Genetics and Molecular Biology, 36, 3, 347-352 (2013)
Copyright © 2013, Sociedade Brasileira de Genética. Printed in Brazil
www.sbg.org.br
Research Article
Karyotypic conservatism in five species of Prochilodus (Characiformes,
Prochilodontidae) disclosed by cytogenetic markers
Tatiana Aparecida Voltolin1, Manolo Penitente1, Bruna Bueno Mendonça1, José Augusto Senhorini3,
Fausto Foresti2 and Fábio Porto-Foresti1
1
Departamento Ciências Biológicas, Faculdade de Ciências,
Universidade Estadual Paulista “Julio de Mesquita Filho”, Bauru, SP, Brazil.
2
Departamento de Morfologia, Instituto de Biociências,
Universidade Estadual Paulista “Julio de Mesquita Filho”, Botucatu, SP, Brazil.
3
Instituto Chico Mendes de Conservação da Biodiversidade,
Centro Nacional de Pesquisa e Conservação de Peixes Continentais, Pirassununga, SP, Brazil.
Abstract
The family Prochilodontidae is considered a group with well conserved chromosomes characterized by their number,
morphology and banding patterns. Thence, our study aimed at accomplishing a cytogenetic analysis with conventional methods (Giemsa staining, silver staining of the nucleolus organizer regions-AgNOR, and C-banding) and fluorescence in situ hybridization (FISH) with 18S and 5S ribosomal DNA probes in five species of the Prochilodus
genus (Prochilodus argenteus, Prochilodus brevis, Prochilodus costatus, Prochilodus lineatus and Prochilodus
nigricans) collected from different Brazilian hydrographic basins. The results revealed conservatism in chromosome
number, morphology, AgNORs 18S and 5S rDNAs location and constitutive heterochromatin distribution patterns.
The minor differences observed in this work, such as an Ag-NOR on a P. argenteus chromosome and a distinct
C-banding pattern in P. lineatus, are not sufficient to question the conservatism described for this group. Future work
using repetitive DNA sequences as probes for FISH will be interesting to further test the cytogenetic conservatism in
Prochilodus.
Keywords: AgNOR, C-banding, hydrographic basins, conserved karyotype, FISH.
Received: November 22, 2012; Accepted: June 9, 2013.
Introduction
Fishes of the family Prochilodontidae are significant
components of the fauna of Neotropical rivers and are considered one of the most important elements of commercial
and subsistence freshwater fisheries in South American environments, except in Chile, where they are not found
(Lowe-McConnell, 1975; Goulding, 1981; Vari, 1983;
Flecker, 1996).
Cytogenetic data on Prochilodus species revealed a
conserved karyotype with 2n = 54 chromosomes and a fundamental number FN = 108 (Pauls and Bertollo, 1983,
1990), suggesting that the family Prochilodontidae exhibits
a predominantly conserved chromosomal evolution (Pauls
and Bertollo, 1990). However, a few populations and/or,
species, such as P. brevis, P. lineatus, P. mariae and P.
nigricans, showed karyotypic variation due to the presence
Send correspondence to Fabio Porto-Foresti. Laboratório de Genética de Peixes, Departamento Ciências Biológicas, Faculdade de
Ciências, Universidade Estadual Paulista “Julio de Mesquita Filho”,
Av. Engenheiro Luiz Edmundo C. Coube s/n, 17033-360,Bauru,
SP, Brazil. e-mail: fpforesti@fc.unesp.br.
of supernumerary chromosomes (Pauls and Bertollo 1983,
1990; Oliveira et al., 1997, 2003; Dias et al., 1998; Venere
et al., 1999; Maistro et al., 2000; Cavallaro et al., 2000; Jesus and Moreira-Filho, 2003; Artoni et al., 2006; Voltolin
et al., 2009).
Conventional cytogenetic markers, such Ag-NORs,
evidenced a single chromosome pair bearing NORs in
some species of Prochilodus (Pauls and Bertollo, 1990;
Venere et al., 1999; Oliveira et al., 2003; Jesus and Moreira-Filho, 2003; Vicari et al., 2006; Voltolin et al.,
2009).
Data on the localization of the 5S and 18S ribosomal
genes by FISH in the genomes of species of
Prochilodontidae are still scarce (Jesus and Moreira-Filho,
2003; Hatanaka and Galetti Jr, 2004; Vicari et al., 2006;
Grass et al., 2007; Terêncio et al., 2012). FISH with the 5S
and 18S ribosomal genes showed that they are syntenic in
P. lineatus and P. argenteus, and evidenced a polymorphism in the number of 18S rRNA genes (Jesus and Moreira-Filho, 2003; Hatanaka and Galetti Jr, 2004; Voltolin
et al., 2009).
348
C-banding analyses carried out in Prochilodontidade
representatives showed that the constitutive heterochromatin is frequently restricted to centromeric blocks in all
chromosomes of the standard (A) complement (Pauls and
Bertollo, 1990; Venere et al., 1999; Cavallaro et al., 2000;
Oliveira et al., 2003; Jesus and Moreira-Filho 2003; Artoni
et al., 2006; Voltolin et al., 2009). Additionally, the supernumerary chromosomes were usually entirely heterochromatic in these species (Pauls and Bertollo 1990; Maistro et
al., 2000; Cavallaro et al., 2000; Jesus and Moreira-Filho,
2003; Artoni et al., 2006; Voltolin et al., 2009).
Therefore, the objective of the current study was to
conduct a comparative analysis with conventional and molecular cytogenetic markers in five species of the genus
Prochilodus collected in different Brazilian hydrographic
basins to look for chromosome differences that may have
accumulated in these populations over the years allowing
their cytogenetic differentiation.
Materials and Methods
We analyzed 20 samples of P. lineatus from the
Mogi-Guaçu river, Pirassununga, (São Paulo); 17 individuals of P. nigricans from the Tocantins Araguaia basin
(Tocantins); 15 individuals of P. costatus acquired from the
Aquicultura Tropical pisciculture, Propiá (Sergipe); six
samples of P. argenteus from the São Francisco basin; and
five individuals of P. brevis, acquired from the Departamento de Nacional de Obras Contra a Seca (DNOCS)
dam, in Natal (Rio Grande do Norte).
Mitotic chromosomes were obtained from anterior
kidney fragments (Foresti et al., 1981) and through lymphocyte culture (Fenocchio and Bertollo.,1988) with some
adjustments. The karyotypes were arranged according to
Levan et al. (1964).
Active NORs were identified after silver nitrate staining (Howell and Black, 1980) and the constitutive heterochromatin was detected after C-banding (Sumner, 1972).
FISH was carried out according to Pinkel et al. (1986)
using 5S rDNA probes obtained by PCR from Prochilodus
genomic
DNA
using
the
primers
A
(5_-TACGCCCGATCTCG TCCGATC-3_) and B
(5_-CAGGCTGGTATGGCCGTAAGC-3_) (Pendás et
al., 1994). The 18S rDNA probe was obtained by PCR using the NS1 (5_-GTAGTCATATGCTTGTCTC-3_) and
NS8 (5_-TCCGCAGGTTCACCTACGGA-3_) primers
(White et al., 1990).
The 5S probe was labeled with biotin-dUTP and the
18S probe was labeled with digoxigenin-dUTP (Roche) by
PCR, according to the manufacturer’s instructions.
The 5S and 18S rDNA probes were denatured in 70%
formamide:2xSSC for 5 min. The hybridization occurred at
37 °C overnight in a moist chamber (0.3 mg of denatured
probe, 50% formamide, 10 mg/mL of dextran sulfate;
2xSSC, 5 mg/mL of salmon sperm DNA).
Voltolin et al.
The 5S and 18S probes were immunodetected with
avidin-FITC and anti-digoxigenin-rhodamine, respectively, and the preparations were counterstained with DAPI
(4-6-diamidino-2-phenylindole) and examined under an
epifluorescence photomicroscope (BX 61, Olympus)
equipped with an Olympus DP70 cooled digital camera.
Photomicrographs were taken using the Pro MC 6.0 software.
Results
All specimens of Prochilodus (P. argenteus, P.
brevis, P. costatus, P. lineatus and P. nigricans) collected
in the different Brazilian hydrographic basins presented a
karyotype with 2n = 54, FN = 108 and metacentric and
submetacentric chromosomes (Figure 1a-e). All specimens
of P. lineatus had supernumerary chromosomes (Figure 2a), whereas one P. nigricans specimen had a single B
chromosome that showed intraindividual variation, with 23
cells out of 30 exhibiting the extra chromosome (Figure 2b).
After Ag-NOR staining, only one homolog of a submetacentric pair presented a NOR in P. brevis, P. costatus,
P. lineatus and P. nigricans (Figure 1b-e, highlighted). In
P. argenteus, the NOR was observed only on one homologue of the second largest submetacentric pair. This AgNORs pattern was found in approximately 40 metaphases
of all P. argenteus specimens.
FISH with the 5S and 18S rDNA probes was performed to confirm if there was a karyotypic difference in
the 18S gene location in P. argenteus. Synteny between
these two genes was observed in all samples tested, including P. argenteus, in which the 18S rDNA labeled both
homologues of the second submetacentric pair, which was
not observed after Ag-NOR (Figure 3a, b, c, d and e). Furthermore, the location of these genes was identical in all the
species, i.e., the 5S gene was located near the terminal region of the long arm of the submetacentric chromosome
pair and the 18S gene, in a pericentromeric position,
syntenic with the 5S gene. Clusters of these ribosomal sequences were not detected in any of the species studied, nor
in the B chromosomes present in the genome of P. lineatus
and P. nigricans.
C-banding was performed to identify the distribution
of constitutive heterochromatin in all the species. The results allowed us to differentiate among the specimens of P.
lineatus and those of the other four Prochilodus species.
Conspicuous heterochromatic blocks present only in the
centromeric regions of the standard A chromosome set
were observed in P. lineatus (Figure 4d). In P. argenteus, P.
brevis, P. costatus and P. nigricans, besides the presence of
heterochromatic regions in the centromere, a large heterochromatic block on the long arm of a submetacentric chromosome pair was also observed (Fig. 4b-e). The supernumerary chromosomes of P. lineatus and P. nigricans were
totally heterochromatic (Figure 4d, e).
Cytogenetic markers in Prochilodus
349
Figure 2 - Metaphases of (a) P. lineatus, with four supernumerary chromosomes (arrows). Supernumerary chromosomes occurred in all individuals of this species; (b) metaphase of P. nigricans with only one supernumerary chromosome (arrow).
Figure 1 - Karyotypes of the five Prochilodus species analyzed in this
work: (a) P. argenteus; (b) P. brevis; (c) P. costatus; (d) P. lineatus and
(e)P. nigricans. In the boxes, the AgNOR-bearing pair.
Discussion
Pioneer studies in Prochilodus cytogenetics conducted by Pauls and Bertollo (1983, 1990) evidenced a con-
spicuous homogeneity in karyotypes. Several studies have
shown that specimens of Prochilodus presented 2n = 54,
FN = 108 and biarmed chromosomes (Pauls and Bertollo,
1983, 1990; Oliveira et al., 1997; Cavallaro et al., 2000, Jesus and Moreira-Filho, 2003; Voltolin et al., 2009). Our results are consistent with these data (Figure 1a-e).
The presence of supernumerary microchromosomes
in some species of Prochilodus enabled us to study aspects
concerning their origin, evolution, structure and maintenance. First described in P. lineatus by Pauls and Bertollo
(1983), up to two B microchromosomes were also identified in P. brevis (=P. cearensis) by these same authors
(Pauls and Bertollo (1990). Venere et al. (1999) described
the occurrence of one or two B chromosomes in P.
nigricans and Oliveira et al. (2003) identified up to three
supernumerary chromosomes in some individuals of P.
mariae from the Orinoco river basin in Venezuela.
The specimens of P. brevis, P. costatus, P. lineatus
and P. nigricans studied herein had only one Ag-NOR situated on the long arm of the second largest submetacentric
pair (inbox in Figure 1b-e), as already described for these
species (Pauls and Bertollo, 1983.1990; Venere et al.,
1999; Maistro et al., 2000; Jesus and Moreira-Filho, 2003;
Hatanaka and Galetti Jr, 2004; Vicari et al., 2006; Artoni et
al., 2006; Voltolin et al., 2009).
In P. argenteus, a single AgNOR was observed on the
long arm of one homologue of a submetacentric chromosome (inbox in Figure 1a). This result is inconsistent with
the literature data for this species, in which the single
AgNOR was observed on the second largest submetacentric pair (Hatanaka and Galetti Jr, 2004).
Silver nitrate does not directly bind to rDNA, but to
the proteins associated with the nucleolar structure, restricting the identification to the NORs that had been active in
the preceding interphase (Miller et al., 1976). This is the
most reasonable hypothesis to explain the single AgNOR in
P. argenteus.
The position of the ribosomal genes was further investigated with FISH with the 18S and 5S rDNA probes.
350
Voltolin et al.
Figure 3 - Fluorescence In situ Hybridization (FISH) with 5S (green) and 18S (red) rDNA probes in: (a) P. argenteus; (b) P. brevis; (c) P. costatus; (d) P.
lineatus with a supernumerary chromosome, and (e) P. nigricans with a supernumerary chromosome.
Figure 4 - C-banded cells of: (a) P. argenteus; (b) P. brevis; (c) P. costatus; (d) P. lineatus and (e) P. nigricans. In P. lineatus, constitutive
heterochromatin was only present in the centromeric regions of all A chromosomes. All supernumerary chromosomes were heterochromatic (arrows).
These probes were syntenic in agreement with previous reports (Jesus and Moreira-Filho, 2003;, Hatanaka and Galetti Jr, 2004; Vicari et al., 2006). No additional18S clusters
were found in this species, as previously reported (Maistro
et al., 2000; Vicari et al., 2006) and neither additional 5S
clusters, as already described by Jesus and Moreira-Filho
(2003) and by Vicari et al. (2006) in specimens of P.
lineatus from the Mogi Guaçu river and Dourada lagoon,
respectively.
The syntenic organization of the 5S and 18S ribosomal genes is a rare event among vertebrates. In addition
to P. lineatus (Jesus and Moreira-Filho, 2003; Vicari et al.,
2006; Voltolin et al., 2009), this synteny was also observed
in Salmo salar (Pendás et al., 1994), Oncorhynchus mykiss
(Móran et al., 1996), Astyanax (Almeida-Toledo et al.,
2002), in amphibians (Lucchini et al., 1993) and, more recently, in Pimelodus britskii (Moraes-Neto et al., 2011). On
the other hand, these loci have been mapped on different
chromosomes in many fish species (Martinez et al., 1996;
Morán et al., 1996; Born and Bertollo, 2000; Ferro et al.,
2001; Vicente et al., 2001; Wasko et al., 2001; Noleto et
al., 2007), representing the most frequent condition in vertebrates (Lucchini et al., 1993; Drouin and Muniz De Sá,
1995, Suzuki et al., 1996).
Neither the 5S nor the 18S ribosomal genes have been
found in the B microchromosomes of P. lineatus (Jesus and
Moreira-Filho, 2003) and of P. nigricans.
C-banding has proven very useful in cytogenetic
studies of fish, permitting the identification of constitutive
heterochromatin regions. Differences in the amount or distribution of heterochromatin identified by C-banding are
considered important for some fish groups and the
distribution of C-bands may characterize genera, species
and populations (Montovani et al., 2000).
Several C-banding studies have been performed in
Prochilodontidae, especially in Prochilodus lineatus. Maistro et al. (2000) reported centromeric and subtelomeric
constitutive heterochromatin in the A complement of curimbatás collected in the Mogi-Guaçu river, Pirassununga,
São Paulo. However, telomeric C-bands were absent in the
chromosome preparations analyzed herein. but the heterochromatic nature of the supernumerary chromosomes was
corroborated (Jesus and Moreira-Filho, 2003; Artoni et al.,
2006; Voltolin et al., 2009).
Specimens of Prochilodus are characterized by the
presence of conspicuous centromeric heterochromatic
blocks in the A complement and in all supernumerary chromosomes (Jesus and Moreira-Filho, 2003; Artoni et al.,
2006; Voltolin et al., 2009).
In this study, we observed different patterns of
heterochromatin distribution in the genomes of some species of Prochilodus. In P. lineatus, constitutive heterochromatin was observed only in the pericentromeric regions of
all chromosomes of the standard (A) complement (Figure
4d) and all B chromosomes were heterochromatic. In P.
argenteus, P. brevis, P. costatus and P. nigricans, in addition to the centromeric region of all A chromosomes, we
also observed a large heterochromatic block on a submetacentric chromosome pair (Figure 4a, b, c, e). In addition
to the centromeric C-bands in specimens of P. lineatus
from the Mogi Guaçu river, Jesus and Moreira-Filho (2003)
also evidenced heterochromatic blocks close to the telo-
Cytogenetic markers in Prochilodus
meric region of a submetacentric pair. Despite these small
differences in the constitutive heterochromatin distribution
in Prochilodus, we can still consider them a cytogenetically
conserved group.
Oliveira et al. (2003) confirmed the conservative nature of the chromosome number and morphology in
Prochilodontidae and reinforced the idea that small structural chromosome rearrangements may be the main cause
of karyotypic diversification in this group. In that study, the
authors observed that in Prochilodus mariae from the Orinoco river basin, Venezuela, in addition to the occurrence
of constitutive heterochromatin in all centromeres of the
autosomes, a heterochromatic block was present in a
submetacentric pair, as observed herein in P. argenteus, P.
brevis, P. costatus and P. nigricans. Oliveira et al. (2003)
also identified differing C-banding patterns between
Semaprochilodus kneri and Semaprochilodus laticeps. In S.
kneri, constitutive heterochromatin was present in the
centromeric regions of all A chromosomes and a conspicuous heterochromatic block occurred on the long arm of pair
24. In S. laticeps, heterochromatin was only found in the
pericentromeric regions, as we also observed in P. lineatus.
The presence of heterochromatin only in centromeric
regions of the standard A chromosome set, as described
herein in P. lineatus, differs from the data published by
Maistro et al. (2000) and Jesus and Moreira-Filho (2003)
for P. lineatus from the Mogi Guaçu River, the same location of our collections. These authors described the presence of heterochromatin in the centromeric region of all
autosomes and in the telomeric regions of some pairs of the
A complement.
Nevertheless, the four species analyzed (P.
argenteus, P. brevis, P. nigricans and P. costatus) exhibited the same heterochromatin distribution pattern, in full
agreement with literature data (Pauls and Bertollo, 1993;
Venere et al., 1999; Hatanaka and Galetti Jr, 2004).
Pauls and Bertollo (1983, 1990) stated that the family
Prochilodontidae, especially the Prochilodus genus, presented a conserved karyotype, resulting from a conservative chromosome evolution. The data obtained herein are in
agreement with this proposed conservatism. The small
cytogenetic variations found herein, such as the constitutive heterochromatin distribution and the position of the
NOR among the studied Prochilodus, are not sufficient to
contradict the strong conservatism proposed for the species
of this genus by various authors of numerous cytogenetic
studies.
Acknowledgments
The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) for financial support.
351
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