Universidade Federal do Paraná, Departamento de Genética, Centro Politécnico, Curitiba, Brazil; fceqyn.unam.edu.ar.

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1 CARYOLOGIA Vol. 56, no. 4: , 2003 Cytogenetic characterization of hybrids offspring between Colossoma macropomum (Cuvier, 1818) and Piaractus brachypomus (Cuvier, 1817) from Caicara del Orinoco, Venezuela M. NIRCHIO 1, A.S. FENOCCHIO 2, A.C. SWARÇA 3, J.E. PÉREZ 4, A. GRANADO 5, A. ESTRADA 5 and E. RON 1 1 Escuela de Ciencias Aplicadas del Mar. Universidad de Oriente, Isla de Margarita, Venezuela. Apartado Postal 630-Porlamar. 2 Universidade Federal do Paraná, Departamento de Genética, Centro Politécnico, Curitiba, Brazil; afenocch@ fceqyn.unam.edu.ar. 3 Universidade Estadual de Londrina, Departamento de Biologia Geral, Caixa Postal 6001, CEP , Londrina, PR, Brasil; szwarcy@sercomtel.com.br. 4 Instituto Oceanográfico de Venezuela, Universidad de Oriente, Apartado 243, Cumaná, Venezuela; jperez@ telcel.net.ve. 5 Instituto Limnológico, Universidad de Oriente, Caicara del Orinoco, Estado Bolívar, Venezuela; limnologico@cantv.net. Abstract - Conventional karyotype and nucleolar organizer regions (NORs) of C. macropomum x P. brachypomus hybrids and parental species are reported. A modal diploid number of 54 chromosomes and a fundamental number (NF) of 108 were found for C. macropomum, P. brachypomus and their hybrids. P. brachypomus shows a pair of silver stained chromosomes, while cells with three and four Ag-NOR bearing chromosomes were observed in C. macropomum. The hybrids consistently presented cells with a single metacentric Ag-NOR bearing chromosome and cells with three Ag-NOR bearing chromosomes. The FISH technique was employed to localize 18S rdna in the chromosomes of the parentals and the hybrids. In P. brachypomus the FITC signals appeared in the SM pair as when stained with silver salts. In C. macropomum the signals were evidenced in six chromosomes. In the hybrids, as expected, the FITC dots were observed in four chromosomes. All the techniques employed in the present work represent good tools to identify the parentals and distinguish them of the hybrids. Key words: Fisheries; Interspecific hybrids; Colossoma macropomum; Piaractus brachypomus; Cytogenetic Monitoring; NORs; in situ hybridization. INTRODUCTION The production of interspecific offsprings to obtain individuals in which the best characteristics of the parental species could converge has been one of the goals of aquaculturists (DUNHAM 1987; CHEVASSUS and DORSON 1990). In Latin America hybridization between different species of the Family Characidae has been * Corresponding author: nirchio@cantv.net achieved with success in several Research Institutes. Particularly Colossoma macropomum and Piaractus brachypomus have been object of attention because of their high production yields, easiness for cultivation and massive production of alevins under controlled reproduction conditions (ALMEIDA-TOLEDO et al. 1988; KOSSOSWKI et al. 1983; MARTINO 1994). The analysis of genome structure of hybrid fishes is necessary to achieve an adequate genetic evaluation in order to get a basic understanding that allows to distinguish the types of genet-

2 406 NIRCHIO, FENOCCHIO, SWARÇA, PÉREZ, GRANADO, ESTRADA and RON ic products resulting from a successful hybridization process (ALMEIDA-TOLEDO FILHO et al. 1994) and to assess the potential biological hazards of the hybrid to the genetic integrity of the parental species, wild or cultivated (PÉREZ and RYLANDER 1998). Cytogenetics provides some techniques that could help to understand better the behavior and structure of the chromosomes that was integrate in the hybrids. So, the analysis of the karyotype and principally of the nucleolus organizer regions (NORs) can be appropriate and sensitive methods to verify the ploidy levels as well as the origin of the chromosomes in fishes (ALMEIDA-TOLEDO FILHO et al. 1994; ZHANG and TIERSCH 1997). Locations of the NORs are easily detected by silver impregnation but this technique has the limitation imposed by their necessary activity in the previous interphase. DNA-DNA in situ hybridization is increasingly important for understanding the physical position of all classes of DNA sequences along chromosomes of both plants and animals (HES- LOP-HARRISON 1991). The fluorescence in situ hybridization (FISH) with rdna probes is a useful tool to reveal precisely all the chromosomes involved in nucleolar organization as well as the physical location and copy number of the ribosomal genes. This paper reports the karyotypic formulaes and organizer nucleolar regions (NORs) of Colossoma macropomum, Piaractus brachypomus and their hybrids by means of conventional and AgN- OR staining and in situ hybridization with rdna biotinilated probes. MATERIALS AND METHODS Five individuals of each parental species (Colossoma macropomum and Piaractus brachypomus) and 25 specimens of the resulting offspring obtained by in vitro fecundation of ovocites of P. brachypomus (female N 2) with sperm of C. macropomum (males N 2 and N 63) were analyzed. Each specimen was injected intraperitonially with a yeast suspension (1cc/100 g of weight) to stimulate the cellular proliferation in the hematopoietic organ (kidney) (LOZANO et al. 1988). After a 18 hour period each fish was injected with colchicine solution (0.5%; 1 ml/100g body weight), and maintained in a well aerated aquarium for 4-6 hours. The preparations for the visualization of the chromosomes, were obtained following the technique described by NIR- CHIO and CEQUEA (1998). For the determination of the conventional karyotype, the preparations were stained during 20 minutes with Giemsa (10%) diluted in buffer phosphate, ph 6.8. The detection of the nucleolar organizer regions (NORs) was performed following the methodology described by HOWELL and BLACK (1980). The 18S rdna segment containing 1700pb of the fish Oreochromis niloticus was used for fluorescence in situ hybridization (FISH) and labeled with biotin-14- datp by nick translation according to the manufacturer s instructions (Gibco cat Nº ). The hybridization technique, post-hybridization washes and visualization were performed according to SWARÇA et al. (2001). The mitotic figures were photographed using green filter and ASA 50 film. The resulting photographs were then scanned (1,200 dpi) and stored as *.tif images Long arm (L), short arm (S) and total length were measured from each chromosome to the nearest 0.01 mm using the measuring tool of ADOBE PHOTO- SHOP Software v Chromosomes were classified as described by LEVAN et al. (1964) and ordered in decreasing size. RESULTS AND DISCUSSION The diploid numbers determined for Colossoma macropomum, Piaractus brachypomus and their hybrids were 54 chromosomes (all bi-armed) and the fundamental number (FN) were 108 (Fig. 1). The chromosome groups were arranged according to the proportion L arm/s arm. The karyotypes of parental species showed a complement constituted by 14 pairs of M and 13 pairs of SM chromosomes, however a clear differentiation among the karyotypes of the species studied here have been observed: the first pair of SM chromosomes of C. macropomum is around 30% longer than the second instead, in P. brachypomus were evidenced a gradual size decrease of all the chromosomes of the complement (Fig. 1a, b). In the hybrid 28 M and 26 SM chromosomes were distinguished (Fig. 1c). In this case these elements were not paired because in some cases they not constitute homologues and in other they would be homeologue chromosomes. This feature is very evident in the group of SM when the first chromosome has not any paireable partner (Fig. 1c). These results are partially in agreement with previous reports that indicate a numerical com-

3 CYTOGENETIC CHARACTERIZATION OF HYBRIDS BETWEEN COLOSSOMA MACROPOMUM AND PIARACTUS BRACHYPOMUS 407 a b c Fig. 1 Conventional karyotype (Giemsa) of Colossoma macropomum (a), Piaractus brachymomus (b) and the hybrid (c). M: metacentric; SM: submetacentric.

4 408 NIRCHIO, FENOCCHIO, SWARÇA, PÉREZ, GRANADO, ESTRADA and RON plement 2n=54 (NF= 108) for C. macropomum and P. brachypomus, but differs respect to the number of M and SM elements. In the case of C. macropomum previous reports indicate 18M + 36 SM (KOSSOWSKI et al. 1983); 20M + 34 SM (ALMEIDA TOLEDO 1987) and 26 M + 28 SM (NAKAYAMA et al. 1990), while for P. brachypomus a complement constituted by 34M + 20 SM (NAKAYAMA et al. 1990) has been described. These differences could be attributed to technical features, as chromosomal condensation. ALMEIDA TOLEDO et al. (1988) found that conventional staining with Giemsa would not allow to differentiate the hybrids from the parental species in the cross C. macropomum x P. mesopotamicus. In the case of the cross C. macropomum ( ) x Mylossoma duriventris ( ) the karyotype of the parental species are numerically equal, but there is an acrocentric marker in M. duriventris which is detected in the hybrids (KOSSOWSKI et al. 1983). P. brachypomus studied in the present work have a pair of SM NOR bearing chromosomes when silver stained. These regions are located at the ends of the long arm (Fig. 2a, b). In C. macropomum were observed cells with four (Fig. 2c) and three (Fig. 2d and 2e) metacentric Ag-NOR bearing chromosomes. The silver salts produce evident dark blocks on terminal regions of these chromosomes (Fig 2f). The hybrids consistently presented cells with a single Ag-NOR bearing chromosome and cells Fig. 2 Chromosomic complement of Colossoma macropomum stained with Silver Nitrate. The arrows show the NOR-bearing chromosomes. In the insets, amplification of silver stained chromosomes with the nucleolar organizer region. Piaractus brachypomus (a, b); Colossoma macropomum (c, d, e, f); hybrid (g, h, i, j, k).

5 CYTOGENETIC CHARACTERIZATION OF HYBRIDS BETWEEN COLOSSOMA MACROPOMUM AND PIARACTUS BRACHYPOMUS 409 Fig. 3 Fluorescence in situ hybridization with biotin-labeled 18S rdna probe in Colossoma macropomum (a, b), Piaractus brachypomus (c, d) and the hybrid (e, f). Arrows indicate rdna FISH.

6 410 NIRCHIO, FENOCCHIO, SWARÇA, PÉREZ, GRANADO, ESTRADA and RON with three Ag-NOR bearing chromosome (Fig. 2g, i, respectively). In the case of the cells with a single silver stained chromosome, it was a SM element like the one of P. brachypomus (Fig. 2h). When three Ag-NOR bearing chromosomes were observed, the conspicuous black dots always was located at the terminal ends of the long arm of an element SM and less evident blocks on the terminal region of two metacentrics (Fig. 2j), like in the parental C. macropomum. The silver staining of the NORs only reveal the position of the genes for the ribosomal RNA in chromosomes that were active in a preceding interphase (HSU et al. 1975; MILLER et al. 1976). An alternative to detect all the chromosomes involved in nucleolar organization as well as the exact location of the ribosomal genes, is the application of the fluorescence in situ hybridization (FISH) with rdna probes. In this work the FISH technique was successfully employed to localize 18S rdna on the chromosomes of Colossoma macropomum, Piaractus brachypomus and their hybrids. In P. brachypomus the FITC signals appeared on the long arm of the submetacentric pair in all analyzed metaphases (Fig. 3c,d), like when the chromosomes preparation were stained with silver salts. Furthermore, in C. macropomum the signals were evidenced on six chromosomes, at the short arm of two metacentric pairs and on the long arm of one submetacentric pair (Fig. 3a, b), but when was applied the Ag-NO 3 were evidenced only three or four NOR-bearing metacentric chromosomes. According to PENDÁS et al. (1993) the silver staining is not recommended to localize rrna genes, but is the best approach to study NOR expression. In accordance with these authors, in C. macropomum the FISH technique have evidenced two chromosomes that were not detectable by AgNO 3 staining (probably inactives or silent during the previous interphase). This inactivation may be due to the presence of some type of gene interaction, like a partial inactivation of the genes. In the hybrids, when was applied the AgNO 3 staining were evidenced cells with a single metacentric chromosome marked and cells with three chromosomes marked, one SM and two M chromosomes. However, in this case, FISH allows to identify the positive FITC signals on four chromosomes, on the short arm of two metacentric pairs and on the long arm of two submetacentric (Fig 3e, f), as expected. On the basis of shape and size, was inferred that one of these two submetacentrics correspond to the Ag-NOR bearing chromosomes of Piaractus brachypomus, and the others (one SM and two M) would correspond to the NOR bearing chromosomes of C. macropomum. This results, in the same way that in C. macropomum confirm that the silver staining only shows NORs that were active in the preceding interphase, once the AgNO 3 react with acid proteins associated with rdna (JORDAN 1987). In this work, conventional coloration helps to identify the chromosome types and allow to distinguish clearly the karyotype of the offspring resulting from the cross between C. macropomum and P. brachypomus, the hybrids also were perfectly differentiated by the number and location of nucleolar genes. Thus, both, conventional karyotype (Giemsa stained) and NOR phenotype (Ag and FISH), can be used as valid methods to identificate parental species and to verify the origin of the hybrids. Acknowledgments The authors are thankful to Dra. Ana Lúcia Dias and Dra. Lúcia Giuliano-Caetano from the University Estadual de Londrina (Brazil) for providing the rdna 18S probe for hybridization experiments and for their continuous cooperation. Swarça, A.C. was supported by fellowship from CAPES. REFERENCES ALMEIDA-TOLEDO S.F., FORESTI L.F., FORESTI F., BERNARDINO G. and CALCAGNOTTO D., 1994 Monitoramento e Conservação genética em projeto de hibridação entre pacu e tambaqui. Cadernos de Ictiogenética, 2 ( ALMEIDA-TOLEDO, L. F., FORESTI F., RAMOS S.M., ORMANEZI R., CAROSFELD V.J.S. and TOLEDO F.S.., 1988 Estudos citogenéticos de híbridos entre fêmeas de Pacu (Piaractus mesopotamicus) e machos de Tambaqui (Colossoma macropomum). B. Téc. CEPTA, Pirassununga, 1 (2): CEHEVASSUS B. and DORSON M., 1990 Genetics of resistance to disease in fish. Aquaculture, 85: DUNHAM R.A., 1987 American catfish breeding program. Proceedings of the World Symposium on Selection, Hybridization and Genetic Engi-

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