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Karyotype and nucleolar organizer regions of pygocentrus cariba (serrasalminae) from Caicara del Orinoco... Article in Interciencia · July 2005 Source: OAI
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KARYOTYPE AND NUCLEOLAR ORGANIZER REGIONS OF Pygocentrus cariba (SERRASALMINAE) FROM CAICARA DEL ORINOCO, VENEZUELA Juan I. Gaviria, Mauro Nirchio, Ángel Granado y Alfonso Estrada 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.
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
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
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.
formula, and locations of nucleolar organizer regions (NOR) in P. cariba from Caicara del Orinoco, Venezuela.
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
Isla Margarita, Venezuela. email:
[email protected] 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.
Materials and Methods
KEYWORDS / Karyotype/ NOR / Pygocentrus cariba / 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), Núcleo Nueva Esparta, Venezuela.
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Mauro Nirchio. M.Sc. in Marine Sciences, UDO, Venezuela. Professor, UDO, Venezuela. Address: Escuela de Ciencias Aplicadas del Mar, Núcleo Nueva Esparta, UDO. Apartado Postal 147 de Porlamar,
0378-1844/05/01/044-04 $ 3.00/0
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RESUMO 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.
TABLE I SUMMARY OF THE CHROMOSOME FINDINGS IN STUDIED PIRANHAS Species
2n
M
SM
ST
A
NF
NOR-BC
Serrasalmus altispinis S. brandti S. compressus S. elongatus S. hollandi S. humeralis S. manuelli S. marginatus S. spilopleura S. spilopleura a S. spilopleura b S. spilopleura c S. rhombeus S. rhombeus S. rhombeus Pygocentrus cariba
60 60 60 60 64 60 60 60 60 60 60 60 60 60 58 60
20 18 18 22 30 16 22 16 24 20 18 19 30 20 22 18
28 24 30 22 26 24 26 20 26 26 26 16 24 24 30
2 8 2 4 16 6 6 6 4 4 4 4 4 6 2 2
10 10 10 12 18 12 8 12 12 10 12 11 10 10 10 10
110 110 110 108 110 108 112 108 108 110 108 109 110 110 106 110
8 8 12 10 12 8 12 10 12 11 8 12 12 12
Reference Nakayama et al., 2002 Cestari and Galetti, 1992 Nakayama et al., 2002 Nakayama et al., 2002 Muramoto et al., 1968 Cestari, unpublished data Nakayama et al., 2002 Cestari and Galetti, 1992 Nakayama et al., 2002 Cestari and Galetti, 1992 Cestari and Galetti, 1992 Cestari and Galetti, 1992 Nirchio et al., 2002 Nakayama et al., 2001 Nakayama et al., 2001 This paper
2n: diploid number, M: metacentric, SM: submetacentric, ST: subtelocentric, A: acrocentric, FN: fundamental number, NOR-BC: maximum number of NORbearing chromosomes
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. Cbands 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
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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 com-
mon 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 ho-
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mus, 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 Figure 1. Karyotype of Pygocentrus cariba. NOR-bearing chromosomes appear in long arm of a the squares (a). Correspondence among NOR-bearing chromosomes when stained m e d i u m - s i z e d metacentric pair, with AgNO3 with the same chromosomes previously stained with Giemsa (b). being more evident in some whereas variation chromosomes than in others, occurred on the is a characteristic that has not acrocentric series. been observed in other piraIn other piranha genera and that has been nhas, NORs have suggested as a probable been detected exsinapomorphy in the genus clusively on the Serrasalmus. Based on a clashort arms of STdistic analysis of the SerA chromosomes rasalminae subfamily, Ma(Table I). Hence, chado-Allison (1982, 1985) NORs on a SM proposed that the genera Pypair in P. cariba gocentrus and Serrasalmus seems to be an are part of a monophyletic apomorphy. To group that also includes the avoid any possiPygopristis and Prystobrycon bility of misclasgenera. The cytogenetic simisification of the larities between P. cariba and chromosome type, the species so far studied of Figure 2. C- banded metaphase of Pygocentrus the index (L/S) the genus Serrasalmus, in the cariba. was determined number and type of chromofor 30 pairs of somes, multiplicity of NORmology of these regions in the chromosomes Nº 20 from bearing chromosomes and the the chromosomal compledifferent individuals, finding distribution of constitutive ment. that the centromeric index heterochromatin located in In order to identify NORwas always, within the SM centromeric and telomeric pobearing chromosomes, serange. sition, reinforces the hypothquential staining (Giemsa Regarding C-banding, this esis of the close phylogenetic followed by AgNO3 impregprocedure shows that heterorelationship between the genchromatic blocks in P. cariba nation) was used. The NORera Pygocentrus and Serrasalwere located at centromeric bearing chromosomes were mus. and telomeric regions of pracrecognized as one pair of tically all chromosomes, some medium sized submetacenREFERENCES being more conspicuous than tric (N° 20), and on all the Bertollo LAC, Takahashi CS, others. Some interstitial five small-sized acrocentric Moreira-Filho O (1978) Cytoheterocromatic segments were chromosome pairs (Nº 26, taxonomic considerations on observed (Figure 2). 27, 28, 29 and 30). The Hoplias lacerdae (Pisces, In other piranha species inNORs on the SM pair Nº 20 Erytrinidae). Rev. Brasil. cluded in the genus Serrasalwer e always visualized Genet. 1: 103-120.
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