Telomere Shortening in the Colonial Coral Acropora digitifera During Development Author(s): Hiroki Tsuta , Chuya Shinzato , Nori Satoh and Michio Hidaka Source: Zoological Science, 31(3):129-134. 2014. Published By: Zoological Society of Japan DOI: http://dx.doi.org/10.2108/zsj.31.129 URL: http://www.bioone.org/doi/full/10.2108/zsj.31.129

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¤ 2014 Zoological Society of Japan

ZOOLOGICAL SCIENCE 31: 129–134 (2014)

Telomere Shortening in the Colonial Coral Acropora digitifera During Development Hiroki Tsuta1, Chuya Shinzato2, Nori Satoh2, and Michio Hidaka1* 1

Department of Chemistry, Biology and Marine Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan 2 Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan

To test whether telomere length can be used in estimating the age of colonial corals, we used terminal restriction fragment (TRF) length analysis to compare the telomere lengths of the coral Acropora digitifera at three developmental stages: sperm, planula larvae, and polyps of adult colonies. We also compared the mean TRF lengths between branches at the center and periphery of tabular colonies of A. digitifera. A significant difference was observed in the mean TRF lengths in sperm, planulae, and polyps. The mean TRF length was longest in sperm and shortest in polyps from adult colonies. These results suggest that telomere length decreases during coral development and may be useful for estimating coral age. However, the mean TRF length of branches at the center of a table-form colony tended to be longer than that of peripheral branches, although this difference was not statistically significant. This suggests that both the chronological age of polyps and cell proliferation rate influence telomere length in polyps, and that estimating coral age based on telomere length is not a simple endeavor. Key words:

Acropora, aging, colony, coral, planula, telomere, TRF

INTRODUCTION Some corals live for hundreds of years under favorable conditions (Hughes and Jackson, 1985; Potts et al., 1985; Lough and Barnes, 1997). Traditionally, coral age is estimated from the size of colonies and annual growth rate of the coral, or by examining skeletal growth bands (e.g., Klein and Loya, 1991; Lough and Barnes, 1997). However, these methods can be applied only in corals with massive growth forms, not branching corals, particularly those species in which asexual reproduction via fragmentation is common. Whether corals that have regenerated from fragments are the same age as the source colony is not known. Furthermore, coral colonies sometimes fuse or divide, making it difficult to estimate the age of corals from their size (Hughes and Jackson, 1980). Telomeres are tandem repeat sequences at the ends of eukaryotic chromosomes that protect chromosome ends from degradation and end-to-end chromosome fusion (Blackburn, 1991). In corals and other cnidarians, the telomeric repeat is composed of the motif TTAGGG (Sinclair et al., 2007; Traut et al., 2007; Ojimi et al., 2009; Zielke and Bodnar, 2010; Nakamichi et al., 2012), as in other basal metazoa. Telomeres shorten during cell division due to incomplete replication of the ends of the double strands, and cell division stops when the telomeres shorten beyond a certain critical length (e.g., Blackburn, 2000; Urquidi et al., * Corresponding author. Tel. : +81-98-895-8547; Fax : +81-98-895-8576; E-mail: [email protected] doi:10.2108/zsj.31.129

2000). In humans and some birds, a negative correlation exists between organism age and the telomere length of blood mononuclear cells (e.g., Haussmann and Vleck, 2002; Kimura et al., 2007; Haussmann and Mauck, 2008), suggesting that telomere length may reflect the age of these organisms. However, telomerase, a reverse transcriptase, extends telomeres by adding the telomere motif at the end of the 3′ end of G-rich strands (Greider and Blackburn, 1985). Telomerase activity has been detected in the somatic tissues of some vertebrates and invertebrates, including corals (Zielke and Bodnar, 2010; Nakamichi et al., 2012), although it is unknown whether this occurs at sufficient levels to counteract telomere shortening. If it were possible to estimate coral age based on telomere length, it would greatly enhance our understanding of coral population biology and demography, as well as various life history traits, such as aging and senescence. In a previous study on the colonial coral Galaxea fascicularis using single telomere length analysis (STELA), we did not detect a significant difference in telomere lengths at different developmental stages. However, Ojimi et al. (2012) reported that sperm of the solitary coral Ctenactis echinata have longer telomeres than somatic tissue using STELA. The telomere dynamics of corals remain poorly understood. Therefore, this study estimated the telomere lengths of Acropora digitifera, a fast-growing colonial coral, at three developmental stages using conventional terminal restriction fragment (TRF) analysis.

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MATERIALS AND METHODS Samples Six and 12 colonies of A. digitifera (10–28 cm in diameter) were collected from a shallow reef off Sesoko Island, Okinawa, Japan, in May 2012 and May 2013, respectively, under Okinawa Prefecture permit. They were maintained in tanks supplied with unfiltered seawater at Sesoko Station, University of the Ryukyus. Two 2-cm-long branches were collected from each colony, one from the center and the other from the periphery of the colony. Each branch was divided into a branch tip (~1 cm long) containing an axial polyp and several radial polyps, and a basal part (~1 cm long) containing radial polyps. The branch samples were immersed in CHAOS solution (4 M guanidine thiocyanate, 0.5% sarkosyl, 25 mM Tris, and 0.1 M 2-mercaptoethanol; modified from Fukami et al., 2004) to preserve the DNA and stored at −20°C until use. To collect sperm and planula larvae, A. digitifera colonies were placed individually in plastic chambers after sunset around the full moon in June 2012 and June 2013. When colonies spawned egg– sperm bundles, these were collected using a pipette and eggs floating at the surface were removed using a pipette. A sample of filtered seawater containing sperm was centrifuged at 3000 rpm for 5 min. The sperm pellets were placed in CHAOS solution and stored at −20°C. To obtain planula larvae, the remaining seawater containing sperm was mixed with eggs released by another colony. Planulae derived from four combinations of parent colonies were prepared in both years. The planulae were fixed using CHAOS solution when they started to swim, three days after fertilization.

Terminal restriction fragment (TRF) length analysis To estimate the lengths of telomeres of A. digitifera, we used TRF length analysis. The integrity of the extracted DNA was checked by electrophoresing the DNA on 1.0% agarose gels and visualizing it with ethidium bromide. The DNA (0.5–2.0 μg) was digested overnight with the restriction enzymes RsaI (10 U) and HinfI (10 U; TaKaRa Bio, Otsu, Japan) in 20 μl digestion buffer at 37°C. The digested DNA was resolved on 0.8% agarose gels of 6 cm length at 3.5 V/cm for 1 h. After electrophoresis, the DNA was depurinated for 10 min in 0.25 M HCl and denatured for 25 min in 0.5 M NaOH and 1.5 M NaCl. The agarose gels were used for Southern blotting. The DNA was transferred overnight to Hybond N+ membrane (GE Healthcare, Tokyo, Japan) using 10 × SSC buffer. TRF samples were hybridized overnight with an alkaline phosphatase-labeled telomeric probe (TTAGGG)10 at 55°C. Hybridized DNA was detected using the AlkPhos Direct Labeling and Detection System with CDP-Star (GE Healthcare). Luminescent images were taken using ImageQuant LAS4010 (GE Healthcare) and the mean TRF length was calculated from the intensity of the TRF signal in the range of 0.5–23 kb using the ImageQuant TL Analysis Toolbox (GE Healthcare). The mean TRF length was calculated as Σ(ODi × Li)/Σ(ODi), where ODi is the optical density at position i and Li is the TRF length at that position (Kimura et al., 2010). In experiments conducted in 2012 and 2013, we respectively analyzed sperm released from six and five colonies, planula larvae prepared from four and four separate crossings, and branches from four and six adult colonies. We repeated TRF analysis on nine sperm and eight planula samples prepared in 2012 and 2013, and five polyp samples (branch tips about 2 cm long) newly collected after the 2013 spawning, using an improved electrophoresis condition for higher resolution. In the additional experiments, lower amounts of DNA (0.2–0.5 μg) were used for restriction enzyme digestion and the digested DNA was electrophoresed on 0.5% agarose gel with a larger size (12 cm long) at lower voltage gradient (2.5 V/cm) for 3.5 h. To confirm that TRFs detected by the telomere probe were derived from terminal telomere fragments, and not due to interstitial telomeric sequences, total DNA was digested with Bal31 exonuclease before TRF analysis. Genomic DNA (2 μg) extracted from sperm was digested with one unit of Bal31 nuclease in a reaction mixture of 250 μl at 30°C. Fifty microliter aliquots of reaction mixture

DNA extraction DNA was extracted using a modified CTAB method (Coffroth et al., 1992). An equal volume of 2 × cetyl trimethyl ammonium bromide (CTAB) buffer [1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl (pH 8.0), 4% CTAB] was added to the CHAOS solution containing coral DNA, and the mixture was incubated at 65°C for 60 min. DNA was extracted using conventional phenol/chloroform extraction and ethanol precipitation. The CTAB-CHAOS mixture was mixed with an equal volume of a 1:1 mixture of TE-saturated phenol and chloroform–isoamyl alcohol (CIA). After rolling for 10 min at 30 rpm, the mixture was centrifuged (14,000 rpm, 15 min, 4°C), and the upper layer was transferred to a new tube and mixed with an equal volume of CIA. The mixture was again rolled and centrifuged (14,000 rpm, 15 min, 4°C), and the upper layer was transferred to a new tube. This upper layer was mixed with one-tenth volume of 3 M NaOAc and an equal volume of isopropanol, and held at 4°C for 1 h. The mixture was then centrifuged (14,000 rpm, 25 min, 4°C), and the supernatant removed. The DNA pellet was rinsed twice with 70% ethanol and then dissolved in 20 μl TE buffer. The DNA content was determined using a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). The extracts were digested by RNase A in a mixture of 10 μl DNA, 90 μl TE buffer, and 100 μg RNase A at 37°C for 30 min. The DNA was re-extracted using phenol/chloroform extraction and ethanol precipitation, and the Fig. 1. Terminal restriction fragment (TRF) length analysis of sperm, planula larvae, DNA samples were stored at −20°C until use. and polyps of Acropora digitifera. (A) TRF patterns detected using the (TTAGGG)10 In some cases, RNase A (100 μg) was added probe. Lanes 1–4, sperm samples; lanes 5–8, planula samples; lanes 9–12, polyp samto the CHAOS solutions containing samples, ples; lane M, λ HindIII molecular marker. (B) Densitometry profile of TRF patterns. Proand the mixtures were incubated at room temfiles of four representative TRF patterns are shown for each developmental stage. The perature for 10 min before DNA extraction. profile line of each lane was standardized to the total intensity of each lane. Black solid lines, sperm samples; dashed lines, planula samples; gray lines, polyp samples.

Telomere length of Acropora digitifera were removed periodically (0, 30, 60, 120, and 180 min), added to 1 μl of 0.5 M EDTA and incubated at 65°C for 10 min to stop the reaction, and stored at −20°C until DNA was extracted using phenol/chloroform method. The TRF pattern of the DNA was detected as described above. Statistical analysis The TRF length data obtained from 2012 and 2013 experiments were pooled. The mean TRF length was transformed logarithmically and tested for normality and homoscedasticity using Shapiro-Wilk test and Bartlett’s test, respectively. The mean TRF lengths at different developmental stages were compared using analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. For the polyps, the mean TRF lengths of four parts (tip and adjacent basal parts of branches from the center and periphery) of a colony were averaged and the mean value was used as statistical unit for comparison among developmental stages. Comparison of the mean TRF lengths between tips and basal parts of the branches and between branches from the center and periphery of the colonies was tested using a t-test. All statistical analyses were performed using STATISTICA 03J (StatSoft Japan, Tokyo, Japan).

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under lower voltage gradient. Similar results were obtained with changes in the telomere length during development of the coral. The mean TRF length of sperm was significantly longer than that of polyps, and planulae exhibited intermediate mean TRF length between sperm and polyps (Fig. 3). However, the TRF patterns of sperm became broader and shifted to shorter fragment sizes when resolved on 0.5% agarose gels. The mean TRF lengths calculated based on the band pattern between 500 bases and 23 kb were 16.1 ± 2.7 kb (n = 11) for sperm, 11.7 ± 3.4 kb (n = 8) for planulae, and 8.9 ± 1.8 kb (n = 10) for polyps when resolved on 0.8% agarose gels. If the TRFs were resolved on 0.5% agarose gels, the mean TRF lengths of sperm, larvae and polyps were 12.2 ± 2.2 kb (n = 9), 10.0 ± 1.5 kb (n = 8), and 8.8 ±

RESULTS The terminal restriction fragments of the DNA extracted from sperm, planula larvae, and adult polyps of A. digitifera exhibited smear patterns with the telomere probe (TTAGGG)10 (Fig. 1). While the TRF patterns of planula larvae and polyps of adult colonies were within the range of the resolution range of the TRF analysis (< 23 kb), the TRF patFig. 2. Mean TRF lengths of Acropora digitifera at different develterns of sperm sometimes extended beyond the limit of resopmental stages and comparison between the center and periphery olution of the TRF analysis (> 23 kb; Fig. 1A). In three of 11 of adult colonies. (A) Comparison of the mean TRF lengths among sperm samples, the peak TRF intensity (the strongest sperm, planula larvae, and polyps (mean ± SD). (B) Comparison of hybridization) occurred beyond the limit of resolution of the the mean TRF lengths between branches from the central and TRF analysis (> 23 kb), while the other samples showed the peripheral areas of adult colonies. The number in parentheses is the strongest hybridization at slightly less than 20 kb. For polnumber of colonies analyzed. Asterisks indicate a significant difference (Tukey’s multiple comparison test, * P < 0.05). yps, the peak TRF intensity was observed around 10 kb. Planulae exhibited the strongest hybridization at 10–20 kb, within the range of resolution of the TRF analysis (< 23 kb). A significant difference was observed in the mean TRF lengths of sperm, planulae, and polyps of adult colonies (Tukey’s multiple comparison test, P < 0.05). The mean TRF length was longest in sperm and shortest in polyps (Fig. 2A). No significant difference was found in the mean TRF length between the tip region (< 1 cm from the tip) and adjacent basal regions (1–2 cm from the tip) of branches of adult colonies (t-test, P = 0.85). Therefore, the mean TRF length of a branch tip and that of an adjacent basal region of a colony were averaged and the mean value was used as a statistical unit for comparison between the center and periphery of the colonies. The mean TRF length of branches from the central part of the coloFig. 3. Terminal restriction fragment (TRF) length analysis of sperm, planula larvae, nies tended to be longer than that of and polyps of Acropora digitifera. (A) TRF patterns resolved on 0.5% agarose gels and branches, although the difference was not detected using the (TTAGGG)10 probe. Lanes 1–4, sperm samples; lanes 5–8, planula significant (t-test, P = 0.08) (Fig. 2B). samples; lanes 9–12, polyp samples; lane M, λ HindIII molecular marker. (B) CompariTo improve the resolution of the TRF son of the mean TRF lengths among sperm, planula larvae, and polyps (mean ± SD). analysis, we performed the TRF analysis The number in parentheses is the number of colonies analyzed. Asterisks indicate a significant difference (Tukey’s multiple comparison test, * P < 0.05). using larger size gels of 0.5% agarose

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Fig. 5. Southern hybridization of Bal31 treated DNA with a telomere probe. The sperm DNA was digested with Bal31 exonuclease for 0, 30, 60, 120, and 180 min before TRF analysis.

clease for various lengths of time before TRF analysis. After 30–60 min of digestion with Bal31, the intensity of the hybridization signal and size of TRFs decreased (Fig. 5). This supports that the fragments detected with the telomere probe were located at the end of chromosomes and that the hybridization signal was specific to telomere sequences. DISCUSSION The results of the present study show that the mean TRF length is Fig. 4. Evaluation of DNA integrity and efficiency of DNA digestion by the restriction enzymes. longest in sperm and shortest in DNA samples in earlier (A) and additional (C) experiments were resolved on a 1% agarose gel adult polyps of the colonial coral and visualized with ethidium bromide. The DNA samples digested with RsaI and HinfI were Acropora digitifera. The planulae resolved on 0.8% agarose gels in earlier experiments (B) or on 0.5% agarose gels in the addihad intermediate TRF lengths tional experiments (D). The DNA samples in (A) and (B), and those in (C) and (D) correspond to between sperm and polyps. This is samples used for TRF analysis in Fig. 1A and Fig. 3A, respectively. Lanes 1–4, sperm samples; the first study to show that telomere lanes 5–8, planula samples; lanes 9–12, polyp samples; lane M, λ HindIII molecular marker. shortening occurs during develop0.5 kb (n = 5), respectively. ment of a colonial coral using conventional TRF analysis. The integrity of extracted DNA and efficiency of restricThe results suggest that the telomerase activity is not suffition enzyme digestion are shown for earlier (Fig. 4A, B) and ciently high to compensate for telomere attrition during cell additional experiments (Fig. 4C, D). More or less crowndivision in this coral. shaped bands appeared in parallel in high molecular areas The finding that telomere length shortens as the coral (> 23.1 kb), indicating the integrity of the DNA samples (Fig. develops suggests the possibility that the age of coral colo4A, C). Complete digestion of the DNA samples by the nies can be estimated using soft tissue. However, estimating restriction enzymes was verified by the absence of smears coral age based on telomere length is not so simple. Telomin the area beyond 2 kb in the additional experiments (Fig. ere length is determined not only by chronological age or 4D), while weak smears remained in the area (> 2 kb) in the cell division history, but also by the level of telomerase activearlier experiments (Fig. 4B). However, we confirmed that ity in the tissue. However, if telomerase activity in colonies the presence of weak smears did not lead to overestimation is absent or stable at a low level, we can estimate the relaof mean TRF length (data not shown). tionship between the size of coral colonies and their mean Sperm DNA samples were digested with Bal31 exonuTRF length. The relationship between the size of colonies

Telomere length of Acropora digitifera

and their chronological age or cell division history should be determined in future research. The annual growth rate data or annual cycle of wave-like outward growth of Acropora colonies (Stimson, 1996) may be useful for this purpose. To study the telomere rate of change (TROC) during early development, fertilized eggs should be used as a starting point, as it is not known whether sperm and egg have the same mean TRF length. Recently modified STELA has been used in an attempt to compare the telomere lengths of corals at different developmental stages (Ojimi et al., 2012; Tsuta and Hidaka, 2013). While sperm had longer telomere lengths (the size of STELA products) than somatic tissues in the solitary coral C. echinata (Ojimi et al., 2012), no significant difference in telomere length was detected among sperm, planula larvae, and adult polyps of the colonial coral G. fascicularis (Tsuta and Hidaka, 2013). The different results between the two colonial corals G. fascicularis and A. digitifera (present study) may be due to different rates of telomere shortening between the two corals. The TROC may differ among coral species, and a higher rate of cell proliferation in rapidly growing branching corals likely leads to a greater rate of telomere shortening. Acropora digitifera is known as rapidly growing, short-lived species, and G. fascicularis may be a long-lived species, as large colonies frequently exceed 5 m across and 2 m in height (Veron, 2000; Dai and Horng, 2009). It is likely that the modified STELA and the preliminary TRF analysis with a small number of samples could not detect developmental changes in telomere length in G. fascicularis due to its low TROC. The TROC of corals might also be influenced by the levels of telomerase activity in somatic tissues. The somatic tissues of G. fascicularis have high levels of telomerase activity (Nakamichi et al., 2012). Further studies are needed to clarify whether the telomerase activity of the coral is high enough to balance telomere attrition, resulting in stable telomere length. It is also important to study telomere length and telomerase activity in extremely long-lived corals such as massive Porites. An A. digitifera colony assumes a table-like form and grows more or less horizontally, so that new branches are formed at the periphery. The edge zone of table-like A. hyacinthus colonies is sterile zone, where polyps bear no gonads (Wallace, 1985). This is probably because the actively extending edge zone consists of young, sexually immature polyps. Meesters and Bak (1995) reported that regeneration capability decreases from the distal end to base of branches of A. palmata, and that the decrease in regeneration capability appears to be related to polyp age. We postulated that the polyps at the center of a colony are older and possess shorter telomeres than the polyps at the periphery or growing edge. Nevertheless, our results did not support this expectation. The polyps (or branches) at the central part of a colony tended to have longer telomeres than peripheral polyps (branches), although the difference was not significant (t-test, P = 0.08). The cell proliferation rate may be higher in the peripheral branches than at the center of a colony, and cells at the periphery likely experienced more cell divisions, hence a greater extent of telomere attrition than those at the central part of the colony. Another possibility is that differentiated somatic cells migrate from the center to the peripheral zone of a colony. It is

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important to understand where and when somatic cells differentiate from somatic stem cells during polyp or branch formation. The analysis window of the TRF pattern is often optimized for the organisms to be studied (Kimura et al., 2007; Haussmann and Mauck, 2008). In our study, the mean TRF length was based on the TRF pattern in the range from 0.5 to 23 kb because of technical limitations. Since the TRF pattern extended beyond the resolution limit (23 kb) in some sperm samples, it is advisable to perform pulsed-field gel electrophoresis and analyze the complete TRF pattern to calculate a reliable mean TRF length for sperm samples in future research. The mean TRF length of A. digitifera sperm (16.1 kb) is comparable to the telomere length of sperm (17.9 kb) of G. fascicularis (Tsuta and Hidaka, 2013) based on conventional TRF analysis, but shorter than the telomere length of gametes (21.0 kb) of Acropora millepora (Zielke and Bodnar, 2010) based on TRF analysis using pulsedfield gel electrophoresis. The mean TRF length (8.9 kb) of adult polyps of A. digitifera is shorter than the telomere length of G. fascicularis colonies (15.6 kb; [Tsuta and Hidaka, 2013]) or 19.2 kb for colonies of Agaricia fragilis and Madracis auretenra determined using pulsed-field gel electrophoresis (Zielke and Bodnar, 2010). The different telomere lengths of adult colonies among coral species may reflect inter-species differences, variation in TROC among coral species, or the actual ages of colonies analyzed, although it is also possible that they are due to differences in the electrophoresis methods used. The mean TRF length of sperm based on 0.5% agarose gel electrophoresis was shorter than the mean TRF length based on 0.8% agarose gel electrophoresis. The TRF pattern resolved on 0.5% agarose gels is more similar to typical TRF patterns (Kimura et al., 2010). The difference in TRF patterns on 0.5 and 0.8% agarose gels might be due to reduced mobility of long, high molecular weight TRFs on 0.8% agarose gels. It is important to choose appropriate electrophoresis conditions and concentrations of DNA samples applied to the gels for TRF analysis of corals in future studies. While telomere shortening with age and telomere length rate of change (TROC) have been reported in some birds and mammals (Nakagawa et al., 2004), there have been only a few studies of telomere dynamics in marine invertebrates (Bodnar, 2009). Francis et al. (2006) reported a lack of age-associated telomere shortening in two species of sea urchins with different lifespans using conventional TRF analysis. Nilsson Sköld et al. (2011) found that parents of the colonial ascidian Diplosoma listerianum had shorter telomeres than their offspring based on telomere fluorescence in situ hybridization (Telomere FISH). The freshwater planarian Schmidtea mediterranea has asexual and sexual strains. Obligate asexual animals have the potential to maintain telomere length during regeneration via increased telomerase activity in adult stem cells, while sexual animals require passage through a sexual reproductive cycle to restore their telomeres (Tan et al., 2012). It is to be studied whether long-lived corals can escape ageing via increased telomerase activity or they also undergo senescence and their telomeres are restored only by a sexual reproduction cycle.

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This study found that telomere length shortens during the development of the colonial coral A. digitifera using TRF analysis. Our results suggest that the telomerase activity was not high enough to compensate for telomere attrition during cell division in the coral, and suggest that estimating the age of some colonial corals is possible based on the relationship between the telomere length and colony size (age). However, we must consider that telomere length may be affected by various intrinsic and extrinsic factors unrelated to chronological time (Dunshea et al., 2011). Telomere length may be determined by telomere attrition at each cell division, the deleterious effect of oxidative stress, and telomere extension mechanisms such as telomerase activity (Nakagawa et al., 2004). Further studies using colonies with a wider age range are needed to detect a possible correlation between telomere length and colony size and to estimate the TROC of coral. ACKNOWLEDGMENTS We would like to thank Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, where part of this study was done.

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Telomere shortening in the colonial coral Acropora digitifera during development.

To test whether telomere length can be used in estimating the age of colonial corals, we used terminal restriction fragment (TRF) length analysis to c...
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