Chromosomal Characterization of Prochilodus Lineatus from Paraná River, Corrientes, Argentina

Chromosomal Characterization of Prochilodus lineatus from Paraná River, Corrientes, Argentina. I. B Chromosomes and NOR Banding

Lilian Cristina Jorge1*, Sebastián Sanchez1 and Orlando Moreira Filho2

1 Instituto de Ictiología del Nordeste, Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste, Sargento Cabral 2139 (3400), Corrientes, Argentina 2 Departamento de Genética e Evolução, Universidade Federal de São Carlos, Via Washington Luiz, km 235, Caixa Postal 676, CEP 13565-905, São Carlos, SP, Brazil

Received July 2, 2010; accepted April 15, 2011

Summary The ﬁsh of the genus Prochilodus, commonly known as sábalo, are of great economic and ecological importance. Different species of this genus are widely distributed throughout all the basins of South America. Cytogenetic analysis performed on specimens of Prochilodus lineatus from Puerto Abras (Paraná River, Argentina), revealed the occurrence of 54 chromosomes of meta- submetacentric type, together with the presence of small supernumerary chromosomes with intra and interindividual variations. The use of the 18S rDNA probe conﬁrmed the results obtained with Ag- NORs, identifying the presence of a metacentric chromosome pair with ribosomal cistrons in interstitial position in the long arm. Through both of these techniques are noted differences in the size of the nucleolar regions, this variation is probably related to the occurrence of events of duplication and/or deletion.

Key words Fish, Prochilodus lineatus, B chromosomes, NOR, 18S rDNA.

The genus Prochilodus is widely distributed in South America (Leccia 1972). Their economic value of inland fisheries in many South American countries and their anatomical peculiarities, point out interesting material for phylogenetic studies within Characiformes. Prochilodus lineatus known as sábalo or curimbatá represents in Argentina the most abundant fish resources from the Paraná River is an illiophagus fish, like other species of the family Prochilodontidae. From an economic point of view, easier cultivation in several systems and water qualities is, and the fish also has high protein meat and a balanced content of essential amino acids (Lessi 1968). Most of the appreciated reports which concern of P. lineatus karyotypes analyzed were specimens from Brazilian territory. Pauls and Bertollo (1983, 1990) found in animals from different rivers of Paraná River basin diploid number of 54 chromosomes, fundamental number (NF) equal to 108 and a karyotype consisting of chromosomes of meta-submetacentric type. Other authors (Oliveira et al. 1997, Cavallaro et al. 2000) analyzed in this species the frequency and inheritance of chromosomes Bs. On the other hand, Jesus et al. (2003) achieved the molecular characterized 2 families of satellite DNA and its chromosomal location. Due to the limited information of the population of P. lineatus in Argentina, the objectives of this study are to obtain information about the karyotypic characteristics of the species and determine the chromosomal location of 18S ribosomal RNA genes as possible genetic marker for this purpose was used fluorescent in situ hybridization (FISH) and staining with silver nitrate.

* Corresponding author, e-mail: [email protected], [email protected] 220 L. C. Jorge et al. Cytologia 76(2)

Materials and methods We analyzed 34 specimens of P. lineatus (11 females and 23 males), which were collected in the Paraná river in the town of Puerto Abras, province of Corrientes (Argentina). Before slaughter the animals were anesthetized with MS222 (tricaine methanesulphonate). Chromosome preparations were obtained from anterior kidney, according to the methodology proposed by Moreira-Filho and Bertollo (1991). Mitotic chromosomes to obtain by procedure suggested by Foresti et al. (1993). The nucleolus organizing regions (NORs) were identified by silver nitrate staining (Ag-NORs) as described by Howell and Black (1980). The 18S rDNA probe was obtained after amplification via PCR using the primers NS1 (5- GTAGTCATATGCTTGTCTC-3) and NS8 (5-TCCGCAGGTTCACCTACGGA-3) (White et al. 1990) of total DNA extracted from Prochilodus argenteus (Hatanaka and Galetti 2004). The 18S rDNA probe was labeled by Bionick Labeling System Kit (Gibco BRL) according to manufacturer’s instructions. The metaphase chromosomes were treated according to the procedure described by Pinkel et al. (1986) and analyzed using an Olympus BX50 epifluorescence microscope. The chromosomes figures were captured using CoolSNAP-Pro software (Media Cybernetics).

Results and discussion Cytogenetic analysis of the species revealed the presence of 54 chromosomes (meta/submetacentric), fundamental number equal to 108 in both gender. Heteromorphism in sex chromosome was not detected (Fig. 1a). Cytogenetic studies of Prochilodontidae family were characterized like conservative from the karyotypic macrostructure aspect species of the genus Prochilodus showed (P. vimboides, P. lineatus, P. affinis, P. marggravii, P. cearensis, P. argenteus and P. nigricans) showed a karyotype constituted by 54 chromosomes with 2 arms (Pauls and Bertollo 1983, 1990). Furthermore, presence of 0 to 2 microchromosomes Bs, was observed variable in number in the interindividual and intraindividual karyotypic complement (Fig. 1a). The occurrence of supernumerary or B chromosomes in P. lineatus from different sampling points of the Paraná River, are being studied in Brazil by several authors, reaching in some populations a number that varies from 0–7 (Pauls and Bertollo 1983, 1990, Cavallaro 1992, Maistro et al. 2000, Artoni et al. 2006, Carvalho et al. 2008, among others). Silver nitrate staining was used to identification of the NORs in interstitial position on the long arm of a metacentric chromosome pair, identified as the second complement (Fig. 1b), according with Pauls and Bertollo (1990). Similar to the observations made by Jesus and Moreira-Filho (2003) showed heteromorphism in relation to the size of Ag-NORs regions, where 1 chromosome was more marked than its homologous. The Prochilodus genus NORs are located an interstitial position in the second pair of chromosomes apparently its a common condition, although in some species (P. affinis, P. marggravii) was found NOR-bearing chromosomes in terminal position (Pauls and Bertollo 1990, Hatanaka and Galetti 2004). Fluorescent in situ hybridization (FISH) with 18S rDNA probe was using by identify a nucleolar chromosome pair revealing the presence of interstitial ribosomal sites located on the second chromosome pair (Fig. 1c), corresponding to Ag-NORs. However, Jesus and Moreira-Filho (2003) obtained in same species similar results, but our observations showed no additional 18S sites. Silver nitrate staining and FISH showed variation in size of the nucleolar regions, 2011 Chromosomal Characterization of Prochilodus lineatus 221

Fig. 1. (a) Karyotype of Prochilodus lineatus, also 1 small B chromosome. (b) Ag-NOR bearing chromosomes (arrows), and B chromosomes (arrowhead) in somatic metaphase. (c) Metaphase submitted to FISH with 18S rDNA showing hybridization signal in 2 chromosomes (arrows), and B chromosomes (arrowhead) demonstrating interchromosomal differences in copy number from rDNA. According to Jesus and Moreira-Filho (2003) this size heteromorphism is due to a structural polymorphism of ribosomal cistrons and not by differential pattern of NORs expression. NORs polymorphic sizes located between homologous chromosomes could be explained by spontaneous events like as duplication, deletion and unequal crossing over (Gold et al. 1990). Heteromorphism in size of NORs has been reported in neotropical ﬁsh frequently (Moreira-Filho et al. 1984, Jesus and Moreira Filho 2000, Wasko and Galetti 2000, Hatanaka and Galetti 2004, among others). Concluding, although Prochilodus lineatus was observed a conservational karyotypic in many different populations from Paraná River, the location and variability in size and number of NORs sites seems to be an unstable condition among differents populations. Regarding to chromosomes B is necessary to enhance studies in other locations along the Paraná river trying to knowledge more about its distribution, origin and evolution. 222 L. C. Jorge et al. Cytologia 76(2)

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