Karyotypic analysis in two species of fishes of the family Curimatidae:

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1 CARYOLOGIA Vol. 61, no. 3: , 2008 Karyotypic analysis in two species of fishes of the family Curimatidae: AgNO 3, CMA 3 and FISH with 18S probe Teribele 1 Rodrigo, Waleska Gravena 2, Kenia Carvalho 2, Lucia Giuliano-Caetano 2 and Ana Lúcia Dias 2 * 1 Universidade Federal de Mato Grosso do Sul, Centro de Ciências Biológicas e da Saúde, Depto de Biologia, , Campo Grande, MS, Brazil. 2 Universidade Estadual de Londrina, Departamento de Biologia Geral, , Londrina, Paraná, Brazil. Abstract Two species of fishes of the family Curimatidae, Cyphocharax modestus and Steindachnerina insculpta, collected in Tibagi river basin (Paraná, Brazil), were cytogenetically analysed and showed a diploid number of 54 chromosomes m-sm. An AgNOR was observed at the terminal position of chromosome pair 2 for Cyphocharax modestus and in pair 7 for Steindachnerina insculpta. Treatment with fluorochrome CMA 3 and fluorescence in situ hybridization (FISH) with an 18S rdna probe showed staining corresponding to the AgNOR pair for both of the species studied. One individual of C. modestus, collected in the Taquari stream, had only one AgNOR chromosome, where silver nitrate impregnation stained a large portion in chromosome long arm, corresponding to the secondary constriction. An identical result was obtained with CMA 3 staining. The FISH technique showed that one of the sites was large and corresponded to NOR and CMA 3 staining and that the other was very small, indicating the possible occurrence of unequal crossing-over between homologous chromosomes. Key words: AgNOR, CMA 3 staining, Curimatidae, FISH. INTRODUCTION * Corresponding author: phone: (43) ; fax: (43) ; anadias@uel.br The curimatids comprise approximately 140 species that occupy a vast variety of freshwater ecosystems, such as small rapid flowing rivers, low-lying rivers, and still-standing rivers, besides quiet lakes (VARI 1988). So far, there is cytogenetic information only for 35 species, where the majority have 2n = 54 chromosomes, all metasubmetacentric (MARTINS et al. 1996; NAVARRETE and JULIO Jr. 1997; BRASSESCO et al. 2004, ROSA et al. 2007). Within the order Characiformes, there are two well-differentiated trends: groups karyotypically stable and groups that show great chromosomal variability. The Curimatidae family can be included in the first group because the karyotype structure of 54 chromosomes metacentric and submetacentric (m-sm) is well conserved in this family. However, despite the conservation of karyotype macrostructure, the presence of supernumerary or B chromosomes have been observed in different populations (VENERE and GALETTI 1985; VE- NERE 1991; OLIVEIRA and FORESTI 1993; MARTINS et al. 1996; GRAVENA et al. 2007). In addition, variations in diploid number have been observed in 8 of the 35 species of curimatids examined until now (VÊNERE 1991; FELDBERG et al. 1992; 1993; NAVARRETE and JÚLIO JR. 1997; BRASSESCO et al. 2004). FELDBERG et al. (1992) suggested that 2n=54 m-sm is an ancestral karyotype of the curimatids and that variations of this condition represent characters derivatives The nucleolar organizing regions (NORs) in Curimatidae family are generally found in only one pair of chromosomes, but in different positions and chromosomes showing that despite the conserved karyotype structure, the speciation process within this family has been accompanied by micro-structural changes (VENERE and GALETTI 1989; BRASSESCO et al. 2004). The aim of the present study was to determine the karyotype and characterize the NORs of Cyphocharax modestus and Steindachnerina insculpta, collected from Tibagi river basin (Paraná state, Brazil), through conventional staining and other cytogenetic methods like AgNO 3, CMA 3 and in situ hybridization (FISH) with 18S rdna probe.

2 212 teribele, gravena, carvalho, giuliano-caetano and dias MATERIALS AND METHODS We analyzed twelve specimens of Cyphocharax modestus (9 males and 3 females) and fourteen specimens of Steindachnerina insculpta (7 males and 7 females) collected from Três Bocas stream; six specimens of C. modestus (2 males, 3 females and 1 individual of unknown sex), and seven specimens (4 males, 2 females and 1 individual of unknown sex) of S. insculpta collected from Taquari stream; eight specimens (5 males and 3 females), and two females of S. insculpta from the Tibagi and Água da Floresta rivers, respectively. All the water bodies belongs to the Tibagi river basin (Paraná state, Brazil). Mitotic chromosome preparations were obtained according to BERTOLLO et al. (1978). Chromosome morphology was determined on the basis of arm ratio (LEVAN et al. 1964). Nucleolar organizer regions (AgNOR) and Chromomycin A 3 (CMA 3 ) staining were performed using HOWELL and BLACK (1980) and SCHWEIZER (1976) methods, respectively. A 18S rdna probe obtained from Prochilodus argenteus (HATANAKA and GALETTI 2004) was used for fluorescent in situ hybridization (FISH), according SWARÇA et al. (2001). RESULTS AND DISCUSSION The two species, Cyphocharax modestus and Steindachnerina insculpta, showed a diploid number of 54 chromosomes meta-submetacentric (m-sm) (Figure 1A and 1B, respectively), where supernumerary chromosomes were observed in C. modestus from Tres Bocas and Taquari stream and in S. insculpta from Tres Bocas stream, as determined previously by GRAVENA et al. (2007). This karyotypic structure of 54 m-sm is very conserved in the curimatids, as well as in the families Anostomidae, Prochilodontidae and Parodontidae who also present a marked karyotypic stability (FELDBERG et al. 1992). These results are suitable with VARI (1983) that considers these families as sisters groups due to presenting some similar morphologic characteristics. The cytogenetic data obtained until the moment confirm the relationship among these families (BERTOLLO et al. 1986; PAULS and BERTOLLO 1990; MARTINS and GALETTI 1998; BRASSESCO et al. 2004). AgNORs in C. modestus and S. insculpta of the present study were observed at the terminal position in one pair of chromosomes, but these regions were localized in different chromosome pairs for each of the species. In C. modestus, the AgNOR was found on the long arm of the pair 2, a submetacentric (Figure 1A, box), and for S. insculpta it was on a metacentric pair of medium size, this probably being pair 7 (Figure 1B, box). Identical results were observed in different populations of C. modestus (VENERE and GALETTI JU- NIOR 1989; MARTINS et al., 1996; ROSA et al. 2007) and S. insculpta (ROSA et al. 2007), reinforcing the idea that NOR in curimatids can be used as species-specific cytogenetic marker, as also suggested by ROSA et al. (2007). However, VENERE and GALETTI JUNIOR (1989) observed in S. insculpta from the Mogi-Guaçu and Passa-Cinco rivers the AgNOR on pair 25. These authors suggested that internal chromosome modifications by translocations and/or inversions can be occurring within the Curimatidae karyotype. Staining with the CMA 3 produced fluorescent signals corresponding to AgNORs for C. modestus (Figure 1A, box) and S. insculpta (Figure 1B, box), demonstrating that these region are rich in GC pairs. Hybridization with the rdna 18S probe confirmed the number and location of Ag- NORS for the two curimatids species, where one pair showed the ribosomal cistrons (Figure 1A and 1B, box). ROSA et al. (2007) observed similar results with CMA 3 and rdna 18S in C. modestus and S. insculpta from the Paranapanema and Tietê rivers. One individual of C. modestus from the Taquari stream, showed strong AgNORs in only one chromosome, possibly a chromosome of pair 2, corresponding to a one secondary constriction (Figure 2B and 2A, respectively). This same individual showed CMA 3 in only one chromosome, with fluorescent signals on a large portion of the chromosome arm corresponding to that which was observed by silver nitrate impregnation (Figure 2C). FISH analysis in this individual demonstrated ribosomal cistrons in two chromosomes, where one of the homologous showed a large block and the other a very small signal (Figure 2D). This results indicates the probable occurrence of unequal crossing-over, resulting in one of the chromosomes receiving a greater amount of ribosomal cistrons than its homologue. In the chromosome that suffered the loss, the small NOR-bearing region that remained probably had become inactive or due to its reduced size was not detectable with silver nitrate impregnation or CMA 3 staining. The data presented contributes with more cytogenetic information on Cyphocharax modestus and Steindachnerina insculpta, confirming the stability in the karyotype macrostructure of this

3 cytogenetic analysis in two species of curimatidae 213 Fig. 1 (A) Karyotype of Cyphocharax modestus with B-chromosome. In box, the pair 2 with NOR, CMA 3 and 18S rdna. (B) Karyotype of Steindachnerina insculpta. In box the pair 7 with NOR, CMA 3 and 18S rdna.

4 214 teribele, gravena, carvalho, giuliano-caetano and dias Fig. 2 Somatic metaphases of an individual of Cyphocarax modestus collected from the Taquari stream: (A) Giemsa; (B) AgNOR (sequential); (C) CMA 3 and (D) FISH. The arrows indicate the secondary constriction and the NOR-bearing chromosome. The arrowhead points to a B chromosome. group of fishes, but showing variation inter-specific in the location of the NORs, indicating that the evolutionary process in this family has been accompanied by microstructural rearrangements. Acknowledgments The authors are grateful to CAPES and PIBIC/CNPq for their financial support. We are also thankful to Dr. Albert Leyva for his help in the preparation of the manuscript. REFERENCES BERTOLLO L.A.C, TAKAHASHI C.S. and MOREIRA-FILHO O., 1978 Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erythrinidae). Braz. J. Genet., 1: BERTOLLO L.A.C., MOREIRA-FILHO O. and GALETTI P.M., 1986 Cytogenetics and taxonomy: considerations based on chromosome studies of freshwater fish. J. Fish Biol., 28: BRASSESCO M.S., PASTORI M.C., RONCATI H.A. and FENOCCHIO A.S., 2004 Comparative cytogenetic studies of Curimatidae (Pisces, Characiformes) from the middle Paraná river (Argentina). Genet. Mol. Res., 3: FELDBERG E., PORTO J.I.R. and BERTOLLO L.A.C., 1992 Karyotype Evolution in Curimatidae (Teleostei, Characiformes) of the Amazon Region. I. Studies on the Genera Curimata, Psectrogaster, Steindachnerina and Curimatella. Braz. J. Genet., 2: FELDBERG E., PORTO J.I.R. and NAKAYAMA C.M., 1993 Karyotype evolution in Curimatidae (Teleostei, Characiformes) from the Amazon Region. II. Centric fissions in the genus Potamorhina. Genome, 36: GRAVENA W., TERIBELE R., GIULIANO-CAETANO L. and DIAS A.L., 2007 Occurrence of B chromosomes in Cyphocharax modestus and Steindachnerina insculpta (Characiformes, Curimatidae) from the basin of the Tibagi River /PR. Braz. Journal of Biology, 67: HATANAKA T. and GALETTI JR P.M., Mapping 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1929 (Characiformes, Prochilodontidae). Genetica, 122:

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