Karyotype Analysis of Two Groupers, Epinephelus Species (Serranidae)

Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics

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Karyotype analysis of two groupers, Epinephelus species (Serranidae)

Kowit Noikotr, Krit Pinthong, Alongklod Tanomtong, Runglawan Sudmoon, Arunrat Chaveerach & Tawatchai Tanee

To cite this article: Kowit Noikotr, Krit Pinthong, Alongklod Tanomtong, Runglawan Sudmoon, Arunrat Chaveerach & Tawatchai Tanee (2014) Karyotype analysis of two groupers, Epinephelus species (Serranidae), Caryologia, 67:1, 63-65, DOI: 10.1080/00087114.2014.892277 To link to this article: https://doi.org/10.1080/00087114.2014.892277

Published online: 13 Mar 2014.

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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tcar20 Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 2014 Vol. 67, No. 1, 63–65, http://dx.doi.org/10.1080/00087114.2014.892277

Karyotype analysis of two groupers, Epinephelus species (Serranidae) Kowit Noikotra, Krit Pinthongb, Alongklod Tanomtongc, Runglawan Sudmoonc, Arunrat Chaveerachc and Tawatchai Taneed* aDepartment of Biology, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand; bDepartment of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Surin 32000, Thailand; cDepartment of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand; dFaculty of Environment and Resource Studies, Mahasarakham University, Mahasarakham 44000, Thailand

This is an initial report of a cytogenetic study in two economically important species of Epinephelus, namely E. bleekeri and E. coeruleopunctatus, using the G-banding technique. Chromosome numbers of the two species are 2n = 48. The differences were found to be karyotype variations, with 48 telocentric chromosomes in E. bleekeri and two submetacentric and 46 telocentric chromosomes in E. coeruleopunctatus. Statistically, there were no karyotype variations between male and female individuals of each of the species. This basic knowledge is highly important for other technol- ogies, such as those used in hybridization programming. Keywords: cytogenetics; Epinephelus bleekeri; Epinephelus coeruleopunctatus; karyotype; Thailand

Introduction evolution appear to have many similar chromosome Groupers are a category of fish with economic impor- pairs, for example, orang-utans (Pongo pygmaeus), goril- tance, used not only as resources for food but also for las (Gorilla gorilla), chimpanzees (Pan troglodytes), and ornamentation and aquariums. They belong to the sub- humans (Homo sapiens). Chromosome numbers and their family Epinephelinae of the family Serranidae. The sub- karyotypes are often used as essential taxonomic data for fi family was classified into 15 genera by Froese and Pauly those animals that are dif cult to classify according to fi (2013). The genus Epinephelus is one of the most impor- their morphological aspects. For example, the identi ca- tant genera. Each genus is identified mainly by morpho- tion of the four genera of the family Hylobatidae is logical characteristics, such as body configuration, size based on each having a different chromosome number and number of body parts, or color patterns. However, (Geissmann 2002). It follows that there are some varia- there are many overlapping morphological characteristics tions of Epinephelus species, including E. malabalicus among grouper species. Compounding these are the (Zou et al. 2005), E. fuscoguttatus (Liao et al. 2006; many intraspecific variations, especially of color patterns. Wei et al. 2009), E. moara (E. bruneus) (Guo et al. This problem makes it difficult to identify species and 2006), and E. coioides (Wang et al. 2010), showing a misidentification is a recurrent issue. chromosome number of 48 with varied karyotypes. Reports on species diversity of groupers in Thailand Cytogenetic studies provide important basic knowledge have classified 20–25 species (Suvatti 1950; which can have applications for many other studies, such fi Duangsawasdi 1964; Froese and Pauly 2013). Due to as for the detection of ploidy in shes (Pradeep et al. their importance, wild species should be collected for the 2011; Pradeep, Srijaya, Bahuleyan, et al. 2012). purposes of studying their germplasm. These studies can This research is an initial study reporting the chromo- lead to more sustainable usage of groupers in a variety some number and karyotypic characters of two species, of ways, such as improving the breeding of cultivars, E. bleekeri and E. coeruleopunctatus. growth rate, hatchling collection for aquaculture, and identification. Cytogenetics has long been an essential Materials and methods tool to examine the transmission of genetic material from one generation of cells to the next. Changes in chromo- Epinephelus bleekeri and E. coeruleopunctatus were col- some structure, which manifest directly on the transmit- lected from the Andaman Sea in Phuket Province, Thai- fi ting genotype, are expressed as specific individual land. Species identi cation was performed based on patterns, which also affect the growth and evolution of morphological characteristics according to Heemstra and life. Therefore, the study of cytogenetics demonstrates Randall (1993). how genetic variations occur in the chromosome of each Chromosomes were directly prepared in vivo (Chen species. When examining specific chromosome numbers and Ebeling 1968; Nanda et al. 1995) as per the follow- and types for each species, some groups with close ing process. Phytohemagglutinin (PHA) solution was

*Corresponding author. Email: [email protected]

© 2014 Dipartimento di Biologia Evoluzionistica, Università di Firenze 64 K. Noikotr et al. injected into abdominal cavities of the fishes. Twenty- computed for the length of that chromosome. The four hours later, colchicine was injected intramuscularly karyotyping arrangement was ranged from the longest to and/or into the abdominal cavities of the fishes, and was the shortest, except for the sex chromosomes, which are left there for 2–4 hours. The fishes were anesthetized in always the last pair and are shown at the bottom left side ice-cold water and dissected. The kidneys, spleens, and/ of the range. The chromosome number is usually shown or gills were cut into small pieces. The pieces were at the bottom of each picture. Regarding the mixed and squashed with 0.075 M KCl. The large pieces arrangement, the short arm is normally shown on top, of tissue were discarded, and then 15 ml samples of the whereas the long arm is situated at the bottom of each cell sediments were transferred to centrifuge tubes and karyotyping. were incubated for 25–35 minutes. The fresh and cool fixative solution (3:1 methanol/glacial acetic acid) was added to stop the samples’ reaction with the KCl, then Results centrifuged at 1200–1500 rpm for 10 minutes. After- The cytogenetic study of the two sample species wards, the supernatant was discarded, 7–8 ml of the fixa- E. bleekeri and E. coeruleopunctatus using G-banding tive solution was again added, and the samples were technique are shown below. centrifuged once again in order to wash the cell sediments. The cell sediments were resuspended in 1 ml of the fixative solution and kept at –20°C for further chro- Epinephelus bleekeri mosomal study. The chromosome number of this species is 2n = 48. The cell suspensions were dropped onto a clean and Karyotypes are present at 48 telocentric chromosomes, cold slide by micropipette, which was followed by the shown in Figure 1. air-dry technique. The slide was conventionally stained with 20% Giemsa solution for 30 minutes. Chromosome counts were performed on the mitotic Epinephelus coeruleopunctatus metaphase cells under a light microscope. Twenty clearly The chromosome number of this species is 2n = 48. observable and well-spread cell chromosomes of both Karyotypes comprise of two submetacentric and 46 male and female individuals were selected and photo- telocentric chromosomes, shown in Figure 2. graphed. The lengths of the short arm of the chromosome (Ls) and the length of the long arm of the chromosome (Ll) were measured and were calculated to Discussion find the total arm length of the chromosome (LT = Ls + There have been many publications concerning cytoge- Ll). The relative length (RL), centromeric index (CI), netic studies on Epinephelus species, starting with a study and standard deviation (SD) of RL and CI were esti- of E. diacanthus in 1974 (Natarajan and Subrahmanyam mated. CI was also computed to classify the types of 1974), up to a study of E. coioides in 2010 (Wang et al. chromosomes according to Chaiyasut (1989). All param- 2010). All publications show the same chromosome eters were used in karyotyping. counting of 2n = 48, also in accordance with the number For karyotyping, the chromosome pictures were used given to E. bleekeri and E. coeruleopunctatus in this for homologous chromosome pairing. The initiation of research. Variations between species have often occurred chromosome pairing was used to determine each chro- in previous karyotyping. For example, Wang et al. (2010) mosome centromere. Then, the lengths of each chromo- reported that E. coioides were comprised of two submeta- some’s long arm and short arm were measured and centric and 46 acrocentric chromosomes; Wei et al.

Figure 1. Metaphase chromosome plate (left) and karyotype (right) of Epinephelus bleekeri, with 48 telocentric chromosomes. Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics 65

Figure 2. Metaphase chromosome plate (left) and karyotype (right) of Epinephelus coeruleopunctatus, with two submetacentric (boxed) and 46 telocentric chromosomes.

(2009) reported two subtelocentric and 46 telocentric Geissmann T. 2002. Taxonomy and evolution of gibbons. Evo- chromosomes; and Guo et al. (2006) reported 48 acrocen- lutionary Anthropology: Issues, News, and Reviews. 11 – tric chromosomes in E. moara. (S1):28 31. Guo F, Wang J, Su YQ, Wang DX, Xu LN. 2006. Study on This study is an initial report demonstrating a the karyotype of Epinephelus moara. Marine Sciences. cytogenetic study of the two sample species E. bleekeri 2006(8):1–3. and E. coeruleopunctatus using the G-banding technique. Heemstra PC, JE.Randall 1993. Groupers of the world (family The results show that the two species show an equal Serranidae, subfamily Epinephelinae). FAO Fisheries Syn- chromosome number of 2n = 48 with karyotype opsis No. 125 Vol. 16. Available from ftp://ftp.fao.org/ docrep/fao/009/t0540e/t0540e00.pdf. variations. Epinephelus bleekeri included 48 telocentric Liao JQ, Yin SW, Chen GH, Huang H, Lei CG, Lou TT. chromosomes, whereas E. coeruleopunctatus comprised 2006. The karyotype of grouper Epinephelus fuscoguttatus. of two submetacentric and 46 telocentric chromosomes. Fisheries Science. 2006(11):567–569. There were statistically no differences between male and Nanda I, Schsrtl M, Fiechtinger W, Schlupp I, Parzefall J, Sch- female individuals of each of the species. That means no mid M. 1995. Chromosomal evidence for laboratory syn- thesis of triploid hybrid between the gynogenetic teleost sex-linked chromosome was observed in the two species. Poecilia formosa and its host species. Journal of Fish Biol- We expected that the results would be the same for all ogy. 47(4):619–623. species in the genus. Besides providing basic knowledge, Natarajan R, Subrahmanyam K. 1974. A karyotype study of cytogenetic studies have been successfully used for some teleosts from Portonovo waters. Proceedings of the – – detection of ploidy in tilapias (genus Oreochromis), Indian Academy of Sciences Section B. 79(5):173 196. Pradeep PJ, Srijaya TC, Bahuleyan A, Renjithkumar CR, Jose D, which were temperature shocked (Pradeep, Srijaya, Papini A, Chatterji AK. 2012. Triploidy induction by heat- Bahuleyan, et al. 2012; Pradeep, Srijaya, Papini, et al. shock treatment in red tilapia. Caryologia. 65(2):152–156. 2012). This technique is very important in hybridization Pradeep PJ, Srijaya TC, Jose D, Papini A, Hassan A, Chatterji programming of fishes or any other animals, as it can AK. 2011. Identification of diploid and triploid red tilapia – detect and correctly identify the induced diploid or trip- by using erythrocyte indices. Caryologia. 64(4):485 492. Pradeep PJ, Srijaya TC, Papini A, Chatterji AK. 2012. Effects loid offspring, which may be further selected as maternal of triploidy induction on growth and masculinization of red and/or paternal material for further breeding. tilapia [Oreochromis mossambicus (Peters, 1852) × Ore- ochromis niloticus (Linnaeus, 1758)]. Aquaculture. 344–349:181–187. Suvatti C. 1950. Fauna of Thailand. Bangkok: Department of References Fisheries. Chaiyasut K. 1989. Cytogenetics and cytotaxonomy of the Wang SF, Su YQ, Ding SX, Cai Y, Wang J. 2010. Cytogenetic genus Zephyranthus. Bangkok: Department of Botany: Fac- analysis of orange-spotted grouper, Epinephelus coioides, using chromosome banding and fluorescence in situ hybrid- ulty of Sci-ence, Chulalongkorn University. – Chen TR, Ebeling AW. 1968. Karyological evidence of female ization. Hydrobiologia. 638(1):1 10. fi fi Wei Y, Fan T, Jiang G, Sun A, Xu X, Wang J. 2009. Establish- heterogamety in the mosquito sh, Gambusia af nis. fi Copeia. 1968(1):70–75. ment of a novel n cell line from Brown-marbled grouper, Epinephelus fuscoguttatus (Forsskål), and evaluation of its Duangsawasdi M. 1964. A study on the groupers (Epinephelidae) – in Thai waters [bachelor thesis in fisheries]. Faculty of Fish- viral susceptibility. Aquaculture Research. 40(13):1523 1531. eries: Kasetsart University, Bangkok, Thailand. Zou JX, Yu QX, Zhou F. 2005. The karyotypes C-bands patterns and Ag-NORs of Epinephelus malabaricus. Shuichan Froese, R, D.Pauly 2013. FishBase [World Wide Web elec- – tronic publication]. www.fishbase.org, version (10/2013). Xuebao. 29(1):33 37.