© 2017 The Mendel Society Cytologia 82(1) Special Issue: 9–15

First Chromosome Analysis of the Humpback Cardinalfish, Fibramia lateralis (Perciformes, )

Wannapa Kasiroek1,2, Chantra Indananda3, Nattawut Luangoon2, Krit Pinthong4, Weerayuth Supiwong5 and Alongklod Tanomtong6*

1 Department of Aquatic Science, Faculty of Science, Burapha University, Muang, Chonburi 20131, Thailand 2 Institute of Marine Science, Burapha University, Muang, Chonburi 20131, Thailand 3 Department of Biology, Faculty of Science, Burapha University, Muang, Chonburi 20131, Thailand 4 Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Muang, Surin 32000, Thailand 5 Faculty of Applied Science and Engineering, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand 6 Toxic Substances in Livestock and Aquatic Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand

Received July 27, 2015; accepted November 23, 2015

Summary The first chromosome analysis and nucleolar organizer region (NOR) pattern of the humpback car- dinalfish (Fibramia lateralis) were studied. Samples from 10 male and 10 female fish were collected from the Andaman Sea and Gulf of Thailand. Mitotic chromosome preparations were prepared directly from kidney tis- sues. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of F. lateralis was 2n=46, and the fundamental numbers (NF) were 54 in both sexes. The karyotype consisted of 8 large acrocentric, 12 large telocentric, 24 medium telocentric and 2 small telocentric chromosomes. Moreover, the results indicated that the region adjacent to the telomere of the short arm of the second acrocentric chromosome pair showed clearly observable nucleolar organizer regions (NORs). Strange size chromosomes related to sex were not observed. The karyotype formula for F. lateralis is as follows: at t t 2n (diploid)46=L8 +L 12 +M 24 +S 2

Key words Fibramia lateralis, Chromosome, Karyotype, Nucleolar organizer region.

Cardinalfishes are a family Apogonidae belonging to toninae including only one species (Eschmeyer and Fong the class (ray-finned fishes), superorder 2015). The Humpback cardinalfish, Fibramia lateralis Percomorpha, order Perciformes and suborder Percoidei (Valenciennes 1832) has a discreet or diffuse midline (Nelson 2006). They are chiefly marine, but some spe- body stripe ending in a basicaudal spot smaller than the cies are found in brackish water and a few are found in pupil of the eye (Fig. 1). fresh water. Several species are kept in the aquarium Up to the present, basic cytogenetic information is and are popular as small, peaceful and colorful fish. available for the family Apogonidae in only 14 of the Most species live in tropical or subtropical waters, where 356 species (Table 1). The 2n varied from 34 to 46 chro- they inhabit coral reefs and lagoons (Johnson and Gill mosomes (mostly 2n=46) and the NF variation occurred 1998). Apogonids are widely distributed from warm between 46 and 92 in this family. Nevertheless, little is temperate to tropical areas in the Pacific, Indian and known about the karyotypic features of apogonids in the Atlantic Oceans. Most species occur in coral or rocky Pacific and Indian Oceans. NOR banding technique was reefs, while some species inhabit seagrass and coral- also studied in only one species to detect nucleolar orga- line algal meadows, soft-bottom communities, estuaries nizer regions (NORs) (Araújo et al. 2010). These regions and lowland freshwater. Eschmeyer and Fong (2015) are parts of chromosomes in which there are ribosomal reported 356 valid species from the listings in the Cata- ribonucleic acid (rRNA) encoding genes (5.8S, 18S, and log of . The family has been traditionally divided 28S). In all eukaryotic organisms, rRNA genes occur in into four subfamilies: including most of the many copies, thus reflecting high cell demand for rRNA. species (333 species), Pseudamiinae including only 20 NORs, as ribosomal gene clusters, which were active species, Amioidinae including only two species and Pax- in previous interphase, form prominent cytogenetic features, namely secondary constrictions (Andraszek * Corresponding author, e-mail: [email protected] et al. 2009). NOR characterization can be a cytogenetic DOI: 10.1508/cytologia.82.9 marker for cytotaxonomic studies and can even aid in 10 W. Kasiroek et al. Cytologia 82(1) Special Issue

Fig. 1. General characteristic of the humpback cardinalfish (Fibramia lateralis); scale bar indicates 1 cm.

Table 1. Review of fish cytogenetic reports in the family Apogonidae (genera; Apogon, Fibramia, Jaydia, Nectamia, , Phaeoptyx and Sphaeramia).

Species 2n NF Karyotype Ag-NORs Locality Reference

Apogon americanus 36 70 12m+6sm+16a+2t 2 (TR) Brazil Araújo et al. (2010) A. binotatus 36 50 14m/sm+22a/t ̶ USA Rivlin et al. (1987) 36 62 26m/sm+10a/t ̶ USA Rivlin et al. (1987) 35 49 14m/sm+21a/t ̶ USA Rivlin et al. (1987) A. imberbis 36 56 ̶ ̶ Spain Alvarez et al. (1991) A. maculatus 34 61 27m/sm+7a/t ̶ Puerto Rico Rivlin et al. (1988) A. pseudomaculatus 36 66 30m/sm+2a+4t ̶ Puerto Rico Rivlin et al. (1986) Fibramia lateralis 46 54 8a+38t 2 (TR) Thailand Present study Jaydia lineata 46 52 2m+4sm+2a+38t ̶ Japan Murofushi (1986) A. lineatus* 46 ̶ 2m+44sm/a/t ̶ Pacific Rivlin et al. (1986) A. nubilus* 46 92 2m+36sm+8a ̶ USA Rivlin et al. (1986) Ostorhinchus doederleini 46 54 2m+6sm+38a/t ̶ Japan Ojima and Kojima (1985) A. doederleini* O. endekataenia 46 46 46a/t ̶ India Rishi (1973) A. endekataenia* 46 52 2m+4sm+16a+24t ̶ Japan Murofushi (1986) O. moluccensis 46 46 46a/t ̶ India Rishi (1973) A. moluccensis* O. notatus 46 52 2m+4sm+40a/t ̶ Japan Ojima and Kojima (1985) A. notatus* 46 53 2m+5sm+39a/t ̶ Japan Ojima and Kojima (1985) 46 52 2m+4sm+40a/t ̶ Japan Ojima and Kojima (1985) 46 52 2m+4sm+40a/t ̶ Japan Murofushi (1986) O. semilineatus 46 52 2m+4sm+20a+20t ̶ Japan Murofushi et al. (1980) A. semilineatus* 46 54 2m+6sm+38a/t ̶ Japan Ojima and Kojima (1985) Phaeoptyx pigmentaria 38 ̶ 6m+32sm/a/t ̶ Atlantic Rivlin et al. (1986) Sphaeramia orbicularis 46 50 4sm+42a/t ̶ Pacific Ojima and Kojima (1985)

Remarks: 2 n=diploid chromosome number, NF=fundamental number (number of chromosome arm), m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric chromosome, NORs=nucleolar organizer regions, TR=subtelomeric region, *=scientific name in the report, and ̶=not available. constructing phylogenetic hypotheses for several fish some evolution studies of Apogonidae. groups. Some fish groups have a simple NOR system characterized by ribosomal cistrons on only a single Materials and methods chromosome pair, whereas others have a multiple NOR system composed of cistrons dispersed over several Sample collection chromosome pairs (Galetti 1998). We collected 10 males and 10 females of F. lateralis The present study is the first report on chromosomal (20 samples) from the Andaman Sea and the Gulf of characteristics of F. lateralis using conventional staining Thailand. All specimens were maintained in aerated, and Ag-NOR banding techniques. The obtained results flowing seawater aquaria at the Institute of Marine Sci- will increase the basic knowledge of the cytogenetics ence, Burapha University, Muang, Chonburi Province of F. lateralis, which could form the basis for future until analysis. research and provide data to ensure their survival. More- over, the knowledge on basic cytogenetics could be ap- Chromosome preparation plied to numerous breeding studies, and this could also Chromosomes were prepared in vivo (Chen and Eb- provide insight into species conservation and chromo- eling 1968, Nanda et al. 1995) as follows. The 0.05% 2017 First Chromosome Analysis of the Humpback Cardinalfish, Fibramia lateralis (Perciformes, Apogonidae) 11 colchicine was injected into the fish’s intramuscular and submetacentric, acrocentric and telocentric chromo- then left for one hour. The kidney was cut into small somes, respectively. The fundamental number, number pieces, and then squash mixed with 0.075 M KCl. After of chromosome arm (NF), was obtained by assigning discarding all large pieces of tissue, 8 mL of cell sedi- a value of two to metacentric, submetacentric and ac- ments were transferred to a centrifuge tube and incu- rocentric chromosomes and one to telocentric chromo- bated for 25–35 min. The KCl was discarded from the somes (Chooseangjaew et al. 2017). supernatant after centrifugation at 1200 rpm for 8 min. Cells were fixed in fresh, cool fixative (3 methanol : 1 Results and discussion glacial acetic acid) to which up to 8 mL of fixative were gradually added before being centrifuged again at Diploid chromosome number, fundamental number and 1200 rpm for 8 min, at which time the supernatant was karyotype of F. lateralis discarded. The fixation was repeated until the superna- This is the first report on F. lateralis cytogenetical tant was clear, and the pellet was mixed with 1 mL of knowledge. The present study revealed that the diploid fixative. The mixture was dropped onto a clean and cold chromosome number of F. lateralis was 2n=46, and slide by a micropipette followed by air-drying. the fundamental numbers (NF) were 54 in both males and females (Fig. 2). The types of chromosomes were 8 Chromosome staining large acrocentric, 12 large telocentric, 24 medium telo- Conventional staining was done using 20% Giemsa’s centric, and 2 small telocentric chromosomes. Compara- solution for 30 min (Chooseangjaew et al. 2017). Ag- tive studies with others in the family Apogonidae have NOR banding technique was performed by adding four shown the same chromosome number as those found drops of 50% silver nitrate and 2% gelatin on slides. The in Jaydia lineata (Murofushi 1986), Nectamia fusca slides were then sealed with cover glasses and incubated (Rivlin et al. 1986), Ostorhinchus doederleini, O. nota- at 60°C for 5 min. Next, the slides were soaked in dis- tus, Sphaeramia orbicularis (Ojima and Kojima 1985), tilled water until the cover glasses were separated. Then, O. endekataenia, O. moluccensis (Rishi 1973) and O. they were stained with 20% Giemsa’s solution for 1 min semilineatus (Murofushi et al. 1980, Ojima and Kojima (Howell and Black 1980, Sangpakdee et al. 2017). 1985). However, it differs from Phaeoptyx pigmentaria (2n=38), A. americanus, A. binotatus, A. imberbis, A. Chromosome analysis pseudomaculatus (2n=36) and A. maculatus (2n=34) Metaphase figures were analyzed according to the (Rivlin et al. 1986, 1987, 1988, Alvarez et al. 1991). chromosome classification of Chaiyasut (1989). The Although F. lateralis has the same 2n as most of the centromeric index (CI) between 0.50–0.59, 0.60–0.69, species, its NF is different except O. doederleini and O. 0.70–0.89 and 0.90–0.99 were described as metacentric, semilineatus (Ojima and Kojima 1985). In addition, the

Fig. 2. Metaphase chromosome plates and karyotypes of male (A.) and female (B.) humpback cardinalfish (Fibramia lateralis), 2n=46 by conventional staining technique (scale bars 10 µm). 12 W. Kasiroek et al. Cytologia 82(1) Special Issue results showed that cytologically distinguishable sex characterized by a karyotype exclusively composed of chromosomes were observed. It is similar to other spe- telocentric chromosomes (Rishi 1973), a symplesio- cies in the family Apogonidae (Araújo et al. 2010). morphic cytogenetic feature widely observed within the The chromosome data of the family Apogonidae order Perciformes (Molina 2006). Accordingly, the avail- revealed that 60% of all species analyzed so far (N=15 able data indicates a great karyotypic diversity in the spp.) present diploid values equal to 2n=46, suggesting evolution of the group, regarding both diploid number this should be an ancestor condition for this family. Ac- and chromosomal formulas, resulting in high funda- cording to the chromosome diploid, the family Apogo- mental numbers (NF=46–92). This scenario indicates nidae is divided into two groups: first, 2n=46 found in a simultaneous occurrence of different mechanisms of the genera Fibramia, Jaydia, Nectamia, Ostorhinchus karyotypic diversification in the family Apogonidae, and Sphaeramia; second, 2n=34–38 found in the gen- mainly Robertsonian rearrangements and pericentric in- era Apogon and Phaeoptyx. It is known that it has par- versions (Araújo et al. 2010). The karyotype formula for ticular cytogenetic features, as they present extremely F. lateralis is as follows: low diploid values in relation to the order Perciformes at t t and, in some species, a remarkable variation in the 2n (diploid)46=L8 +L 12 +M 24 +S 2 karyotype formulae is also found. Such reduction in the diploid number might be as low as 2n=34, as reported Chromosome markers of F. lateralis in A. maculatus (Rivlin et al. 1988). Nevertheless, the This is the first report on F. lateralis accomplished by chromosomal numbers are reduced, suggesting a high the Ag-NOR banding technique. The technique shows incidence of centric fusions, and high fundamental num- dark bands (NOR positions) on the subtelomeric short bers (NF) are also reported, like in N. fusca (2n=46, arm of the second acrocentric chromosome pair in both NF=92), which indicates that other rearrangements, males and females (Fig. 3). For other comparative stud- such as pericentric inversions, have also played a major ies of the species in the family Apogonidae, A. ameri- role in the chromosomal diversification of this fish group canus had a NOR on the subtelomeric short arm of the (Araújo et al. 2010). submetacentric chromosome pair 8 (Araújo et al. 2010). Among species in the family Apogonidae, some spe- Our obtained results indicated that the subtelomeric cies, such as O. endekataenia and O. moluccensis, are short arm of the second acrocentric chromosome pair

Fig. 3. Metaphase chromosome plates and karyotypes of male (A.) and female (B.) humpback cardinalfish (Fibramia lateralis), 2n=46 by Ag-NOR banding technique; scale bars indicate 10 µm. 2017 First Chromosome Analysis of the Humpback Cardinalfish, Fibramia lateralis (Perciformes, Apogonidae) 13

Fig. 4. Standardized idiogram showing lengths and shapes of chro- Fig. 5. Standardized idiogram of chromosomes of the humpback mosomes of the humpback cardinalfish (Fibramia lateralis), cardinalfish (Fibramia lateralis), 2n=46 by Ag-NOR band- 2n=46 by conventional staining technique. ing technique. The arrow indicates nucleolar organizer re- gions on the short arm of acrocentric chromosome pair 2.

Table 2. Mean length of short arm chromosome (Ls), length long arm chromosome (Ll), length total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from 20 metaphase cells of the male and female humpback cardinalfish (Fibramia lateralis), 2n=46.

Chro. pair Ls Ll LT RL±SD CI±SD Chro. type Chro. size

1 0.333 1.054 1.388 0.0268±0.0017 0.753±0.049 Acrocentric Large 2* 0.305 0.996 1.301 0.0254±0.0020 0.761±0.033 Acrocentric Large 3 0.307 0.951 1.258 0.0243±0.0013 0.753±0.033 Acrocentric Large 4 0.296 0.870 1.166 0.0225±0.0015 0.743±0.035 Acrocentric Large 5 0.000 1.585 1.585 0.0307±0.0025 1.000±0.000 Telocentric Large 6 0.000 1.347 1.347 0.0260±0.0011 1.000±0.000 Telocentric Large 7 0.000 1.275 1.275 0.0246±0.0009 1.000±0.000 Telocentric Large 8 0.000 1.231 1.231 0.0238±0.0007 1.000±0.000 Telocentric Large 9 0.000 1.201 1.201 0.0232±0.0008 1.000±0.000 Telocentric Large 10 0.000 1.178 1.178 0.0227±0.0007 1.000±0.000 Telocentric Large 11 0.000 1.157 1.157 0.0223±0.0006 1.000±0.000 Telocentric Medium 12 0.000 1.134 1.134 0.0219±0.0006 1.000±0.000 Telocentric Medium 13 0.000 1.112 1.112 0.0215±0.0004 1.000±0.000 Telocentric Medium 14 0.000 1.090 1.090 0.0211±0.0004 1.000±0.000 Telocentric Medium 15 0.000 1.072 1.072 0.0207±0.0005 1.000±0.000 Telocentric Medium 16 0.000 1.050 1.050 0.0203±0.0004 1.000±0.000 Telocentric Medium 17 0.000 1.026 1.026 0.0198±0.0005 1.000±0.000 Telocentric Medium 18 0.000 0.981 0.981 0.0190±0.0008 1.000±0.000 Telocentric Medium 19 0.000 0.945 0.945 0.0183±0.0007 1.000±0.000 Telocentric Medium 20 0.000 0.911 0.911 0.0177±0.0008 1.000±0.000 Telocentric Medium 21 0.000 0.875 0.875 0.0170±0.0010 1.000±0.000 Telocentric Medium 22 0.000 0.828 0.828 0.0161±0.0011 1.000±0.000 Telocentric Medium 23 0.000 0.734 0.734 0.0144±0.0021 1.000±0.000 Telocentric Small

Remarks: * NOR-bearing chromosome and chro.=chromosome 14 W. Kasiroek et al. Cytologia 82(1) Special Issue showed clearly observable NORs in all of 20 examined difference between the largest and the smallest chromo- fish (10 male and 10 female fish). The number and loca- somes is approximately twofold. The chromosome mark- tion of NORs in chromosomes can be variable among ers of F. lateralis are chromosome pairs 5 and 23, which populations and/or closely related species, thus repre- are the largest and the smallest telocentric chromo- senting a useful cytotaxonomic marker for phyloge- somes, respectively. Data of the chromosomal checks on netic reconstruction in some groups (Cross et al. 2006). mitotic metaphase cells of the F. lateralis is shown in the Structural rearrangements might be a cause of variation Table 2. Further studies in other species of the Apogo- in either chromosomal location or frequency of these re- nidae family are required in order to provide a better gions (Gu and Hua 2003, Shan et al. 2003). Although the understanding about the dynamic scenario of karyotype karyotypes in Apogonidae have been related to a great diversification in this family. number of pericentric inversions and Robertsonian fu- sions, variation in the number or position of NORs were Acknowledgements absent between both analyzed populations of F. lateralis. The lack of information in other species restrains any This work was supported by the doctoral thesis further studies about the evolutionary pattern of these support grant from the Faculty of Science, Burapha regions within Apogonidae. University, fiscal year 2015, the Institute of Marine Sci- NORs play an important role in displaying the perfect ence, Burapha University and the Toxic Substances in markers to display chromosomal polymorphism within Livestock and Aquatic Animals Research Group, Khon and between species in many groups of fish. This variety Kaen University. may affect NOR number, its localization on the chromo- some, size, and active numbers in each genome. The References previous studies of NOR exhibited variations between species, within species, and even between individuals Alvarez, M. C., Otis, J., Amores, A. and Guise, K. 1991. Short-term (Castro et al. 1996). NORs on different homologous cell culture technique for obtaining chromosomes in marine and freshwater fish. J. Biol. 39: 817–824. chromosomes may have different sizes. Some fish may Amemiya, C. T. and Gold, J. R. 1988. 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