First Report of Chromosome Analysis of Two Dragonets (Perciformes, Callionymidae)

Wannapa Kasiroek1, Nattawut Luangoon1, Weerayuth Supiwong2, Bundit Tengjaroenkul3,6, Krit Pinthong4 and Alongklod Tanomtong5,6*

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

Received December 6, 2015; accepted April 8, 2016

Summary We report the first chromosome analysis in the ocellated dragonet (Synchiropus ocellatus) and picturesque dragonet (S. picturatus) from Thailand. Kidney cell samples were taken from five male and five female fishes. The mitotic chromosome preparation was prepared directly from kidney cells. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of S. ocellatus and S. picturatus were 2n=40, and the fundamental number (NF) was 40 in both males and females. The chromosomes were present as large telocentric, medium telocentric and small telocentric chromosomes in numbers of 16–18–6 and 14–24–2, respectively. No morphological difference was identified between sex chromosomes of male and female specimens. After Ag-NOR banding technique, a single pair of nucleolar organizer regions (NORs) was observed on the long arm centromeric region of large telocentric chromosome pair 5 in S. ocellatus and chromosome pair 4 in S. picturatus. The karyotype formulas could be deduced as: t tt S. ocellatus 2 n () 40 =L16 +M 18 +S 6 t tt S. picturatus 2 n () 40 =L14 +M 24 +S 2

Key words Dragonet, Synchiropus ocellatus, Synchiropus picturatus, Karyotype, Chromosome.

Dragonets are small, perciform, marine fish of the 74). Sawada and Sakamoto (1980) reported cytogenetics diverse family Callionymidae, found mainly in the tropi- of the Eleutherochir mirabilis at Japan. The results show cal waters of the western Indo-Pacific. They are benthic that the diploid chromosome number was 2n=36 and the organisms, spending most of their time near the sandy NF was 36 in both male and female. The chromosomes bottoms at a depth of roughly 200 m. There exist 182 consist of 36 telocentric chromosomes. Murofushi et al. species of the fish in 10 genera. Due to similarities in (1983) reported the chromosome of the Repomucenus morphology and behavior, dragonets are sometimes con- beniteguri, R. huguenini, R. ornatipinnis and R. richard- fused with members of the goby family. However, male sonii from Japan, which were investigated by using con- dragonets can be differentiated from the goby by their ventional staining and Ag-NOR staining techniques. The very long dorsal fins and females by their protruding diploid chromosome numbers were 2n=37–38, 32, 37–38 lower jaws. The Draconettidae may be considered a sis- and 38, respectively and the NFs were 38, 34, 38 and ter family, whose members are very much alike, though 38, respectively. The chromosome presences of meta- rarely seen (Fricke 2002, 2006, Motomura and Mukai centric and telocentric autosomes were 0–38 (2n=38) or 2006, Yoshigou et al. 2006). 1–36 (2n=37), 2–30, 0–38 (2n=38) or 1–36 (2n=37) and Karyotypes of five species of dragonets have been 0–38, respectively. After Ag-NOR banding technique, reported previously. Their karyotypes are characterized a single pair of NORs was observed on the long arm of by the presence of 32–38 diploid chromosomes (NF=34– telocentric chromosome in R. beniteguri, R. huguenini, R. ornatipinnis and R. richardsonii. In addition, Ojima * Corresponding author, e-mail: [email protected] and Kikuno (1987) reported the karyotype of R. rich- DOI: 10.1508/cytologia.82.59 ardsonii from Japan. The results showed that the diploid 60 W. Kasiroek et al. Cytologia 82(1) Special Issue

Fig. 1. General characteristic of the ocellated dragonet (Synchiropus ocellatus) and picturesque dragonet (S. picturatus); scale bars indicate 1 cm.

Table 1. Review of cytogenetic publications of dragonets in the family Callionymidae (genera; Eleutherochir, Repomucenus and Synchiropus).

Species 2n NF Karyotype formula NOR banded Locality Reference

Eleutherochir mirabilis 36 36 36t ̶ Japan Sawada and Sakamoto (1980) Repomucenus beniteguri 38 38 38t ̶ Japan Murofushi et al. (1983) 37 38 1m+36t 2 Japan Murofushi et al. (1983) R. huguenini 32 34 2m+30t 2 Japan Murofushi et al. (1984) R. ornatipinnis 38 38 38t ̶ Japan Murofushi et al. (1983) 37 38 1m+36t 2 Japan Murofushi et al. (1983) R. richardsonii 38 38 38t 2 Japan Murofushi et al. (1984) 38 74 36sm+2t ̶ Japan Ojima and Kikuno (1987) Synchiropus ocellatus 40 40 40t 2 Thailand Present study S. picturatus 40 40 40t 2 Thailand Present study

Remarks: 2 n=diploid chromosome number, NF=fundamental number (number of chromosome arms), m=metacentric chromosome, t=telocentric chromosome, and ̶=not available. chromosome number was 2n=38, the NF was 74 in both onet (S. picturatus) have been published until the pres- male and female. The types of chromosomes were 36 ent. The present study is the ﬁrst report of cytogenetic submetacentric and two telocentric chromosomes. studies of S. ocellatus and S. picturatus accomplished Although, a large number of 13000 marine ﬁsh spe- with the conventional staining and Ag-NOR banding cies have been recorded (Nelson 2006), fewer than 2% techniques. The results obtained will increase our basic of these have been studied cytogenetically (Brum 1996). knowledge of the cytogenetics of S. ocellatus and S. pic- No study describing the karyotypes of the ocellated turatus, which could form the basis for future research dragonet (Synchiropus ocellatus) and picturesque drag- and provide data to ensure their survival. 2017 First Report of Chromosome Analysis of Two Dragonets (Perciformes, Callionymidae) 61

Fig. 2. Metaphase chromosome plates and karyotypes of male (A. and C.) and female (B. and D.) ocellated dragonet (Synchiropus ocellatus), 2n (diploid)=40 by conventional staining (A. and B.) and Ag-NOR banding techniques (C. and D.); scale bars indicate 5 µm. There is no observation of strange size chromosomes related to sex.

1980, Sangpakdee et al. 2017). The metaphase figures Materials and methods were analyzed according to the chromosome classifica- Five males and five females of S. ocellatus and S. tion after Chaiyasut (1989). The centromeric index (CI) picturatus were obtained from Thailand (Fig. 1). The between 0.50–0.59, 0.60–0.69, 0.70–0.89, and 0.90–0.99 fish were transferred to laboratory aquaria and were were described as metacentric, submetacentric, acro- kept under standard conditions for seven days prior to centric and telocentric chromosomes, respectively. The the experiment. Procedures for fish chromosome were fundamental number, NF (number of chromosome arm), prepared directly from kidney cells (Chen and Ebel- is obtained by assigning a value of two to metacentric, ing 1968, Nanda et al. 1995, Kasiroek et al. 2017). The submetacentric and acrocentric chromosomes and one to chromosome preparations were stained with 10% Giem- telocentric chromosomes (Chooseangjaew et al. 2017). sa’s for 30 min (Chooseangjaew et al. 2017) and NORs were identified by Ag-NOR staining (Howell and Black 62 W. Kasiroek et al. Cytologia 82(1) Special Issue

Fig. 3. Metaphase chromosome plates and karyotypes of male (A. and C.) and female (B. and D.) picturesque dragonet (Synchiropus picturatus), 2n (diploid)=40 by conventional staining (A. and B.) and Ag-NOR banding techniques (C. and D.); scale bars indicate 5 µm. No apparent sex chromosomal heteromorphic was identiﬁed.

ocellatus and S. picturatus was 40 in both males and fe- Results and discussion males. The comparative studies with others in the family Diploid number, fundamental number and karyotype of Callionymidae have shown different NFs, such as those S. ocellatus and S. picturatus found in E. mirabilis, NF=36 (Sawada and Sakamoto The diploid chromosome number (2n) found in S. 1980); R. beniteguri, NF=38 (Murofushi et al. 1983); R. ocellatus and S. picturatus was 40 chromosomes in both huguenini, NF=34 (Murofushi et al. 1984); R. ornatipin- males and females (Table 1, Fig. 2). In comparison with nis, NF=38 (Murofushi et al. 1983) and R. richardsonii, the family Callionymidae, it is a different diploid chro- NF=38 or 74 (Murofushi et al. 1983, Ojima and Kikuno mosome number compared with Eleutherochir mirabilis, 1987). 2n=36 (Sawada and Sakamoto 1980); Repomucenus The karyotypes of C. decussatus and C. lineolatus beniteguri, 2n=37–38 (Murofushi et al. 1983); R. hu- consisted of 48 telocentric chromosomes (mono-armed guenini, 2n=32 (Murofushi et al. 1984); R. ornatipinnis, chromosomes). The comparative studies with others in 2n=37–38 (Murofushi et al. 1983) and R. richardsonii, the family Callionymidae have shown different karyo- 2n=38 (Murofushi et al. 1983, Ojima and Kikuno 1987). types; the studies by Murofushi et al. (1983, 1984) found We found that the fundamental number (NF) of S. that in R. beniteguri, R. huguenini, R. ornatipinnis and 2017 First Report of Chromosome Analysis of Two Dragonets (Perciformes, Callionymidae) 63

Table 2. Mean of length short arm chromosome (Ls), length long arm chromosome (Ll), length total arm chromosome (LT), centromeric index (CI), relative length (RL) and standard deviation (SD) of CI and RL from metaphase chromosome of 10 cells in ocellated dragonet (Synchiropus ocellatus), 2n=40.

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

1 0.000 6.557 6.557 1.000±0.000 0.076±0.002 Large Telocentric 2 0.000 6.076 6.076 1.000±0.000 0.070±0.002 Large Telocentric 3 0.000 5.842 5.842 1.000±0.000 0.068±0.002 Large Telocentric 4 0.000 5.669 5.669 1.000±0.000 0.066±0.002 Large Telocentric 5* 0.000 5.456 5.456 1.000±0.000 0.063±0.002 Large Telocentric 6 0.000 5.134 5.134 1.000±0.000 0.059±0.002 Large Telocentric 7 0.000 4.628 4.628 1.000±0.000 0.054±0.002 Large Telocentric 8 0.000 4.358 4.358 1.000±0.000 0.050±0.001 Large Telocentric 9 0.000 4.232 4.232 1.000±0.000 0.049±0.001 Medium Telocentric 10 0.000 4.157 4.157 1.000±0.000 0.048±0.001 Medium Telocentric 11 0.000 4.081 4.081 1.000±0.000 0.047±0.002 Medium Telocentric 12 0.000 3.929 3.929 1.000±0.000 0.046±0.002 Medium Telocentric 13 0.000 3.809 3.809 1.000±0.000 0.044±0.001 Medium Telocentric 14 0.000 3.711 3.711 1.000±0.000 0.043±0.001 Medium Telocentric 15 0.000 3.570 3.570 1.000±0.000 0.041±0.001 Medium Telocentric 16 0.000 3.468 3.468 1.000±0.000 0.040±0.001 Medium Telocentric 17 0.000 3.346 3.346 1.000±0.000 0.039±0.001 Medium Telocentric 18 0.000 3.208 3.208 1.000±0.000 0.037±0.001 Small Telocentric 19 0.000 3.027 3.027 1.000±0.000 0.035±0.001 Small Telocentric 20 0.000 2.120 2.120 1.000±0.000 0.024±0.016 Small Telocentric

Remarks: *=NOR-bearing chromosome and chro.=chromosome.

Table 3. Mean of length short arm chromosome (Ls), length long arm chromosome (Ll), length total arm chromosome (LT), centromeric index (CI), relative length (RL) and standard deviation (SD) of CI and RL from metaphase chromosome of 10 cells in picturesque dragonet (Synchiropus picturatus), 2n=40.

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

1 0.000 6.085 6.085 1.000±0.000 0.072±0.002 Large Telocentric 2 0.000 5.893 5.893 1.000±0.000 0.069±0.002 Large Telocentric 3 0.000 5.556 5.556 1.000±0.000 0.065±0.001 Large Telocentric 4* 0.000 5.368 5.368 1.000±0.000 0.063±0.003 Large Telocentric 5 0.000 4.936 4.936 1.000±0.000 0.058±0.002 Large Telocentric 6 0.000 4.551 4.551 1.000±0.000 0.054±0.002 Large Telocentric 7 0.000 4.748 4.748 1.000±0.000 0.056±0.003 Large Telocentric 8 0.000 4.288 4.288 1.000±0.000 0.051±0.002 Medium Telocentric 9 0.000 4.312 4.312 1.000±0.000 0.051±0.002 Medium Telocentric 10 0.000 4.050 4.050 1.000±0.000 0.048±0.004 Medium Telocentric 11 0.000 4.195 4.195 1.000±0.000 0.049±0.002 Medium Telocentric 12 0.000 3.713 3.713 1.000±0.000 0.044±0.002 Medium Telocentric 13 0.000 3.880 3.880 1.000±0.000 0.046±0.005 Medium Telocentric 14 0.000 3.643 3.643 1.000±0.000 0.043±0.002 Medium Telocentric 15 0.000 3.736 3.736 1.000±0.000 0.044±0.002 Medium Telocentric 16 0.000 3.458 3.458 1.000±0.000 0.041±0.004 Medium Telocentric 17 0.000 3.286 3.286 1.000±0.000 0.039±0.001 Medium Telocentric 18 0.000 3.190 3.190 1.000±0.000 0.038±0.003 Medium Telocentric 19 0.000 3.118 3.118 1.000±0.000 0.037±0.002 Medium Telocentric 20 0.000 2.830 2.830 1.000±0.000 0.033±0.001 Small Telocentric

Remarks: *=NOR-bearing chromosome and chro.=chromosome.

R. richardsonii from Japan, the chromosome presences Kikuno 1987). It is possible that the ﬁsh’s sex chromo- of metacentric and telocentric chromosomes were 0–38 somes are dependent on an initiation of differentiation. (2n=38) or 1–36 (2n=37), 2–30, 0–38 (2n=38) or 1–36 Therefore, chromosomes containing sex-determination (2n=37) and 0–38, respectively. Meanwhile, Sawada and gene cannot be found by cytogenetic analyses (Bertollo Sakamoto (1980) found that in E. mirabilis from Japan, et al. 2004). The karyotype formulas for S. ocellatus and the chromosomes consist of 36 telocentric chromosomes. S. picturatus are as follows: Both species investigated have no cytologically distin- S. ocellatus 2 n () 40 =Lt +M tt +S guishable sex chromosome. This characteristic is simi- 16 18 6 t tt lar to others in the family Callionymidae (Sawada and S. picturatus 2 n () 40 =L14 +M 24 +S 2 Sakamoto 1980, Murofushi et al. 1983, 1984, Ojima and 64 W. Kasiroek et al. Cytologia 82(1) Special Issue

Fig. 4. Idiograms showing lengths and shapes of chromosomes Fig. 5. Idiograms of the ocellated dragonet (Synchiropus ocellatus, of the ocellated dragonet (Synchiropus ocellatus, A.) and A.) and picturesque dragonet (S. picturatus, B.), 2n (dip- picturesque dragonet (S. picturatus, B.), 2n (diploid)=40 by loid)=40 by Ag-NOR banding technique; arrows indicate conventional staining technique. nucleolar organizer region (NOR).

Chromosome markers of S. ocellatus and S. picturatus which may be caused by the translocation between some Our present study was accomplished by using the part of a chromosome having NOR and another chromo- Ag-NOR banding technique. The objective of this tech- some. Furthermore, NOR is usually located close to the nique was to present nucleolar organizer regions (NORs) telomere of the chromosome arm (Sharma et al. 2002). representing the location of genes (loci) that function The asymmetrical karyotype of S. ocellatus and S. in ribosome synthesis (18S and 28S ribosomal RNA) picturatus with only one type of telocentric chromosome (Sharma et al. 2002). The regions adjacent to the long found in this study is the important chromosome marker. arm centromeric region of large telocentric chromo- The idiogram shows continuous length gradation chro- some pair 5 in S. ocellatus and chromosome pair 4 in S. mosomes. The largest and smallest chromosomes show picturatus showed clearly observable NOR (Figs. 2, 3). size differences (approximately threefold in S. ocellatus Analyses of chromosomes from Callionymid revealed and twofold in S. picturatus). As for the chromosome positive Ag-NOR marks on a single pair of homologous marker of S. ocellatus and S. picturatus, the first chro- chromosomes (Murofushi et al. 1983, 1984). In fish, mosome pair is the largest telocentric and chromosome the location of NORs in a terminal position and close to pair 20 is the smallest telocentric chromosome. The data the centromeres is also considered a primitive feature. of the chromosomal checks on mitotic metaphase cells of Therefore, these species of the Callionymidae family the S. ocellatus and S. picturatus are shown in Tables 2 (genera Eleutherochir, Repomucenus and Synchiropus) and 3. Figures 4 and 5 show the standardized idiograms retain the plesiomorphic condition for major ribosomal of S. ocellatus and S. picturatus from conventional and genes. However, some fishes have more than two NORs, Ag-NOR staining techniques. 2017 First Report of Chromosome Analysis of Two Dragonets (Perciformes, Callionymidae) 65

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