Regular Article
Karyotype Differentiation Characterized by C-banded Heterochromatin and Nucleolus Organizer Regions in Anemonefish (Pomacentridae)
2021 Volume 86 Issue 2 Pages 175-179
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2021 Volume 86 Issue 2 Pages 175-179
Karyotypes, C-banded heterochromatin, and silver-positive nucleolus organizer regions (Ag-NORs) of two anemonefish species, Amphiprion polymnus and Premnas biaculeatus, were studied and compared with those of previously reported species, A. clarkii, A. frenatus, and A. perideraion. The chromosome number was 2n=48 for all five species. The fundamental numbers (NF) in A. polymnus (NF=84) and P. biaculeatus (NF=90) were different from the three species (NF=86). Karyotypic differences among the species were found in the distribution of C-banded heterochromatin for large submetacentric chromosomes. Moreover, A. polymnus showed a peculiar C-banding pattern with telomeric C-bands distributed in all chromosomes. The Ag-NORs were commonly located in the terminal regions of the short arms of an acrocentric pair; however, the NOR-bearing chromosomes were different in size among the species. This study demonstrated that the karyotypes of A. polymnus and P. biaculeatus had different conditions from those of the three previously studied species and that the C-band patterns in large submetacentric chromosomes could be a potential reliable marker to distinguish the anemonefish.
Anemonefish live commensally with large sea anemones in coral reefs. They are classified under the family Pomacentridae and the subfamily Amphiprioninae. This subfamily is comprised of 30 anemonefish species: one species belonging to the genus Premnas, while the other 29 species belonging to the genus Amphiprion (Nelson 2006).
Arai and Inoue (1976), and Arai et al. (1976) reported the first cytogenetic studies on three anemonefish species, A. clarkii, A. frenatus, and A. ocellaris. These studies characterized anemonefish as having the same chromosome number of 2n=48 and NF between 78 and 86. Karyotypic data for two species, A. polymnus and A. perideraion, were added later by Tanomtong et al. (2012) and Takai and Kosuga (2013), respectively.
Takai and Kosuga (2007) reported that the karyotypic constitutions were very similar in A. clarkii and A. frenatus, however, the distributions of C-banded heterochromatin and Ag-NORs were different. Furthermore, Takai and Kosuga (2013) revealed a similar observation in A. perideraion. These studies have demonstrated that the karyotypes of previously studied anemonefish can be differentiated by C-bands and NORs. Thus, the C-bands of two large submetacentric chromosomal pairs could be reliable markers for distinguishing anemonefish species. It is interesting to observe whether these findings are widely applicable to other anemonefishes.
In this paper, karyotypes characterized by C-bands and Ag-NORs in two anemonefish species, A. polymnus and P. biaculeatus, are reported. Whether reliable chromosome markers occur to distinguish the species are mainly discussed; primarily in comparison with those of A. clarkii, A. frenatus, and A. perideraion previously reported by Takai and Kosuga (2007, 2013).
Three specimens of A. polymnus and four of P. biaculeatus obtained from a fish dealer in Japan were used for this study. The sexes of these specimens were unknown.
Chromosome slides were prepared by the direct method using kidney tissues (Takai and Ojima 1987) and the fin culture method (Takai and Ojima 1993, 1996). In the direct method, kidney tissues were removed, placed on a Petri dish with 0.5 mL of Eagle’s minimum essential medium (MEM), and squashed gently with the wide end of tweezers before 4 mL of medium was added. Tissue fragments were gently pipetted, and fragment-free cell suspensions were used for chromosome preparation. The remaining tissue fragments were discarded. In the fin culture method, fins were sterilized by immersing in Dakin’s solution for 30 s. Dakin’s solution contained 40 mL 10% Antiformin, 6 mL of 1 M HCl, and 4 g (1 L) NaHCO3. The fins were rinsed thrice with Ca2+- and Mg2+-free phosphate-buffered saline (CMF-PBS) and then cut into small pieces. Fin pieces were transferred to a flask containing a magnetic stirrer bar, and approximately 8 mL of 0.1% trypsin (1 : 250, Difco) and 0.01% 2Na·EDTA solution dissolved in CMF-PBS. The flask was placed on a magnetic stirrer and stirred for approximately 20 min. Liberated cells were harvested, suspended in α-modified Eagle’s MEM with 10% fetal bovine serum and kanamycin (60 mg L−1), transferred to a culture dish, and cultured at 25°C (Takai and Ojima 1996). As an additional simple method, fin pieces were placed on culture dishes at a density of about 10 pieces per dish; culture medium was added to the dishes, and the fin pieces were cultured at 25°C (Takai and Ojima 1993). After 2–4 passages, the cultured cells were used for chromosome preparation.
The directly prepared kidney cells and the cultured fin cells were treated with 0.1 µg mL−1 colchicine for 2 h, hypotonized with 68 mM KCl for 20 min, and fixed with a freshly prepared mixture of acetic acid and methanol (1 : 3). A drop of this fixed cell suspension was placed on a clean slide and air-dried.
The slides were stained with 2% Giemsa in 1/15 M phosphate buffer (pH 6.8) and observed microscopically. The slides were then destained with 70% ethanol and subjected to C-banding and Ag-NOR staining, which was carried out according to the BSG method by Sumner (1972) and the one-step method by Howell and Black (1980), respectively.
The chromosome number for each specimen was determined based on the counts of over 20 cells in metaphase. Karyotype analysis was performed based on the chromosome classification by Levan et al. (1964). To calculate NF, metacentric and submetacentric chromosomes were assigned a score of 2 and, subtelocentric and acrocentric chromosomes were assigned a score of 1.
In A. polymnus, the chromosome number was 2n=48, and the karyotype consisted of six pairs of metacentric chromosomes, 12 pairs of submetacentric chromosomes, and six pairs of subtelocentric or acrocentric chromosomes. The NF was 84 (Table 1, Figs. 1, 2).
| Species | CN | NF | Karyotype* | Ag-NORs** | References |
|---|---|---|---|---|---|
| Amphiprion clarkii | 48 | 86 | 12M+26SM+10ST-A | 2, MS A, T | (1) |
| A. frenatus | 48 | 86 | 12M+26SM+10ST-A | 2, LS A, T | (1) |
| A. perideraion | 48 | 86 | 12M+26SM+10ST-A | 2, MS A, T | (2) |
| A. polymnus | 48 | 84 | 12M+24SM+12ST-A | 2, MS A, T | This study |
| Premnas biaculeatus | 48 | 90 | 12M+30SM+6ST-A | 2, SS A, T | This study |
*M, metacentric; SM, submetacentric; ST, subtelocentric; A, acrocentric. **The number of Ag-NORs, Ag-NOR-bearing chromosomes (LS: large-size, MS: middle-size, SS: small-size), locations (T: terminal region). (1) Takai and Kosuga (2007), (2) Takai and Kosuga (2013).
The C-banded chromosomes showed two different conditions, where centromeric regions were more deeply stained (Fig. 1A) and where telomeric regions were more deeply stained (Fig. 1B). These two different conditions were observed in the same individual. Centromeric C-bands were observed in many chromosomes, and prominent centromeric C-bands were observed in three pairs of large submetacentric chromosomes. Telomeric C-bands were observed in the long arms of all chromosomes and short arms of many chromosomes. Ag-NORs were observed in the terminal regions of the short arms of a pair of middle-sized acrocentric chromosomes. In one specimen, Ag-NORs were observed in only one member of the chromosomal pair. Chromosomal regions showing Ag-NORs often appeared as slightly stained secondary constrictions (Fig. 2).
The karyotype and NORs in A. polymnus were previously reported by Tanomtong et al. (2012). The results reported by Tanomtong et al. (2012) and those observed in this study concur in the number of NORs but differ slightly in the karyotypic constitution. This difference may stem from the conditions of the metaphase chromosomes studied as this study dealt with more elongated chromosomes than those by Tanomtong et al. (2012).
In P. biaculeatus, the chromosome number was 2n=48, and the karyotype was composed of six pairs of metacentric chromosomes, 15 pairs of submetacentric chromosomes, and three pairs of subtelocentric or acrocentric chromosomes. The NF was 90 (Fig. 3).
The C-bands were observed in the centromeric regions of many chromosomes, and prominent centromeric C-bands were observed in two pairs of large submetacentric chromosomes. Telomeric C-bands were observed in some chromosomes, and noticeable telomeric C-bands were observed in the short arms of a pair of large submetacentric chromosomes without prominent centromeric C-band (Fig. 3). Ag-NORs were located in the terminal regions of the short arms of a pair of small-sized acrocentric chromosomes. Chromosomal regions showing Ag-NORs often appeared as slightly stained secondary constrictions (Fig. 4) and stained by C-banding (Fig. 3).
The chromosome number was 2n=48 for A. polymnus and P. biaculeatus, as well as the three other species, A. clarkii, A. frenatus, and A. perideraion previously studied. The fundamental numbers in A. polymnus (NF=84) and P. biaculeatus (NF=90) were different from the three other species (NF=86). Karyotypic differences among the species were found in the distribution of C-banded heterochromatin for large submetacentric chromosomes (Fig. 5). Moreover, A. polymnus showed a peculiar C-banding pattern with telomeric C-bands distributed in all chromosomes. Such distinct C-bands did not occur in the other species. The two different conditions in C-banded chromosomes might result from the structural difference between centromeric and telomeric heterochromatin (Hartley 1991). The Ag-NORs were commonly located in the terminal regions of the short arms of an acrocentric pair; however, the NOR-bearing chromosomes were different in size among the species.
The molecular phylogeny by Santini and Polacco (2006) revealed that Amphiprioninae was a monophyletic group. This suggests that the karyotypes of anemonefishes have diversified from a common ancestral karyotype. According to the molecular phylogeny, the genus Premnas would have been differentiated from the genus Amphiprion, and later the four Amphiprion species may have been differentiated in the order of A. clarkii, A. perideraion, and A. polymnus/A. frenatus. It was difficult to infer the karyotypic differentiation of the anemonefish species based on the molecular phylogeny.
This study demonstrated that in anemonefish various karyotypic changes, including changes in C-bands and Ag-NORs, have occurred and C-band patterns in the large submetacentric chromosomes could be a potential reliable marker to distinguish the anemonefish (Fig. 5).
Thus, anemonefish are cytogenetically attractive fish. Further detailed investigation, using several other banding and fluorescence in situ hybridization techniques, will provide interesting insights and clarify the process of complicated karyotypic differentiation discussed in this study.
I would like to thank Editage (www.editage.com) for English language editing.
