Aquaculture 424–425 (2014) 228–233

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Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Effects of natural biofilms on settlement of plantigrades of the mussel

Mytilus coruscus
Jin-Long Yang a,b,c,d,⁎, Xiang Li a, Xiao Liang a, Wei-Yang Bao e, He-Ding Shen a,b,c,d, Jia-Le Li a
a
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
b
Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai 201306, China
c
Shanghai Engineering Research Center of Aquaculture, Shanghai 201306, China
d
Shanghai University Knowledge Service Platform, Shanghai Ocean University Aquatic Animal Breeding Center (ZF1206), Shanghai 201306, China
e
Institute of Marine Science and Technology, Yangzhou University, Jiangsu 225009, China

a r t i c l e i n f o a b s t r a c t

Article history: The effect of natural biofilms on settlement of plantigrades of Mytilus coruscus was investigated in the
Received 16 July 2013 laboratory. Plantigrades settled in response to natural biofilms, and the percentages of plantigrade settlement
Received in revised form 3 January 2014 increased with biofilm age. The settlement-inducing activity of biofilms was positively correlated with age-
Accepted 7 January 2014
related characteristics of the biofilm, such as dry weight, thickness, chlorophyll a concentration and densities
Available online 16 January 2014
of bacteria and diatoms. Cluster analysis of denaturing gradient gel electrophoresis revealed high similarity
Keywords:
between bacterial communities in biofilms according to biofilm age, indicating that bacterial community
Mytilus coruscus structure may not play an important role in settlement of plantigrades in this species. Therefore, natural biofilms
Settlement may be used to enhance settlement of plantigrades for the Chinese aquaculture industry.
Plantigrade © 2014 Elsevier B.V. All rights reserved.
Natural biofilms
Biofilm age

1. Introduction plantigrades, post-larvae, spat or juveniles could detach from primary

In the marine environment, biofilms exist on all surfaces and play a
key role in mediating biotic interactions and biogeochemical activities
(Qian and Dahms, 2009). Additionally, they mediate larval settlement
and metamorphosis of many marine invertebrates (Hadfield, 2011;
Hadfield and Paul, 2001; Qian et al., 2007), provide a suitable food
source to newly metamorphosed juveniles, and serve as biofilters to
absorb and biodegrade excessive nutrients (Qian et al., 2007). Yu et al.
(2010), for example, demonstrated that natural biofilms could promote
larval settlement of the pearl oyster Pinctada fucata, and Campbell
et al. (2011) showed that biofilms on hard surfaces enhanced larval
settlement of the native Eastern oyster Crassostrea virginica.
Mussels possess a planktonic larval phase preceding a benthic adult
phase. Larvae will survive to the stage of competence for settlement
if suitable environmental conditions are available, but they can delay
settlement until they found a suitable substrate (Crisp, 1974), such as
biofilms (Alfaro et al., 2011; Bao et al., 2007a,b; Dobretsov, 1999;
Ganesan et al., 2010; Satuito et al., 1995, 1997). However, mussel settle-
ment is not permanent and the metamorphosed individuals, termed
⁎ Corresponding author at: College of Fisheries and Life Science, Shanghai Ocean
University, Shanghai 201306, China. Tel.: +86 21 61900440; fax: +86 21 61900405.
E-mail address: jlyang@shou.edu.cn (J.-L. Yang).
settlement sites and reattach on alternative habitats (Bayne, 1964;
Buchanan and Babcock, 1997; Kavouras and Maki, 2003). The secondary
settlement behavior of mussels is important for the distribution of
populations and could contribute to local recruitment dynamics
(Corre et al., 2013). Despite the long-recognized role of biofilms as
potential inducers of mussel larval settlement and metamorphosis,
knowledge of the interactions between marine biofilms and resettle-
ment of metamorphosed plantigrades of mussels is limited.
The mussel, Mytilus coruscus Gould, 1860, which inhabits the
temperate zone along the coastal waters of East Asia (Chang, 2007),
is an important aquaculture species in China (Chang and Wu, 2007).
Recently, due to the overexploitation of wild resources of this species,
it is difficult to meet the market demands (Chang and Wu, 2007).
Thus, development of hatchery techniques has become of economic
interest in this species. Efforts have been made to understand the role
of biofilms on larval settlement and metamorphosis. However, there
has been no effort to clarify the effects of biofilms on settlement of
plantigrades beyond the larval settlement, and the patterns that medi-
ate the mechanism of settlement of plantigrades remain unknown for
this species. In the present study, we investigated the effects of natural
biofilms on settlement of plantigrades of the mussel M. coruscus. The
characteristics of biofilms of different ages were investigated according
to their dry weight, thickness, chlorophyll a (chl a) concentration,
bacterial and diatom densities and bacterial community structure.
The purpose of this study was to gain insights into the settlement

0044-8486/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaculture.2014.01.007
 J.-L. Yang et al. / Aquaculture 424–425 (2014) 228–233
mechanism of plantigrades of M. coruscus, and to provide valuable
information for artificial culture of plantigrades of this species.
2. Materials and methods
Details of the bioassays used in this study are provided in Table 1 to
facilitate understanding of the experimental design. Briefly, the
settlement-inducing activities of natural biofilms with different ages
(7 days, 14 days, 21 days and 28 days) on plantigrades were investigat-
ed using 9 replicates. The characteristics of those biofilms including dry
weight, thickness, chl a concentration, bacterial and diatom densities
and bacterial community structure were analyzed.
2.1. Preparation of natural biofilms
Biofilm slips were prepared by immersing clean glass slips (half
portions of microscope glass slides; 38 mm × 26 mm) in coastal
seawater at Gouqi Island (122°46′E; 30°43′N), Zhejiang, China. The
glass slips were placed on PVC holders and immersed at a depth of
0.5–1.0 m below the water surface for 7–28 days. Slips were brought
back to the laboratory on the same day of the bioassay and were
thoroughly washed with autoclaved 1.2 μm-filtered sea water
(AFSW) before the assays.
2.2. Measurement of biofilm dry weight
Dry weight was measured by the method of Bao et al. (2007a).
The biofilms on each glass slip were scraped off using a sterile glass
slide and separately suspended in AFSW. Each suspension was collected
on a pre-weighed GF/C filter (Whatman glass fiber filter; pore size:
1.2 μm) by filtration. Each filter paper holding the biofilm was washed
with 50 ml of 0.22 μm filtered distilled water, dried for 48 h in an
oven at 80 °C and cooled to room temperature in a desiccator before
weighing. The dry weight of the biofilm was determined after
subtracting the weight of the filter.
2.3. Quantifications of densities of bacteria and diatoms
Biofilms were fixed in 5% formalin for a maximum of 3 weeks.
Samples were washed with AFSW and stained with acridine orange
(AO, 0.1%) for 5 min. Bacterial densities of stained samples were
counted under the microscope at 1000 × magnification using an
Olympus BX51 epifluorescence microscope. Diatoms were counted
directly at 200× magnification under a light microscope immediately
after samples were brought back to the laboratory. The densities of
bacteria and diatoms in each sample were quantified from ten random
fields of view.
Table 1
Schematic representation of experimental design.
General Experimental setup
Biofilm work Preparation of natural biofilms
The characteristics of biofilms of different ages
Measurement of biofilm dry weight
Quantifications of densities of bacteria and diatoms
Determination of biofilm thickness
Chl a concentration in biofilms
Bacterial community analysis
DNA extraction
PCR amplification of 16S rDNA
Bacterial community profiling by denaturing gradient
gel electrophoresis
Mussel work Culture of plantigrades
Settlement bioassays
229
2.4. Determination of biofilm thickness
Biofilm thickness was measured by the method of Yang et al. (2013).
The biofilms with four ages were fixed in 5% formalin solution for 24 h.
Biofilms were stained with propidium iodide (5 μg ml−1) and incubated
for 15 min in the dark. Slides were washed three times and then ob-
served at 400 × magnification under an Olympus FluoView™ FV1000
Laser-Scanning Confocal microscope. Three replicate biofilms were
examined for each age of biofilms. Ten random fields of view of each
biofilm were selected for imaging and analysis. Thirty image stacks
of varying thickness were generated to determine the full thickness
of the biofilms in each field of view.
2.5. Chl a concentration in biofilms
Chl a concentration was measured by the method of Wang et al.
(2012). Biofilms were scraped from 3 replicate glass slides using sterile
glass slides, filtered through membranes and preserved at −20 °C. Chl a
extraction was conducted at 4 °C 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 and 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):
½12:12  ðD664−D750Þ−1:58  ðD647−D750Þ−0:08  ðD630−D750Þ  Ve  d
chl a ¼
A
where, chl a is chl a concentration (μg cm− 2) in the biofilm; D630,
D647, D664 and D750 are the absorptions at 630, 647, 664 and
750 nm; Ve, the extraction volume; A, the area of substratum
surface; d, the optical length of the cuvette.
2.6. DNA extraction
Biofilms were scraped from 3 replicate glass slides as above and
centrifuged for 5 min at 10,000 g. The supernatant was discarded
and genomic DNA was extracted using a 3S DNA Isolation Kit
for Environmental Samples V2.2 following the manufacturer's
instructions (Shenergy Biocolor Bioscience and Technology Company,
Shanghai, China).
2.7. PCR amplification of 16S rDNA
Bacterial 16S rRNA genes were amplified using the primers 357F,
which contains a GC clamp (5′-C GCC CGC CGC GCG CGG CGG GCG
GGG CGG GGG CAC GGG GGG CCT ACG GGA GGC AGC AG-3′) and
518R (5′-ATT ACC GCG GCT GCT GG-3′) (Muyzer et al., 1993). PCR am-
plification was performed in a 25 μl reaction mixture containing 0.5 μl of
each primer (10 μM), 1 μl of template DNA (40–80 ng), 0.25 μl of Ex Taq
(5 U/μl), 2.5 μl of 10 × PCR buffer, 2 μl of MgCl2 (25 mM), 0.5 μl of
deoxynucleotide triphosphates (10 mM each) and sterile distilled
water to a final volume of 25 μl. PCR cycling was carried out in an
Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) thermocycler
under the following conditions: an initial denaturing step at 94 °C for
5 min, a touch-down thermal cycling of denaturation at 94 °C for
1 min, annealing at 65–55 °C for 1 min (reducing 0.5 °C per cycle) and
elongation at 72 °C for 0.5 min. Then 15 additional PCR cycles were con-
ducted, each cycle consisting of 1 min denaturation at 94 °C, 1 min an-
nealing at 55 °C and 0.5 min synthesis at 72 °C. Finally, an extension
step was carried out at 72 °C for 8 min. PCR products were verified by
agarose gel electrophoresis (1.2% weight/volume agarose) with
ethidium bromide staining and visualized using an ultraviolet (UV)
transilluminator.
230 J.-L. Yang et al. / Aquaculture 424–425 (2014) 228–233
2.8. Bacterial community profiling by denaturing gradient gel electrophoresis
Denaturing gradient gel electrophoresis (DGGE) of the PCR
amplified 16S rDNA was carried out using the D-Code™ Universal
Mutation Detection System (Bio-Rad, Hercules, CA, USA). PCR products
were resolved on a vertical gel containing 8% (w/v) polyacrylamide
(acrylamide:bisacrylamide 37.5:1) and a denaturing gradient ranging
from 40 to 55%. One hundred percent denaturant is defined as a 7 M
urea and 40% (v/v) deionized formamide. Electrophoresis (60 V, 12 h)
was performed in 1 × TAE buffer (40 mM Tris–acetate, 20 mM acetate,
1 mM Na2-EDTA) at 60 °C. 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 num-
ber of bands observed in all biofilm bacterial communities and the pres-
ence 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.
2.9. Culture of plantigrades
Plantigrades (shell length/height = 1.28 ± 0.01 mm/0.85 ±
0.01 mm) of the mussel M. coruscus were supplied by Shengsi
Service Center of Marine Science and Technology Development,
Zhoushan, China, and shipped to Shanghai Ocean University,
Shanghai, China. Plantigrades were transferred and cultured in 2 l aerat-
ed glass beakers with filtered seawater (acetate-fiber filter: 1.2 μm pore
size) at an initial density of 10 plantigrades ml−1. Plantigrades were
fed a diet of Chaetoceros gracilis at 10–20 × 104 cells ml−1day−1. The
culture water was changed every day and the temperature maintained
at 18 °C. Plantigrades were cultured for more than 1 week and were
used in settlement bioassays.
2.10. Settlement bioassays
Ten plantigrades were transferred into individual glass Petri dishes
(Ø64 mm × 19 mm height) containing 20 ml AFSW and one biofilm
slip. The inducing activity of the biofilms (i.e. biofilm effect) was
evaluated by the percentage of settled plantigrades after 24 h. Settled
plantigrades were verified after 24 h when they attached to the surface
with byssal threads. Petri dishes, each containing 20 ml of AFSW, 10
plantigrades and a clean (non-biofilmed) glass slip were set up as
negative controls in all assays. Assays were conducted at 18 °C in
darkness with 9 replicates for each condition.
2.11. Data analysis
Settlement-inducing activities of the biofilms were evaluated by the
percentages of settled plantigrades. Prior to statistical analysis, all data
expressed in percentages were arcsine-transformed and tested for
normality and homogeneity of variance. Normality of distribution was
assessed using Shapiro–Wilk test. Homogeneity of variance was verified
using O'Brien test. The effects of age on biofilm effect, dry weight,
bacterial and diatom densities, biofilm thickness and chl a were
analyzed using the Kruskal–Wallis test because these data did not
meet parametric assumption even after transformation. Correlations
between biofilm age or biofilm effect and dry weight, bacterial and
diatom densities, biofilm thickness and chl a were analyzed using a
Spearman's rank correlation test. The DGGE profiles were analyzed
with Smart View and bacterial diversity was calculated with the
Shannon diversity index. All statistical computations were per-
formed using JMP™ software. Differences were considered significant
at p b 0.05.
3. Results
3.1. Effects of natural biofilms on settlement of plantigrades
The percentages of settled plantigrades on natural biofilms of dif-
ferent ages are shown in Fig. 1. In the negative controls, only 10 ± 2%
plantigrades were observed to settle on non-biofilmed glass slips. In
general, the percentages of settlement significantly increased with
the age of biofilms (Kruskal–Wallis: p b 0.0001). The maximum
percentage of settlement was observed with 28 day biofilms. The
relationship between biofilm effect, bacterial density and diatom
density is shown in Table 2. Dry weight was significantly correlated
with the biofilm effect (Spearman's rank correlation test: p b 0.0001).
Similarly, the densities of bacteria and diatoms, the thickness and
the chl a concentration were also correlated with the biofilm effect
(Spearman's rank correlation test: p b 0.0001).
3.2. Dry weight, bacterial and diatom densities, thickness and chl a at
different ages
The dry weights, the bacterial and diatom densities, the thickness
and the chl a concentrations of biofilms at different ages are shown in
Fig. 2. There was no significant difference between the mean dry weight
of the 7 day and 14 day biofilms (Kruskal–Wallis test: p N 0.05, Fig. 2A),
and both were lower than the weight of the 28 day biofilms (Kruskal–
Wallis test: p b 0.05, Fig. 2A). Biofilm age significantly affected the
bacterial and diatom densities. The bacterial density increased with
the biofilm age until day 21 and no significant increase was observed
between 21 day biofilms and 28 day biofilms (Fig. 2B). By contrast,
diatom density significantly increased with the biofilm age (p b 0.05)
until day 28 (Fig. 2C). Biofilm age significantly affected the thickness
of the biofilm (Fig. 2D). The biofilm thickness increased with the
increasing biofilm age (p b 0.05), the maximum being obtained with
28 day biofilms (Fig. 2D). No significant increase in chl a was observed
until day 21 and the chl a concentration was maintained stable after
that (Fig. 2E).
The relationship between the biofilm age and dry weight, densities
of bacteria and diatoms, thickness and chl a concentration is shown
in Table 2. The dry weight, the density of bacteria and diatoms, the
thickness and the chl a concentration were all positively correlated to
biofilm age (Spearman's rank correlation test: p b 0.0001).
3.3. Bacterial community analysis by DGGE
A representative DGGE gel of biofilms of different ages is shown in
Fig. 3. Comparative analysis of bacterial DGGE patterns using cluster
analysis is shown in Fig. 4. In Batch 1, cluster analysis revealed that 21
Fig. 1. Percentages of settlement of M. coruscus plantigrades on natural biofilms of
different ages. Data with significant difference are indicated by different letters.
Data are means (± SE) of 9 replicates.
 J.-L. Yang et al. / Aquaculture 424–425 (2014) 228–233 231

Table 2
Correlation analysis run between biofilm activity or biofilm age and five factors (dry weight, bacterial and diatom densities, thickness and chlorophyll a).

Dry weight Bacterial density Diatom density Thickness Chlorophyll a

rs p rs p rs p rs p rs p

Biofilm effect 0.878⁎ b 0.0001 0.897⁎ b 0.0001 0.940⁎ b 0.0001 0.961⁎ b 0.0001 0.878⁎ b 0.0001
Biofilm age 0.878⁎ b 0.0001 0.897⁎ b 0.0001 0.940⁎ b 0.0001 0.961⁎ b 0.0001 0.878⁎ b 0.0001

rs: Spearman's rank order correlation analysis; p: p-value.

⁎ Significant at p b 0.05.

day biofilms and 28 day biofilms shared 78% similarity and that these
biofilms shared 74% and 68% with 7 day biofilms and 14 day biofilms, re-
spectively. A similar tendency of clustering was observed in Batch 3. In
Batch 2, clustering of bacterial DGGE patterns was observed between
the 7 day biofilms and 14 day biofilms as well as between the 21 day
biofilms and 28 day biofilms.
4. Discussion and conclusions
The results of the present investigation demonstrate that natural
biofilms promoted the settlement of plantigrades of M. coruscus. Biofilm
activity was positively correlated with the biofilm dry weight, thickness,
chl a concentration and bacterial and diatom densities, variables all
which co-vary with biofilm age. Previous studies also have shown
that other factors such as water flow (Alfaro, 2005, 2006), air bubbles
(Alfaro, 2006), light (Carl et al., 2011; Kobak, 2001), macroalgae (Davis
and Moreno, 1995), adult mussel beds (Bayne, 1964), macroscopic or-
ganisms (Ank et al., 2009), and microtopography (Carl et al., 2012)
also mediate the settlement of plantigrades or juveniles of mussels.
Biofilms are surface-associated communities of microorganisms and
of their mucilaginous extracellular products (Decho, 2000; Flemming
and Wingender, 2010; Shikuma and Hadfield, 2010). Biofilms have
been reported to enhance or inhibit larval settlement of many marine
invertebrates (Dobretsov, 2009; Dobretsov et al., 2013; Hadfield,
2011; Hadfield and Paul, 2001; Qian et al., 2007; Wieczorek and Todd,
1998). However, the role of the biofilms on the settlement of planti-
grades or juveniles of mussels remains little known. Ank et al. (2009)
showed that biofilms had no significant effect on settlement of juveniles
of the brown mussel Perna perna. In contrast, the results of the present
investigation show that biofilms significantly enhanced settlement
of plantigrades of M. coruscus, indicating that different mussel species
exhibit varying responses to the biofilms. In addition, all tested char-
acteristics of biofilms co-varied with the biofilm age in our present
experiment design. Therefore, effects of single factor of the biofilm,
such as bacteria and diatoms, on plantigrade settlement need to be
further investigated.
Biofilm age has been shown to be positively correlated to larval
settlement of mussels (Bao et al., 2007a; Toupoint et al., 2012; Wang

Fig. 2. Dry weights (A), bacterial density (B), diatom density (C), thickness (D) and
chlorophyll a concentration (E) of natural biofilms of different ages. Data with significant
difference are indicated by different lower-case letters. Dry weights are means (±SE) of 9
replicates. Bacterial and diatom densities are means (±SE) of 10 random fields of view.
Biofilm thicknesses are means (±SE) of 30 replicates. Chlorophyll a concentrations are Fig. 3. DGGE fingerprint of the bacterial 16S rRNA gene fragments from natural biofilms of
means (±SE) of 6 replicates. different age.
232 J.-L. Yang et al. / Aquaculture 424–425 (2014) 228–233
Fig. 4. Dendrogram generated from the DGGE profiles, showing the similarities of bacterial
community composition.
et al., 2012). However, the effect of interaction between biofilm age and
plantigrade settlement remains little known. In the present study,
settlement rates of plantigrades of M. coruscus increased with the
biofilm age and this finding is consistent with previous reports on
mussel larval settlement (Bao et al., 2007a; Toupoint et al., 2012;
Wang et al., 2012). Further research needs to be conducted on
a broader screening of more mussel species for the purpose of
obtaining a good understanding of interaction between biofilm age
and plantigrade settlement.
Previous studies have reported that the inducing activity of biofilms
on mussel larval settlement was positively related with dry weight as
well as bacteria and diatom densities (Bao et al., 2007a; Wang et al.,
2012), which was confirmed by our results. Wang et al. (2012) sug-
gested that larval settlement of M. coruscus might not be directly
influenced by the biomass of photosynthetic organism in biofilms.
By contrast, Toupoint et al. (2012) suggested that the overall influ-
ence of biofilms on larval settlement of the mussel M edulis
appeared to be related to the concentration of photosynthetic cells.
The present results demonstrated that the chl a concentration did
not increase until day 21 and no further increase was observed
even after 28 days. This finding indicates that chl a might not be
related to diatom density, but might be an indicator of other algae
colonizing the biofilm.
The results of the present study showed that plantigrade settlement
is positively correlated to the thickness of the biofilm, indicating that
thickness is one of the important drivers inducing the settlement
of M. coruscus plantigrade. By contrast, Yang et al. (2013) suggested
that larval settlement of M. coruscus is not related to the thickness of
the bacterial biofilms.
Bacterial communities of natural biofilms have been known to be
important for settlement of larvae of some marine invertebrates
(Chung et al., 2010; Wang et al., 2012; Yu et al., 2010). In the present
investigation, comparative cluster analysis of bacterial DGGE patterns
showed that there was little age distinction in biofilm community struc-
ture in two batches, indicating that biofilm community structure may
not be crucial in the process of plantigrade settlement in this species.
By contrast, previous reports have demonstrated that the community
composition in biofilms shifts with age (Chung et al., 2010; Huggett
et al., 2009; Wang et al., 2012). Huggett et al. (2009) suggested that
this shift in community composition over time may be responsible
for higher larval settlement of Hydroides elegans on the surface of
older biofilms.
In conclusion, natural biofilms enhanced settlement of plantigrades
of M. coruscus, and the settlement rates of plantigrades increased
with biofilm age. This is the first study to investigate settlement of
plantigrades of M. coruscus in response to natural biofilms, and provides
useful information for the artificial culture of M. coruscus.
Acknowledgments
This study was supported by the National Natural Science
Foundation of China (No. 31101885), the Shanghai Municipal Science
and Technology Commission (12230502100), the Innovation Program
of Shanghai Municipal Education Commission (14ZZ143), the Shanghai
Rising-Star Program (10QA1403200) and the Ecological Remediation
Project on the Adjacent Water of Qingcaosha Reservoir.
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