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Mar Biol (2007) 152:11211132 DOI 10.

1007/s00227-007-0759-0

R ES EA R C H A R TI CLE

Larval settlement and metamorphosis of the mussel Mytilus galloprovincialis on diVerent macroalgae
Jin-Long Yang Cyril Glenn Satuito Wei-Yang Bao Hitoshi Kitamura

Received: 21 April 2007 / Accepted: 21 June 2007 / Published online: 17 July 2007 Springer-Verlag 2007

Abstract Settlement of mussels is commonly associated with macroalgae. The eVects of 19 macroalgal species on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Settlement and metamorphosis inducing activities of macroalgae Chlorodesmis fastigiata and Ceramium tenerrimum collected each month during the period between January 2005 and April 2006 were also investigated. Furthermore, C. fastigiata and C. tenerrimum were subjected to various treatments to investigate the roles of bacteria and diatoms on the algal surface in the induction of larval settlement and metamorphosis of M. galloprovincialis and the characteristics of the cues in these two macroalgae. Pediveliger larvae of M. galloprovincialis settled and metamorphosed in high percentages on Cladophora sp., Chlorodesmis fastigiata, Centroceras clavulatum, and Ceramium tenerrimum, all of which were Wlamentous in morphology. Macroalgae that were cylindrical, phylloid, Xabellate, palmate and pinnate all showed low (<8%) percentages of post-larvae but four other Wlamentous macroalgae also had low mussel larval settlement, suggesting that chemical factors may also be involved. Seasonal variation had no eVect on inductive activities of C. fastigiata and

C. tenerrimum. Treatment of C. fastigiata and C. tenerrimum with formalin, ethanol and heat resulted in the signiWcant decrease or loss of their inductive activities. Survival of algal cells within treated macroalgae also decreased signiWcantly. Treatment of the two macroalgae with antibiotics and GeO2 reduced the numbers of bacteria and diatoms on their surface but did not aVect their inductive activities, indicating that the cue was produced by macroalgae and not by coexisting bacteria and diatoms. However, conditioned water and crude extracts of these two macroalgae did not induce larval settlement and metamorphosis. Thus, larvae of M. galloprovincialis settled and metamorphosed on speciWc Wlamentous macroalgae. The chemical cues produced by C. fastigiata and C. tenerrimum were susceptible to ethanol and heat treatments and were not recovered in the conditioned water nor in the extracts. The Wnding that inactive C. tenerrimum can be produced from culturing its apical segments provides a new tool to elucidate the chemical cue(s) from macroalgae through manipulation of their culture conditions.

Introduction Mussels are a dominant species in the inter-tidal zones of most of Japan (Sakaguchi 1987; Sakaguchi and Kajihara 1988). It is estimated that the mussel Mytilus galloprovincialis accounts for ca. 80% (in biomass) of all fouling organisms on submerged man-made structures in Tokyo Bay, Japan (Kajihara 1985). In cooling water systems of Japanese electric power stations, antifouling paints and chlorination are widely used to control biofouling (Sakaguchi 2003), including the pervasive mussel, M. galloprovincialis. EVorts are being made to develop new antifouling technologies that also take into consideration the impact on

Communicated by S. Nishida. J.-L. Yang (&) C. G. Satuito W.-Y. Bao Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki-shi, 852-8521 Nagasaki, Japan e-mail: d705155j@cc.nagasaki-u.ac.jp H. Kitamura Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki-shi, 852-8521 Nagasaki, Japan

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the environment. EVorts are also being made to understand factors controlling their settlement behavior (HadWeld and Paul 2001), since this is important to the development of new antifouling technologies. Mussel larvae settle on a wide variety of substrates, e.g., macroalgae (Seed 1969; Eyster and Pechenik 1987; Dobretsov 1999; Bulleri et al. 2006), mussel beds (Petersen 1984; McGrath et al. 1988; Cceres-Martnez et al. 1993, 1994), surfaces with bioWlms (Satuito et al. 1995, 1997; Bao et al. 2007), rocks and rugous hard surfaces (Petersen 1984; Cceres-Martnez et al. 1994) and Wlamentous ropes (Kajihara and Oka 1980; Katsuyama 1995; Ramrez and CceresMartnez 1999). Field observations conducted by numerous researchers showed mussel settlement to be commonly associated with macroalgae, in particular Wlamentous macroalgae (e.g., Bayne 1964; Davis and Moreno 1995; Dobretsov 1999). Petersen (1984) observed that larvae of M. californianus settled on several species of macroalgae, M. edulis beds, M. californianus beds and bare rocks with scattered barnacles but preferred Wlamentous macroalgae and settled on them in high densities. In Ra de Vigo, NW Spain, competent larvae of M. galloprovincialis settled directly on Wlamentous nylon ropes, byssus and other materials in mussel beds, macroalgae and on rugous hard surfaces (Cceres-Martnez et al. 1994). Filamentous macroalgae appear to be important for primary settlement of M. edulis (Bayne 1964) and may also be important for M. galloprovincialis. Recruitment of the mytilid Choromytilus chorus on the robust Wlamentous red alga Gymnogongrus furcellatus was also reported by Davis and Moreno (1995). The importance of Wlamentous macroalgae and Wlamentous structures on the settlement and recruitment of mussel has been postulated to be a means of protection from predators (Moreno 1995) and adaptation to avoid intense competition from established adult clumps of the same or diVerent species (Petersen 1984), as well as, dislodgment by hydrodynamic forces (Alfaro and JeVs 2002; Alfaro et al. 2004; Alfaro 2005, 2006). One source of cue suggested to be responsible for settlement induction of mussel larvae by macroalgae are chemical cues. Dobretsov (1999) demonstrated that while the bioWlm and wash-outs from the Wlamentous green alga Cladophora rupestris attracted settlement, those from the brown alga Laminaria saccharina repelled M. edulis larvae. Cooper (1981) reported that M. edulis larvae can be induced to settle and metamorphose using seawater extract from Platythamnion villosum. Chemical cues derived from the algae Scytothamnus australis and Melanthalia abscissa signiWcantly increased larval settlement of the mussel Perna canaliculus (Alfaro et al. 2006). On the other hand, Cceres-Martnez et al. (1994) observed that post-larvae of M. galloprovincialis attached on byssal Wlaments and thalli of C. rubrum but only under moving water condition and that these indi-

viduals did not search for a speciWc substrate under static water condition. Furthermore, larvae of M. galloprovincialis have been shown to settle and metamorphose in the laboratory on microbial bioWlms formed either in aquariums (Satuito et al. 1995, 1997) or in the sea (Bao et al. 2007), in the absence of Wlamentous algae. Davis and Moreno (1995) also tested the response of juvenile C. chorus to surface extracts of three algal species which co-occurred with C. chorus, and they concluded that chemical cue(s) are unlikely to be important in the formation of aggregations of this mytilid species. It is apparent from these observations that diVerent mussel species exhibit varying responses to macroalgae and that the role of macroalgae on mussel larval settlement needs further investigation. In order to clarify the mechanism of larval settlement and metamorphosis of M. galloprovincialis, the authors have been investigating the role of natural chemical cues on mussel larval settlement and metamorphosis. Recently, the authors have demonstrated that larvae of M. galloprovincialis settled and metamorphosed in response to a cue produced by living bacteria in microbial bioWlms (Bao et al. 2007). In the current study, the authors investigated the settlement and metamorphosis response of M. galloprovincialis larvae to 19 diVerent macroalgal species. The two species that induced settlement, Chlorodesmis fastigiata and Ceramium tenerrimum, were tested in the laboratory for changes in inductive activity each month during the period between January 2005 and April 2006. Moreover, potential settlement and metamorphosis cues of C. fastigiata and C. tenerrimum were examined with and without treatment of the macroalgae with formalin, ethanol, heat, antibiotic drugs and germanium dioxide (GeO2). Simultaneously, the eVects of these treatments on algal cell survival were investigated. Activities of conditioned water and crude extracts of these two algal species were also investigated. The settlement inducing activity of C. tenerrimum cultured in the laboratory from apical segments was also investigated.

Materials and methods Spawning and larval culture Adult M. galloprovincialis used for spawning were either collected from populations growing on the wharf adjacent to the Nagasaki Prefectural Institute of Fisheries, Tairamachi, Nagasaki (12951E; 3243N), and on a pontoon bridge of Nagasaki City Fisheries Center, Makishima, Nagasaki (12959E; 3245N), or purchased from a culture farm in Matoya, Isobe-machi, Mie (13652E; 3422N), Japan. Spawning in the laboratory was induced following the method described by Satuito et al. (2005). Mussels were cleaned by brushing oV materials attached on the

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shell surfaces and rinsing in seawater, packed in ice overnight and then transferred to a 30 l polycarbonate tank with Wltered sea water (Whatman glass-Wber Wlter, Middlesex, UK, GF/C: 1.2 m, FSW) at ca. 24C Wnal water temperature was ca. 21C. Mussels that started spawning were immediately separated from the group and transferred to 2 l glass beakers. Eggs derived from a single female were fertilized by sperms from a single male. Fertilized eggs were washed thoroughly with FSW and left undisturbed for 2 days inside an incubator maintained at 17C. After 2 days, swimming straight-hinge veliger larvae were collected, washed gently with FSW and cultured following the methods described by Satuito et al. (2005). Brie Xy, straight-hinge veliger larvae were cultured in 2 l glass beakers at an initial stocking density of 5 larvae ml1. Larvae were fed a diet of Chaetoceros gracilis at 5 104 cells ml1 day1. Culture water was changed every other day and the temperature maintained at 17 1C. Larvae were cultured to the pediveliger stage of growth and were used in settlement and metamorphosis assays when shell height and shell length reached >288 and >309 m, respectively (Satuito et al. 2005). Straight-hinge veliger larvae were also stored inside a refrigerator for a maximum period of 3 months and were cultured inside an incubator to the pediveliger stage when needed in assays. This ensured the supply of pediveligers almost all year round. Refrigeration had no adverse eVects on the survival, growth, settlement behavior and metamorphosis of larvae (Satuito et al. 2005). Conditions for storing larvae in the refrigerator were the same as that reported by Satuito et al. (2005). Conditions for culturing the refrigerated larvae were the same as described above. Larval settlement and metamorphosis bioassays Twenty pediveliger larvae were released to each glass Petri dish ( 60 mm 20 mm height) containing 20 ml of 0.22 m Millipore Wltered seawater (0.22 m FSW) and a piece of the test macroalga (fresh alga, treated alga, etc.). Settlement inducing activities of these macroalgae were evaluated by the percentage of metamorphosis to post-larvae obtained after 48 h from the start of assays. Post-larvae were veriWed under the microscope as individuals with post-larval shell growth. Petri dishes, each containing 20 larvae and 20 ml of 0.22 m FSW were also set as controls during assays. All assays were conducted at 17 1C in a dark environment. In assays to screen the settlement inducing activities of the diVerent algal species, wet weights of macroalgae used were from 0.025 to 0.2 g per Petri dish. In the other assays to test the eVect of treatments (formalin, ethanol, heat, and antibiotics) and culture conditions (culturing with GeO2 added into the culture medium and culturing the apical segments of

the alga) on C. fastigiata and C. tenerrimum activities, the wet weight of the treated or cultured alga used per Petri dish was 0.1 g. For each algal species, treatment, and culture condition, at least six replicate assays were conducted using larvae from at least two separate culture batches. In assays to investigate the eVects of the conditioned water and crude extracts of C. fastigiata and C. tenerrimum at least six replicate assays were also conducted using larvae from at least two separate culture batches. During assays with treated C. fastigiata and C. tenerrimum and with their respective conditioned water and crude extracts, algae collected on the same day of the assays were also set for comparison. Collection of macroalgae Nineteen diVerent macroalgal species including six green algae (Enteromorpha compressa, Ulva pertusa, Cladophora sp., Chlorodesmis fastigiata, Codium fragile, and Bryopsis plumosa), Wve brown algae (Dilophus okamurae, Padina arborescens, Myelophycus simplex, Hizikia fusiformis, and Sargassum thunbergii) and eight red algae (Amphiroa zonata, Corallina pilulifera, Gelidium elegans, Grateloupia Wlicina, Grateloupia turuturn, Centroceras clavulatum, Ceramium tenerrimum, and Heterosiphonia pulchra) were collected from a rocky shore in Koe-machi, Nagasaki (12950E; 3245N), and a wharf of the Nagasaki Prefectural Institute of Fisheries, Taira-machi, Nagasaki (12951E; 3243N), Japan, and evaluated for their ability to induce larval metamorphosis in M. galloprovincialis. Thalli that appeared healthy and clean were selected for the assays. These were brought back to the laboratory on the day of the assay and were thoroughly washed with FSW prior to use in assays. Young bivalves (SL: <1 cm) were sometimes observed in the algal samples. In such cases, the bivalves including the algal portion where these were attached were removed and the remaining algal pieces washed thoroughly with FSW prior to use in assays. During the period between January 2005 and April 2006, C. fastigiata and C. tenerrimum were collected every month for assay purposes, except on those months when these algae were absent at the sampling sites. The temperature of the seawater at the sampling site was measured each time algae were collected. In cases when algae were collected on more than one occasion in 1 month, all seawater temperature data measured for that month was averaged. Preparation of treated C. fastigiata and C. tenerrimum Chlorodesmis fastigiata and C. tenerrimum were exposed to formalin, ethanol and heat treatments following the procedure used by Bao et al. (2007) to treat microbial bioWlms. Algal pieces were soaked in 5% formalin solution for 24 h and then rinsed with 500 ml of FSW for six times each day

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for 3 days using a total volume of 9 l FSW to prepare formalin treated (FA) algae. Ethanol treated (10E, 20E, 50E, and 100E) algae were prepared by soaking algae in 10, 20, 50 or 100% ethanol solutions for 30 min. Heat treated (30H, 35H, 40H, and 100H) algae were prepared by heating algae in 30, 35, 40 or 100C for 30 min. After ethanol or heat treatments, treated algae were rinsed six times using a total volume of 2 l FSW for each treatment condition. Antibiotic treated (1 AB and 10 AB) C. fastigiata and C. tenerrimum were prepared by soaking each of these two algae in diVerent concentrations of antibiotic solutions for 48 h after washing in FSW. Antibiotic solution used in 1 AB algae contained 20 mg l1 of streptomycin sulphate, 10 mg l1 of penicillin G, 2 mg l1 of neomycin and 10 mg l1 of kamamycin (Huggett et al. 2005). For 10 AB algae, the concentration of the antibiotic solution used was ten-fold of that used in 1 AB algae. Each of these antibiotics treated algae were rinsed six times using a total volume of 2 l FSW prior to use in assays. Culture of C. fastigiata and C. tenerrimum thalli in the laboratory Culture of C. fastigiata and C. tenerrimum in culture media with and without addition of germanium dioxide (GeO2) Freshly collected C. fastigiata and C. tenerrimum thalli were thoroughly washed with 0.22 m FSW and pieces (ca. 0.5 g wet weight) of these algae were separately cultured for one and 3 weeks in 200 ml Xasks each Wlled with 100 ml SW culture medium (Iwasaki 1961) either with or without GeO2, to investigate the eVect of diatoms present on algal surfaces on the settlement inducing activities of these two algae. Cultures were maintained at 17C in a 14:10 h light : dark environment and the culture media changed once every 34 days. Germanium dioxide was added into SW culture medium at a concentration of 5 mg l1 (Tatewaki 1992). Culture of C. tenerrimum apical segments In a separate investigation, freshly collected C. tenerrimum thalli were washed with 0.22 m FSW and 0.51 mm segments with apices were excised from the algal pieces under a dissecting microscope using needles (modiWed from Tatewaki 1992). Excised apical segments were then washed with 0.22 m FSW and individually cultured in wells of 24-well tissue culture plates (Falcon, Guilford, CT, USA), each well containing 2 ml of SW culture medium, for 1 week at 20C in a 14:10 h light : dark environment. After 1 week of culture, these apical segments were transferred to 200 ml Xasks each Wlled with 100 ml of SW culture medium, further cultured for 2, 3, and 4 weeks under condi-

tions similar to that described above to culture apical segments in tissue culture plates, and then used in assays. Preparation of the conditioned water and crude extracts of C. fastigiata and C. tenerrimum Freshly collected C. fastigiata and C. tenerrimum were thoroughly washed with FSW, placed in Xasks containing FSW and then left undisturbed for 24 h at 17C in a dark environment. After 24 h, FSW conditioned with either C. fastigiata or C. tenerrimum was Wltered through a Whatman GF/C Wlter to obtain the conditioned water of the alga. One hundred percent concentration of the conditioned water was prepared by adding 0.1 g algal pieces to every 20 ml of FSW (modiWed from Li et al. 2004a). The amount of alga placed in the Xask with FSW was adjusted to prepare other concentrations. For example, 0.01 g of the alga was added to every 20 ml of FSW to prepare 10% concentration of the conditioned water, while 1 g of the alga was added to every 20 ml of FSW to prepare ten-fold concentration of the 100% conditioned water (1,000% concentration). C. fastigiata and C. tenerrimum conditioned water were either used immediately or frozen at 30C in a freezer prior to use in assays. One-gram of C. fastigiata was crushed in 2 ml of FSW in a mortar cooled on crushed ice, the resulting suspension collected, and centrifuged at 8,000 rpm for 60 min at 4C. After centrifugation, the supernatant was Wltered through a Whatman GF/C Wlter to obtain the concentrate crude extract of C. fastigiata. One hundred percent concentration of the crude extract of C. fastigiata was prepared by adding 0.2 ml of the concentrate crude extract to 20 ml of 0.22 m FSW. DiVerent concentrations were prepared by adjusting the volume of the concentrate crude extract added to FSW. Concentrate crude extract of C. tenerrimum was also prepared following the protocol described above for preparation of C. fastigiata concentrate crude extract. Concentrate crude extract of C. fastigiata and C. tenerrimum were either used immediately or frozen at 30C in a freezer prior to use in assays. Enumeration of bacteria and diatoms and determination of the cell survival of C. fastigiata and C. tenerrimum Algal pieces (0.1 g) were added to 10 ml of 0.22 m FSW and vortexed for 60 s to remove bacteria from the surface of algae. Prior to vortexing, pieces of the treated and untreated macroalgae were washed in the same manner as these were washed prior to use in assays. Five-milliliter of the bacterial suspension was Wxed with 5 ml of 5% formalin solution (2.5% Wnal concentration). Bacteria in the suspension were then stained by adding acridine orange solution (AO 0.1%) and the suspension Wltered through 0.2 m polycarbonate Nuclepore (Whatman) Wlters. Numbers of bacteria on Nuclepore Wlters were counted directly at

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1,000 magniWcation under an Olymplus BH-2 epiXuorescent microscope. AO was purchased from Sigma Co., St. Louis, MO, USA. Numbers of diatoms on surfaces of 0.5 mm length algal pieces were counted directly at 200 magniWcation under a light microscope. The wet weights of 5 cm length of C. fastigiata and C. tenerrimum were also determined in order to estimate numbers of diatoms per 0.1 g algae. Numbers of bacteria and diatoms were enumerated from ten random Welds of view. Treated and untreated pieces of C. fastigiata and C. tenerrimum were stained with 0.05% (w/v) erythrosine solution in FSW and incubated at 17C for 30 min (modiWed from Saga 1989). Stained algae were thoroughly washed with FSW for 30 min, and cells that were not stained red by the dye were counted as living cells. The percentage of surviving algal cells was calculated from a total of 500 (living and dead) cells counted at 100 magniWcation under a light microscope. Erythrosine was purchased from Merck, Daimastch, Germany. Data analysis Settlement and metamorphosis inducing activities of the diVerent macroalgae, conditioned water and crude extract were evaluated by the percentages of post-larvae. Activities of the 19 species of macroalgae were assessed using the KruskalWallis Test. EVects of season, treatments and culture conditions on settlement and metamorphosis inducing activities, algal cell survival, and numbers of bacteria and diatoms of C. fastigiata and C. tenerrimum were assessed by one-way Analysis of Variance (ANOVA) followed by the TukeyKramer Honestly SigniWcant DiVerence (HSD) test, using the JMP software. All data expressed in percentage were arcsine-transformed prior to analysis with one-way ANOVA and TukeyKramer HSD test. DiVerences were considered signiWcant at P < 0.05.

(C. fastigiata and Cladophora sp.) and two red (C. clavulatum and C. tenerrimum) species, all Wlamentous in morphology, induced >75% metamorphosis to post-larvae. The other 15 species all showed low settlement and metamorphosis inducing activities. The other four Wlamentous macroalgae (B. plumose, M. simplex, G. elegans, and H. pulchra) induced <14% metamorphosis to post-larvae after 48 h exposure to the algae, even though algal amounts were increased to 0.2 g of alga per Petri dish. This indicates that not all Wlamentous algae induce larval settlement and metamorphosis in M. galloprovincialis. All Wve Phaeophyta species investigated induced <10% metamorphosis to post-larvae. Percentages of post-larvae on C. fastigiata and C. tenerrimum collected on diVerent months during the period between January 2005 and April 2006, and water temperatures at the sampling site during the same period are as shown in Fig. 1. Percentages of post-larvae on C. fastigiata ranged between 75 and 91% throughout the experimental period, while that on C. tenerrimum were between 71 and 92%. Larval settlement and metamorphosis on C. fastigiata and C. tenerrimum remained constantly high throughout the experimental period regardless of the month these were collected and assayed (C. fastigiata: P > 0.05, C. tenerrimum: P > 0.05, ANOVA, Fig. 1), implying that activities of these two algae were not inXuenced by seasonal variations. EVects of treatments on activities of C. fastigiata and C. tenerrimum Percentages of post-larvae and corresponding cell survivals of C. fastigiata and C. tenerrimum treated with formalin, ethanol and heat are as shown in Fig. 2. The average percentage of post-larval metamorphosis on UT C. fastigiata was 78% (Fig. 2a). However, percentages of post-larvae on FA, 10E, 20E, 50E, 100E, 35H, 40H, and 100H C. fastigiata signiWcantly decreased (P < 0.001, ANOVA), suggesting that the cue in C. fastigiata may have been signiWcantly or completely destroyed by the above treatment conditions. Algal cell survival in UT C. fastigiata was 94%. Algal cell survival in treated (FA, 10E, 20E, 50E, 100E, 35H, 40H, and 100H) C. fastigiata where percentages of post-larvae signiWcantly decreased, also decreased and were between 0 and 37% (P < 0.001, ANOVA, Fig. 2a). A similar tendency was observed in the case of C. tenerrimum where the UT alga, which induced 77% post-larval metamorphosis, had 96% algal cell survival (Fig. 2b). Moreover, treated (FA, 10E, 20E, 50E, 100E, 35H, 40H, and 100H) C. tenerrimum that had signiWcantly lower percentages of post-larvae (P < 0.001, ANOVA) also had signiWcantly lower percentages of algal cell survivals (056%) than the UT alga (P < 0.05, ANOVA followed by Tukey Kramer HSD test: UT alga versus each treated alga, Fig. 2b). These results imply that settlement inducing activities

Results Throughout all assays, the percentage of metamorphosis to post-larvae of M. galloprovincialis in Petri dishes containing only 0.22 m FSW was always 0%. Larval settlement and metamorphosis in response to macroalgae Morphologies and settlement and metamorphosis inducing activities, expressed in percentage of post-larvae, of the 19 macroalgal species are summarized in Table 1. Larvae of M. galloprovincialis responded diVerently to the 19 diVerent species of macroalgae (P < 0.001, KruskalWallis test, Table 1). Among the macroalgae tested, two green

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1126 Table 1 Taxonomic classiWcation of the 19 species of macroalgae and their settlement and metamorphosis inducing activities expressed in percentages of post-larvae

Mar Biol (2007) 152:11211132

Phyla Algal species CHLOROPHYTA Enteromorpha compressa Ulva pertusa Cladophora sp. Chlorodesmis fastigiata

Morphology

Weight (g)

Post-larvae (%)

KruskalWallis (rank sums)a

c ph W W

0.1 0.1 0.1 0.2 0.025 0.05 0.1 0.2

6 8 9 6 6 9 21 6 10 14 6 7 7 7 7 7 10 7 11 6 9 9 6 6 6 6 30 6 9 6

8 4 81 89 12 53 83 90 7 13 13 4 1 2 2 6 3 1 1 2 2 1 48 76 26 55 78 85 3 6

6 8 3

2 7 5

Codium fragile Bryopsis plumosa PHAEOPHYTA Dilophus okamurae Padina arborescens Myelophycus simplex Hizikia fusiformis Sargassum thunbergii RHODOPHYTA Amphiroa zonata Corallina pilulifera Gelidium elegans Grateloupia Wlicina Grateloupia turuturu Centroceras clavulatum Ceramium tenerrimum The letters of c, ph, W, X, pa, pi indicate cylindrical, phylloid, Wlamentous, Xabellate, palmate, pinnate, respectively a Score sums were ranked from 1 to 19

c W

0.1 0.1 0.2

X X W c c c pa W pi ph W W

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.2 0.025 0.05 0.1 0.2

9 15 14 11 10 17 13 18 16 19 4

1 12

Heterosiphonia pulchra

0.1 0.2

of these two algae were signiWcantly aVected by their cell survival and that these algae induced mussel larval settlement and metamorphosis only when alive. Alternatively, results also suggest that treatments possibly altered the surface chemistry of these algae either by cross-linking (formalin treatment), denaturing (heat treatment) or extracting lipid components (ethanol treatment) of extracellular products on the surface of algae. Activities of conditioned water and crude extracts of C. fastigiata and C. tenerrimum Percentages of post-larval metamorphosis on the conditioned water and crude extracts of C. fastigiata were all 0%

at all concentrations (10, 100, and 1,000%) tested (Table 2). Percentages of post-larval metamorphosis on the conditioned water and crude extracts of C. tenerrimum were between 0 and 2% at all concentrations (10, 100, and 1,000%) tested (Table 2). EVects of bacteria and diatoms on the settlement inducing activities of C. fastigiata and C. tenerrimum Percentages of post-larvae on C. fastigiata and C. tenerrimum and their corresponding numbers of bacteria after treatment with antibiotic solution are as shown in Fig. 3. In C. fastigiata, the percentage of post-larvae on the UT alga was 79% and the corresponding number of bacteria was

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Temperature (c ) 30 20 10
60 100

1127

(a)

100 80 60 40

80

0 100 80 Post-larvae ( % ) 60 40 20 0
Ja n Fe b M ar A pr M ay Ju n Ju l A ug Se p O ct N ov D ec Ja n Fe b M ar A pr
40 20

20

100 80 60 40 20 0

(b)

100 80 60 40 20 0

2005

2006

Fig. 1 Percentages of post-larvae (Mytilus galloprovincialis) on Chlorodesmis fastigiata and Ceramium tenerrimum collected each month from January 2005 to April 2006 and the corresponding seawater temperature. Open and shaded boxes indicate C. fastigiata and C. tenerrimum, respectively. Shaded circles indicate temperature of seawater measured at the time of the sampling. Data are means (+SD) of six replicates

UT

FA

10

20

50

10

0E 30H 35H 40H 00H 1

7.7 106 cells per 0.1 g alga. Treatment of C. fastigiata with diVerent concentrations of the antibiotic mixture signiWcantly reduced the number of bacteria (P < 0.001, ANOVA). The number of bacteria in 10 AB C. fastigiata was 1.3 106 cells per 0.1 g alga; 80% reduction from that of the UT alga (P < 0.05). However, antibiotic treatment did not aVect the inducing activity of the alga and percentages of post-larvae in 1 AB and 10 AB algae were at the same level as that of the UT algae (P > 0.05). Similarly, treatment of C. tenerrimum with antibiotic solution signiWcantly reduced the numbers of bacteria in treated algae (P < 0.001, ANOVA), and was 1.3 106 cells per 0.1 g alga in the 10 AB alga; 91% reduction in number as compared to the control (UT alga). However, percentages of post-larvae on antibiotic treated algae remained constantly high (P > 0.05, ANOVA); 62 and 65% post-larvae for 1 AB and 10 AB algae, respectively. Percentages of post-larvae on C. fastigiata and C. tenerrimum cultured in SW media with and without GeO2, and their corresponding numbers of diatoms are as shown in Fig. 4. Freshly collected alga induced 80% post-larval metamorphosis and had 3.2 104 diatom cells per 0.1 g of the alga. When cultured in the laboratory for 3 weeks, the number of diatoms per 0.1 g of the cultured alga increased nearly seven-fold in SW media without GeO2 but decreased signiWcantly to 2.0 103 cells per 0.1 g alga in the SW medium containing GeO2 (P < 0.05); a nearly 94% decrease as compared to that of the freshly collected alga. In contrast, no signiWcant diVerence in percentages of post-larvae was observed between algae cultured in media

Treatments
Fig. 2 Percentages of post-larvae (Mytilus galloprovincialis) and algal cell survivals in treated Chlorodesmis fastigiata (a) and Ceramium tenerrimum (b). Shaded squares indicate algal cell survival. UT indicates algae that were not treated. FA indicates 5% formalin treated algae. 10E, 20E, 50E, and 100E indicate algae treated with ethanol at concentrations of 10, 20, 50, and 100%, respectively. 30H, 35H, 40H, and 100H indicate algae heated at 30, 35, 40, and 100C, respectively. Data are means (+SD) of 615 replicates Table 2 Settlement and metamorphosis inducing activities, expressed in percentages of post-larvae, of conditioned water and crude extracts prepared from Chlorodesmis fastigiata and Ceramium tenerrimum Algal species Concentration (%) n Post-larvae (%)

Chlorodesmis fastigiata UT Conditioned water Crude extracts Ceramium tenerrimum UT Conditioned water Crude extracts 10, 100, 1,000 10 100 1,000 36 629 14 25 16 75 13 00 13 24 13 10, 100, 1,000 10, 100, 1,000 21 612 6 83 7 00 00

Hundred percentage of concentration means 0.1 g alga per 20 ml FSW

with and without GeO2 even after 3 weeks of culture (1 week: algae cultured with GeO2 versus without GeO2, P > 0.05; 3 weeks: algae cultured with GeO2 versus without

123

Algal cell survival (% )

Post-larvae (% )

1128
100 80 10 8

Mar Biol (2007) 152:11211132

60 10 7 40 20 0 10 6 UT 1 AB 10 AB

Antibiotic treatments
Fig. 3 Percentages of post-larvae (Mytilus galloprovincialis) and the corresponding numbers of bacteria in untreated algae (UT) and antibiotic solution treated (AB) algae. Open and shaded boxes indicate Chlorodesmis fastigiata and Ceramium tenerrimum, respectively. Closed triangles indicate numbers of bacterial cells. 1 AB and 10 AB indicate algae treated with antibiotics solution. 1 AB = streptomycin sulphate 20 mg l1, penicillin G 10 mg l1, neomycin 2 mg l1, and kanamycin 10 mg l1, 10 AB = 1 AB concentration increased tenfold. Data are means (+SD) of six replicates

GeO2, P > 0.05). In addition, the percentage of post-larvae on C. fastigiata cultured for 3 weeks in SW medium with GeO2 remained constantly high and was at the same level as that of the fresh alga (P < 0.05, Fig. 4a). A similar tendency was observed when C. tenerrimum thalli were cultured in SW media with and without GeO2. After 3 weeks of culture, the number of diatoms per 0.1 g C. tenerrimum cultured in SW medium with GeO2 decreased by 92% as compared to that of the freshly collected alga (P < 0.05). However, percentages of post-larvae on all cultured C. tenerrimum remained constantly high and were at the same level as that of the fresh alga (P > 0.05, Fig. 4b). In addition, no signiWcant diVerence in percentages of post-larvae was observed between algae cultured in media with and without GeO2 for 3 weeks (P > 0.05). These results suggest that bacteria and diatoms may not play a crucial role in the production of the potential cue of C. fastigiata and C. tenerrimum. The activity of C. tenerrimum cultured from apical segments Percentages of post-larvae on C. tenerrimum cultured from naturally grown thalli and those from apical segments, and their corresponding numbers of bacteria are as shown in Fig. 5. The percentage of post-larvae on fresh C. tenerrimum was 77%. When naturally grown thalli of C. tenerrimum were brought to the laboratory and cultured in SW medium, the settlement inducing activity of this cultured alga remained constantly high until after 4 weeks of culture (P > 0.05). However, the percentage of post-larvae on this cultured alga gradually decreased to 63% after 5 weeks (P < 0.05). In contrast, C. tenerrimum cultured from apical segments for 3 weeks showed no inducing activity and the percentage of post-larvae remained 0% even after 5 weeks of culture. Addition of bacteria, obtained from freshly collected naturally grown alga, into the SW medium containing C. tenerrimum cultured for 4 weeks and then culturing it for another week had no eVect and post-larval metamorphosis on this cultured alga remained 0% (Fig. 5a). The number of bacteria on fresh C. tenerrimum was 1.3 107 cells per 0.1 g alga. This number gradually increased with the culture period and reached 6.6 107 cells after 5 weeks. After 5 weeks, the numbers of bacteria on the two types of algae cultured from apical segments were 2.5 to 4.4 107 cells per 0.1 g alga. These numbers were signiWcantly higher than that of the fresh alga (P < 0.001, ANOVA, Fig. 5b).

100 80

(a)

10 6

10 5 60 40 10 4

20 0 100 80 10 5 60 40 20 10 3 0 0 1 2 3 10 3

(b)

10 6

10 4

Culture Periods (weeks)


Fig. 4 Percentages of post-larvae (Mytilus galloprovincialis) and the corresponding numbers of diatoms in Chlorodesmis fastigiata (a) and Ceramium tenerrimum (b) cultured in SW media with and without GeO2. Shaded diamond shaped marks indicate numbers of diatom cells. Open and shaded boxes indicate fresh C. fastigiata and C. tenerrimum, respectively. Striped boxes indicate C. fastigiata and C. tenerrimum cultured in SW without GeO2; grid boxes, C. fastigiata and C. tenerrimum cultured in SW with GeO2. Data are means (+SD) of 612 replicates

Diatoms (cells . 0.1g)

Post-larvae (% )

Bacteria (cells . 0.1g)

P o s t - l ar v a e ( % )

Discussion and conclusions Macroalgae had been reported to aVect larval settlement and metamorphosis of many invertebrates, e.g., two bryozoan

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Mar Biol (2007) 152:11211132


100

1129

(a)

80 60 40 20 0

Bacteria (cells . 0.1g)

10 8

(b)

10 7

10

20 30 Culture Periods (days)

40

Fig. 5 Percentages of post-larvae (a) (Mytilus galloprovincialis) and the corresponding numbers of bacteria (b) in Ceramium tenerrimum cultured in the laboratory from naturally growth thalli (open triangle) and apical segments (open circle, open square). Open triangles, circles, and squares indicate naturally grown alga, cultured apical segments and cultured apical segments with the addition of bacteria from fresh alga on the 4th week of culture, respectively. Shaded triangles, circles, and squares indicate corresponding bacterial numbers of fresh alga, cultured apical segments and cultured apical segments with the addition of bacteria from fresh alga on the 4th week of culture, respectively. Data are means (+SD) of six replicates

species (Walters et al. 1996; Schmitt et al. 1998), a polychaete species (Walters et al. 1996; Harder et al. 2004), mollusks (Morse and Morse 1984; Yvin et al. 1985; Dobretsov 1999; Krug and Manzi 1999; Daume et al. 2000; Huggett et al. 2005) and echinoderms (Pearce and Scheibling 1991; Kitamura et al. 1993; Takahashi et al. 2002; Swanson et al. 2004; Li et al. 2004a, b). Mussel larval settlement has also been associated with macroalgae, and a large volume of literature is available on this subject (e.g., Bayne 1964, 1965; Eyster and Pechenik 1987; CceresMartnez et al. 1994; Davis and Moreno 1995; Dobretsov 1999; Alfaro et al. 2006; Bulleri et al. 2006). In the present investigation, the authors have demonstrated through laboratory experiments that larvae of the mussel M. galloprovincialis responded diVerently to diVerent macroalgae and settled and metamorphosed in high percentages on the algae C. fastigiata, Cladophora sp., C. clavulatum and C. tenerrimum, all of which were Wlamentous in morphology. In addition, the settlement and metamorphosis inducing activities of C. fastigiata and C. tenerrimum remained constantly high throughout the investigation, regardless of the season. The Wnding that M. galloprovincialis settled and metamorphosed on Wlamentous algae is consistent with

reports on M. edulis larvae (Bayne 1964; Petersen 1984; Eyster and Pechenik 1987; Dobretsov 1999). CceresMartnez et al. (1994) concluded from Weld observations that M. galloprovincialis used Wlamentous, thallus and membranous algae among other substrates for direct settlement. In the present investigation, algae that were not Wlamentous all showed low (<10%) settlement inducing activities. Interestingly, not all Wlamentous algae tested in this investigation showed high settlement and metamorphosis inducing activities, suggesting that the Wlamentous morphology of algae may not be a crucial factor to the induction of mussel larval settlement and metamorphosis by macroalgae. Other factors, such as chemical cues that may be present or produced in the active algae (C. fastigiata, Cladophora sp., C. clavulatum, and C. tenerrimum) may have induced mussel larval settlement and metamorphosis. Dobretsov (1999) observed that M. edulis larvae were attracted to bioWlms and wash-outs from C. rupestris and he concluded that chemical cues produced by the alga aVected larval behavior. The present investigation also demonstrated that treatment of C. fastigiata and C. tenerrimum with formalin, ethanol and heat resulted in the signiWcant reduction or loss of settlement inducing activities of these algae. Formalin treatment of microbial or bacterial bio Wlms have been used in previous reports as a technique to kill constituent organisms in microbial bioWlms without causing large damage to their surface chemistry, in order to investigate the presence of surface bound cues (Kirchman et al. 1982; Satuito et al. 1995; Unabia and HadWeld 1999; Lau and Qian 2001; Bao et al. 2007). Solvents and heat have also been used to investigate whether cues in microbial bioWlms were susceptible to these treatments (Bao et al. 2007). The results obtained from subjecting C. fastigiata and C. tenerrimum to the above treatments were similar to those reported by Bao et al. (2007) for treated microbial bioWlms in that the cue from the two algae were not surface bound and were susceptible to ethanol and heat treatments. Conditioned water and crude extracts prepared from these two algae also showed no inducing activities for larvae of M. galloprovincialis, suggesting that the cues were unstable and were destroyed during extraction. On the other hand, Cooper (1981) reported that precipitates and supernatants from the seawater homogenate of the Wlamentous red alga P. villosum induced settlement and metamorphosis of M. edulis larvae. Alfaro et al. (2006) also showed that solvent extract prepared from S. australis and M. abscissa induced larval settlement in P. canaliculus, although it was not speciWed in this case whether larvae that settled on algae metamorphosed to the post-larval stage. Simultaneous enumeration of surviving algal cells in these treated algae showed that treatments employed signiWcantly or completely killed the algal cells. These

Post-larvae (% )

123

1130

Mar Biol (2007) 152:11211132

Wndings suggest that the viability of algal cells is important for the production of the cue that induced settlement and metamorphosis of M. galloprovincialis larvae. Bao et al. (2007) also proposed that the viability of bacterial cells in microbial bioWlms is a requirement for the production of the cue for settlement and metamorphosis of M. galloprovincialis larvae. Treatment of C. fastigiata and C. tenerrimum with antibiotics or culturing these algae in media with GeO2 resulted in the signiW cant reduction in the numbers of bacteria and diatoms on these algae but settlement and metamorphosis inducing activities of these treated algae were not aVected and remained constantly high. Bacteria on surfaces of algae induce settlement and metamorphosis in the polychaete Janua (Dexiospira) brasiliensis Grube (Kirchman et al. 1982), crown-of-thorns star Wsh Acanthaster planci (Johnson and Sutton 1994), and sea urchin Heliocidaris erythrogramma (Huggett et al. 2006). BioWlms from C. rupestris were reported to attract M. edulis larvae in the same way as did the alga itself (Dobretsov 1999). Moreover, bacteria from microbial bioWlms have been reported to play a crucial role in the settlement and metamorphosis of M. galloprovincialis (Satuito et al. 1995, 1997; Bao et al. 2007), while Dobretsov and Railkin (1994) also reported that diatoms played a predominant role in M. edulis larval settlement. In the present study, the two antibiotic treated macroalgae (10 AB C. fastigiata and 10 AB C. tenerrimum) both had 1.3 106 cells of bacteria per 0.1 g alga; equivalent to ca. 1.3 105 cells cm2 in density of bacteria on the algal surface. This bacterial density is 38 times lower than the density of bacteria (5 106 cells cm2) in multispecies bioWlms that induced 6080% metamorphosis to post-larvae in the report by Bao et al. (2007). Similarly, diatom densities on surfaces of the two GeO2 treated macroalgae were between 200 and 400 cells cm2, and were signiWcantly lower than those in multispecies bioWlms with equally high inductive activities (Bao et al. 2007). Although treatment of these macroalgae with antibiotics and GeO2 possibly resulted in the alteration of community composition on algal surface, results strongly suggest that bacteria and diatoms on surfaces of C. fastigiata and C. tenerrimum played no direct role in the induction of M. galloprovincialis settlement and metamorphosis and that the cue was produced by the macroalgae themselves. For Haliotis rubra , Huggett et al. (2005) reported that bacteria and diatoms on surfaces of the algae Ulva australis, Amphiroa anceps, and Corallina oYcinalis did not induce larval settlement and they further suggested that macroalgae rather than bacteria or diatoms may be responsible for the production of the settlement cue. The loss of the settlement inducing activity of C. tenerrimum when cultured in the laboratory from apical

segments provides evidence that the culture condition can aVect macroalgal activity. Unlike treated C. tenerrimum, where treatment either by formalin, ethanol or heat resulted in the death of algal cells, the cultured alga was alive and its surface chemistry would have been similar with that of the fresh alga. A possible explanation for the loss in the settlement inducing activity of the cultured alga could be that nutrients necessary to synthesize the potential cue was not available in the culture medium. This result also provides evidence that chemical cues produced by the algae, and not merely their Wlamentous morphology or physical properties, play an important role in the induction of settlement and metamorphosis of M. galloprovincialis larvae. Cooper (1981), Dobretsov (1999), and Alfaro et al. (2006) also showed that chemical cues are involved during the settlement of other Mytilus species. On the other hand, diVerence in composition of the bacterial community between cultured and fresh algae may have resulted in the diVerent settlement inducing activities. The Wnding that inactive C. tenerrimum can also be produced in the laboratory by culturing its apical segments is noteworthy, since this can be used as a tool to elucidate the chemical cue produced by the alga through manipulation of its culture condition, i.e., addition or elimination of certain nutrients in the culture medium may lead to the identiWcation of speciWc nutrients required to produce the potential cue. The fact that M. galloprovincialis larvae respond to a wide range of cues including those in red and green Wlamentous macroalgae (the present study) and in bioWlms (Satuito et al. 1995, 1997; Bao et al. 2007) gives larvae of this species ecological advantage in natural systems. Where Wlamentous macroalgae are available, larvae can avoid inter- and intra-species competition as proposed by Petersen (1984) as well as dislodgement by hydrodynamic forces as discussed by Alfaro (2005, 2006) for P. canaliculus. Larvae can also settle on substrates suitable for survival by responding to cues from speciWc bacteria in bioWlms (Satuito et al. 1995, 1997; Bao et al. 2007) in the absence of Wlamentous macroalgae. In conclusion, larval settlement and metamorphosis of M. galloprovincialis was induced by the Wlamentous algae C. fastigiata, Cladophora sp., C. clavulatum, and C. tenerrimum. Chemical cues produced by these algae, and not coexisting bacteria nor diatoms, were directly involved in the induction of M. galloprovincialis larval settlement and metamorphosis. The cues in C. fastigiata and C. tenerrimum were susceptible to ethanol and heat treatments and were not recovered in the extracts nor in the conditioned water. The authors are now conducting further investigations to elucidate the chemical cue in C. tenerrimum by manipulating culture conditions of its apical segments.

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Mar Biol (2007) 152:11211132 Acknowledgements The authors are grateful to the Nagasaki Prefectural Institute of Fisheries for their cooperation in the collection of adult mussels and macroalgae. The authors are also grateful to Dr. K. Kuwano (Nagasaki University) for identiWcation of macroalgae and to Mr. M. Hirano for technical assistance. All experiments complied with the current Japanese laws.

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