Effects of Calcification Inhibitors on the Viability of the Coralline Algae Lithophyllum yessoense and Corallina pilulifera

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    Coralline algae, the algal whitening phenomenon-causing seaweeds, are characterized by calcareous deposits in the cell wall. The viability of the coralline algae Lithophyllum yessoense and Corallina pilulifera was quantitated using a triphenyltetrazolium chloride assay and eight calcification inhibitors. Among these inhibitors, ferric citrate showed the strongest inhibition of coralline algae viability. The concentrations of ferric citrate conferring 50% inhibition were 1.7 and 3.8 mM for L. yessoense and C. pilulifera, respectively. Thus, at a specific concentration and in a localized area, ferric citrate may be used to prevent the blooming of coralline algae.


    Algal whitening , Anti-fouling , Calcification inhibitor , Ferric citrate , Lithophyllum yessoense , Corallina pilulifera

  • Introduction

    Many rocky seashore areas of Korea and Japan are dominated by coralline algae such as Lithophyllum yessoense (Suzuki et al., 1998; Kim, 2000). However, as calcareous algae cover the surfaces of rocks in a pink-colored crust, the area covered by seaweed flora decreases. This algal whitening phenomenon is observed in barren ground, coralline flats, and deforested areas, and is associated with specific species of coralline algae (Tokuda et al., 1994). Since 1990, the area affected by algal whitening, which runs from south Cheju Island to the middle East Sea, has expanded (Chung et al., 1998). In this area, most of the fleshy seaweed has disappeared from the rocks because of algal whitening, which reduces food sources and spawning locations for fish and shellfish. This phenomenon is now considered a natural hazard adversely affecting marine ecosystems and damaging commercial fishing areas. Although biological (Agateuma et al., 1997; Daume et al., 1999) and physical (Masaki et al., 1984; Johnson and Mann, 1986) factors may be sufficient to prevent the recruitment of fleshy seaweeds, allelopathic bromoform (Ohsawa et al., 2001) and fatty acid (Kim et al., 2004; Luyen et al., 2009) substances inhibit the settlement or germination of seaweed spores. One approach to restore fleshy seaweed colonization in these areas is the removal or inhibition of living coralline algae. Before applying chemicals in the field, it is necessary to test the in vitro inhibitory activity of several calcification inhibitors against coralline algae to identify compounds suitable for coralline species inhibition in areas affected by algal whitening. Alternatively, coralline algae may be used to predevent fouling by fleshy seaweeds. When a biomimetic coralline algal material is prepared, it can be used as an environmentally friendly anti-fouling material. To generate biomimetic coralline material, it is necessary to add a calcification inhibitor to the material to prevent additional attachment and the blooming of living coralline algae on the product. Thus, a calcification inhibitor can be used to remediate algal whitening and prepare a biomimetic coralline material. In this report, inhibitors were quantitatively screened using a triphenyltetrazolium chloride (TTC) assay.

    Materials and Methods

      >  Tissue preparation

    The coralline algae L. yessoense and Corallina pilulifera were collected from the rocky intertidal area at Cheongsapo (35˚09ʹ28ʺ N, 129˚11ʹ47ʺ E), on the east coast of Busan, Korea. Stones covered with coralline algae were transported in a container of seawater to the laboratory. After rinsing well with autoclaved seawater to remove epiphytes and debris, the encrusted or non-articulated tissues of L. yessoense were sonicated three times with 30-s pulses of an ultrasonic water bath (low-intensity frequency of 40 kHz) to remove other microepiphytes. The tissue was then scraped off the stones using a saw and thoroughly washed at least six times by centrifugation at 1,000 g for 30 s (Kang et al., 2005). Articulated coralline tissues were cleaned by brushing thoroughly and sonicating (40 kHz) twice for 1 min in autoclaved seawater, and then immersed in 1% Betadine for 2 min to eliminate epiphytes (Jin et al., 1997). The articulated coralline tissues were then rehabilitated at 18°C in Provasoli’s enriched seawater (PES) (Provasoli, 1968) for 1 day before use.

      >  Tissue culture

    In the tissue cultures of coralline algae, six hydroxyapatite inhibitors (alendronate sodium trihydrate, AlCl3, dichloromethylene diphosphonic acid, etidronic acid, ferric citrate, and FeCl3) and one bicarbonate channel blocker (4,4´-diisothiocyanatostilbene-2,2´-disulfonic acid) as calcium inhibitors were dissolved in distilled water and added to the tissue culture solution at concentrations of 10 or 1 mM, respectively. In addition, an impermeable carbonic anhydrase inhibitor (acetazolamide) was dissolved in dimethyl sulfoxide (DMSO) and added to the solution. To measure tissue viability, 25 μL of each inhibitor was added to 5 mL of PES medium containing 0.1 g of L. yessoense or 0.05 g of C. pilulifera. The mixture was cultured for 5 days at 16°C with rotation at 20 rpm under a photon flux density (fluorescent light) of 40 μmol m-2 s-1 and on a light cycle of 16-h light/8-h dark. A reference culture was prepared by mixing 25 μL of distilled water or DMSO in the same medium. After harvesting the tissues by centrifugation at 3,000 g for 30 s, cell viability was measured using the TTC assay. The relative viability (%) was calculated as: (S/C) × 100, where S equals the absorbance of the tissue with inhibitors and C equals the absorbance of the reference culture.

      >  Viability assay

    The TTC assay described by Park et al. (2006) was used. A total of 1 mL of 0.8% TTC solution in seawater containing 50 mM Tris-HCl buffer (pH 8.0) was added to the tissue in a microtube and incubated in the dark for 1.5 h at 20°C under a drop of mineral oil (M-3516; Sigma, St. Louis, MO, USA). The tissue was then rinsed four times by centrifugation at 3,000 g for 30 s with sterilized seawater. Triphenylformazan (TPF) that formed in the tissues was extracted with 0.6 mL of 0.2 N NaOH in 75% ethanol by heating for 15 min at 60°C. Next, TPF was partitioned by adding 0.6 mL of hexane followed by vortexing. After centrifugation for 1 min, the amount of TPF from the top phase was quantified by measuring the absorbance at 475 nm.

      >  Statistical analysis

    For each assay with calcification inhibitors and control samples, the experiments were repeated at least three times. The mean values of the index were compared to the control using Student’s t-test.


    To determine the effects of the calcification inhibitors on coralline viability, we compared eight commercially available inhibitors, including six hydroxyapatite inhibitors, one bicarbonate channel blocker, and one carbonic anhydrase inhibitor. Each compound at 1 and 10 mM, respectively, was added to the coralline culture, and the viability of the culture after 5 days was measured using the TTC assay. For crustose L. yessoense tissues, reference cultures without calcification inhibitors reached an absorbance of 1.24. Ferric citrate showed an absorbance of 0.02, which corresponded to 2% relative viability at 10 mM (Table 1). At 1 mM, ferric citrate inhibited viability to 76% compared to the control. Next, FeCl2 inhibited viability to 54% at 10 mM. The effects of the calcification inhibitors on the viability of articulated coralline C. pilulifera were also determined using the TTC assay after 5 days of culture. A reference culture lacking calcification inhibitors reached an absorbance of 1.11. Among the inhibitors, ferric citrate, AlCl3, and FeCl3 inhibited viability to 15%, 20%, and 29% compared to the control, respectively, at 10 mM (Table 2). Overall, ferric citrate most significantly suppressed the viability of L. yessoense and C. pilulifera. To explore treatment concentrations, we used a dose-response curve to determine the concentration resulting in 50% inhibition (IC50) and the minimum concentration resulting in 100% inhibition (MIC). For ferric citrate against L. yessoense, a typical gradient of viability inhibition ranged sigmoidally with IC50 and MIC values of 1.7 and 10 mM, respectively (Fig. 1A). For C. pilulifera, a typical gradient of inhibition showed IC50 and MIC values of 3.8 and 20 mM, respectively (Fig. 1B).


    Algal whitening can devastate marine forests. However, algal whitening-causing coralline species may be used to prevent fouling by fleshy seaweed. Thus, biomimetic coralline algae may be applicable as an environmentally friendly material for anti-fouling coating treatment. To generate biomimetic coralline algae, it is necessary to include calcification inhibitors in the material to prevent the additional attachment and growth of living coralline species. Calcification is a critical process in plants because calcareous skeletons support and protect the soft parts of organisms, and secreted proteins play major roles in the photosynthetic assimilation of bicarbonate and nutrient acquisition (McConnaughey and Whelan, 1997). commercial calcification inhibitors were compared using TTC assays. Among them, ferric citrate or Fe(III) citrate showed the strongest inhibition against coralline cell viability. Ferric citrate as a hydroxyapatite inhibitor is also known to be a non-protein-bound iron transporter in plants (Solti et al., 2012) and animals (Baker et al., 1998). Part of the ferric ion undergoes reduction to ferrous iron mediated by ferric chelate reductases (Jeong et al., 2008). Ferrous citrate or Fe(II) citrate induces oxidative damage in mitochondria through lipid peroxidation and alterations in membrane proteins (Castilho et al., 1994). Under natural conditions, iron can be present as divalent or trivalent cations depending on the chemical environment, which makes it a good cofactor for oxidoreductase-type enzymes. Nevertheless, free ferrous ions are dangerous to living organisms as they can catalyze the Fenton reaction and produce reactive radicals (Winterbourn, 1995). Thus, at specific concentrations and in a localized area, ferric citrate may be used to prevent the additional settlement of coralline algae on biomimetic materials or to prevent the blooming of coralline algae.

  • 1. Agateuma Y, Mateuyama K, Nakata A, Kawai T, Nishikawa N 1997 Marine algal succession on coralline flats after removal of sea urchins in Suttsu bay on the Japan Sea coast of Hokkaido, Japan [Nippon Suisan Gakkaishi] Vol.63 P.672-680 google doi
  • 2. Baker E, Baker SM, Morgan EH 1998 Characterisation of non-transferrin-bound iron (ferric citrate) uptake by rat hepatocytes in culture [Biochim Biophys Acta] Vol.1380 P.21-30 google doi
  • 3. Castilho RF, Meinicke AR, Almeida AM, Hermeslima M, Vercesi AE 1994 Oxidative damage of mitochondria induced by Fe(II) citrate is potentiated by Ca2+ and includes lipid peroxidation and alterations in membrane proteins [Arch Biochem Biophys] Vol.308 P.158-163 google doi
  • 4. Chung HS, Cho KW, Chung KH, Kim JH, Shin JH, Seo YW, Kang JS, Lee IK 1998 Ecological characteristics of algal whitening in coastal zone of Seogwipo area, Cheju Island [Algae] Vol.13 P.362-374 google
  • 5. Daume S, Brand-Gardner S, Woelkerling WJ 1999 Settlement of abalone larvae (Haliotis laevigata Donovan) in response to non-geniculate coralline red algae (Corallinales, Rhodophyta) [J Exp Mar Biol Ecol] Vol.234 P.125-143 google doi
  • 6. Jeong J, Cohu C, Kerkeb L, Pilon M, Connolly EL, Guerinot ML 2008 Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions [Proc Natl Acad Sci USA] Vol.105 P.10619-10624 google doi
  • 7. Jin HJ, Seo GM, Cho YC, Hwang EK, Sohn CH, Hong YK 1997 Gelling agents for tissue culture of the seaweed Hizikia fusiformis [J Appl Phycol] Vol.9 P.489-493 google
  • 8. Johnson CR, Mann KH 1986 The crustose coralline alga, Phymatolithon Foslie, inhibits the overgrowth of seaweeds without relying on herbivores [J Exp Mar Biol Ecol] Vol.96 P.127-146 google doi
  • 9. Kang SE, Park SM, Choi JS, Ahn DH, Kim YD, Hong YK 2005 Effect of Seaweed Extracts on the Viability of the Crustose Coralline Lithophyllum yessoense [Fisheries and aquatic sciences] Vol.8 P.243-246 google doi
  • 10. Kim JH 2000 Taxonomy of the Corallinales, Rhodophyta, in Korea. Ph.D. Dissertation google
  • 11. Kim MJ, Choi JS, Kang SE, Cho JY, Jin HJ, Chun BS, Hong YK 2004 Multiple allelopathic activity of the crustose coralline alga Lithophyllum yessoense against settlement and germination of seaweed spores [J Appl Phycol] Vol.16 P.175-179 google doi
  • 12. Luyen QH, Cho JY, Choi JS, Kang JY, Park NG, Hong YK 2009 Isolation of algal spore lytic C17 fatty acid from the crustose coralline seaweed Lithophyllum yessoense [J Appl Phycol] Vol.21 P.423-427 google doi
  • 13. Masaki T, Fujita D, Hagen NT 1984 The surface ultrastructure and epithallium shedding of crustose coralline algae in an Isoyake area of southwestern Hokkaido, Japan [Hydrobiologia] Vol.116/117 P.218-223 google doi
  • 14. McConnaughey TA, Whelan JF 1997 Calcification generates protons for nutrient and bicarbonate uptake [Earth-Sci Rev] Vol.42 P.95-117 google doi
  • 15. Ohsawa N, Ogata Y, Okada N, Itoh N 2001 Physiological function of bromoperoxidase in the red marine alga, Corallina pilulifera: production of bromoform as an allelochemical and the simultaneous elimination of hydrogen peroxide [Phytochemistry] Vol.58 P.683-692 google doi
  • 16. Park SM, Kang SE, Choi JS, Cho JY, Yoon SJ, Ahn DH, Hong YK 2006 Viability assay of coralline algae using triphenyltetrazolium chloride [Fish Sci] Vol.72 P.912-914 google doi
  • 17. Provasoli L, Watanabe A, Hattori A 1968 Media and prospects for the cultivation of marine algae;Cultures and Collections of Algae P.63-75 google
  • 18. Solti A, Kovacs K, Basa B, Vertes A, Sarvari E, Fodor F 2012 Uptake and incorporation of iron in sugar beet chloroplasts [Plant Physiol Biochem] Vol.52 P.91-97 google doi
  • 19. Suzuki Y, Takabayashi T, Kawaguchi T, Matsunaga K 1998 Isolation of an allelophatic substance from the crustose coralline algae, Lithophyllum spp., and its effect of on the brown alga, Laminaria religiosa Miyabe (Phaeophyta) [J Exp Mar Biol Ecol] Vol.225 P.69-77 google doi
  • 20. Tokuda H, Kawashima S, Ohno M, Ogawa H 1994 Seaweeds of Japan google
  • 21. Winterbourn CC 1995 Toxicity of iron and hydrogen peroxide: the Fenton reaction [Toxicol Lett] Vol.82/83 P.969-974 google doi
  • [Fig. 1.] Effects of ferric citrate on the viability of tissues from the crustose alga Lithophyllum yessoense (A) and articulated alga Corallina pilulifera (B). Viability was measured based on the absorbance at 475 nm; the values are expressed as the mean ± SD of at least three independent assays.
    Effects of ferric citrate on the viability of tissues from the crustose alga Lithophyllum yessoense (A) and articulated alga Corallina pilulifera (B). Viability was measured based on the absorbance at 475 nm; the values are expressed as the mean ± SD of at least three independent assays.
  • [Table 1.] Effects of calcification inhibitors on the viability of the crustose coralline alga Lithophyllum yessoense
    Effects of calcification inhibitors on the viability of the crustose coralline alga Lithophyllum yessoense
  • [Table 2.] Effects of calcification inhibitors on the viability of the coralline alga Corallina pilulifera
    Effects of calcification inhibitors on the viability of the coralline alga Corallina pilulifera