Report on the Chromosomes of Liza Klunzingeri (Day, 1888) in Zyarat Estuary, Persian Gulf

Journal of the Persian Gulf (Marine Science)/Vol. 4/No. 12/June 2013/8/23-30

Report on the Chromosomes of Liza klunzingeri (Day, 1888) in Zyarat Estuary, Persian Gulf

Faqih Ahmadani, Ahmad1; Hosseini, Seyed Javad*2,3, Qasemi, Seyed Ahmad2; Mohammadi, Gholam Hossein4

1- Dept. of Aquaculture, Faculty of Agriculture and Natural Resources, Islamic Azad University, Ahvaz, IR Iran 2- Inistitute of Persian Gulf, Persian Gulf University, Busheher, IR Iran 3- Dept. of Cell and Molecular Biology, Faculty of Science, Persian Gulf University, Busheher, IR Iran 4- South of Iran Aquaculeare Research Center, Ahvaz, IR Iran

Received: Febrmary 2012 Accepted: December 2012

Abstract This paper reports on the karyotyping of mullet fish species, Liza klunzingeri, endemic of the Persian Gulf. The metaphase chromosome spreads, obtained from kidney cells of 13 specimens were examined. The diploid chromosome number was found to be 2n=48. The karyotype was discovered to be consisted of 48 acrocentric chromosome pairs. The arm number was determined to be NF=48. The chromosome number observed in this fish is the same as the majority of Mugil fish, thus confirming the commonality of this feature in the family.

Keywords: Liza klunzingeri, Chromosome, Karyotype, Persian Gulf

1. Introduction coasts of the Khuzestan province (Kashy et al., 2007). This fish is dispersed from the Persian Gulf to Liza klunzingeri belongs to Mugilidae family, the Indian Ocean, Arabian Sea and the Gulf of Oman which is distributed in various coastal aquatic (Randall, 1995). Catching is done by beach seines, habitats of the world’s tropical, subtropical and set nets, and gill nets (Ismail et al., 1998). temperate regions. This family includes 17 genera and Studies worldwide on Liza klunzingeri are done 72 species (Nelson, 2006). The fish habitat is various primarily based on morphological and anatomical coastal substrata, brackish waters, and lagoons with traits, respectively (Ismail et al., 1998; Abou-Seedo high salinity (Golani et al., 2002). Because of the wide and Dadzie., 2004; Kashi et al., 2007; Valinasab et al., variety and small differences between mullet species, 2005). Fish chromosome studies is one of the various considerable divergences exist in their systematic applications of genetics in fishery research and has classification. Liza klunzingeri is one of the mullet several applications in taxonomic studies, genetics, fish species that has valuable fisheries catch over the breeding and biotechnology. Many morphologically * Email: [email protected] similar fish may have different types and numbers of

23 Faqih Ahmadani et al. / Report on the Chromosomes of Liza klunzingeri… chromosomes. Therefore, knowledge of normal identified (Table 1), yet there is no report on the type karyotype of aquatic species can be instrumental in and number of chromosomes of Liza klunzingeri. The such studies. Before applying the method of breeding aim of this study is to determine the number and type fish, acquiring enough information about the type and of the chromosomes of Liza klunzingeri, along with its number of chromosomes of the species is necessary karyotype, the number of chromosomal arms (NF), (Gold et al., 1990). Karyotype of many mullet fish bases along the chromosomes, and the ratios of long species of the genus Mugil and Liza have been and short arms.

Table 1. Chromosome number in 19 Mugilidae species Chromosomal Species 2n NF Source formula Oedalechilus labeo 48 2St +46a 48 Rossi et al., 2000 Mugil cephalus 48 48a 48 Rossi et al., 1996 M.trichodon 48 48a 48 Nircho et al., 2005 M.speigleri 48 48a 48 Rishi and Singh., 1982 M.platanus 48 48a 48 Jordao et al., 1992 M.parsia 48 48a 48 Khuda-Buksh and manna., 1974 M.liza 48 48a 48 Nirchio and Cequea., 1998 M.gaimardianus 48 48a 48 Nirchio et al., 2003 M.curema 24 22m+2Sm 48 Nirchio and Cequea., 1998 M.corsula 48 48a 48 Khuda-Buksh and manna., 1974 M. curema 28 20m+4St+4a 48 Le Grande and Fitzsimons., 1976 Liza aurata 48 2St +46a 48 Choudry et al., 1979 L.saliens 48 2St +46a 48 Gornung et al., 2001 L.saliens 48 46a +2Sm 50 Arefyev., 1989 L.ramada 48 2Sm +46a 48 Rossi et al., 1997 L.ramada 48 46a +2Sm 50 Delgado et al., 1992 Paramugil parmatus 48 48a 48 Choudry et al., 1979 Chelon macrolepis 48 48a 48 Choudry et al., 1979 L.aurata 48 46a+2St 48 Cano et al., 1982 L.aurata 48 46a+2Sm 48 Delgado et al., 1991 L. saliens 48 46a +2St 48 Cataudella et al., 1974 L. ramada 48 46a+2St 48 Cataudella et al., 1973 L. ramada 48 46a +2St 48 Cataudella et al., 1974 L. ramada 48 46a +2Sm 48 Delgado et al., 1991 Chelon labrosus 48 2St+46a 48 Cataudella et al., 1974 Liza klunzingeri 48 48a 48 Present Study Agonostomus 48 46a+2st 48 Nirchio et al., 2008 monticola M.incilis 48 48a 48 Hett et al., 2011

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2. Materials and Methods treatments were determined to be 50 – 60 and 30 min, respectively. Both the types of slide preparation Thirteen specimens of Liza klunzingeri were were effective in obtainiing well-spread metaphase caught from Zyarat Estuary and transported alive to a chromosomes. Analyses of chromosome spreads well-aerated aquarium in the laboratory with room revealed that modal chroomosome number contained temperature, before chromosomal analysis. 48 (2n=48) (Table 2) (Fig. 1). Karyological study was carried out according to Table 2. Frequencies distributiions of chromosome counts from kidney cells of Liza klunzingerii. Nirchio and Colleagues (2005) with some Species 2n Total modifications. Each fish was injected with 25µgr/gr 45 46 47 48 49 body weightht colchicine solution, and scarified afftter 60 Liza klunzingeri 3 4 3 64 2 76 ± 10 min. Kidney tissue was then removed and placed in physiological serum, and cell suspension was obtained by repeated pipetting. The suspension was centrifuged at 1100 rpm for 10 min at 4 °C. Cell pellets were treated by suspension in hypotonic 0.075 molar KCl solution and tested for 20, 25, 30 and 35 minutes at room temperature. Samples were then centrifuged, and hypotonic solution was replaced with freshly prepared methanol-glacial acetic acid (3:1) as a fixative. Samples were fixed for 40 to 60 minutes with 2 or 3 time changes each. The suspension obtained was centrifuged, and the supernatant was removed. Afterwards, the cell pellets were resuspended in the few remaining drops. For chromosomal spreads preparation, the cool cell suspension was dropped onto pre-warmed clean slide (45-50 °C). In another method, cell suspension was dropped onto cold clean slide. Staining was performed with 10 and 20% Gimsa in a Sorenson buffer (pH 6.8) for 10 – 20 min. Microscopic observations were done with a Nikon Light microscope. Total length of each chromosome, short and long arms length and arm ration were determined using MicroMeasure software V3.3 according to the Levan method (Levan et al., 1964).

3. Results

Each of the steps involved in the chromoosome study is important in attaining large number of well spread metaphase chromosomes. Among these steps, the colchiciine and hypotonic treatment were more important. The optimal colchicines and hypotonic Fig. 1: Chromosomal Spreads (1100X) of Liza klunzingeri, 2n = 48. 25 Faqih Ahmadani et al. / Report on the Chromosomes of Liza klunzingeri…

The chromosomes of 76 chromosomal spreads data are summarized in Table 3. The chromosomal were counted, and 64(84.2%) had 48 chromosomes. spreads obtained allowed us to propose that karyotype

Chromosome number varied from 45 to 49 (Table 2). of Liza klunzingeri consist 48 acrocentric and NF=48

Figure 1a shows karyotype analysis using (Fig. 2). The ideogram of the L.klunzingeri was made MicroMeasure V3.3. The morphological and numerical on the basis of the karyotype (Fig. 3). Table 3: Morphometric data of the chromosomes of Liza klunzingeri (2n=48). Relative lengths (RL) (% of the set) and centromeric indices (CI). Length Relative lengths Short Arm Ratio Cent. Index No Long arm Classification each % of set arm (L/S) (S/(L+S)) 1 33.5086 2.8434% 33.5086 0 N/A 0 Acrocentric 2 32.2939 2.7404% 32.2939 0 N/A 0 Acrocentric 3 31.9234 2.7089% 31.9234 0 N/A 0 Acrocentric 4 30.0397 2.5491% 30.0397 0 N/A 0 Acrocentric 5 28.5103 2.4193% 28.5103 0 N/A 0 Acrocentric 6 28.3604 2.4066% 28.3604 0 N/A 0 Acrocentric 7 28.2633 2.3983% 28.2633 0 N/A 0 Acrocentric 8 27.5744 2.3399% 27.5744 0 N/A 0 Acrocentric 9 27.4587 2.3301% 27.4587 0 N/A 0 Acrocentric 10 27.3895 2.3242% 27.3895 0 N/A 0 Acrocentric 11 27.3293 2.3191% 27.3293 0 N/A 0 Acrocentric 12 27.3200 2.3183% 27.3200 0 N/A 0 Acrocentric 13 27.0466 2.2951% 27.0466 0 N/A 0 Acrocentric 14 26.9051 2.2831% 26.9051 0 N/A 0 Acrocentric 15 26.5476 2.2527% 26.5476 0 N/A 0 Acrocentric 16 26.4511 2.2446% 26.4511 0 N/A 0 Acrocentric 17 25.5193 2.1655% 25.5193 0 N/A 0 Acrocentric 18 25.3447 2.1507% 25.3447 0 N/A 0 Acrocentric 19 25.2866 2.1457% 25.2866 0 N/A 0 Acrocentric 20 25.2676 2.1441% 25.2676 0 N/A 0 Acrocentric 21 25.2364 2.1415% 25.2364 0 N/A 0 Acrocentric 22 24.4698 2.0764% 24.4698 0 N/A 0 Acrocentric 23 24.3814 2.0689% 24.3814 0 N/A 0 Acrocentric 24 24.0288 2.0390% 24.0288 0 N/A 0 Acrocentric 25 23.9604 2.0332% 23.9604 0 N/A 0 Acrocentric 26 23.8563 2.0244% 23.8563 0 N/A 0 Acrocentric 27 23.6541443 2.0072% 23.6541 0 N/A 0 Acrocentric 28 23.4824 1.9926% 23.4824 0 N/A 0 Acrocentric 29 23.4562 1.9904% 23.4562 0 N/A 0 Acrocentric 30 23.3939 1.9851% 23.3939 0 N/A 0 Acrocentric 31 23.3040 1.9775% 23.3040 0 N/A 0 Acrocentric 32 23.2080 1.9694% 23.2080 0 N/A 0 Acrocentric 33 23.0900 1.9593% 23.0900 0 N/A 0 Acrocentric 34 22.6092 1.9186% 22.6092 0 N/A 0 Acrocentric 35 22.2277 1.8862% 22.2277 0 N/A 0 Acrocentric 36 22.2101 1.8847% 22.2101 0 N/A 0 Acrocentric 37 22.1953 1.8834% 22.1953 0 N/A 0 Acrocentric 38 22.0883 1.8743% 22.0883 0 N/A 0 Acrocentric 39 21.8451 1.8537% 21.8451 0 N/A 0 Acrocentric 40 21.6987 1.8413% 21.6987 0 N/A 0 Acrocentric 41 21.1448 1.7943% 21.1448 0 N/A 0 Acrocentric 42 20.9747 1.7799% 20.9747 0 N/A 0 Acrocentric 43 20.8279 1.7674% 20.8279 0 N/A 0 Acrocentric 44 20.3817 1.7295% 20.3817 0 N/A 0 Acrocentric 45 19.7821 1.6786% 19.7821 0 N/A 0 Acrocentric 46 19.0369 1.6154% 19.0369 0 N/A 0 Acrocentric 47 17.8996 1.5189% 17.8996 0 N/A 0 Acrocentric 48 15.6710 1.3298% 15.6710 0 N/A 0 Acrocentric

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1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 23 24 Fig. 2: Karyotype (100X) Liza klunzingeri, 2n = 48.

Fig. 3: Ideogram of Liza klunzingeri chromosomes

4. Discussion obtained with 30 min hypotonic incubation. Fixative treatment was not found to be as important as Chromosome studies are basically similar in colchicine and hypotoniic treatment in obtaining methodology, almost all including colchicine, chromosome preparation;; but personal experience hypotonic, fixative treatments, slide preparation, and showed that overnight maaiintenance of cell suspension staining, respectively. A simple method for in fixative solution in reeffrigerator resulted in better Karyological study of fish is the direct chromosome slide preparation. preparation from kidney of live specimens because The chromosome number of most of the species in kidney tissue in most fishes contains the hematopoietic the genus Liza is 2n=448, 46 pairs of which are elements, which provide numerous blood cells in active acrocentric. However, alll 48 chromosomes of the division (Gold et al., 1990a). Liza klunzingeri in this study, are acrocentric. In a In this study, colchicine treatment was found to be review study conducted by Sola et al. (2008), three important in obtaining metaphase chromosomes. The Cytotypes of chromosomal formula are defined for best time for colchicine treatment was determined to mullet fish: Cytotype A, which consists of 48 pairs of be 50 to 60 min. Decreasing time of colchicines acrocentric chromosomes,, accounted for the greatest treatment reesulted in low metaphase chromosomes number (10 species belonging to four genera); and longer treatment led to more condensed Cytotype B which consistts of 46 pairs of acrocentric chromosomes. The best chromosome spreads were chromosomes, plus two pairs of subtelocentric

27 Faqih Ahmadani et al. / Report on the Chromosomes of Liza klunzingeri…

(observed in five species belonging to three genera) and that great metacentric or submetacentric chromosomes Cytotype C which consists exclusively of biarmed have been generated through the reduction in number chromosomes. M. curema from Louisiana and Brazil of the chromosomes by the Robertsonian fusion (2n=28, NF=48) and from Venezuela (2n=24, NF=48) (Doucette and Fitzsimous., 1998). Diversity in the display these cytotypes. The karyotype of Liza karyotype of the genus Mugil may be due to the klunzingeri can be assigned to cytotype A. It is likely centromere fusions of the acrocentric chromosomes (Le that other studies around the world on Liza klunzingeri Grande and Fitzsimons., 1976). In a report by Legrande and comparison of those studies yield different results. & Fitzsimons (1976), the number of the chromosomes For example, the chromosomal formulae for L. Ramada of M.curema in the Caribbean was 2n=24, while in was 2n= 48= 46a+ 2st (Cataudella et al.,1973, 1974); another report by Nirchio & Cequea (1998), this yet for the same species, Delgado et al. (1992) obtained number for the same fish in the Gulf of Mexico was 2n=48=46a+ 2sm. The same is true about L. saliens, for 2n=28. Since the arms of chromosomes in M.curema which the two chromosomal formulae were 2n= were longer than those in grey mullet and M.liza, it 48=46a+2st (Gornug et al., 2001; Cataudella et al., seemed reasonable that the karyotype of this fish might 1974) and 2n=48=46a + 2sm (Arefyev et al., 1989). have evolved more than other species of the mullet Two different chromosomal formulae for L. aurata are family (Legrande and Fitzsimous., 1976; Nirchio and reported, 2n=48= 46a+ 2sm (Cataudella et al., 1974; Cequea, ., 1998) Delgado et al., 1992) and 2n=48=26a+ 2st (Cano et al., Despite their systematic similarities, cytogenetic 1982). On the one hand, high concentration and long- information distinguishes M. curema and M. term effects of colchicine causes high compression, rubrioclalus (Nirchio et al., 2007). Therefore, we can shrinkage and shortening of the chromosome arms conclude that the mullet family, except for the M. (Beck et al., 1980); This makes it difficult to detect the curema, possess 48 pairs of chromosomes, most of short arms of the chromosomes; and consequently, the which are acrocentric (Sola et al., 2007, 2008). Like all mistakes in distinguishing different chromosomsl arms other species of the genus Liza, all the chromosomes of leads to differences observed in the karyotypes (Nirchio the Liza klunzingeri were determined to be acrocentric. and Cequea., 1998). On the other hand, due to their Complementary information can be obtained through small size and high compression, detection of further research on this species in the Persian Gulf, chromosome arms is very difficult and sometimes it is Gulf of Oman, Indian Ocean. impossible to detect homologous chromosomes. To overcome this problem, method of chromosome Acknowledgements banding could be employed. Provided that the facilities are available, rating band method could be very useful This work was granted by the Persian Gulf in identifying the chromosomes more precisely; since University, Grant of 19/63. We would like to thank all without such patterns in band rating chromosomal those who assisted in the field collection and laboratory arms, detecting the chromosomal similarities is only processing of samples for this project. We are grateful possible based on the chromosome size and the position to Mr. Ali Nakisa for editing the manuscript. of the centromere (Nirchio et al., 2008). Recent studies using chromosomal markers through realignment of References chromosomes have shown that the Chromosomal evolution of mullet is more complex than it was Abou-Seedo, F.S. and Dadzie, S., 2004. believed (Sola et al., 2008). Karyologic study of twenty Reproductive cycle in the male and female grey species of Clupeifromes and Elopifromes has shown mullet, Liza klunzingeri in the Kuwaiti waters of

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