Karyotype and nucleolar organizer regions of Pygocentrus cariba (SERRASALMINAE) from Caicara del Orinoco, Venezuela

Juan I. Gaviria, Mauro Nirchio, Ángel Granado y Alfonso Estrada

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

Mauro Nirchio. M.Sc. in Marine Sciences, UDO, Venezuela. Professor, UDO, Venezuela. Address: Escuela de Ciencias Aplicadas del Mar, Núcleo Nueva Esparta, UDO. Apar-tado Postal 147 de Porlamar, Isla Margarita, Venezuela. e- mail: nirchio@cantv.net

Ángel Granado. M.Sc. in Marine Sciences, UDO, Venezuela. Professor, Instituto Limnológico, UDO, Caicara del Orinoco, Venezuela.

Alfonso Estrada. T.S.U. in Fishery and Aquaculture, IUTEMAR, Fundación La Salle, Venezuela. Laboratory Assistant, Instituto Limnológico, UDO, Caicara del Orinoco, Venezuela.

Resumen

Se estudió el cariotipo de Pygocentrus cariba de Caicara del Orinoco, Venezuela. El número diploide de cromosomas 2n=60 estuvo compuesto de 18 metacéntricos, 30 submetacéntricos, 2 subtelocéntricos y 10 acrocéntricos (NF= 110). Un máximo de 12 cromosomas portadores de NOR fueron detectados, estando las NOR localizadas en la región terminal de los brazos cortos de un par de cromosomas metacéntricos de mediano tamaño y de cinco pares acrocéntricos pequeños. La heterocromatina constitutiva se observó localizada en las regiones centroméricas y teloméricas de casi todos los cromosomas, algunas más conspicuas que otras. Algunos segmentos heterocromáticos intersticiales fueron también observados. Las similaridades entre P. cariba y las especies del género Serrasalmus estudiadas hasta ahora, en términos de número y tipo de cromosomas, multiplicidad de cromosomas portadores de NOR y la distribución de heterocromatina constitutiva localizada en posiciones centromérica y telomérica, refuerzan la hipótesis de la relación filogénética cercana entre los géneros 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 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.

Resumen

Estudou-se o cariótipo de Pygocentrus cariba de "Caicara del Orinoco", Venezuela. O número diplóide de cromossomos 2n=60 esteve composto de 18 metacêntricos, 30 submetacêntricos, 2 subtelocêntricos e 10 acrocêntricos (NF= 110). Um máximo de 12 cromossomos portadores de NORs foram detectados, estando as NORs localizadas na região terminal dos braços curtos de um par de cromossomos metacêntricos de tamanho médio e de cinco pares acrocêntricos pequenos. A heterocromatina constitutiva se observou localizada nas regiões centroméricas e teloméricas de quase todos os cromossomos, algumas mais conspícuas que outras. Alguns segmentos heterocromáticos intersticiais foram também observados. As similaridades entre P. cariba e as espécies do gênero Serrasalmus estudadas até agora, em termos de número e tipo de cromossomos, multiplicidade de cromossomos portadores de NORs e a distribuição de heterocromatina constitutiva localizada em posições centromérica e telomérica, reforçam a hipótese da relação filogenética próxima entre os gêneros Pygocentrus e Serrasalmus.

KEYWORDS / Karyotype/ NOR / Pygocentrus cariba /

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

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; Cestari 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, Bolívar 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 (1ml/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 (1ml/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 20min 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 AgNO₃ and the same chromosomes previously stained with Giemsa. C-bands were obtained according to the methods described by Sumner (1972).

Mitotic chromosomes were photographed using a Nikon Coolpix® 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.

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 NOR-bearing chromosomes, sequential staining (Giemsa followed by AgNO₃ impregnation) was used. The NOR-bearing chromosomes were recognized as one pair of medium sized submetacentric (N° 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 ST-A 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).

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.

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