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Cariotipo y regiones nucleolares organizadoras (nor) en Pygocentrus cariba (serrasalminae) de Caicara del Orinoco, Venezuela.

RESUMEN

Se estudio el cariotipo de Pygocentrus cariba de Caicara del Orinoco, Venezuela. El numero diploide de cromosomas 2n=60 estuvo compuesto de 18 metacentricos, 30 submetacentricos, 2 subtelocentricos y 10 acrocentricos (NF= 110). Un maximo de 12 cromosomas portadores de NOR fueron detectados, estando las NOR localizadas en la region terminal de los brazos cortos de un par de cromosomas metacentricos de mediano tamano y de cinco pares acrocentricos pequenos. La heterocromatina constitutiva se observo localizada en las regiones centromericas y telomericas de casi todos los cromosomas, algunas mas conspicuas que otras. Algunos segmentos heterocromaticos intersticiales fueron tambien observados. Las similaridades entre P. cariba y las especies del genero Serrasalmus estudiadas hasta ahora, en terminos de numero y tipo de cromosomas, multiplicidad de cromosomas portadores de NOR y la distribucion de heterocromatina constitutiva localizada en posiciones centromerica y telomerica, refuerzan la hipotesis de la relacion filogenetica cercana entre los generos Pygocentrus y Serrasalmus.

SUMMARY

Cytogenetic studies were conducted on Pygocentrus cariba from Caicara del Orinoco, Venezuela. The diploid chromosome number 2n=60 consisted of 18 metacentric, 30 submetacentric, 2 subtelocentric and 10 acrocentric chromosomes (NF= 110). A maximum of 12 nucleolar organizer regions (NOR) bearing chromosomes were visualized, with the NORs located terminally on the short arms of one pair of medium sized metacentric chromosomes, and on five small-sized acrocentric chromosome pairs. Constitutive heterochromatin blocks were located at centromeric and telomeric regions of practically all chromosomes, some being more conspicuous than others. Similarities between P. cariba and the species of the genus Serrasalmus so far studied, in terms of the number and type of chromosomes, multiplicity of NORbearing chromosomes and the distribution of constitutive heterochromatin located in centromeric and telomeric position, reinforces the hypothesis of the close phylogenetic relationship between the genera Pygocentrus and Serrasalmus.

RESUMO

Estudou-se o cariotipo de Pygocentrus cariba de "Caicara del Orinoco", Venezuela. 0 numero diploide de cromossomos 2n=60 esteve composto de 18 metacentricos, 30 submetacentricos, 2 subtelocentricos e 10 acrocentricos (NF= 110). Um maximo de 12 cromossomos portadores de NORs foram detectados, estando as NORs localizadas na regiao terminal dos bracos curtos de um par de cromossomos metacentricos de tamanho medio e de cinco pares acrocentricos pequenos. A heterocromatina constitutiva se observou localizada nas regioes centromericas e telomericas de quase todos os cromossomos, algumas mais conspicuas que outras. Alguns segmentos heterocromaticos intersticiais foram tambem observados. As similaridades entre P. cariba e as especies do genero Serrasalmus estudadas ate agora, era termos de numero e tipo de cromossomos, multiplicidade de cromossomos portadores de NORs e a distribuicao de heterocromatina constitutiva localizada em posicoes centromerica e telomerica, reforfam a hipotese da relacao filogenetica proxima entre os generos Pygocentrus e Serrasalmus.

KEYWORDS / Karyotype/NOR / Pygocentrus cariba /

Introduction

The Serrasalminae is a highly diversified subfamily of Neotropical fishes, consisting of at least 60 species contained in 14 genera that include the well-known predatory "piranhas" or "caribes" (Machado-Allison, 1983). In spite of numerous chromosome studies of piranhas (Muramoto et al., 1968; Prado and Galetti, 1986; Cestafi and Galetti, 1992; Nakayama, 1997; Nakayama et al., 2000; 2001; 2002; Centofante et al., 2002) the species present in Venezuela remain poorly known from a cytogenetic perspective, except for a report on Serrasalmus rhombeus (Nirchio et al., 2002).

Pygocentrus cariba is one of the 16 species of piranhas that have been recognized in Venezuela. It is commonly found in the main stream, small secondary channels and flooded areas of the Orinoco basin (Machado-Allison and Fink, 1996).

This paper describes the diploid number, chromosome formula, and locations of nucleolar organizer regions (NOR) in P. cariba from Caicara del Orinoco, Venezuela.

Materials and Methods

Twenty-eight individuals (7 males, 21 females) of Pygocentrus cariba (Humboldt & Valenciennes, 1821) (Pisces: Serrasalminae) collected in the Laguna de Castillero, Caicara del Orinoco, Bolivar State, Venezuela, were analyzed on November-December, 2003. Voucher specimens were deposited in the fish collection of the Escuela de Ciencias Aplicadas del Mar, Universidad de Oriente, Venezuela.

Mitotic stimulation of the hematopoietic organ (kidney) was induced by injecting i.p. two doses (1 ml/100g body weight each, with a 24h interval) of yeast suspension, previous to the injection of colchicine (Lozano et al., 1988). After a 48h period each fish was injected with a 0.025% colchicine solution (1 ml/100g body weight) and maintained in a well-aerated aquarium for 1-2 hours. The mitotic chromosome preparations followed the technique described by Bertollo et al. (1978).

For conventional karyotype the preparations were stained during 20ruin with 5% Giemsa in phosphate buffer pH 6.88. Detection of NOR followed the silver staining method (Howell and Black, 1980). Staining was performed sequentially in order to identify the correspondence between NOR-bearing chromosomes when stained with AgN[O.sub.3] and the same chromosomes previously stained with Giemsa. Cbands were obtained according to the methods described by Sumner (1972).

Mitotic chromosomes were photographed using a Nikon Coolpix[R] 995 Digital Camera following the microscopy settings recommended by the manufacturer (http://www. nikonusa.com/pdf/ CoolpixSeriesMtngInstructions). Images were stored as .tif format files and digitally processed with Adobe Photoshop v. 7.0. This software was also used for measuring long (L) and short (S) arms, as well as whole chromosome lengths, and for constructing karyograms based on arm length ratios (Levan et al., 1964). Metacentric (M), submetacentric (SM) and subtelocentric (ST) chromosomes were considered to be biarmed, and acrocentric (A) chromosomes were considered to be uniarmed.

Results and Discussion

The available cytological information on piranhas shows that diploid number ranges from 2n= 58 to 2n= 64 (Table I) with 2n= 60 being the most common diploid number among the species of the group, which has led to propose 2n= 60 as the probable basic diploid number (Porto et al, 1991, Nakayama et al., 2002).

P. cariba displayed a karyotype 2n= 60, comprised of 9 pairs of metacentric (M), 15 pairs of submetacentric (SM), 1 pair of subtelocentric (ST) and 5 pairs of acrocentric chromosomes. The chromosome arm number (NF value) equaled 110 (Figure 1a). No heteromorphic sex chromosomes were observed.

[FIGURA 1 OMITIR]

As in the other species of piranhas so far karyotyped (Table I), P. cariba exhibited multiple NORs, varying from a minimum of 6 to a maximum of 12. This is a common feature in Serrasalminae (Nakayama et al., 2001), which has been recognized as a synapomorphic characteristic by Porto et al. (1992). This variation in the number of NOR-bearing chromosomes has been explained as a result of genetic activity regulation of the ribosomal cistrons (Cestari and Galetti, 1992). Given that silver stainability of NORs requires the transcriptional activity of the ribosomal genes during the preceding interphase (Hubbel, 1985), and taking into account that a variable number of NORs, between 6 and 12, are expressed, it was impossible to establish whether these NOR signals are homologous or homeologous. Fluorescent in situ Hybridization (FISH) with specific probes would be useful in establishing homology of these regions in the chromosomal complement.

In order to identify NORbearing chromosomes, sequential staining (Giemsa followed by AgN[O.sub.3] impregnation) was used. The NOR-bearing chromosomes were recognized as one pair of medium sized submetacentric (No 20), and on all the five small-sized acrocentric chromosome pairs (No 26, 27, 28, 29 and 30). The NORs on the SM pair NO 20 were always visualized whereas variation occurred on the acrocentric series.

In other piranhas, NORs have been detected exclusively on the short arms of STA chromosomes (Table I). Hence, NORs on a SM pair in P. cariba seems to be an apomorphy. To avoid any possibility of misclassification of the chromosome type, the index (L/S) was determined for 30 pairs of the chromosomes NO 20 from different individuals, finding that the centromeric index was always, within the SM range.

Regarding C-banding, this procedure shows that heterochromatic blocks in P. cariba were located at centromeric and telomeric regions of practically all chromosomes, some being more conspicuous than others. Some interstitial heterocromatic segments were observed (Figure 2).

[FIGURA 2 OMITIR]

In other piranha species included in the genus Serrasalmus, such as S. spilopleura (Cestari and Galletti, 1992), S. rhombeus (Nakayama et al., 2001), S. altispinis, S. compressus, S. elongatus and S. manuelli (Nakayama et al., 2002) heterochromatin is present in the centromeric and telomeric regions of practically all chromosomes. Also, the common occurrence of an interstitial segment in the long arm of a medium-sized metacentric pair, being more evident in some chromosomes than in others, is a characteristic that has not been observed in other piranha genera and that has been suggested as a probable sinapomorphy in the genus Serrasalmus. Based on a cladistic analysis of the Serrasalminae subfamily, Machado-Allison (1982, 1985) proposed that the genera Pygocentrus and Serrasalmus are part of a monophyletic group that also includes the Pygopristis and Prystobrycon genera. The cytogenetic similarities between P cariba and the species so far studied of the genus Serrasalmus, in the number and type of chromosomes, multiplicity of NOR-bearing chromosomes and the distribution of constitutive heterochromatin located in centromeric and telomeric position, reinforces the hypothesis of the close phylogenetic relationship between the genera Pygocentrus and Serrasalmus.
TABLE I
SUMMARY OF THE CHROMOSOME FINDINGS IN STUDIED PIRANHAS

Species                    2n   M    SM   ST   A    NF    NOR-BC

Serrasalmus altispinis     60   20   28    2   10   110      8
S. brandti                 60   18   24    8   10   110      8
S. compressus              60   18   30    2   10   110     12
S. elongatus               60   22   22    4   12   108     10
S. hollandi                64   30   --   16   18   110     --
S. humeralis               60   16   26    6   12   108     --
S. manuelli                60   22   24    6    8   112     12
S. marginatus              60   16   26    6   12   108      8
S. spilopleura             60   24   20    4   12   108     12
S. spilopleura a           60   20   26    4   10   110     10
S. spilopleura b           60   18   26    4   12   108     12
S. spilopleura c           60   19   26    4   11   109     11
S. rhombeus                60   30   16    4   10   110      8
S. rhombeus                60   20   24    6   10   110     12
S. rhombeus                58   22   24    2   10   106     12
Pygocentrus cariba         60   18   30    2   10   110     12

Species                         Reference

Serrasalmus altispinis     Nakayama et al., 2002
S. brandti                 Cestari and Galetti, 1992
S. compressus              Nakayama et al., 2002
S. elongatus               Nakayama et al., 2002
S. hollandi                Muramoto et al., 1968
S. humeralis               Cestari, unpublished data
S. manuelli                Nakayama et al., 2002
S. marginatus              Cestari and Galetti, 1992
S. spilopleura             Nakayama et al., 2002
S. spilopleura a           Cestari and Galetti, 1992
S. spilopleura b           Cestari and Galetti, 1992
S. spilopleura c           Cestari and Galetti, 1992
S. rhombeus                Nirchio et al., 2002
S. rhombeus                Nakayama et al., 2001
S. rhombeus                Nakayama et al., 2001
Pygocentrus cariba         This paper

2n: diploid number, M: metacentric, SM: submetacentric, ST:
subtelocentric, A: acrocentric, FN: fundamental number, NOR-BC:
maximum number of NOR-bearing chromosomes


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Received: 04/20/2004. Modified: 12/22/2005. Accepted: 01/04/2005.

Juan I. Gaviria. M.Aq., Auburn University, USA. Professor, Escuela de Ciencias Aplicadas del Mar, Universidad de Oriente (UDO), Nucleo Nueva Esparta, Venezuela.

Mauro Nirchio. M.Sc. in Marine Sciences, UDO, Venezuela. Professor, UDO, Venezuela. Address: Escuela de Ciencias Aplicadas del Mar, Nucleo Nueva Esparta, UDO. Apartado Postal 147 de Porlamar, Isla Margarita, Venezuela. email: nirchio@cantv.net

Angel Granado. M.Sc. in Marine Sciences, UDO, Venezuela. Professor, Instituto Limnologico, UDO, Caicara del Orinoco, Venezuela.

Alfonso Estrada. T.S.U. in Fishery and Aquaculture, IUTEMAR, Fundacion La Salle, Venezuela. Laboratory Assistant, Instituto Limnologico, UDO, Caicara del Orinoco, Venezuela.
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Title Annotation:Comunicaciones; piranas
Author:Gaviria, Juan I.; Nirchio, Mauro; Granado, Angel; Estrada, Alfonso
Publication:Interciencia
Date:Jan 1, 2005
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