Nucleolar organizer regions as markers of chromosomal polymorphism in Apareiodon affinis (Pisces, Parodontidae)

CARYOLOGIA Vol. 57, no. 2: 195-199, 2004 Nucleolar organizer regions as markers of chromosomal polymorphism in Apareiodon affinis (Pisces, Parodontidae) Lilian Cristina Jorge 1 * and Orlando Moreira Filho 2 1 Instituto de Ictiología del Nordeste, Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste. Sargento Cabral 2139, C.P. 3400, Corrientes, Argentina. Fax: 54 03783 425753 int.113, e-mail: liliancj@vet.unne.edu.ar. 2 Departamento de Genética e Evolução,Universidad Federal de SãoCarlos, Rod. Washington Luis, Km 235, CEP. 13565-905, São Carlos, SP, Brazil. Abstract Specimens of Apareiodon affinis collected from four sites of the Paraná River (called Lower Paraná, Argentina) were analyzed. This species presents a diploid number of 54 chromosomes, without any apparent sex chromosomal heteromorphism. However, it was possible to verify the occurrence of 10 cytotypes, some of them common to all collected sites, characterizing an structural chromosomal polymorphism. Data obtained from chromosomal banding methods, results further suggest an inter-individual NOR polymorphism. Such variation is possibly caused by pericentric inversions and other chromosomal rearrangements. The 5S rdna was separated from 18S rdna clusters, these cistrons seems to present variation, suggesting that other chromosomes could also be involved in the polymorphism. The results obtained confirmed the structural polymorphism in A. affinis. Key words: Apareiodon affinis, Argentina, cytogenetics, fish, NOR polymorphism. INTRODUCTION *Corresponding author: e-mail: liliancj@vet.unne.edu.ar. Since Parodon species were firstly described, Parodontidae species were included in many families and subfamilies. Initially, there were included in Characidae, and lately within Hemiodontidae (Pavanelli 1999). Only after Roberts (1974) the identity of the family Parodontidae was recognized. This family is composed by three genera: Parodon, Apareiodon, and Saccodon. The first two genera are distributed throughout South America and Saccodon spp. are restricted to Northern of South America. The difference between Parodon and Apareiodon is based on the presence or absence of mandibular teeth. Cytogenetical data are available for Parodon and Apareiodon species; P. tortuosus, P. hilarii, Paradon sp., A. affinis, A. ibitiensis, A. piracicabae, A. vittatus, Apareiodon sp. A, Apareiodon sp. B and Apareiodon sp. C(Moreira-Filho et al. 1980, 1984, 1985, 1993; Jesus 1996; Jesus et al. 1999, Jesus and Moreira- Filho 2000 a, b). All analyzed specimens were originally collected in rivers located in the Brazilian territory. These studies ranged from karyotype identification to banding patterns. Therefore, different banding procedures applied on Parodontidae allowed a better characterization of its species (Moreira- Filho et al. 1984; Jesus 1996; Jesus et al. 1999; Jesus and Moreira-Filho 2000 a, b). Previous conventional cytogenetical analyses in A. affinis from Paraná River, Argentina, detected the occurrence of 10 cytotypes (Jorge and Moreira- Filho 2000). In the present work, a more detailed analysis is performed, involving banding and FISH procedures to establish chromosomal markers, possibly indicating the nature of the rearrangements related to the present polymorphism. MATERIAL AND METHODS The specimens were collected at four different sites from Paraná River, Lower Paraná (Argentina). The Paraná River basin is an important hydrographic system situated on the center-eastern of South America and comprises an area of 1,600,000 km 2 (Petri and Fúlfaro 1983). The localities were: Ituzaingo (56 42 15 W 27 31 04 S) (Corrientes Province); Ita Ibate (57 16 25 W 27 28 32 S); Corrientes (58 50 08 W 27 28 12 S); Reconquista (59 34 45 W 29 13 36 S) (Santa Fe Province). Twenty-four individuals were analyzed (11 females and 13 males). Mitotic chromosomes were obtained from kidney cells, following the method of Bertollo et al. (1978). C-banding procedure was performed according to Sumner (1972). The nucleolar organizer regions

196 jorge and moreira filho (NORs) were evidenced by silver nitrate staining (Ag- NOR) and by distamycin/chromomycin (DA/CMA 3 )as described by Howell and Black (1980) and Schweizer (1980), respectively. The 18S rdna probe was obtained after amplification via PCR using the primers NS1 (5 -GTAGTCAT- ATGCTTGTCTC-3 ) and NS8 (5 -TCCGCAGGT- TCACCTACGGA-3 ) (White et al. 1990) of total DNA extracted from Prochilodus affinis (Characiformes). The amplification products were kindly provided by Dr. HATANAKA (UFSCar). The 5S rdna probe was composed by the recombinant plasmid p-bs/ks containing the 5S rrna gene from Leporinus elongatus (Martins and Galetti Jr. 1999). The fragments of 5S (about 200 bp) and 18S (about 1800 bp) rdna were purified with Kit Sephaglass Band Prep (Pharmacia Biotech). The probes were marked with biotinylated adenine (BdATP) according to manufacturer s instructions. The in situ hybridization were performed according to Pinkel et al. (1986). Fresh slides were incubated with RNase 0,004% in a moist chamber at 37 C. The chromosomal denaturation was carried out with formamide 70%/ 2X SSC for 5 minutes at 70 C. The hybridization solution (1µl of denatured probe, 50% formamide, 10% dextrane sulphate and 2X SSC) were added to each slide, and kept overnight at 37 C in a moist chamber. The hybridization signals were detected by using Avidine-Fluorescein conjugate (FITC) and biotinylated anti-avidin antibody. The chromosomes were counterstained with propidium iodide (50µg/ml) and examined with a Olympus BX50 epifluorescence microscope. Photographs were taking using 400 ASA Kodak Gold Ultra film. RESULTS AND DISCUSSION Apareiodon affinis from Paraná River (Lower Paraná, Argentina) presents a diploid number of 54 chromosomes, for both sexes, without any apparent sex chromosomal heteromorphism. Although the chromosomal number is constant, an intrapopulational variation was found involving the number of meta-submetacentric (48 to 50), subtelocentric (0 to 4) and acrocentric (0 to 6) chromosomes, consequently altering the fundamental number (102 to 108). Thus, it was possible to determine 10 cytotypes, some of them common to all localities, characterizing a structural chromosomal polymorphism (Jorge and Moreira-Filho 2000). This polymorphism and the presence of acrocentric chromosomes in the cytotypes of A. affinis from Argentina can be considered a derived condition, representing a novelty for the family Parodontidae. The origin of these variations could be explained by pericentric inversions of bi-armed chromosomes giving rise to one-armed elements, thus altering the fundamental number. This hypothesis is reinforced by the absence of acrocentric chromosomes in most of Parodon and Apareiodon species (Moreira-Filho et al. 1980, 1984, 1985, 1993; Jesus 1996; Jesus and Moreira-Filho 2000 a, b), excepting A. affinis from Cuiabá River (Jesus et al. 1999). Besides that, other families related to Parodontidae (Prochilodontidae, Curimatidae, Anostomidae, Chilodontidae) present 2n=54 and NF=108, with M/SM chromosomes (Galetti Jr. et al. 1981 a, b; Pauls and Bertollo 1983, 1990; Feldberg et al. 1987; Feldberg et al. 1992; Venere and Galetti Jr. 1989; Martins and Galetti Jr. 1997, 1998). Intrapopulational variations determined by pericentric inversions were observed in other fish populations such as Ilyodon furcidens from Coahuaya River in Mexico (Turner et al. 1985), Hoplerythrinus unitaeniatus from Negro River in Brazil (Giuliano-Caetano and Bertollo 1988), Astyanax altiparanae from Claro River in Brazil (Pacheco et al. 2001). By silver nitrate and CMA 3 staining, and fluorescence in situ hybridization (FISH) with 18S rdna probe, the nucleolar chromosomal pair was identified, being directly involved with the chromosomal polymorphism observed in A. affinis from Argentina. In most of the analyzed samples, the NORs were evidenced at terminal position on the long arms, coincident with the secondary constrictions observed in the 26 th pair. These chromosomes were present in two structural displays: subtelocentric and acrocentric, in three different combinations: ST/ST, ST/A and A/A (Figure 1a, b, c). It was also possible to identify, more rarely, ST chromosomes bearing bitelomeric NORs. These chromosomes were not observed in homozygosis, but paired with ST or A chromosomes bearing NORs on the long arms (Figure 1a, b, c). The presence of nucleolar organizer regions at terminal position in both arms of a subtelocentric chromosome (see Figure 1a, b, c) could be a result of a pericentric inversion or other rearrangements such as translocation or gene conversion. Variation in NOR position among nucleolar chromosomes due to the occurrence of these rearrangements was also reported in other fish species (Galetti Jr. et al. 1995; Almeida-Toledo et al. 1996; Rodrigues and Collares-Pereira 1996; Castro et al. 1996; Jesus 1996). In the present work, preliminary analyses with fluorescence in situ hybridization with 5S rdna showed positive signals in a pair of heteromorphic medium-sized chromosomes: one of them is characterized as metacentric, bearing marks at interstitial position and the other one is a submetacentric with interstitial marks on both arms (Figure 1d). The 5S

nors as markers of chromosomal polymorphism 197 Fig. 1 Somatic metaphases of A. affinis after staining by silver nitrate (a), CMA 3 (b), FISH with 18S rdna (c) and 5S rdna (d) probes. The arrows indicate the marked chromosomes in each methodology. The chromosomal pairs bearing ribosomal cistrons from different individuals are shown on the right of each metaphase plate. Note that, by silver nitrate staining, three patterns are observed: two acrocentric chromosomes bearing telomeric NORs on the long arms; one subtelocentric chromosome and an acrocentric one bearing terminal NORs on the long arms; and two subtelocentric chromosomes with NORs located terminally on the long arms, besides an additional mark in one of them at the end of the short arms (a). After CMA 3 staining, the same chromosomes can be observed, besides a subtelocentric pair bearing terminal marks on the long arms (b). By FISH with 18S rdna probe, the previously described pairs are found, plus a subtelocentric chromosome carrying NORs on the telomeres of both arms and an acrocentric bearing marks at the end of the long arms (c). FISH results with 5S rdna probe revealed a single (heteromorphic) pair bearing 5S rrna genes, comprising a metacentric chromosome interstitially marked in one arm and a subtelocentric bearing interstitial signals on both arms (d). rrna genes are not linked to 18S rdna sites. The interstitial position of these genes was also observed in other fish groups such as Anguilla anguilla (Martínez et al. 1996), Coregonus arditi and C. zenithicus (Sajdak et al. 1998), Leporinus spp. (Martins and Galetti Jr. 1999), and Hoplias malabaricus (BORN and Bertollo 2000). The presence of interstitial 5S ribosomal genes on both arms of the subtelocentric chromosome can be related to pericentric inversion of an original metacentric chromosome, suggesting a polymorphism for this region and constituting, as well as NORs, a marker for the rearrangements occurred in this species. By now, this interpretation remains speculative and a sample increasing is required to provide conclusive data. The constitutive heterochromatin was evidenced on the pericentric region of most of the chromosomes and on telomeric portion of some pairs. Conspicuous terminal blocks were verified in the long arm of the 26 th pair (acrocentric), adjacent to the secondary constriction (Figure 2). Such heterochromatin pattern was also observed in the first ST pair in several species from Parodon and Apareiodon genera (Jesus 1996; Jesus et al. 1999; Jesus and Moreira- Filho 2000 a, b). The location of heterochromatic blocks adjacents to NORs from the 26 th pair and their variants confers

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