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Biofouling: The Journal of Bioadhesion and Biofilm

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Larval settlement and metamorphosis of the mussel

Mytilus coruscus in response to natural biofilms
a

Chong Wang , Wei-Yang Bao , Zhong-Qi Gu , Yi-Feng Li , Xiao Liang , Yun Ling ,
Sheng-Li Cai , He-Ding Shen & Jin-Long Yang

College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China

Institute of Marine Science and Technology, Yangzhou University, Yangzhou, China

Shengsi Service Center of Marine Science and Technology Development, Zhoushan, China

Faculty of Fisheries, Nagasaki University, Nagasaki, Japan

Version of record first published: 21 Mar 2012.

To cite this article: Chong Wang, Wei-Yang Bao, Zhong-Qi Gu, Yi-Feng Li, Xiao Liang, Yun Ling, Sheng-Li Cai, He-Ding Shen &
Jin-Long Yang (2012): Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms,
Biofouling: The Journal of Bioadhesion and Biofilm Research, 28:3, 249-256
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Biofouling
Vol. 28, No. 3, March 2012, 249256

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biolms
Chong Wanga,1, Wei-Yang Baob, Zhong-Qi Guc, Yi-Feng Lia, Xiao Liangd, Yun Linga, Sheng-Li Caia,
He-Ding Shena and Jin-Long Yanga*,1
a

College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China; bInstitute of Marine Science and Technology,
Yangzhou University, Yangzhou, China; cShengsi Service Center of Marine Science and Technology Development, Zhoushan,
China; dFaculty of Fisheries, Nagasaki University, Nagasaki, Japan

Downloaded by [Universidad De Concepcion] at 12:39 29 November 2012

(Received 30 September 2011; nal version received 15 February 2012)

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biolms was
investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biolms and post-larval
settlement and metamorphosis increased with biolm age. The activity of the biolm was positively correlated with
biolm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a
concentration. The change in bacterial community composition corresponding to biolm age may explain dierences
in the age-dependent inducing activities of biolms, which in turn may play an important role in larval settlement in
this species.
Keywords: mussel; larval settlement and metamorphosis; biolm; bacterial community structure

Introduction
Many marine invertebrate larvae have been reported to
recognize environmental cues via external chemoreceptors, which transduce the cues into internal
processes with neural and/or hormonal elements
moderating settlement and metamorphosis (Crisp
1974; Morse 1990; Qian 1999; Hadeld and Paul
2001; Hadeld 2011). Understanding the environmental factors controlling larval settlement and metamorphosis is therefore important in the elds of biofouling,
antifouling research and aquaculture (Hadeld and
Paul 2001; Yang et al. 2007; Carl et al. 2011). Natural
cues that inuence larval settlement and metamorphosis are often associated with biolms attached to
substrata (Hadeld and Paul 2001; Anderson and
Epifanio 2009).
Biolms are surface-associated communities of
microorganisms along with the mucilaginous extracellular polymers they secrete (Decho 2000; Shikuma
and Hadeld 2010). Biolms exist on almost all
exposed substrata in the marine environment (Thiyagarajan et al. 2006). Biolms have been shown to
mediate larval settlement and metamorphosis of many
invertebrates, eg the polychaete, Hydroides elegans
(Huggett et al. 2009; Chung et al. 2010); the bryozoan,
Bugula neritina (Kitamura and Hirayama 1987; Dobretsov and Qian 2006); the sea urchins, Strongylocentrotus droebachiensis (Pearce and Scheibling 1991),
Pseudocentrotus depressus (Rahim et al. 2004) and
*Corresponding author. Email: jlyang@shou.edu.cn
1
These two authors contributed equally.
ISSN 0892-7014 print/ISSN 1029-2454 online
2012 Taylor & Francis
http://dx.doi.org/10.1080/08927014.2012.671303
http://www.tandfonline.com

Anthocidaris crassispina (Rahim et al. 2004); the

barnacles Balanus amphitrite (Maki et al. 1990;
Wieczorek et al. 1995; Harder et al. 2001; Hung et al.
2008) and B. trigonus (Lau et al. 2005) and the
ascidian, Ciona intestinalis (Wieczorek and Todd
1997). With respect to molluscs, Zhao et al. (2003)
demonstrated that biolms induced larval settlement
of the silver (gold-lip) pearl oyster Pinctada maxima
while Yu et al. (2010) showed that biolms induced
larval settlement and metamorphosis of the pearl
oyster P. fucata. Chiu et al. (2007) found that biolms
formed under dierent environmental conditions had
dierent eects on metamorphosis of larvae of the
slipper limpet, Crepidula onyx. Campbell et al. (2011)
investigated whether biolms that formed on hard
surfaces placed in Little Wicomico River had an
inhibitive eect on the native Eastern oyster, Crassostrea virginica, but their results suggested that biolms
developed in the eld had no inhibitory eect on larval
settlement.
Mussel larvae also settle and metamorphose in
response to biolms including natural multi-species
biolms (Dobretsov 1999; Bao et al. 2007a) and
monospecies bacterial or diatom biolms (Satuito
et al. 1995, 1997; Bao et al. 2007b; Ganesan et al.
2010). For natural multi-species biolms, Dobretsov
(1999) demonstrated that Mytilus edulis larvae were
attracted to swim towards the biolms formed on the
lamentous green alga Cladophora rupestris, but were

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250

C. Wang et al.

repelled by biolms on the brown alga Laminaria

saccharina. Bao et al. (2007a) showed that biolms
formed in the eld induced larval settlement and
metamorphosis of M. galloprovincialis. These reports
indicate that mussel larvae may be able to distinguish
between biolms formed under dierent environmental
conditions. Although the role of biolms in biofouling
by macroorganisms has been recognized, knowledge of
the interaction between biolms and mussel settlement
and metamorphosis is limited.
The mussel, Mytilis coruscus, is a common species
inhabiting the temperate zone along the coastal water
of East Asia (Chang 2007). In China, this species is an
important commercial marine bivalve, but it is also an
important fouling organism, especially in the East
China Sea (Cai et al. 1994; Chang et al. 2008). In the
present study, the authors investigated the eects of
natural multi-species biolms on settlement and
metamorphosis of larvae of M. coruscus. Various
aspects of biolm biology were investigated including
age, dry weight, chlorophyll a (chl a) concentration,
bacterial and diatom densities and bacterial community structure. The correlation between the inducing
activity of the biolms and dry weight, bacterial and
diatom densities, and chl a concentration was also
investigated.
Materials and methods
Spawning and larval culture
Adults of M. coruscus were collected from populations
growing *2 miles from the coast of Shengsi, Zhoushan (1228440 E; 308730 N), China. Spawning in the
laboratory was induced following a method modied
described by Yang et al. (2008). Mussels were cleaned
by brushing o material attached on the shell surfaces
and rinsing in seawater, packed in ice overnight then
transferred to a 10 l polycarbonate tank with ltered
seawater (acetate-ber lter: 1.2 mm pore size, FSW) at
ca 218C and a nal water temperature of ca 188C.
Mussels that started spawning were transferred to
individual 2 l glass beakers. Eggs were collected using a
glass pipette and transferred to a beaker containing
FSW. Fertilization was achieved by gently mixing with
a sperm suspension in FSW; the suspension was then
left undisturbed for 20 min. Fertilized eggs were
ltered onto a nylon plankton net (mesh size: 20 mm)
to remove excess sperm, washed thoroughly with FSW
and left undisturbed for 2 days in an incubator
maintained at 188C. After 2 days, swimming straighthinge veliger larvae were collected, washed gently with
FSW and cultured in 2 l glass beakers at an initial
density of 5 larvae ml71. Larvae were fed a diet of
Chaetoceros gracilis at 5 6 104 cells ml71 day71. The
culture water was changed every other day and the

temperature maintained at 18 + 18C. Larvae were

cultured to the pediveliger stage of growth and were
used in settlement and metamorphosis bioassay.
Preparation of natural biolms
Biolm slips were prepared by immersing clean glass
slips (half portions of microscopic glass slides;
38 mm 6 26 mm) in coastal seawater at Gouqi Island
(1228460 E; 308430 N), Zhejiang, China. The glass slips
were placed on PVC holders and immersed at a depth
of 0.51.0 m below the surface for 728 days. The slips
were brought back to the laboratory and thoroughly
washed with autoclaved ltered seawater (AFSW)
prior to use in assays on the same day.
Larval settlement and metamorphosis bioassays
Twenty pediveliger larvae were transferred into individual glass Petri dishes (64 mm 6 19 mm height)
containing 20 ml of AFSW and one biolm slip. The
settlement inducing activity of the biolms was
evaluated microscopically by determining the percentage of metamorphosis to post-larvae (ie individuals
with post-larval shell growth) obtained after 48 h. Petri
dishes, each containing a clean (non-biolmed) glass
slip, 20 larvae and 20 ml of AFSW were set up as
negative controls in all assays. Petri dishes, each
containing 20 ml of 100 mm epinephrine (Yang et al.
2011) and 20 larvae were set up as positive controls in
all assays. Assays were conducted at 18 + 18C in
darkness. Three to six replicates were used in assays.
Each assay was repeated three times.
Measurement of biolm dry weight
Dry weight was measured by the method of Bao et al.
(2007a). The biolms on each glass slip were scraped
o by using a sterile glass slide and separately
suspended in AFSW. Each suspension was collected
on a pre-weighed GF/C lter (Whatman glass ber
lter; pore size: 1.2 mm) by ltration. Each lter paper
with biolm was washed with 50 ml of 0.22 mm ltered
distilled water, dried for 48 h in an oven at 808C and
cooled to room temperature in a desiccator before
weighting. The dry weight of the biolm was determined after subtracting the weight of the lter.
Enumeration of densities of bacteria and diatoms
Biolms were xed in 5% formalin solution for a
maximum duration of 3 weeks. Samples were washed
with AFSW and stained with acridine orange (AO,
0.1%) for 5 min. The bacterial densities of the stained
samples were determined by counting directly at

Biofouling
10006 magnication under an Olympus BX51 epiuorescence microscope. The densities of the diatoms
were determined by counting directly at 200 6 magnication under a light microscope immediately after
samples were brought back to the laboratory. The
densities of the bacteria and diatoms in each sample
were enumerated from 10 random elds of view.

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Chl a concentration in biolms

Biolms were scraped from three replicate glass slides
using sterile glass slides, ltered through membranes
and preserved at 7208C. Chl a extraction was
conducted at 48C using 90% acetone for 14 h in
darkness. To ensure complete extraction of chlorophyll, samples were vortexed for 1 h at the end of the
extraction period, then centrifuged for 10 min at 3000
rpm. The chl a concentration of the supernatants was
determined spectrophotometrically (UNIC 2100 spectrophotometer). The wavelengths measured were 630,
647, 664 and 750 nm, respectively. The chl a concentration was calculated using the following equation
(Ma et al. 2011):

chl a

251

buer, 2 ml of MgCl2 (25 mM) and 0.5 ml of

deoxynucleotide triphosphates (10 mM each) and
sterile distilled water to a nal volume of 25 ml. PCR
cycling was carried out in an Eppendorf Mastercycler
(Eppendorf, Hamburg, Germany) thermocycler under
the following conditions: an initial denaturing step at
948C for 5 min, a touch-down thermal cycling of
denaturation at 948C for 1 min, annealing at 65558C
for 1 min (reducing 0.58C per cycle) and elongation at
728C for 0.5 min. Then another 15 PCR cycles were
conducted, each cycle consisting of 1 min denaturation
at 948C, 1 min annealing at 558C and 0.5 min synthesis
at 728C. Finally, an extension step was carried out at
728C for 8 min. PCR products were veried by agarose
gel electrophoresis (1.2% weight/volume agarose) with
ethidium bromide staining and visualized using an
ultraviolet (UV) transilluminator.
Bacterial community proling by denaturing gradient gel
electrophoresis
Denaturing gradient gel electrophoresis (DGGE) of
the PCR amplied 16S rDNA was carried out using

12:12  D664  D750  1:58  D647  D750  0:08  D630  D750  Ve  d

A

where chl a is chl a concentration (mg cm72) in the

biolm; D630, D647, D664 and D750 are the absorption at 630, 647, 664 and 750 nm; Ve, the extraction
volume; A, the area of the substratum surface; and d,
the optical length of the cuvette.
DNA extraction
Biolms were scraped from three replicate glass slides
as above and centrifuged for 5 min at 10,000g. The
supernatant was discarded and genomic DNA was
extracted using a 3S DNA Isolation Kit for Environmental Samples V2.2 following the manufacturers
instructions (Shenergy Biocolor Bioscience and Technology Company, Shanghai, China).
PCR amplication of 16S rDNA
Bacterial 16S rRNA genes were amplied using the
primers 357F, which contains a GC clamp (50 -C GCC
CGC CGC GCG CGG CGG GCG GGG CGG GGG
CAC GGG GGG CCT ACG GGA GGC AGC
AG-30 ) and 518R (50 -ATT ACC GCG GCT GCT
GG-30 ) (Muyzer et al. 1993). PCR amplication was
performed in a 25 ml reaction mixture containing 0.5 ml
of each primer (10 mm), 1 ml of template DNA (4080
ng), 0.25 ml of Ex Taq (5 U/ml), 2.5 ml of 10 6 PCR

the D-CodeTM Universal Mutation Detection System

(Bio-Rad, Hercules, CA, USA). PCR products were
resolved on a vertical gel containing 8% (w/v)
polyacrylamide (arylamide:bisacrylamide 37.5:1) and
a denaturing gradient ranging from 4070%. One
hundred percent denaturant is dened as a 7 M urea
and 40% (v/v) deionized formamide. Electrophoresis
(60 V, 14 h) was performed in 1 6 TAE buer
(40 mM Tris-acetate, 20 mM acetate, 1 mM Na2EDTA) at 608C. After electrophoresis, the gel was
stained in ethidium bromide for 20 min and photographed under UV illumination. DGGE gel images
were analyzed using Quantity One analysis software
(Bio-Rad). A similarity matrix was constructed based
on the total number of bands observed in all biolm
bacterial communities and the presence or absence of
these bands in each community. Agglomerative hierarchical clustering was performed using UPGMA
(unweighted pair group method using arithmetic
averages) and the similarities among communities
were displayed as a dendrogram.
Data analysis
The settlement and metamorphosis inducing activities
of the biolms were evaluated by the percentages of
post-larvae. Prior to statistical analysis, all data

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252

C. Wang et al.

expressed in percentages were arcsine-transformed and

tested for normality. The eects of age on biolm
activity and chl a concentration were assessed using
one-way Analysis of Variance (ANOVA) followed by
the TukeyKramer Honestly Signicant Dierence
(HSD) test. Correlations between biolm age or
biolm activity and dry weight, bacterial and the
diatom densities of the biolms were analyzed using a
Spearmans rank correlation test or a Pearson correlation test. The DGGE proles were analyzed with
Smart View and bacterial diversity was calculated with
the Shannon diversity index. All statistical computations were performed using JMPTM software. Dierences were considered signicant at p 5 0.05.

settlement and metamorphosis (ANOVA: p 5 0.0001),

reaching a maximum of 93 + 6% after 28 days. The
relationship between biolm activity and dry weight,
and bacterial and diatom densities is shown in Table 1.
There was a signicant positive correlation between
biolm activity and dry weight (Pearson correlation
test: p 5 0.05). A positive correlation was observed
between bacterial densities and the percentages of postlarvae [bacterial density vs post-larvae (%): Spearmans rank correlation test: p 5 0.0001]. Similarly, a
positive correlation was also observed between diatom
densities and the percentages of post-larvae [diatom
density vs post-larvae (%): Spearmans rank correlation test: p 5 0.0001].

Results

Dry weight, bacterial and diatom densities at dierent

biolm ages

Larval settlement and metamorphosis in response to

biolms

The dry weights, and the bacterial and diatom densities

of biolms of dierent ages are as shown in Figure 2.
The dry weight gradually increased with age, the
maximum being obtained after 28 days. Biolm age
signicantly aected both bacterial (KruskalWallis
test: p 5 0.0001) and diatom density (p 5 0.0001).
Bacterial densities increased with biolm age reaching
a maximum cell density of 3.5 6 107 + 7.4 6 106
cells cm72 after 21 days, which subsequently decreased
to 2.8 6 107 + 7.4 6 106 cells cm72 at 28 days. The
cell densities of diatoms increased with increasing age
of the biolms; cell densities ranged from 1.1 6
104 + 3.7 6 103 cells cm72 to 3.0 6 105 +
1.0 6 105 cells cm72. The relationship between
biolm age and dry weight, and bacterial and diatom
densities is shown in Table 1. Dry weight was
signicantly correlated with biolm age (Pearson
correlation test: p 5 0.05). Similarly, the density of
bacteria and diatoms was also signicantly correlated
with biolm age (Spearmans rank correlation test:
p 5 0.0001).

The percentages of post-larvae of M. coruscus on

biolms of dierent ages are shown in Figure 1. In all
experiments, no metamorphosed post-larvae were
found in the negative controls. In the positive controls,
epinephrine induced 475% metamorphosis of postlarvae. Biolm of increasing age signicantly increased

Figure 1. Percentage of post-larvae of M. coruscus on

biolms of dierent ages. Data are means (+SD) of three to
six replicates. Data with signicant dierence are indicated
by dierent letters (TukeyKramer HSD test, a 0.05).

Chl a analysis
The chl a concentrations in biolms of dierent ages
are shown in Figure 3. The maximum chl a

Table 1. Correlation analysis run between biolm activity or biolm age and three factors (dry weight, bacterial and diatom
densities).
Dry weight

Biolm activity
Age

Bacterial density

Diatom density

rp

rs

rs

0.745*
0.742*

0.005
0.006

0.840
0.840*

5 0.0001
5 0.0001

0.958*
0.958*

5 0.0001
5 0.0001

rp Pearsons correlation coecient; rs Spearmans rank order correlation analysis; p p-value; *: signicant at p 5 0.05.

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Biofouling

253

Figure 3. Chl a concentration in biolms of dierent ages.

Data are means (+SD) of six replicates. Data with
signicant dierence are indicated by dierent letters
(TukeyKramer HSD test, a 0.05)

Figure 2. Dry weights (A), bacterial densities (B) and

diatom densities (C) of biolms of dierent ages. Dry weights
are means (+SD) of three replicates. Bacterial and diatom
densities are means (+SD) of 10 random elds of view.

Figure 4. DGGE ngerprint of the bacterial 16S rRNA

gene fragments from biolms of dierent age.

concentration was obtained in biolms after 14 days

but then there was no signicant change in older
biolms (ANOVA: p 4 0.05). No signicant correlation was observed between chl a concentration and
biolm activity (Pearson correlation test: p 4 0.05).

day-biolms and 14 day-biolms as well as between the

21 day-biolms and the 28 day-biolms. A similar
tendency of clustering was also found in batch 3. In
batch 2, cluster analysis revealed that 14 day-biolms
and 21 day-biolms shared 81% similarity and that
these two biolms shared 77% and 69% with 7 daybiolms and 28 day-biolms, respectively.

Bacterial community analysis by DGGE

Discussion

A representative DGGE gel from dierent ages of

biolms is shown in Figure 4 and a comparative
analysis of bacterial DGGE patterns using cluster
analysis is shown in Figure 5. In batch 1, clustering of
bacterial DGGE patterns was observed between the 7

In the present study, the authors have demonstrated

that larvae of the mussel M. coruscus settled and
metamorphosed in response to natural biolms.
Biolm activity was correlated with biolm age, and
dry weight as well as bacterial and diatom densities,

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254

C. Wang et al.

Figure 5. Dendrogram generated from the DGGE proles,

showing the similarities of bacterial community composition.

but there was no signicant correlation between

biolm activity and chl a concentration. In addition,
no mortality was observed after larvae were exposed to
biolms of dierent ages (data no shown), indicating
that natural biolms could be used as non-toxic
inducers of settlement and metamorphosis of M.
coruscus for studies relating to biofouling and aquaculture. Biolms have been recognized as being
important for larval settlement and metamorphosis
of many marine invertebrates including polychaetes,
bryozoans, molluscs, sea urchins, barnacles and
ascidians. Interest in marine biolms and their role in
recruitment of benthic marine invertebrates has
burgeoned over the past decades (Hadeld 2011).
The aim of such research is to improve understanding
of the factors that moderate larval settlement (Rahim
et al. 2004; Bao et al. 2007a, 2007b; Hadeld 2011),
especially in relation to the control of biofouling and
to enhance production in aquaculture hatcheries (Zhao
et al. 2003; Dobretsov 2009; Ganesan et al. 2010; Yu
et al. 2010; Campbell et al. 2011).
A number of studies have shown that biolm age
aects the settlement and metamorphosis of larvae of
many marine invertebrates (Keough and Raimondi
1995; Rahim et al. 2004; Yu et al. 2010; Campbell et al.
2011). The results of the present investigation show
that settlement and metamorphosis of larvae of M.
coruscus increased with increasing age of the biolm.
This nding is consistent with previous reports on P.
maxima (Zhao et al. 2003), P. fucata (Yu et al. 2010),
Crassostrea virginica (Campbell et al. 2011), M.
galloprovincialis (Bao et al. 2007a), S. droebachiensis

(Pearce and Scheibling 1991), P. depressus (Rahim

et al. 2004) and B. amphitrite (Hung et al. 2008). On
the other hand, settlement by larvae of other organisms such as A. crassispina (Rahim et al. 2004) can be
independent of age. Furthermore, Balanus variegatus
exhibited the opposite pattern, with settlement negatively correlated with age of the biolm (Keough and
Raimondi 1995).
In the present study, the inducing activity of
biolms was positively correlated with dry weight as
well as with bacterial and diatom densities. Bao et al.
(2007a) demonstrated that the inducing activity of
biolms for the mussel M. galloprovincialis was also
positively related with dry weight and the density of
bacteria and diatoms. Wieczorek et al. (1995) suggested that enhanced settlement of cyprids of B.
amphitrite by older lms could be explained by an
increase in microbial density and overall diversity, as
well as by changes in the metabolic activities of the
lms. On the other hand, some researchers have
suggested that extracellular products rather than
biolm composition (ie density of bacteria and
diatoms) might mediate larval settlement and metamorphosis (Qian et al. 2003; Yu et al. 2010). Qian et al.
(2003) demonstrated that there was no correlation
between settlement of barnacle cypris larvae (B.
amphitrite) and the abundance of bacteria and
diatoms, but suggested that that structure of the
bacterial community in the biolms was more important in mediating larval settlement. Yu et al. (2010)
also showed that larval settlement and metamorphosis
of P. fucata was not correlated with the density of
bacteria and diatoms, but suggested that the bacterial
community might play an important role in larval
settlement.
Earlier studies have shown that isolated bacterial
strains can induce larval settlement and metamorphosis of marine invertebrates such as Hydroides elegans
and M. galloprovincialis (Satuito et al. 1995; Unabia
and Hadeld 1999; Lau et al. 2002; Bao et al. 2007b).
However, the presence of inducing bacterial strains in a
natural biolm does not necessarily explain the
inducing activity of the biolms (Chung et al. 2010).
It has been hypothesized that the composition of the
biolm community is one of the most important
factors determining whether a biolm inhibits or
facilitates settlement of the larvae of marine invertebrates (Wieczorek and Todd 1998; Thiyagarajan et al.
2006). In the present study, biolm community
structure was assessed by chl a analysis and DGGE
analysis of bacterial diversity, in addition to enumeration of bacterial and diatom densities. Chl a has been
used as an indicator of the biomass of photosynthetic
organisms in biolms (Thompson et al. 1999; von der
Meden et al. 2010). The present results showed that

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Biofouling
there was no signicant correlation between biolm
activity and chl a content, indicating that the biomass
of photosynthetic organism in natural biolms does
not directly inuence larval settlement of larvae of M.
coruscus. Comparison of the bacterial community
structure of biolms of dierent ages showed that the
community composition shifted with age, which is
consistent with previous reports (Webster et al. 2004;
Huggett et al. 2009; Chung et al. 2010; Bacchetti De
Gregoris et al. 2011). Comparative cluster analysis of
bacterial DGGE patterns also revealed that age
distinctions existed in biolm structure. The change
in community composition corresponding to biolm
age may explain dierences in the inducing activities of
biolms, further supporting the fact that bacterial
structure may play an important role in larval
settlement for this species. Huggett et al. (2009)
suggested that the shift in bacterial community
composition over time may be responsible for higher
settlement of larvae of H. elegans on the surface of old
biolms.
Some attention has been paid to the inuence of
the composition of the bacterial community on
the settlement and metamorphosis of larvae (eg
Thiyagarajan et al. 2006; Chung et al. 2010; Campbell
et al. 2011), but few studies have addressed the
importance of diatom community structure. Chiu
et al. (2007) demonstrated that the composition of
the diatom community may be as important as that of
the bacterial community with respect to metamorphosis of larvae of Crepidula onyx. Thus, the structure of
the diatom community may also be important in
relation to settlement and metamorphosis of larvae of
M. coruscus and this warrants further investigation.
In conclusion, settlement and metamorphosis of
larvae of M. coruscus were induced by natural biolms,
and the inducing activity was aected by biolm age.
Furthermore, the data suggest that the bacterial
community structure of the biolms may also play an
important role. The present study extends knowledge
of the interaction between biolms and settlement and
metamorphosis of mussels. It also provides useful
information for studies relating to biofouling and
aquaculture research.

Acknowledgements
This study was supported by the National Natural Science
Foundation of China (No. 31101885), the Innovation
Program of the Shanghai Municipal Education Commission
(10YZ123), the Chen Guang project (09CG54) supported
by the Shanghai Municipal Education Commission and the
Shanghai Education Development Foundation, the Shanghai
Rising-Star Program (10QA1403200), the Leading Academic
Discipline Project of the Shanghai Municipal Education
Commission (J50701, Marine Biology), and the Special

255

Research Funds for Selection and Cultivation of Outstanding Young Teachers of Shanghai Universities (SSC09002).

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