredeiras do rio Xingu, na região de Altamira, Pará. Tese de Doutorado Universidade Estadual de Campinas, Instituto de Biologia. 199p. Received September 15, 2003; accepted November 3, 2004