Karyological analysis of Cyprinion macrostomum Heckel, 1843, from Godarkhosh River, Ilam Province, Iran Item Type article Authors Nasri, M.; Keivany, Y.; Dorafshan, S. Download date 02/10/2021 18:07:24 Link to Item http://hdl.handle.net/1834/37566 Iranian Journal of Fisheries Sciences 14(3) 786-796 2015 Karyological analysis of Cyprinion macrostomum Heckel, 1843, from Godarkhosh River, Ilam Province, Iran Nasri M.; Keivany Y.*; Dorafshan S. Received: May 2012 Accepted: December 2014 Abstract In this study, for the first time in Iran, the karyotype of bigmouth Lotak, Cyprinion macrostomum Heckel, 1843, was investigated through examining metaphase chromosomes of seven fish with mean weight 30±5g caught by electrofishing from Godarkhosh River in Ilam Province. To stimulate cell divisions, fish were injected intraperitoneally two times by phytohemagglutinin (PHA). The cell divisions were arrested in metaphase stage by intraperitoneal injection of colchicine. Well-separated cells were obtained from kidney and gill filament and chromosome spreads were prepared and stained with giemsa. Karyotype was obtained as 2n=50. The karyotype consisted of 5 metacentric, 12 submetacentric and 8 telocentric chromosome pairs. Centromeric index, arm ratio and Fundamental Number (FN) were determined as 0-50, 1-∞, and 84, respectively. Keywords: Bigmouth lotak, Cyprinion macrostomum, Godarkhosh River; Iran, Karyotype. Downloaded from jifro.ir at 1:12 +0330 on Thursday February 22nd 2018 Department of Natural Resources (Fisheries Division), Isfahan University of Technology, Isfahan 84156-83111, Iran. *Corresponding author's email: [email protected] 787 Nasri et al., Karyological analysis of Cyprinion macrostomum Heckel, 1843, from… Introduction 1983). In addition, we can pursuit ancestral The genus Cyprinion (Cyprinidae) karyological changes and fixation in comprises nine species, among which five various new species (Winkler et al., 2004). are reported from Iran and three from Karyological study of fishes has several Tigris-Euphrates basin (C. kais, C. usages in aquaculture (e.g., to identify macrostomumand C. tenuiradius). The first chromosome-manipulated fish, fish two species are well distributed in inland breeding and the rapid production of waters of Iran, Iraq, Turkey, and Syria inbreed lines) (Chingjiang et al., 1986; Gül (Coad, 1995, 1996, 2015; Epler et al., 2001; et al., 2004). Due to their smaller and more Eschmeyer and Fricke, 2014; Froese and contracted chromosomes, the main Pauly, 2015; Keivany et al. 2015). In Iran, difficulty in working with fish C. macrostomum is named Lotak-e Dahan chromosomes is to obtain high quality Bozorg (Big mouth Lotak) (Figure 1). metaphase spreads (Gül et al., 2004). Bigmouth Lotak is edible and fished by Howes (1982) reviewed the genus and natives of the region and considered a Durand et al. (2002)conducted some valuable species for sport fishing (Abdoli, phylogenetic and biogeographical studies 2000). on C. macrostomum and C. kais in the There are some uncertainties about the Middle East. Patimar and Nasri (2007) taxonomy and phylogenetic status of studied the age structure and growth of C. Cyprinion species and several authors macrostomum in Ilam Province, Iran. Nasri considered the systematic status of (2008) studied the taxonomy and Nasri el Cyprininae species and genera with their al. (2013) investigated the osteology of C. phylogenetic links still doubtful (Howes, macrostomum and C. kais in Karkheh River 1982). Some researchers considered C. kais basin. Karyological analyses of C. and C. macrostomum as synonyms (Berg, macrostomum by Gaffaroğlu and Yüksel 1949), but Bianco and Banarescu (1982) (2004), Yilmaz et al. (2005) and Yüksel and Downloaded from jifro.ir at 1:12 +0330 on Thursday February 22nd 2018 denoted that they were wrongly considered Gaffaroğlu (2008) were conducted in as synonymous. Turkey, but karyological study on this Karyology is a useful tool to study the genus in Iran was restricted to C. taxonomy and phylogenetic relationships tenuiradius (Esmaeili and Piravar, 2006) among fishes. The study of fish and C. kais (Nasri et al., 2010). chromosomes is a routine activity in This study is the first karyological studying fish biology and taxonomy analysis of C. macrostomum in Iran. The nowadays (Kalbassi et al., 2006; Esmaeiliet result of this study would shed light on the al., 2010; Nasri et al., 2010; Okonkwo and systematics and taxonomy of the genus and Obiakor, 2010; Nezamoleslami et al., 2013; could be used to differentiate between Singh et al., 2013). By karyological studies, similar species which are morphologically we can obtain basic information including hard to recognize. number and morphology of chromosomes to study systematic and evolutionary states Materials and methods of the animals (Macgregor and Varley, Iranian Journal of Fisheries Sciences 14(3) 2015 788 In November 2007, seven individuals of stage and left for 7 hours before sacrificing. bigmouth Lotak (mean weight 30±5 g and Kidney and gill filament cells were mean length 12±3 cm) were caught in removed, homogenized and hypotonized Godarkhosh River (45°54'3"E and simultaneously by tri–sodium citrate 1% for 33°30'16"N) in Ilam Province. through 45 minutes at room temperature. Because of electrofishing. Fish were transferred alive their tiny tissues, the obtained tissues from to the Ichthyology Laboratory at Isfahan each group were mixed. Then, samples University of Technology and stored in a were centrifuged at 1300 rpm for 10 50-liter aquarium with continuous minutes and supernatant was removed and aerationat water temperatures of 15°C for cold fresh carnoy (3:1 methanol and glacial adaptation to laboratory conditions. acetic acid) was added to fix the cells. To study karyotype, the air-dried Samples were stored at 4°C for 30 minutes chromosome preparation method as then centrifuged. This process was repeated described by Thorgaard and Disney (1990) three times and carnoy was replaced in 30- was used with some modifications. To minute intervals. After the last stimulate mitotic divisions, the fish were centrifugation, cold and fresh carnoy was injected intraperitoneally with added and samples were stored at 4°C. Phytohemagglutinin (PHA) (4 μg.g–1 b.w) Smears were prepared using splash method in two steps with a 20-hour interval at 20°C. (cold lamella) and air dried for 24 hours, Eight hours after the second PHA injection, then, stained with giemsa 10%. Metaphasic fish were divided into two groups (four and chromosomes were analyzed and three fish) and colchicine was injected photographed using a Nikon microscope intraperitoneally (25 and 50 μg.g–1 b.w, in model Fujix Digital Camera, HC–300zi by the first and second group, respectively) to 100x magnification lens, immersion oil, depress the mitotic division at metaphase and blue photo filter. Downloaded from jifro.ir at 1:12 +0330 on Thursday February 22nd 2018 Figure 1: Cyprinion macrostomum from Godarkhosh River (Karkheh River basin). 789 Nasri et al., Karyological analysis of Cyprinion macrostomum Heckel, 1843, from… Figure 2: Map of the study area showing the Godarkhosh River (sampling region) and its position in Ilam Province in Western Iran. About 120 metaphasic plates were counted submetacentric and telocentric and a proper plate was selected to obtain chromosomes were denoted. karyotype formulae and karyogram. Measurements were performed by Adobe Results Photoshop CS5 professional software. One hundred and twenty metaphase plates Downloaded from jifro.ir at 1:12 +0330 on Thursday February 22nd 2018 Calculation of data and drawing the of the seven specimens of C. macrostomum ideogram were performed in Microsoft were counted. The diploid number per each Office Excel 2010 software. metaphase plate ranged between 35 and 57. For each chromosome, centromeric Diploid number of 2n=50 constituted 60% index (I=100 S/C), (S: short arm length &C: and 2n=48 constituted 18.33% of the total length of chromosome), arm ratio (R = metaphase plates (Table 1). Using a proper L/S), (L: long arm) and relative metaphase plate (Figure 3A) and based on chromosomes length (R=100×C/L), (L: chromosomal indicators (Table 2), summation of all chromosomes length) chromosomal formulae was obtained as 5 were calculated as described by Levan et al. metacentric, 12 submetacentric and 8 (1964) and the Fundamental Number (FN) telocentric. Centromeric index, arm ratio was calculated. Preparation and ranking of and Fundamental Number (FN) were chromosomes were performed using Levan determined as 0-50, 1-∞, and 84, et al. (1964) method, with some respectively. The largest chromosome was modifications, and metacentric, a submetacentric (5.62 μm) and the smallest Iranian Journal of Fisheries Sciences 14(3) 2015 790 was a telocentric one (2.23 μm) (Figure 3). 2n=50 (Table 1), are usually the result of Based on the chromosomal indicators losses or additions from nearby cells during (Figure 3 and Table 2), a karyogram (Figure preparation or other artifacts as reported in 3B) was drawn and an ideogram was other studies (Gül et al., 2004; Esmaeili and depicted. The diploid numbers, rather than Piravar, 2006). Table 1: Abundance of chromosomes in the counted plaques of Cyprinion macrostomum. Number of Chromosomes 35 45 47 48 49 50 51 52 54 57 in Each Plaque Number of Metaphase Plates 2 3 5 22 2 72 6 5 2 1 Frequency % 1.66 2.5 4.16 18.33 1.66 60 5 4.16 1.66 0.83 Table 2: Centromeric index in Cyprinion macrostomum (m: metacentric; sm: sub metacentric; t: telocentric). Short Long Chromosome Arm Centromeric