Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

Gaviria, Juan I.; Nirchio, Mauro; Granado, Ángel; Estrada, Alfonso Karyotype and nucleolar organizer regions of cariba (serrasalminae) from Caicara del Orinoco, Venezuela Interciencia, vol. 30, núm. 1, enero, 2005, pp. 44-47 Asociación Interciencia Caracas, Venezuela

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative KARYOTYPE AND NUCLEOLAR ORGANIZER REGIONS OF (SERRASALMINAE) FROM CAICARA DEL ORINOCO, VENEZUELA

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

SUMMARY

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

RESUMEN

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

Introduction 2002) the species present in formula, and locations of Voucher specimens were de- Venezuela remain poorly nucleolar organizer regions posited in the fish collection The Serrasalminae is a known from a cytogenetic (NOR) in P. cariba from of the Escuela de Ciencias highly diversified subfamily perspective, except for a re- Caicara del Orinoco, Venezu- Aplicadas del Mar, Universi- of Neotropical fishes, consist- port on Serrasalmus rhom- ela. dad de Oriente, Venezuela. ing of at least 60 species con- beus (Nirchio et al., 2002). Mitotic stimulation of the tained in 14 genera that in- Pygocentrus cariba is one Materials and Methods hematopoietic organ (kidney) clude the well-known preda- of the 16 species of was induced by injecting i.p. tory “piranhas” or “caribes” that have been recognized in Twenty-eight individuals (7 two doses (1ml/100g body (Machado-Allison, 1983). In Venezuela. It is commonly males, 21 females) of Pygo- weight each, with a 24h inter- spite of numerous chromo- found in the main stream, centrus cariba (Humboldt & val) of yeast suspension, pre- some studies of piranhas small secondary channels and Valenciennes, 1821) (Pisces: vious to the injection of (Muramoto et al., 1968; Pra- flooded areas of the Orinoco Serrasalminae) collected in the colchicine (Lozano et al., do and Galetti, 1986; Cestari basin (Machado-Allison and Laguna de Castillero, Caicara 1988). After a 48h period and Galetti, 1992; Nakayama, Fink, 1996). del Orinoco, Bolívar State, each fish was injected with a 1997; Nakayama et al., 2000; This paper describes the Venezuela, were analyzed on 0.025% colchicine solution 2001; 2002; Centofante et al., diploid number, chromosome November-December, 2003. (1ml/100g body weight) and

KEYWORDS / Karyotype/ NOR / Pygocentrus cariba / Received: 04/20/2004. Modified: 12/22/2005. Accepted: 01/04/2005.

Juan I. Gaviria. M.Aq., Auburn Mauro Nirchio. M.Sc. in Marine Isla Margarita, Venezuela. e- Alfonso Estrada. T.S.U. in Fish- University, USA. Professor, Sciences, UDO, Venezuela. mail: [email protected] ery and Aquaculture, IUTE- Escuela de Ciencias Aplicadas Professor, UDO, Venezuela. Ángel Granado. M.Sc. in Marine MAR, Fundación La Salle, del Mar, Universidad de Address: Escuela de Ciencias Sciences, UDO, Venezuela. Venezuela. Laboratory Assis- Oriente (UDO), Núcleo Nueva Aplicadas del Mar, Núcleo Professor, Instituto Limno- tant, Instituto Limnológico, Esparta, Venezuela. Nueva Esparta, UDO. Apar- lógico, UDO, Caicara del Ori- UDO, Caicara del Orinoco, tado Postal 147 de Porlamar, noco, Venezuela. Venezuela.

44 0378-1844/05/01/044-04 $ 3.00/0 JAN 2005, VOL. 30 Nº 1 RESUMO

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

TABLE I SUMMARY OF THE CHROMOSOME FINDINGS IN STUDIED PIRANHAS Species 2n M SM ST A NF NOR-BC Reference Serrasalmus altispinis 60 20 28 2 10 110 8 Nakayama et al., 2002 S. brandti 60 18 24 8 10 110 8 Cestari and Galetti, 1992 S. compressus 60 18 30 2 10 110 12 Nakayama et al., 2002 S. elongatus 60 22 22 4 12 108 10 Nakayama et al., 2002 S. hollandi 64 30 - 16 18 110 - Muramoto et al., 1968 S. humeralis 60 16 26 6 12 108 - Cestari, unpublished data S. manuelli 60 22 24 6 8 112 12 Nakayama et al., 2002 S. marginatus 60 16 26 6 12 108 8 Cestari and Galetti, 1992 S. spilopleura 60 24 20 4 12 108 12 Nakayama et al., 2002 S. spilopleura a 60 20 26 4 10 110 10 Cestari and Galetti, 1992 S. spilopleura b 60 18 26 4 12 108 12 Cestari and Galetti, 1992 S. spilopleura c 60 19 26 4 11 109 11 Cestari and Galetti, 1992 S. rhombeus 60 30 16 4 10 110 8 Nirchio et al., 2002 S. rhombeus 60 20 24 6 10 110 12 Nakayama et al., 2001 S. rhombeus 58 22 24 2 10 106 12 Nakayama et al., 2001 Pygocentrus cariba 60 18 30 2 10 110 12 This paper 2n: diploid number, M: metacentric, SM: submetacentric, ST: subtelocentric, A: acrocentric, FN: fundamental number, NOR-BC: maximum number of NOR- bearing chromosomes maintained in a well-aerated settings recommended by the shows that diploid number mon feature in Serrasalminae aquarium for 1-2 hours. The manufacturer (http://www. ranges from 2n= 58 to 2n= (Nakayama et al., 2001), mitotic chromosome prepara- nikonusa.com/pdf/ 64 (Table I) with 2n= 60 be- which has been recognized tions followed the technique CoolpixSeriesMtngInstructions). ing the most common diploid as a synapomorphic charac- described by Bertollo et al. Images were stored as .tif number among the species of teristic by Porto et al. (1978). format files and digitally the group, which has led to (1992). This variation in the For conventional karyotype processed with Adobe propose 2n= 60 as the prob- number of NOR-bearing the preparations were stained Photoshop v. 7.0. This soft- able basic diploid number chromosomes has been ex- during 20min with 5% Giemsa ware was also used for mea- (Porto et al, 1991, Nakayama plained as a result of genetic in phosphate buffer pH 6.88. suring long (L) and short et al., 2002). activity regulation of the ri- Detection of NOR followed (S) arms, as well as whole P. cariba displayed a bosomal cistrons (Cestari and the silver staining method chromosome lengths, and for karyotype 2n= 60, comprised Galetti, 1992). Given that sil- (Howell and Black, 1980). constructing karyograms of 9 pairs of metacentric (M), ver stainability of NORs re- Staining was performed se- based on arm length ratios 15 pairs of submetacentric quires the transcriptional ac- quentially in order to identify (Levan et al., 1964). Meta- (SM), 1 pair of subtelocentric tivity of the ribosomal genes the correspondence between centric (M), submetacentric (ST) and 5 pairs of acrocen- during the preceding inter- NOR-bearing chromosomes (SM) and subtelocentric tric chromosomes. The chro- phase (Hubbel, 1985), and when stained with AgNO3 and (ST) chromosomes were mosome arm number (NF taking into account that a the same chromosomes previ- considered to be biarmed, value) equaled 110 (Figure variable number of NORs, ously stained with Giemsa. C- and acrocentric (A) chromo- 1a). No heteromorphic sex between 6 and 12, are ex- bands were obtained according somes were considered to be chromosomes were observed. pressed, it was impossible to to the methods described by uniarmed. As in the other species of establish whether these NOR Sumner (1972). piranhas so far karyotyped signals are homologous or Mitotic chromosomes were Results and Discussion (Table I), P. cariba exhibited homeologous. Fluorescent in photographed using a Nikon multiple NORs, varying from situ Hybridization (FISH) Coolpix® 995 Digital Cam- The available cytological a minimum of 6 to a maxi- with specific probes would era following the microscopy information on piranhas mum of 12. This is a com- be useful in establishing ho-

JAN 2005, VOL. 30 Nº 1 45 mus, such as S. Centofante L, Porto JIR, Feldberg spilopleura E (2002) Chromosomal poly- morphism in Serrasalmus (Cestari and Gal- spilopleura Kner, 1858 letti, 1992), S. (Characidae, Serrasalminae) rhombeus (Naka- from Central . yama et al., Caryologia 55: 37-45. 2001), S. altis- Cestari MM, Galetti PM (1992) pinis, S. com- Chromosome evolution in the genus Serrasalmus and cyto- pressus, S. elon- taxonomic considerations about gatus and S. Serrasalminae (Characidae, Pi- manuelli (Naka- sces). Brazil. J. Genet. 15: yama et al., 555-567. 2002) heterochro- Howell WM, Black DA (1980) matin is present Controlled silver staining of in the centro- nucleolus organizer regions with a protective colloidal de- meric and te- veloper: a 1-step method. lomeric regions Experientia 3: 1014-1015. of practically all Hubel HR (1985) Silver staining as chromosomes. an indicator of active riboso- Also, the com- mal genes. Stain Technol. 60: mon occurrence 285-294. of an interstitial Levan A, Fredga K, Sandberg AA segment in the (1964) Nomenclature for cen- tromeric position on chromo- Figure 1. Karyotype of Pygocentrus cariba. NOR-bearing chromosomes appear in long arm of a somes. Hereditas 52: 201-220. medium-sized the squares (a). Correspondence among NOR-bearing chromosomes when stained Lozano R, Ruiz RC, Ruiz RM metacentric pair, with AgNO3 with the same chromosomes previously stained with Giemsa (b). (1988) A method for increas- being more evi- ing the number of mitoses dent in some available for cytogenetic analy- whereas variation chromosomes than in others, sis in rainbow trout. Stain occurred on the is a characteristic that has not Technol. 63: 335-338. acrocentric series. been observed in other pira- Machado-Allison A (1982) Studies on the systematics of the sub- In other pira- nha genera and that has been family Serrasalminae (Pisces: nhas, NORs have suggested as a probable Characidae). Thesis. The been detected ex- sinapomorphy in the genus George Washington University, clusively on the Serrasalmus. Based on a cla- Washington DC, USA. 267 pp. short arms of ST- distic analysis of the Ser- Machado-Allison A (1983) Estudios A chromosomes rasalminae subfamily, Ma- sobre la sistemática de la (Table I). Hence, chado-Allison (1982, 1985) Subfamilia Serrasalminae (Teleostei, Characidae) Parte NORs on a SM proposed that the genera Py- II. Discusión sobre la con- pair in P. cariba gocentrus and Serrasalmus dición monofilética de la seems to be an are part of a monophyletic subfamilia. Acta Biol. Venez. apomorphy. To group that also includes the 11: 145-195. avoid any possi- Pygopristis and Prystobrycon Machado-Allison A (1985) Estudios bility of misclas- genera. The cytogenetic simi- sobre la Subfamilia Serrasal- minae (Teleostei-Characidae). sification of the larities between P. cariba and Parte III: Sobre el estatus chromosome type, the species so far studied of genérico y relaciones filoge- Figure 2. C- banded metaphase of Pygocentrus the index (L/S) the genus Serrasalmus, in the néticas de los géneros Pygo- cariba. was determined number and type of chromo- pristis, Pygocentrus, Pristo- for 30 pairs of somes, multiplicity of NOR- brycon y Serrasalmus (Te- leostei-Characidae-Serrasal- mology of these regions in the chromosomes Nº 20 from bearing chromosomes and the minae), Acta Biol. Venez. 12: the chromosomal comple- different individuals, finding distribution of constitutive 19-42. ment. that the centromeric index heterochromatin located in Machado-Allison A, Fink W (1996) In order to identify NOR- was always, within the SM centromeric and telomeric po- Los peces caribes de Venezue- bearing chromosomes, se- range. sition, reinforces the hypoth- la: diagnosis, claves, aspectos quential staining (Giemsa Regarding C-banding, this esis of the close phylogenetic ecologicos y evolutivos. Colec- ción Monografias Nº52. Con- followed by AgNO3 impreg- procedure shows that hetero- relationship between the gen- sejo de Desarrollo Científico y nation) was used. The NOR- chromatic blocks in P. cariba era Pygocentrus and Serrasal- Humanístico. Universidad Cen- bearing chromosomes were were located at centromeric mus. tral de Venezuela. 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