Mar Biol (2007) 152:1121–1132
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 Cera-
mium tenerrimum collected each month during the period
between January 2005 and April 2006 were also investi-
gated. 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. galloprovin-
cialis and the characteristics of the cues in these two macro-
algae. 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%) per-
centages of post-larvae but four other Wlamentous macroal-
gae 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
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
C. tenerrimum. Treatment of C. fastigiata and C. tenerri-
mum with formalin, ethanol and heat resulted in the signiW-
cant decrease or loss of their inductive activities. Survival
of algal cells within treated macroalgae also decreased sig-
niWcantly. 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 spe-
ciWc Wlamentous macroalgae. The chemical cues produced
by C. fastigiata and C. tenerrimum were susceptible to eth-
anol and heat treatments and were not recovered in the con-
ditioned 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 cul-
ture 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 galloprovin-
cialis 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 (Sakagu-
chi 2003), including the pervasive mussel, M. galloprovin-
cialis. EVorts are being made to develop new antifouling
technologies that also take into consideration the impact on
123
1122
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; Dobret-
sov 1999; Bulleri et al. 2006), mussel beds (Petersen 1984;
McGrath et al. 1988; Cáceres-Martínez et al. 1993, 1994),
surfaces with bioWlms (Satuito et al. 1995, 1997; Bao et al.
2007), rocks and rugous hard surfaces (Petersen 1984; Các-
eres-Martínez et al. 1994) and Wlamentous ropes (Kajihara
and Oka 1980; Katsuyama 1995; Ramírez and Cáceres-
Martínez 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 Ría 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 (Cáceres-Martínez 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 Choromy-
tilus chorus on the robust Wlamentous red alga Gymnogong-
rus furcellatus was also reported by Davis and Moreno
(1995). The importance of Wlamentous macroalgae and Wla-
mentous 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, dis-
lodgment by hydrodynamic forces (Alfaro and JeVs 2002;
Alfaro et al. 2004; Alfaro 2005, 2006).
One source of cue suggested to be responsible for settle-
ment 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 Platytham-
nion villosum. Chemical cues derived from the algae Scyto-
thamnus australis and Melanthalia abscissa signiWcantly
increased larval settlement of the mussel Perna canaliculus
(Alfaro et al. 2006). On the other hand, Cáceres-Martínez
et al. (1994) observed that post-larvae of M. galloprovin-
cialis attached on byssal Wlaments and thalli of C. rubrum
but only under moving water condition and that these indi-
123
Mar Biol (2007) 152:1121–1132
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 labora-
tory on microbial bioWlms formed either in aquariums (Satu-
ito 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. galloprovin-
cialis settled and metamorphosed in response to a cue pro-
duced by living bacteria in microbial bioWlms (Bao et al.
2007). In the current study, the authors investigated the set-
tlement and metamorphosis response of M. galloprovincialis
larvae to 19 diVerent macroalgal species. The two species
that induced settlement, Chlorodesmis fastigiata and Cera-
mium tenerrimum, were tested in the laboratory for changes
in inductive activity each month during the period between
January 2005 and April 2006. Moreover, potential settle-
ment and metamorphosis cues of C. fastigiata and C. tenerr-
imum 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, Taira-
machi, Nagasaki (129°51⬘E; 32°43⬘N), and on a pontoon
bridge of Nagasaki City Fisheries Center, Makishima,
Nagasaki (129°59⬘E; 32°45⬘N), or purchased from a culture
farm in Matoya, Isobe-machi, Mie (136°52⬘E; 34°22⬘N),
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
Mar Biol (2007) 152:1121–1132
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, Middle-
sex, UK, GF/C: 1.2
m, FSW) at ca. 24°C Wnal water tem-
perature was ca. 21°C. 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 17°C. After
2 days, swimming straight-hinge veliger larvae were col-
lected, washed gently with FSW and cultured following
the methods described by Satuito et al. (2005). BrieXy,
straight-hinge veliger larvae were cultured in 2 l glass beak-
ers at an initial stocking density of 5 larvae ml¡1. Larvae were
fed a diet of Chaetoceros gracilis at 5 £ 104 cells ml¡1 day¡1.
Culture water was changed every other day and the temper-
ature maintained at 17 § 1°C. 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 metamor-
phosis 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 refriger-
ated larvae were the same as described above.
Larval settlement and metamorphosis bioassays
Twenty pediveliger larvae were released to each glass Petri
dish (Ø 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-lar-
vae 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 § 1°C 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 anti-
biotics) and culture conditions (culturing with GeO2 added
into the culture medium and culturing the apical segments of
1123
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 con-
dition, at least six replicate assays were conducted using lar-
vae 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 repli-
cate 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, Clado-
phora sp., Chlorodesmis fastigiata, Codium fragile, and
Bryopsis plumosa), Wve brown algae (Dilophus okamurae,
Padina arborescens, Myelophycus simplex, Hizikia fusifor-
mis, 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 (129°50⬘E; 32°45⬘N), and a wharf of the Naga-
saki Prefectural Institute of Fisheries, Taira-machi, Naga-
saki (129°51⬘E; 32°43⬘N), Japan, and evaluated for their
ability to induce larval metamorphosis in M. galloprovin-
cialis. 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. fastig-
iata 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 pro-
cedure 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
123
1124
for 3 days using a total volume of 9 l FSW to prepare for-
malin 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 100°C 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 l¡1 of streptomycin sulphate,
10 mg l¡1 of penicillin G, 2 mg l¡1 of neomycin and
10 mg l¡1 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 antibiot-
ics 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 17°C in a
14:10 h light : dark environment and the culture media
changed once every 3–4 days. Germanium dioxide was
added into SW culture medium at a concentration of
5 mg l¡1 (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 0.5–1 mm seg-
ments with apices were excised from the algal pieces under
a dissecting microscope using needles (modiWed from Tate-
waki 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 20°C 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-
123
Mar Biol (2007) 152:1121–1132
tions similar to that described above to culture apical seg-
ments 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 thor-
oughly washed with FSW, placed in Xasks containing FSW
and then left undisturbed for 24 h at 17°C in a dark environ-
ment. 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 hun-
dred percent concentration of the conditioned water was pre-
pared 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 concen-
trations. For example, 0.01 g of the alga was added to every
20 ml of FSW to prepare 10% concentration of the condi-
tioned water, while 1 g of the alga was added to every 20 ml
of FSW to prepare ten-fold concentration of the 100% con-
ditioned water (1,000% concentration). C. fastigiata and C.
tenerrimum conditioned water were either used immediately
or frozen at ¡30°C 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 col-
lected, and centrifuged at 8,000 rpm for 60 min at 4°C. After
centrifugation, the supernatant was Wltered through a What-
man 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. DiVer-
ent 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 ¡30°C 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 suspen-
sion 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
Mar Biol (2007) 152:1121–1132
1,000£ magniWcation under an Olymplus BH-2 epiXuores-
cent 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 enumer-
ated from ten random Welds of view.
Treated and untreated pieces of C. fastigiata and C. ten-
errimum were stained with 0.05% (w/v) erythrosine solu-
tion in FSW and incubated at 17°C 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 sur-
viving 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, Dai-
mastch, 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
Kruskal–Wallis Test. EVects of season, treatments and cul-
ture 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 Tukey–Kramer Honestly SigniWcant DiVerence (HSD)
test, using the JMP™ software. All data expressed in per-
centage were arcsine-transformed prior to analysis with
one-way ANOVA and Tukey–Kramer HSD test. DiVer-
ences were considered signiWcant at P < 0.05.
Results
Throughout all assays, the percentage of metamorphosis to
post-larvae of M. galloprovincialis in Petri dishes contain-
ing 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, Kruskal–Wallis
test, Table 1). Among the macroalgae tested, two green
1125
(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 induc-
ing activities. The other four Wlamentous macroalgae (B. plu-
mose, 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. gal-
loprovincialis. All Wve Phaeophyta species investigated
induced <10% metamorphosis to post-larvae.
Percentages of post-larvae on C. fastigiata and C. tenerr-
imum collected on diVerent months during the period
between January 2005 and April 2006, and water tempera-
tures 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 col-
lected 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 per-
centage 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. fastigi-
ata signiWcantly decreased (P < 0.001, ANOVA), suggest-
ing 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. ten-
errimum where the UT alga, which induced 77% post-lar-
val 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 per-
centages of post-larvae (P < 0.001, ANOVA) also had sig-
niWcantly lower percentages of algal cell survivals (0–56%)
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
123
1126 Mar Biol (2007) 152:1121–1132

Table 1 Taxonomic
Phyla Morphology Weight n Post-larvae Kruskal–Wallis
classiWcation of the 19 species
(g) (%) (rank sums)a
of macroalgae and their Algal species
settlement and metamorphosis
inducing activities expressed CHLOROPHYTA
in percentages of post-larvae Enteromorpha compressa c 0.1 6 8 6
Ulva pertusa ph 0.1 8 4 8
Cladophora sp. W 0.1 9 81 3
0.2 6 89
Chlorodesmis fastigiata W 0.025 6 12
0.05 9 53
0.1 21 83 2
0.2 6 90
Codium fragile c 0.1 10 7 7
Bryopsis plumosa W 0.1 14 13 5
0.2 6 13
PHAEOPHYTA
Dilophus okamurae X 0.1 7 4 9
Padina arborescens X 0.1 7 1 15
Myelophycus simplex W 0.1 7 2 14
Hizikia fusiformis c 0.1 7 2 11
Sargassum thunbergii c 0.1 7 6 10
RHODOPHYTA
Amphiroa zonata c 0.1 10 3 17
Corallina pilulifera pa 0.1 7 1 13
Gelidium elegans W 0.1 11 1 18
0.2 6 2
Grateloupia Wlicina pi 0.1 9 2 16
Grateloupia turuturu ph 0.1 9 1 19
Centroceras clavulatum W 0.1 6 48 4
0.2 6 76
Ceramium tenerrimum W 0.025 6 26
0.05 6 55
The letters of c, ph, W, X, pa, pi
indicate cylindrical, phylloid, 0.1 30 78 1
Wlamentous, Xabellate, palmate, 0.2 6 85
pinnate, respectively Heterosiphonia pulchra W 0.1 9 3 12
a
Score sums were ranked from
0.2 6 6
1 to 19

of these two algae were signiWcantly aVected by their cell
survival and that these algae induced mussel larval settle-
ment and metamorphosis only when alive. Alternatively,
results also suggest that treatments possibly altered the sur-
face chemistry of these algae either by cross-linking (for-
malin treatment), denaturing (heat treatment) or extracting
lipid components (ethanol treatment) of extracellular prod-
ucts on the surface of algae.
at all concentrations (10, 100, and 1,000%) tested (Table 2).
Percentages of post-larval metamorphosis on the condi-
tioned 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

Activities of conditioned water and crude extracts
of C. fastigiata and C. tenerrimum
Percentages of post-larval metamorphosis on the condi-
tioned water and crude extracts of C. fastigiata were all 0%
Percentages of post-larvae on C. fastigiata and C. tenerri-
mum and their corresponding numbers of bacteria after treat-
ment 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

123
Mar Biol (2007) 152:1121–1132 1127

30 100
Temperature (°c )
(a) 100

20 80
80

10
60 60
0
100 40 40

Algal cell survival (% )

80 20 20
Post-larvae ( % )

Post-larvae (% )
60 0 0
40
100 (b) 100
20
80 80
0
n

n
ay

ec
n

ug

pr
ar

ct
ov

ar
b

pr

l
Ja

Se

Ja
Fe
Ju
Fe

Ju

D
M

M
M

A
A

N
A

60 60
2005 2006

Fig. 1 Percentages of post-larvae (Mytilus galloprovincialis) on Chlo- 40 40

rodesmis fastigiata and Ceramium tenerrimum collected each month
from January 2005 to April 2006 and the corresponding seawater tem-
20 20
perature. Open and shaded boxes indicate C. fastigiata and C. tenerri-
mum, respectively. Shaded circles indicate temperature of seawater
0 0
measured at the time of the sampling. Data are means (+SD) of six rep-
licates UT FA 10
E
20
E
50
E
10
0E 30H 35H 40H 00H
1
Treatments
7.7 £ 106 cells per 0.1 g alga. Treatment of C. fastigiata with
Fig. 2 Percentages of post-larvae (Mytilus galloprovincialis) and al-
diVerent concentrations of the antibiotic mixture signiWcantly gal cell survivals in treated Chlorodesmis fastigiata (a) and Ceramium
reduced the number of bacteria (P < 0.001, ANOVA). The tenerrimum (b). Shaded squares indicate algal cell survival. UT indi-
number of bacteria in 10 AB C. fastigiata was 1.3 £ 106 cells cates algae that were not treated. FA indicates 5% formalin treated
per 0.1 g alga; »80% reduction from that of the UT alga algae. 10E, 20E, 50E, and 100E indicate algae treated with ethanol at
concentrations of 10, 20, 50, and 100%, respectively. 30H, 35H, 40H,
(P < 0.05). However, antibiotic treatment did not aVect the and 100H indicate algae heated at 30, 35, 40, and 100°C, respectively.
inducing activity of the alga and percentages of post-larvae in Data are means (+SD) of 6–15 replicates
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 Table 2 Settlement and metamorphosis inducing activities, expressed
with antibiotic solution signiWcantly reduced the numbers in percentages of post-larvae, of conditioned water and crude extracts
of bacteria in treated algae (P < 0.001, ANOVA), and was prepared from Chlorodesmis fastigiata and Ceramium tenerrimum
1.3 £ 106 cells per 0.1 g alga in the 10 AB alga; 91% reduc- Algal species Concentration n Post-larvae
tion in number as compared to the control (UT alga). How- (%) (%)
ever, percentages of post-larvae on antibiotic treated algae
Chlorodesmis fastigiata
remained constantly high (P > 0.05, ANOVA); 62 and 65%
UT 21 83 § 7
post-larvae for 1 AB and 10 AB algae, respectively.
Conditioned water 10, 100, 1,000 6–12 0§0
Percentages of post-larvae on C. fastigiata and C. tenerr-
Crude extracts 10, 100, 1,000 6 0§0
imum cultured in SW media with and without GeO2, and
Ceramium tenerrimum
their corresponding numbers of diatoms are as shown in
Fig. 4. Freshly collected alga induced 80% post-larval UT 36 75 § 13
metamorphosis and had 3.2 £ 104 diatom cells per 0.1 g of Conditioned water 10, 100, 1,000 6–29 0§0
the alga. When cultured in the laboratory for 3 weeks, the Crude extracts 10 14 1§3
number of diatoms per 0.1 g of the cultured alga increased 100 25 2§4
nearly seven-fold in SW media without GeO2 but 1,000 16 1§3
decreased signiWcantly to 2.0 £ 103 cells per 0.1 g alga in Hundred percentage of concentration means 0.1 g alga per 20 ml FSW
the SW medium containing GeO2 (P < 0.05); a nearly
94% decrease as compared to that of the freshly collected with and without GeO2 even after 3 weeks of culture
alga. In contrast, no signiWcant diVerence in percentages of (1 week: algae cultured with GeO2 versus without GeO2,
post-larvae was observed between algae cultured in media P > 0.05; 3 weeks: algae cultured with GeO2 versus without

123
1128 Mar Biol (2007) 152:1121–1132

100 10 8 GeO2, P > 0.05). In addition, the percentage of post-larvae

on C. fastigiata cultured for 3 weeks in SW medium with

Bacteria (cells . 0.1g)

80 GeO2 remained constantly high and was at the same level
P o s t - l ar v a e ( % )

60
40
20
0
UT 1 AB
Antibiotic treatments
Fig. 3 Percentages of post-larvae (Mytilus galloprovincialis) and the
corresponding numbers of bacteria in untreated algae (UT) and antibi-
otic solution treated (AB) algae. Open and shaded boxes indicate Chlo-
rodesmis 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 l¡1, penicillin G 10 mg l¡1, neomycin 2 mg l¡1, and kanamycin
10 mg l¡1, 10 AB = 1 AB concentration increased tenfold. Data are
means (+SD) of six replicates
as that of the fresh alga (P < 0.05, Fig. 4a). A similar ten-
dency was observed when C. tenerrimum thalli were cul-
10 7 tured 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).
10 6 However, percentages of post-larvae on all cultured C. ten-
10 AB errimum remained constantly high and were at the same
level as that of the fresh alga (P > 0.05, Fig. 4b). In addi-
tion, 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

100 (a) 10 6
Percentages of post-larvae on C. tenerrimum cultured from
naturally grown thalli and those from apical segments, and
80 their corresponding numbers of bacteria are as shown in
10 5
Fig. 5. The percentage of post-larvae on fresh C. tenerrimum
60 was 77%. When naturally grown thalli of C. tenerrimum
were brought to the laboratory and cultured in SW
40 medium, the settlement inducing activity of this cultured
Diatoms (cells . 0.1g)

10 4
alga remained constantly high until after 4 weeks of culture
Post-larvae (% )

20 (P > 0.05). However, the percentage of post-larvae on this

10 3 cultured alga gradually decreased to 63% after 5 weeks
0
(P < 0.05). In contrast, C. tenerrimum cultured from apical
segments for 3 weeks showed no inducing activity and the
100 (b) 10 6
percentage of post-larvae remained 0% even after 5 weeks
80
of culture. Addition of bacteria, obtained from freshly col-
10 5 lected naturally grown alga, into the SW medium contain-
60 ing C. tenerrimum cultured for 4 weeks and then culturing it
for another week had no eVect and post-larval metamorpho-
40
10 4
sis on this cultured alga remained 0% (Fig. 5a). The number
of bacteria on fresh C. tenerrimum was 1.3 £ 107 cells per
20 0.1 g alga. This number gradually increased with the culture
10 3 period and reached 6.6 £ 107 cells after 5 weeks. After
0 5 weeks, the numbers of bacteria on the two types of algae
0 1 2 3
Culture Periods (weeks) cultured from apical segments were 2.5 to 4.4 £ 107 cells
per 0.1 g alga. These numbers were signiWcantly higher than
Fig. 4 Percentages of post-larvae (Mytilus galloprovincialis) and the
corresponding numbers of diatoms in Chlorodesmis fastigiata (a) and that of the fresh alga (P < 0.001, ANOVA, Fig. 5b).
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. tenerr- Discussion and conclusions
imum, respectively. Striped boxes indicate C. fastigiata and C. tenerr-
imum cultured in SW without GeO2; grid boxes, C. fastigiata and
C. tenerrimum cultured in SW with GeO2. Data are means (+SD) of Macroalgae had been reported to aVect larval settlement
6–12 replicates and metamorphosis of many invertebrates, e.g., two bryozoan

123
Mar Biol (2007) 152:1121–1132
100 (a)
Post-larvae (% )
80
60
40
20
0 sis inducing activities, suggesting that the Wlamentous mor-
10 8
Bacteria (cells . 0.1g)
(b)
10 7
0 10 20 30
Culture Periods (days)
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, cir-
cles, and squares indicate naturally grown alga, cultured apical seg-
ments 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, respec-
tively. Data are means (+SD) of six replicates
species (Walters et al. 1996; Schmitt et al. 1998), a poly-
chaete 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 Schei-
bling 1991; Kitamura et al. 1993; Takahashi et al. 2002;
Swanson et al. 2004; Li et al. 2004a, b). Mussel larval set-
tlement 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; Cáceres-
Martínez 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 labo-
ratory experiments that larvae of the mussel M. gallopro-
vincialis 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 con-
stantly high throughout the investigation, regardless of the
season. The Wnding that M. galloprovincialis settled and
metamorphosed on Wlamentous algae is consistent with
1129
reports on M. edulis larvae (Bayne 1964; Petersen 1984;
Eyster and Pechenik 1987; Dobretsov 1999). Cáceres-
Martínez et al. (1994) concluded from Weld observations
that M. galloprovincialis used Wlamentous, thallus and
membranous algae among other substrates for direct settle-
ment. In the present investigation, algae that were not Wla-
mentous all showed low (<10%) settlement inducing
activities. Interestingly, not all Wlamentous algae tested in
this investigation showed high settlement and metamorpho-
phology 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. fastigi-
ata, Cladophora sp., C. clavulatum, and C. tenerrimum)
may have induced mussel larval settlement and metamor-
phosis. Dobretsov (1999) observed that M. edulis larvae
40
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 treat-
ment 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. For-
malin treatment of microbial or bacterial bioWlms have
been used in previous reports as a technique to kill constit-
uent 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 bio-
Wlms 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 sur-
face bound and were susceptible to ethanol and heat treat-
ments. 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. canalicu-
lus, although it was not speciWed in this case whether lar-
vae 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
123
1130
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 signiWcant 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 starWsh Acanth-
aster planci (Johnson and Sutton 1994), and sea urchin
Heliocidaris erythrogramma (Huggett et al. 2006). Bio-
Wlms 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 cm¡2
in density of bacteria on the algal surface. This bacterial
density is »38 times lower than the density of bacteria
(5 £ 106 cells cm¡2) in multispecies bioWlms that induced
60–80% 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 cm¡2, 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 settle-
ment 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 sur-
faces 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. ten-
errimum when cultured in the laboratory from apical
123
Mar Biol (2007) 152:1121–1132
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 poten-
tial 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 induc-
tion of settlement and metamorphosis of M. galloprovin-
cialis 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 com-
munity 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 note-
worthy, 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 cer-
tain nutrients in the culture medium may lead to the identi-
Wcation 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 Wla-
mentous 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 Peter-
sen (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 (Satu-
ito et al. 1995, 1997; Bao et al. 2007) in the absence of Wla-
mentous 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.
Mar Biol (2007) 152:1121–1132
Acknowledgements The authors are grateful to the Nagasaki Pre-
fectural 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.
References
Alfaro AC (2005) EVect of water Xow and oxygen concentration on
early settlement of the New Zealand green-lipped mussel, Perna
canaliculus. Aquaculture 246:285–294
Alfaro AC (2006) Byssal attachment of juvenile mussels, Perna cana-
liculus, aVected by water motion and air bubbles. Aquaculture
255:357–361
Alfaro AC, Copp BR, Appleton DR, Kelly S, JeVs AG (2006) Chemi-
cal cues promote settlement in larvae of the green-lipped mussel,
Perna canaliculus. Aquacult Int 14:405–412
Alfaro AC, JeVs AG (2002) Small-scale mussel settlement patterns
within morphologically distinct substrata at Ninety Mile Beach,
northern New Zealand. Malacologia 44:1–15
Alfaro AC, JeVs AG, Creese RG (2004) Bottom-drifting algal/mussel
spat associations along a sanday coastal region in northern New
Zealand. Aquaculture 241:269–290
Bao WY, Satuito CG, Yang JL, Kitamura H (2007) Larval settlement
and metamorphosis of the mussel Mytilus galloprovincialis in re-
sponse to bioWlms. Mar Biol 150:565–574
Bayne BL (1964) Primary and secondary settlement in Mytilus edulis
L. (Mollusca). J Anim Ecol 33:513–523
Bayne BL (1965) Growth and the delay of metamorphosis of the larvae
of Mytilus edulis (L.). Ophelia 2:1–47
Bulleri F, Airoldi L, Branca GM, Abbiati M (2006) Positive eVects of
the introduced green alga, Codium fragile ssp. tomentosoides, on
recruitment and survival of mussels. Mar Biol 148:1213–1220
Cáceres-Martínez J, Robledo JAF, Figueras A (1993) Settlement of
mussels Mytilus galloprovinvialis on an exposed rocky shore in
Ría de Vigo, NW Spain. Mar Ecol Prog Ser 93:195–198
Cáceres-Martínez J, Robledo JAF, Figueras A (1994) Settlement and
post-larve behaviour of Mytilus galloprovinvialis: Weld and labo-
ratory experiments. Mar Ecol Prog Ser 112:107–117
Cooper K (1981) A model to explain the induction of settlement and
metamorphosis of planktonic eyed-pediveligers of the blue mus-
sel Mytilus edulis L. by chemical and tactile cues (Abstract).
J ShellWsh Res 2:117
Daume S, Krsinich A, Farrell S, Gervis M (2000) Settlement, early
growth and survival of Haliotis rubra in response to diVerent al-
gal species. J Appl Phycol 12:479–488
Davis AR, Moreno CA (1995) Selection of substrata by juvenile Chor-
omytilus chorus (Mytilidae): are chemical cues important? J Exp
Mar Biol Ecol 191:167–180
Dobretsov SV (1999) EVects of macroalgae and bioWlm on settlement
of blue mussel (Mytilus edulis L.) larvae. Biofouling 14:153–165
Dobretsov SV, Railkin AI (1994) Correlative relationships between ma-
rine microfouling and macrofouling. Russ J Mar Biol 20:87–90
Eyster LS, Pechenik JA (1987) Attachment of Mytilus edulis L. larvae
on algal and byssal Wlaments is enhanced by water agitation.
J Exp Mar Biol Ecol 114:99–110
HadWeld MG, Paul VG (2001) Natural chemical cues for settlement
and metamorphosis of marine-invertebrate larvae. In: McClintock
JB, Baker JB (eds) Marine chemical ecology. CRC, Boca Raton,
pp 431–461
Harder T, Dobretsov S, Qian PY (2004) Waterborne polar macromol-
ecules act as algal antifouling in the seaweed Ulva reticulate. Mar
Ecol Prog Ser 274:133–141
1131
Huggett MJ, de Nys R, Williamson JE, Heasman M, Steinberg PD
(2005) Settlement of larval blacklip abalone, Haliotis rubra, in
response to green and red macroalgae. Mar Biol 147:1155–1163
Huggett MJ, Williamson JE, de Nys R, Kjelleberg S, Steinberg PD
(2006) Larval settlement of the common Australian sea urchin
Heliocidaris erythrogramma in response to bacteria from the sur-
face of coralline algae. Oecologia 149:604–619
Iwasaki H (1961) The life-cycle of Porphyra tenera in vitro. Biol Bull
121:173–187
Johnson CR, Sutton DC (1994) Bacteria on the surface of crustose cor-
alline algae induce metamorphosis of the crown-of-thorns starWsh
Acanthaster planci. Mar Biol 120:305–310
Kajihara T (1985) The mussel Mytilus galloprovincialis—the valiant
invader of coastal sea area. In: Okiyama M, Suzuki K (eds) Japa-
nese marine organisms—Ecology of invasion and disturbance
(Nihon no kaiyo seibutsu shinryaku to kakuran noseitaigaku, In
Japanese). Tokai University Press, Tokyo, pp 49–54
Kajihara T, Oka M (1980) Seasonal occurrence of marine mussel
plantigrades in Tokyo Harbor. Bull Jpn Soc Sci Fish 46:145–148
Katsuyama I (1995) Filamentous organisms observed with the blue
mussel. Marine Fouling 11:9–13
Kirchman D, Graham S, Reish D, Mitchell R (1982) Bacteria induce
settlement and metamorphosis of Janua (Dexiospira) brasiliensis
Grube (Polychaeta: Spirorbidae). J Exp Mar Biol Ecol 56:153–
163
Kitamura H, Kitahara S, Koh HB (1993) The induction of larval settle-
ment and metamorphosis of two sea urchins, Pseudocentrotus
depressus and Anthocidaris crassispina, by free fatty acids
extracted from the coralline red alga Corallina pilulifera. Mar
Biol 115:387–392
Krug PJ, Manzi AE (1999) Waterborne and surface-associated carbo-
hydrates as settlement cues for larvae of the specialist marine Her-
bivore Alderia modesta. Biol Bull 197:94–103
Lau SCK, Qian PY (2001) Larval settlement in the serpulid polychaete
Hydroides elegans in response to bacterial Wlms: an investigation
of the nature putative larval settlement cue. Mar Biol 138:321–
328
Li JY, Rahim SAKA, Satuito CG, Kitamura H (2004a) Combination of
macroalgae-conditionded water and periphytic diatom Navicula
ramosissima as an inducer of larval metamorphosis in the sea
urchins Anthocidaris crassispina and Pseudocentrotus depressus.
Sessile Organisms 21:1–6
Li JY, Rahim SAKA, Satuito CG, Kitamura H (2004b) Characteriza-
tion of the active substances in water conditioned by the coralline
red alga Corallina pilulifera as inducers of metamorphosis in lar-
vae of the sea urchin Anthocidaris crassispina (in Japanese with
English abstract). Sessile Organisms 21:41–46
McGrath D, King PA, Gosling EM (1988) Evidence for the direct set-
tlement of Mytilus edulis larvae on adult mussel beds. Mar Ecol
Prog Ser 47:103–106
Moreno CA (1995) Macroalgae as a refuge from predation for recruits
of the mussel Choromytilus chorus (Molina, 1782) in Southern
Chile. J Exp Mar Biol Ecol 191:181–193
Morse ANC, Morse DE (1984) Recruitment and metamorphosis of
Haliotis larvae induced by molecules uniquely available at the
surfaces of crustose red algae. J Exp Mar Biol Ecol 75:191–215
Pearce CM, Scheibling RE (1991) EVect of macroalgae, microbial
Wlms, and conspeciWcs on the induction of metamorphosis of
the green sea urchin Strongylocentrotus droebachiensis (Müller).
J Exp Biol Ecol 147:147–162
Petersen JH (1984) Larval settlement behavior in competing species:
Mytilus californianus Conrad and M. edulis L. J Exp Mar Biol
Ecol 82:147–159
Ramírez SC, Cáceres-Martínez J (1999) Settlement of blue mussel
Mytilus galloprovincialis Lamarck on artiWcial substrates in Bahía
De Todos Santos B.C., México. J shellWsh Res 18:33–39
123
1132
Sakaguchi I (1987) Bivavles. In: Kajihara T (ed) Attaching organisms
and aquaculture (in Japanese). Kouse Press, Tokyo, pp 100–107
Sakaguchi I (2003) A review of antifouling technologies in power
plant cooling water systems (in Japanese with English abstract).
Sessile Organisms 20:15–19
Sakaguchi I, Kajihara T (1988) Recruitment from larvae to fouling
community of marine mussel (in Japanese with English abstract).
Marine Fouling 7:23–29
Satuito CG, Bao WY, Yang JL, Kitamura H (2005) Survival, growth,
settlement and metamorphosis of refrigerated larvae of the mussel
Mytilus galloprovincialis Lamarck and their use in settlement and
antifouling bioassays. Biofouling 21:217–225
Satuito CG, Natoyama K, Yamazaki M, Fusetani N (1995) Induction
of attachment and mtamorphosis of laboratory cultured mussel
Mytilus edulis galloprovincialis larvae by microbial Wlm. Fish Sci
61:223–227
Satuito CG, Shimizu K, Fusetani N (1997) Studies on the factors inXu-
encing larval settlement in Balanus amphitrite and Mytilus gallo-
provincialis. Hydrobiologia 358:275–280
Schmitt TM, Lindquist N, Hay ME (1998) Seaweed secondary metab-
olites as antifoulants: eVects of Dictyota spp. diterpenes on survi-
vorship, settlement, and development of marine invertebrate
larvae. Chemoecology 8:125–131
123
Mar Biol (2007) 152:1121–1132
Seed R (1969) The ecology of Mytilus edulis L. (Lamellibranchiata) on
the exposed rocky shores. I. breeding and settlement. Oecologia
(Berlin) 3:277–316
Swanson RL, Williamson JE, de Nys R, Kumar N, Bucknall MP,
Steinberg PD (2004) Induction of settlement of larvae of the sea
urchin Holopneustes purpurascens by histamine from a host alga.
Biol Bull 206:161–172
Takahashi Y, Itoh K, Ishii M, Suzuki M, Itabashi Y (2002) Induction
of larval settlement and metamorphosis of the sea urchin Strong-
ylocentrotus intermedius by glycoglycerolipids from the green
alga Ulvella lens. Mar Biol 140:763–771
Tatewaki M (1992) Isolation and culture of macroalgae. In: Nisizawa
K, Tihara M (eds) Methods in phycological studies (in Japanese).
Kyouritu Press, Tokyo, pp 53–106
Unabia CRC, HadWeld MG (1999) Role of bacteria in larval settlement
and metamorphosis of the polychaete Hydroides elegans. Mar
Biol 133:55–64
Walters LJ, HadWeld MG, Smith CM (1996) Waterborne chemical
compounds in tropical macroalgae: positive and negative cues for
larval settlement. Mar Biol 126:383–393
Yvin JC, Chevolot L, Chevolot-Magueur AM, Cochard JC (1985) First
isolation of jacaranone from an alga, Delesseria sanguinea. A
metamorphosis inducer of Pectin larvae. J Nat Prod 48:814–816