Imparfinis aff. schubarti (Siluriformes, Pimelodidae) of

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1 CARYOLOGIA Vol. 57, no. 4: , 2004 Analyses of karyotype and nucleolus organizer regions of Imparfinis aff. schubarti (Siluriformes, Pimelodidae) of the Tibagi river basin, Paraná, Brazil Stolf Renata, Ana Claudia Swarca, Lucia Giuliano-Caetano and Ana Lúcia Dias* Universidade Estadual de Londrina, Departamento de Biologia Geral, Londrina, Paraná, Brazil. Abstract Were analysed cytogenetically 53 specimens of Imparfinis aff. schubarti of the basin of the Tibagi river (Paraná, Brazil). The diploid number of 58 chromosomes was observed and a karyotype of 28M+28SM+2ST with a fundamental number (FN) of 116. The nucleolus organizer regions (NORs) were studied by means of AgNO 3 and CMA 3 staining, as well as by FISH using an 18S rdna probe. NORs were found to be located at the interstitial position on the long arm of the first metacentric chromosome pair, corresponding to a secondary constriction observed in the conventional staining. AgNO 3 and CMA 3 evidenced a size heteromorphism between the NORs of the homologous chromosomes. However, after FISH no difference was observed in the size of the homologous NORs. The first M pair. showed a pale C-banding in the pericentromeric region. Key words: Imparfinis, karyotype, AgNOR, CMA 3, rdna 18S INTRODUCTION The genus Imparfinis belongs to the order Siluriformes, family Pimelodidae, comprising approximately 28 species and may be considered the most complex and the least understood genus of this family (Lundberg et al. 1991; Bockmann 1994). They are endemic neotropical fishes, found in the headwaters of many rivers in Central and South Americas. Their representatives are small in size showing benthonic and nocturnal habits (Castro and Cassati, 1997). To date, there are few cytogenetic data on Imparfinis species, the diploid number ranging from 2n=56 chromosomes in Imparfinis cf. piperatus (Vissotto et al. 2002; Fenocchio et al. 2003a), and Imparfinis piperatus (Vicente et al. 1994), to 2n=58 chromosomes in Imparfinis mirini (Vissotto et al. 1997), Imparfinis piperatus (Vissotto et al. 2002) and Imparfinis aff. schubarti (Fenocchio et al. 2003a). In all the examined species, the nucleolar organizing regions are located in the interstitial region of at least one chromosome pair (Vissotto et al. 2002), indicating that this position and a single NOR system are common features in Imparfinis. *Corresponding author: Universidade Estadual de Londrina, CCB, Depto. de Biologia Geral; , Caixa Postal 6001, Londrina, Paraná, Brazil; anadias@uel.br. The aim of this study was to improve the chromosomal data in this fish group. Different populations of Imparfinis aff. schubarti, were studied by conventional Giemsa staining and other cytogenetical methods, as AgNO 3, CMA 3 and DAPI staining, as well as fluorescence in situ hybridization (FISH). MATERIALS AND METHODS Were studied 22 males, 30 females plus one specimen with an indeterminate sex of Imparfinis aff. schubarti, from six tributary of the Tibagi river basin (Paraná, Brazil): Marrequinhas and Três Bocas stream, Água da Floresta, Vinícius, Taquari and Jataizinho rivers. Mitotic chromosomes were obtained according to Bertollo et al. (1978). Chromosome morphology was determined on the basis of the arm ratios, as proposed by Levan et al. (1964). Metacentric (M), submetacentric (SM) and subtelocentric (ST) chromosomes were considered as biarmed ones. NOR silver staining (AgNOR) was performed according to Howell and Black (1980), and chromomycin (CMA 3 ) and DAPI staining as described by Schweizer (1976, in Verma and Babu, 1995). C-banding was made using the methods of Sumner (1972). The 18S rdna probe (1700pb), obtained from the fish Oreochromis niloticus, was used for FISH. The probe was labeled with biotin- 14-dATP by nick translation (Gibco cat N

2 analyses of karyotype and nors of imparfinis aff. schubarti ), according to the manufacturer s instructions. Hybridization and post-hybridization steps, and the visualization of the chromosomal signals were carried out as reported by Swarca et al. (2001a). RESULTS AND DISCUSSION All Imparfinis aff. schubarti specimens analysed had a 2n=58 chromosome number and a karyotype of 28M + 28SM + 2ST, with a fundamental number (FN=number of chromosome arms) of 116 (Figure 1). A conspicuous secondary constriction was seen in the first metacentric chromosome pair. Another population of I. aff. schubarti, from Itirapina, São Paulo State, showed this same diploid number (2n=58) but different karyotypic formula, 22M + 18SM + 10ST + 8A and fundamental number, FN= 98 (Fenocchio et al. 2003a). The genus Imparfinis is characterized by a high FN, greater than 90, with the karyotype comprised mainly of meta and submetacentric chromosomes. A high FN is characteristic of the order Siluriformes and, according to Oliveira and Gostonyi (2000), corresponds to a plesiomorphic condition (primitive traits) widely distributed in this fish group. In Imparfinis aff. schubarti from the Tibagi river basin, the fundamental number of 116 was found to be constant in individuals from all the locations sampled, demonstrating a fixed karyotype. This fundamental number is the most common in the genus, being also observed in Imparfinis piperatus (Vissotto et al. 2002) and Imparfinis mirini (Vissotto et al. 1997), showing the predominance of biarmed chromosomes in the karyotype of these species. Differences in karyotypic formula and in FN have been made evident in different species and populations of Imparfinis. Although this may be due in part to the different criteria used in the chromosomal classification and/or to differences in the degree of chromosome condensation, it is very probable that chromosomal rearrangements have been Fig. 1 Giemsa-stained karyotype of Imparfinis aff. schubarti.

3 350 stolf, swarca, giuliano-caetano and dias occurred during the evolutionary process of this fish group, allowing to real karyotypic differentiations (Vissotto et al. 2002). Pimelodidae fishes have, in general, one pair of chromosomes bearing NOR, with a preferential location at telomeric regions. In Imparfinis aff. schubarti, AgNORs were observed in the interstitial region of the long arm of the largest metacentric pair (Figure 2A), corresponding to the secondary constriction observed by conventional staining. Cetopsorhamdia (Fenocchio et al. 2003a; Vissotto et al. 1999) and Imparfinis are the only two Pimelodidae genera that have NORs at an interstitial position in the chromosomes. Imparfinis species with 2n=58 chromosomes had NORs in the first metacentric chromosome pair, while species with 2n=56 had Fig. 2 Imparfinis aff. schubarti chromosomes. (A) AgNOR, (B) CMA 3 staining, (C) DAPI staining, (D) Fluorescence in situ hybridization with biotin-labeled 18S rdna probe, (E) The NOR-bearing pairs C-banding. The arrows indicate the nucleolar organizing regions.

4 Bertollo L.A.C., Takahashi C.S. and Moreira- Filho O., 1978 Cytotaxonomic considerations in Hoplias lacerdae (Pisces, Erythrinidae). Rev Brasil Genet., 1: Bockman F. A., 1994 Description of Mastiglanis asopos, a new pimelodid catfish from northern Brazil, with comments on phylogenetic relationships inside the subfamily Rhamdiinae (Siluriformes: Pimelodidae). Proc Biol Soc Wash, 107: Castro R. M. C. and Casatti L., 1997 The fish fauna from a small forest stream of the upper Paraná river basin, southeastern Brazil. Ichth Explor Freshw, 7: Fenocchio A. S., Bertollo L. A. C., Takahashi C. S., Dias A. L. and Swarca A. C., 2003a Cytogenetic studies and correlate considerations on Rhamdiinae relationships (Pisces, Siluroidei, Pimelodidae) Cytologia, 68: Fenocchio A. S., Swarca A. C., Cestari M. M. and Dias A. L., 2003b Karyotypic characterization and NOR analysis by different banding techniques of Rhamdia quelen (Pisces, Pimelodidae) from the First Plateau of the Iguaçu river (Brazil). Folia Biol., 51: 3-4. Howell W. M. and Black D. A., 1980 Controled silver staining of nucleous organizer regions with a protective coloidal developer: a one-step method. Experientia, 36: Levan A., Fredga K. and Sandberg A. A., 1964 Nomenclature for centromeric position on chromosome. Hereditas, 52: Lundberg J. G., Bornbush A. H. and Mago-Leccia F., 1991 Gladioglanis conquistator N. sp. from Ecuador with diagnoses of the subfamilies Rhamdiianalyses of karyotype and nors of imparfinis aff. schubarti 351 NORs in the largest acrocentric pair. Vissotto et al. (2002) suggested that this acrocentric chromosome probably arose from a deletion or translocation concerning the short arm of the metacentric chromosome since chromosomes with NORs in species with 56 and 58 chromosomes differ basically in the size of the chromosomes, as well as in the size of the short arms. However, other chromosomal rearrangements, such as pericentric inversions or NOR transpositions, can also be related to the NORs locations in Imparfinis. An evident heteromorphism in the size of the Ag- NORs was observed between the homologous chromosomes of Imparfinis aff. schubarti, asalso reported for Imparfinis piperatus (Vissotto et al. 2002). These heteromorphic NORs were also seen with chromomycin A 3 (CMA 3 ) staining (Figure 2B). Such correspondence between CMA 3 and AgNOR is frequently observed in fish, demonstrating that NORs are rich in GC base pairs. Besides the nucleolar chromosome pair, other chromosomes also showed weakly CMA 3 positive signals, indicating some other GC-rich regions in the complement, as observed by Swarca et al. (1999) in another Pimelodidae species, Pinirampus pirinampu. Treatment with the fluorochrome DAPI (ATspecific) generally leads to an uniform staining pattern in fish chromosomes. In Imparfinis aff. schubarti, the secondary constriction region appeared totally negative, that is, it was not stained by DAPI, confirming that this region is practically devoid of AT base pairs, and reinforcing its GC-rich nature (Figure 2C). C-banding of I. aff schubarti was poor, showing a pale pericentromeric mark in the first metacentric pair, that is the NOR-bearing (Figure 2E). In spite of the large size of secondary constriction facilitate their detection by means of CMA 3, these regions were not entirely C-positive, reinforcing the idea that not all repetitive sequences are C-banded. In situ hybridization with 18S rdna probe confirmed the NOR location in the secondary constriction of the first metacentric chromosome (Figure 2D), as also occur in the Pimelodidae species, Zungaro zungaro (Swarca et al. 2001b), Pimelodella aff. avanhandavae (Swarca et al. 2003) and Rhamdia + quelen (Fenocchio et al. 2003b). The other CMA 3 signals on Imparfinis aff. schubarti chromosomes must correspond to heterochromatic GC-rich regions and not to silent (inactive) nucleolar organizing regions. Swarca et al. (2001a) showed that this is also true for P. pirinampu. The heteromorphism evidenced by AgNORs has a probable functional origin, and is not related to the number of rdna gene copies in the homologous chromosomes as observed by Vinas et al. (1996) and Swarca et al. (2003). So, the heteromorphism seen with the techniques applied, except FISH, could be due to a differential degree of condensation of the secondary constriction, that is more relaxed in one of the homologue chromosomes. According to Stack and Anderson (2001) the most decondensed regions were not spiralized probably due to an accumulation of proteins when the NORs were active in the previous interphase. The data now presented allow the chromosomal characterization of Imparfinis aff. schubarti from the Tibagi river basin. They reinforce that the taxonomy of the genus Imparfinis needs to be revised. Indeed, some species present distinct karyotypic populations, besides a confused classification. Acknowledgments The authors are grateful to CAPES and PIBIC/CNPq for financial support. We are thankful to Dr. Oscar A. Shibatta for the identification of the species and to Dr. Alberto S. Fenocchio for a critical reading of the manuscript. REFERENCES

5 352 stolf, swarca, giuliano-caetano and dias nae Bleeker and Pseudopimelodinae N. subf. (Siluriformes: Pimelodidae). Copeia, 1: Oliveira C. and Gosztonyi A. E., 2000 A cytogenetic study of Diplomystes mesembrinus (Teleostei, Siluriformes, Diplomystidae) with a discussion of chromosome evolution in Siluriformes. Caryologia, 53: Schweizer D., 1976 Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma, 58: Stack S. M. and Anderson, L. K A model for chromosome structure during the mitotic and meiotic cell cycles. Chrom. Res., 9: Sumner,A.T A simple technique for demonstrating centromeric heterochromatin. Expl. Cell Res. 75: Swarca A. C., Giuliano-Caetano L. and Dias A. L., 1999 Cytogenetic characterization through chromosomic banding of Pinirampus pirinampu (Pisces, Pimelodidae) from the Tibagi river basin PR/Brazil. Caryologia, 1-2: Swarca A. C., Giuliano-Caetano L., Vanzela A. L. L., Dias A. L., 2001a Polymorphism of rdna genes in Pinirampus pirinampu (Pisces, Pimelodidae) detected by in situ hybridization. Cytologia, 66: Swarca A. C., Giuliano-Caetano L. and Dias A. L., 2001b Cytogenetic characterization of the large South American siluriform fish species Zungaro zungaro (Pisces, Pimelodidae). Chrom. Science, 5: Swarca A. C, Vidotto A. P. and Dias A. L., 2003 Cytogenetic characterization of Pimelodella aff. avanhandavae (Siluriformes, Pimelodidae) from Tibagi River (Paraná State, Brazil). Caryologia, 56: Verma R.S. and Babu A., 1995 Human Chromosomes: Principles and Techniques. 2 nd ed United States of America. International Edition. 419 p. Vicente M. R. R., Margarido V. P. e Galetti Jr P. M., 1994 Estudos cromossômicos em Imparfinis piperatus (Pisces, Siluriformes) do riacho São João (Bacia do Leste). Resumos: 17. V Simpósio de Citogenética Evolutiva e Aplicada de Peixes Neotropicais, Botucatu, Brazil. Vinas A., Gomez C., Martinez P. and Sanchez L., 1996 Localization of rdna genes in European eel (Anguilla anguilla) by FISH. Genome, 39: Vissotto P. C., Foresti F. and Oliveira C., 1997 A ZZ/ZW sex chromosome system in Imparfinis mirini (Pisces, Siluriformes). Cytologia, 62: Vissotto P. C., Foresti F. and Oliveira C., 1999 Karyotype description of five species of Pimelodidae (Teleostei, Siluriformes). Chrom. Science, 3: 1-7. Vissotto P. C., Foresti F. and Oliveira C., 2002 Karyotypic characterization of two species of the genus Imparfinis (Teleostei, Siluriformes, Heptapteridae). Chrom. Science, 5: