LJournal of Experimental Marine Biology and Ecology,225 (1998) 69–77

Isolation of an allelopathic substance from the crustosecoralline algae, Lithophyllum spp., and its effect on thebrown alga, Laminaria religiosa Miyabe (Phaeophyta)

a b c ,*Yoshihiro Suzuki , Tadashi Takabayashi , Tomohiro Kawaguchi ,bKatsuhiko Matsunaga

aDepartment of Civil and Environmental Engineering, Faculty of Engineering, Miyazaki University,Miyazaki 889-21, Japan

bDepartment of Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate 041, JapancDepartment of Environmental Health Sciences, School of Public Health, University of South Carolina,

Columbia, S.C. 29208, USA

Received 10 April 1997; received in revised form 27 July 1997; accepted 7 August 1997

Abstract

An allelopathic substance that destroys zoospores of the brown alga, Laminaria religiosaMiyabe, was isolated by several separation techniques from the ethanol extract of the crustosecoralline alga, Lithophyllum spp. A bioassay was used to test the effect of isolated fractionsagainst the dinoflagellate, Heterosigma akashiwo Hada, and zoospores of L. religiosa. Furtherpurification of the allelopathic substance in ether phase by silica gel column chromatographyrevealed that the isolated active substance had major absorption peaks at 220, 263 and 283 nm inthe UV region and exhibited characteristics consistent with a nonpolar group. The results suggestthat allelopathy may play an important role for reducing epiphyte growth on crustose corallinealgal surfaces and may partially explain their predominance in the coastal region of the northernJapan Sea. 1998 Elsevier Science B.V.

Keywords: Allelopathy; Crustose coralline algae; Heterosigma akashiwo; Laminaria religiosa;Lithophyllum spp.; Zoospores

1. Introduction

Many parts of the rocky shoreline of the Japan Sea, once populated by vast

*Corresponding author. Tel.: 181 803 7776994; fax: 181 803 7773391; e-mail:tkawaguchi@sophe.sph.sc.edu

0022-0981/98/$19.00 1998 Elsevier Science B.V. All rights reserved.PII S0022-0981( 97 )00208-6

70 Y. Suzuki et al. / J. Exp. Mar. Biol. Ecol. 225 (1998) 69 –77

communities of macroalgae (e.g. Laminaria japonica Areschong, Laminaria religiosaand Undaria pinnatifida (Harvey) Suringer), are now dominated by crustose corallinealgae (Lithophyllum yessoense). The change in algal composition is a critical problem toJapanese fisheries which depend on the macroalgal communities as habitat forcommercial fishes. This change, termed ‘Isayake’ (Barren of the rocky shore), hasdecimated a large proportion of macroalgal populations in the northern Japan Sea in thenearshore of Hokkaido Island in the past decade, and is continuing to spread in thesouthern region (Noro et al., 1983).

Although the cause of Isoyake phenomenon is not well understood, Suzuki et al.(1995) proposed that it resulted from decreased bioavailable iron supply by the clearanceof coastal forest, a source of iron fluvate. Suzuki et al. (1994) and Matsunaga et al.(1994) have shown that coralline algae have competitive edge over other algae due totheir low iron requirement for the optimal growth in iron-deficient coastal waters. Thereduction in iron bioavailability may have favored the growth of coralline algae overmacroalgae, but temporal and spatial differences in the rapidity with which macroalgaedeclined suggests the influence of other stressors (Matsuyama, 1991). One possiblefactor for the decline is the production of allelopathic substances by coralline algaewhich are inhibitory to macroalgal growth. Allelopathy has been hypothesized as one ofthe causes of the relative lack of epiphytes on crustose algae (Masaki et al., 1981;Breitburg, 1982). Chemical substances that inhibit herbivore growth were found in somecoralline algae (Lithothamnium glaciale Kjellman, and Lithophyllum spp.; Morse andMorse, 1984). However, the involvement of allelopathy in competition between crustosecoralline algae and macroalgae has received little attention (Ohta, 1979). Here we reportthe isolation of an allelopathic substance from the coralline alga, Lithophyllum spp., andits effect on zoospores of the brown macroalga, Laminaria religiosa and the di-noflagellate, Heterosigma akashiwo.

2. Materials and methods

2.1. Extraction of allelopathic substances

Specimens of Lithophyllum spp. were collected from a coastal region of Southern2Hokkaido, Japan. After measuring the surface area (cm ), samples were washed with a

brush to carefully remove attached organisms. Allelopathic substances were extracted2from the samples (ca. 650 cm of total surface area) with 1 l of ethanol (pure reagent

grade) for 24 h at 208C. The extract was concentrated to 50 ml at 208C under reducedpressure in an evaporator. The concentrated extract (50 ml) was filtered through aWhatman GF/C filter and further purified by the following methods (Fig. 1).

2.2. Isolation of allelopathic substance

2.2.1. Sephadex LH-20 gel filtration chromatography of the algal extractThe algal extract was separated by a gel-filtration chromatography with a Sephadex

LH-20 column (molecular exclusion range; 100–4000; 1.8 cm diameter, 35 cm height).

Y. Suzuki et al. / J. Exp. Mar. Biol. Ecol. 225 (1998) 69 –77 71

Fig. 1. A flow chart of isolation and bioassay.

Sephadex LH-20 was applied to fractionate the allelopathic substance containing activesites such as OH 2 , aromatic, and basic groups due to its unique binding capacity(Sakata and Ina, 1985). Five ml sample was injected onto the top of the column, theneluted with 150 ml ethanol at a flow-rate of 10 ml /h. Collection of the 5 ml fraction wasstarted immediately after the addition of ethanol. Fractions showing allelopathic activitywere determined by a bioassay using H. akashiwo, pooled, and the activity was furtherconfirmed by zoospores of L. religiosa. The separation procedure was repeated 10 timesto obtain enough quantity for further analysis. The pooled samples (150 ml) were thenconcentrated to 50 ml for further purification.

2.2.2. Separation of liquid and ether phasesFifty ml of the active mixture was transferred to a 100-ml separation funnel, and 10

ml distilled water and 20 ml petroleum ether were added to estimate the polarity of theactive substance. The mixture was shaken vigorously for 5 min and allowed to stand for5 min. The procedure was repeated three times. The aqueous fraction was concentratedto 50 ml at 208C. The ether phase was evaporated to dryness and the residue wasre-dissolved in 50 ml ethanol. The effects of aqueous and ether phases on H. akashiwo

72 Y. Suzuki et al. / J. Exp. Mar. Biol. Ecol. 225 (1998) 69 –77

were monitored. As a control, the same separation procedure and assay were applied ona ethanol solvent without the algal extract.

2.2.3. Isolation of active fraction by silica gel column chromatographyA silica gel (Nacalai tesque Silica gel 60, 70–130 mesh, 30 g) column was washed

with petroleum ether containing 1% (v/v) n-propanol. Five ml of ether phase wasinjected on the top of the column and eluted with 200 ml petroleum ether with 1%n-propanol at a flow-rate of about 1.5 ml /min. Collection of 10 ml fractions startedimmediately. A solvent gradient was used at 1% n-propanol to 3%, 6%, 10%, and 15%n-propanol in petroleum ether. UV absorption spectra of all fractions were determined ata range of 210 nm to 400 nm.

2.2.4. BioassayCrude extracts and isolated fractions of Lithophyllum spp. were monitored by a

bioassay using H. akashiwo and/or zoospores of L. religiosa according to the method ofKakisawa et al. (1988). Natural seawater from Funka Bay, Japan was collected inprecleaned polyethylene tanks and filtered through an acid-cleaned 0.45 mm Milliporemembrane filter.

H. akashiwo was continuously cultivated in modified Grund media (Brown et al.,221977) under 80 mE.cm (light: dark 5 12 h: 12 h) at 208C. When the cell density

5 21reached ca. 10 cells.ml , cells were used in the bioassay (25 ml assay samples addedto 2 ml medium). After 1 h, the number of dead cells was counted under a microscope.Appearance of cells (e.g. cell swelling) and termination of the motility were used todetermine cell condition. A control solution was prepared by the addition of 25 mlethanol. Allelopathic activity (A) of the fractions is defined by the following equation(Kakisawa et al., 1988),

A(%) 5 N /N 3 100t 0

where N and N are number of the initial cells and number of dead cells after 1 h,0 t

respectively.Zoospores were obtained from mature sporophytes of L. religiosa, which were

harvested from a coastal region in the northern Japan Sea, Hokkaido, Japan, according tothe macroalgal cultivation method (Tatewaki, 1979). The zoospores were adhered onto

4 22glass plates (2.532.5 cm, ca. 10 cells.cm ) in 25 ml of Grund media. Isolatedfractions (25, 50, 100, 250 or 400 ml) were added to test media containing zoospores and

22then cultivated under 80 mE.cm (12 h light:12 h dark) at 108C for 20 days. As acontrol, only ethanol (25, 50, 100, 250 or 400 ml) as added to the culture. With anassumption of 100% recovery during the extraction and the separation, it is estimatedthat the allelopathic substance in 400 ml active fraction is equivalent to the amount ofthe allelopathic substance extracted from Lithophyllum spp. grown on the total area of

2approximately 2.1 cm on the rock. The media was changed once a week. The growth ofzoospores (gamephytes formation) was observed under a microscope.

Y. Suzuki et al. / J. Exp. Mar. Biol. Ecol. 225 (1998) 69 –77 73

3. Results

3.1. Effect of crude extracts

The allelopathic activity of each fraction separated by gel-filtration chromatographyon H. akashiwo is shown in Fig. 2. Fractions 15 to 19 exhibited the allelopathic activity,and fractions 16 to 18 had 100% effect. In these media, the cell stopped moving, beganto swell and degenerated. Accordingly, fractions 16 to 18 were mixed, and the effect ofthis mixture on zoospores of L. religiosa was investigated. In controls, zoospores grewwell and became sporophytes (0.2–0. 5 mm length) (Fig. 3; a-1, b-1, c-1). In contrast,the growth of zoospores in the medium containing 50 ml of the ethanol extract mixturewas inhibited (Fig. 3; a-2), and all zoospores degenerated and did not survive (Fig. 3;b-2 and c-2) in media containing 100 and 250 ml of the mixture, respectively.

3.2. Effect of ether phase fraction on H. akashiwo

The activity of petroleum ether and aqueous phases from the active mixture weretested by a bioassay using H. akashiwo. The ether fraction was found to be lethal(A5100%) while the aqueous fraction and control tests were not. This result indicatedthat the ether phase contained an allelopathic substance. Therefore, further isolations ofthe ether phase were carried out by silica gel column chromatography.

3.3. Effect of fraction isolated by silica gel column chromatography

Four substances were isolated by measuring UV spectrum (210–400 nm). All of thesesubstances had absorption spectra peak at 210 nm (Chromatogram at 210 nm shown inFig. 4). Fractions 5–8 resulted in almost 100% effect against H. akashiwo (Table 1). In

Fig. 2. The effect of each fraction separated by Sephadex LH-20 on Heterosigma akashiwo.

74 Y. Suzuki et al. / J. Exp. Mar. Biol. Ecol. 225 (1998) 69 –77

Fig. 3. Allelopathic activity of the mixture of active fractions separated by Sephadex LH-20 on the growth ofLaminaria religiosa zoospores. a-1, b-1 and c-1 contained 50, 100 and 250 ml of ethanols as control in 25 mlmedium, respectively. All zoospores grew well and became young sporophytes. a-2, b-2 and c-2 contained 50,100 and 250 ml of the mixture of active fractions in 25 ml medium, respectively. The growth of zoospores wasinhibited (a-2). All zoospores degenerated and did not survive (b-2 and c-2). Scale bar shows 200 mm.

Y. Suzuki et al. / J. Exp. Mar. Biol. Ecol. 225 (1998) 69 –77 75

Fig. 4. Elution pattern of the ether phase from ‘the mixture of active fractions’ at UV 210 nm by Silica gel 60column chromatography. The arrows indicate where the solvent (n-propanol) concentration was changed.

contrast, significant differences were not found (P,0.05) in the effect between the otherfractions and the control. When thin layer chromatography was used for the separation ofthe same fraction four separate spots were also fractionated on the plate (result notshown).

When the active fraction no. 5–8 was tested with L. religiosa zoospores, growth ormaturation was not affected by the addition of 25 or 50 ml of the extract, but maturationwas inhibited by 20 days of incubation when 100 ml was added, and zoospores growthwas completely inhibited within 24 in media containing 400 ml of the extract (Table 2).In contrast, zoospores grew well and became young sporophytes within 20 days incontrol media. This active fraction showed the major peaks at 220, 263 and 283 nm inthe UV region.

4. Discussion

The crude ethanol extract of the crustose coralline alga, Lithophyllum spp., had aninhibitory effect on the growth of L. religiosa zoospores. The bioassay result of the

Table 1Activity of the fractions separated by a silica gel column chromatography against H. akashiwo

Fraction number (No.) Cells killed (%)Mean6S.D. (n53)

5–8 99.760.438–39 2.060.961 2.361.1100 3.660.8Control 4.060.4

76 Y. Suzuki et al. / J. Exp. Mar. Biol. Ecol. 225 (1998) 69 –77

Table 2Allelopathic activity of the extracts separated by silica gel column chromatography against zoospores of L.religiosa

Volume added (m l) Cell viability

10 days 20 days 10 days 20 days (Control)

25 1 11 1 11

50 1 11 1 11

100 1 11 1 11

250 1 2 1 11

400 2 2 1 11

11: Some gametophytes became young sporophytes.1: All gametophytes continued only vegetative growth.2: All zoospores (gametophytes) were degenerated.

active substance isolated by silica gel column chromatography is consistent with thecharacteristics of an original ethanol crude extract that affects the growth of H. akashiwoand zoospores of L. religiosa. Kakisawa et al. (1988) reported that brown alga,Cladosiphon okamuranus Tokida, produced an allelopathic substance identified as 6z,9z, 12z, 15z-octadecatetraenoic acid, an unsaturated fatty acid. Unsaturated fatty acidsproduce free radicals when oxidized in seawater, and they may attach to algae as toxins(Murata et al., 1989). The substance from Lithophyllum spp., however would not be anunsaturated fatty acid because the oxidized dried sample had still an effect on L.religiosa. In addition, the compound was classified as nonpolar because of its dissolutionin ether phase and the very weak absorption on silica gel ((SiO ) ) with 1% n-propanol2 x

in petroleum ether. We could not identify the allelopathic substance by using NMR andIR methods because the amount collected was less than the milligram required for theanalysis. Further characterization of the structure of the compound and determination ofits effective concentration for macroalgae will be carried out in future.

Masaki et al. (1981) showed that the zoospore germination of macroalga, L. japonica,on Lithophyllum spp. was inhibited in culture. It has been suggested that crustosecoralline algae have an anti-fouling mechanism that prevents growth of kelp over theirsurfaces by sloughing of their epithelial cells (Masaki et al., 1984; Johnson and Mann,1986). Therefore, if allelopathy acts on epiphytes on the crustose surface, thismechanism may assist the sloughing by inhibiting epiphytic growth until the sloughingof epithelial cells of crustose coralline algal surface occurs.

In short, the crustose coralline algae, Lithophyllum spp., may use allelopathy as achemical defense mechanism and sloughing as a physical defense mechanism incompetition with other alga. These double defense mechanisms may explain, at least inpart, the crustose coralline alga’s predominance in some of the coastal Japan Searegions.

Acknowledgements

The authors thank Dr. A.J. Lewitus and Dr. N. Watabe for their comments on thismanuscript. This work was supported in part by NOAA Grant No. NA90AA-D-SG672.

Y. Suzuki et al. / J. Exp. Mar. Biol. Ecol. 225 (1998) 69 –77 77

References

Breitburg, D.L., 1982. Development of a subtidal epibenthic community: effects of grazing, temporalvariability and prior residency. M.A. thesis, University of California, Santa Barbara, 148 pp.

Brown, V., Ducker, S.C., Roman, K.S., 1977. The effect of orthophosphate concentration on the growth ofarticulated coralline alga. Phycologia 16, 125–131.

Johnson, C.R., Mann, K.H., 1986. The crustose coralline alga, Phymatolithon Foslie, inhibits the overgrowthof seaweeds without relying on herbivores. J. Exp. Mar. Biol. Ecol. 96, 127–146.

Kakisawa, H., Asari, F., Kusumi, T. et al., 1988. An allelopathic fatty acid from the brown alga Chladosiphonokamuranus. Phytochem. 27, 731–735.

Masaki, T., Fugita, D., Akioka, H., 1981. Observation on the spore germination of Laminaria japonica onLithophyllum yessoense (Rhodophyta, Corallinaceae). Bull. Fac. Fish. Hokkaido Univ. 32, 349–356.

Masaki, T., Fugita, D., Hagen, N.T., 1984. The surface ultrastructure and epithallium shedding of crustosecoralline algae in an ‘Isoyake’ area of southwestern Hokkaido, Japan. Hydrobiologia 116-117, 218–223.

Matsunaga, K., Suzuki, Y., Kuma, K., Kudo, I., 1994. Diffusion of Fe(II) from an iron propagation cage and itseffect on tissue iron and pigments of macroalgae on the cage. J. Appl. Phycol. 6, 197–403.

Matsuyama, K., 1991. Management strategy for Isoyake in Hokkaido, Japan. (In Japanese). Mariculture 217,2–12.

Morse, A.N., Morse, D.E., 1984. Recruitment and metamorphosis of Haliotis larvae induced by moleculesuniquely available at the surfaces of crustose red algae. J. Exp. Mar. Biol. Ecol. 75, 191–215.

Murata, H., Sakai, T., Endo, M., Kuroki, A., Kimura, M., Kumada, K., 1989. Screening of removal agents of ared tide plankton Chatonella marina with special reference to the ability of the free radicals derived fromthe hydrogen peroxide and polyunsaturated fatty acids. Bull. Japan. Soc. Sci. Fish. 55, 1075–1082.

Noro, T., Masaki, T., Akioka, H., 1983. Sublittoral distribution and reproductive periodicity of coralline algae(Rhodophyta, Cryptonemiales) in southern Hokkaido. Japan. Bull. Fac. Hokkaido Univ. 34, 1–10.

Ohta, K., 1979. Chemical studies on biologically active substances in seaweeds. Proc. Int. Seaweed Symp. 9,401–411.

Sakata, K., Ina, K., 1985. Digalactosyldiacylglycerols and phosphatidylcholines isolated from a brown alga aseffective phagostimulants for young abalone. Bull. Japan. Soc. Sci. Fish. 51 (4), 659–665.

Suzuki, Y., Kuma, K., Matsunaga, K., 1994. Effect of iron on oogonium formation, growth rate and pigmentsynthesis of Laminaria japonica (Phaeophyta). Fish. Sci. 60, 373–378.

Suzuki, Y., Kuma, K., Kudo, I., Matsunaga, K., 1995. Iron requirement of the brown macroalgae Laminariajaponica, Undaria pinnatifda (Phaeophyta) and the crustose coralline alga Lithophyllum yessoense(Rhodophyta), and their competition in the northern Japan Sea. Phycologia 34 (3), 201–205.

Tatewaki, M., 1979. Cultivation of macroalgae. In: Nishizawa S., Chihara, M. (Eds.), Study of algae, KyoritsuShuppan, Tokyo, pp. 53–165. (In Japanese).