Effects of Biofilms on Zebra Mussel Postveliger Attachment to Artificial Surfaces

Author(s): Jerry H. Kavouras and James S. Maki

Source: Invertebrate Biology, Vol. 122, No. 2 (Spring, 2003), pp. 138-151
Published by: Wiley on behalf of American Microscopical Society
Stable URL: http://www.jstor.org/stable/3227124 .
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Invertebrate Biology 122(2): 138-151.
? 2003 American Microscopical Society, Inc.

Effects of biofilms on zebra mussel postveliger attachment to artificial surfaces

Jerry H. Kavouras and James S. Makia

Department of Biological Sciences, Marquette University, P.O. Box 1881,
Milwaukee, WI 53201-1881, USA

Abstract. The zebra mussel is an introduced fouling organism in North American inland
waters. This field study tested whether natural biofilms, formed by covering substrata with a
100-tLm mesh that allows microorganisms to attach and films to develop in the absence of
postveligers, influenced the attachment of zebra mussel postveligers to artificial surfaces. Low-
wettable polycarbonate and wettable glass surfaces were used in the experiments over four field
seasons to study biofilm formation (1997-1998) and mussel attachment (1998-2000). The pres-
ence of the mesh did not quantitatively affect biofilm development on either substratum as
determined by microscopic direct counts and colony-forming units on R2A agar. Natural bio-
films on polycarbonate surfaces positively influenced postveliger attachment compared to sub-
strata that initially had no film (ANOVA, p-values ranged from ?.05 to -.001). Biofilms did
not influence postveliger attachment to glass surfaces (ANOVA, p>.05). Attachment to both
substrata was similar on surfaces with and without previously settled postveligers. Based on
these results, we conclude that biofilms can enhance postveliger attachment to some but not
all artificial surfaces.

Additional key words: biofouling, biofilms, Dreissena polymorpha

The zebra mussel, Dreissena polymorpha (PALLAS
1771), is a recent addition to the freshwater inverte-
brate communities of North America. It colonizes nat-
ural, solid substrata and fouls (see Clare et al. 1992)
artificial structures. Since its initial discovery in Lake
St. Clair (Hebert et al. 1989), it has spread rapidly to
all of the Great Lakes, along with the Mississippi, Il-
linois, and Ohio rivers (New York Sea Grant 1999a).
Fouling by zebra mussels in North America causes
millions of dollars of damage each year (Morton
1997). The initial event in fouling by this animal is
the attachment of planktonic larvae to a solid surface.
Determination of factors that influence zebra mussel
attachment in the environment is an important step to-
wards developing environmentally acceptable methods
of control.
In marine habitats, invertebrate larvae use environ-
mental cues to determine a suitable substratum for at-
tachment (Rittschof et al. 1998). The nature of these
cues can be chemical, including both inorganic and
organic molecules, or physical, including flow, surface
energy, vibration, light, and texture (Crisp 1984; Ritts-
chof et al. 1998). Generally, there are two types of
a Authorfor correspondence.
E-mail:james.maki@marquette.edu
chemical cues involved in larval recruitment from the
plankton to a surface: conspecific and associative
(Crisp 1984; Maki 1999). Conspecific cues induce lar-
vae to settle and metamorphose on or near individuals
of their own species, while associative cues are pro-
duced by other organisms and provide information on
the choice of habitat (Crisp 1984; Maki 1999). One
source of associative cues can be the microorganisms
present in a biofilm (Maki 1999).
Biofilms have been defined as matrix-enclosed mi-
croorganisms attached to each other and/or surfaces or
interfaces (Costerton et al. 1995) and they can have a
role in the recruitment of marine larval invertebrates.
The literature contains many examples of interactions
including stimulation, inhibition, or no effect by bio-
films and/or microorganisms on the settlement and
metamorphosis of larvae of sessile marine inverte-
brates (see reviews: Wieczorek & Todd 1998; Holms-
trom & Kjelleberg 1999; Maki 1999). Biofilms have
been shown to influence the attachment of larvae of
some marine bivalves; studies on the attachment of the
marine mussel Mytilus edulis to polystyrene plates
demonstrated that the settling larvae attached to algae,
possibly a biofilm, and not directly to the surface
(Bohle 1971). Biofilms have also been shown to be a
source of cues in the recruitment of the oysters Cras-

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Biofilms and zebra mussel settlement 139

Fig. 1. Map showing the location of

the sampling site. a. The state of
Wisconsin. The asterisk indicates
the city of Milwaukee. b. The Mil-
waukee harbor.The circle indicates
the inner harbor.c. The Greenfield
Slip and surroundingarea of the in-
ner harborat Milwaukee, WI. The
arrows indicate the GreenfieldSlip
(43.01805?N, 87.9054?W) and the
Kinnickinnicriver.(Coordinatesand
maps provided by Tiger Map Ser-
vice, http://tiger.census.gov)

sostrea virginica and C. gigas (Weiner et al. 1985, (Claudi & Mackie 1994), so it was assumed that the
1989; Fitt et al. 1989). attached dreissenid larvae were Dreissena polymor-
In the aquatic environment, biofilms rapidly develop pha.
on artificial and natural substrata (Maki & Mitchell
2002). Zebra mussel attachment has been examined on Substratum wettability
many artificial and natural substrata (Walz 1973, 1975;
Surface wettability can be considered as the extent
Lewandowski 1982; van Diepen & Davids 1986; Kil-
that polar liquids spread across a solid surface (Baier
gour & Mackie 1993; Yankovich & Haffner 1993;
et al. 1968). The more wettable a surface is, the further
Wainman et al. 1996; Marsden & Lansky 2000). How-
a liquid spreads. One goal was to determine the effect,
ever, the effects of biofilms developed on artificial sur-
if any, of substrata of contrasting wettability on zebra
faces have not been thoroughly studied. The primary
mussel postveliger attachment because the initial sur-
purpose of this field study was to specifically examine
face chemistry, as indicated in this case by wettability,
the role biofilms play in the attachment of zebra mus-
can affect the adhesion of microorganisms (Baier
sel postveligers to artificial surfaces. This was done by
1980; Shea et al. 1991; Dalton et al. 1994; Busscher
statistical comparison of mussel attachment between
& van der Mei 1997), which in turn may influence
filmed and initially clean surfaces. Second, the meth-
biofilm development (Wimpenny et al. 2000), and sub-
odology also allowed the opportunity to examine
the settlement of larval invertebrates. In
whether postveliger conspecific cues might be in- sequently,
general, plastics have a low wettability and glass is
volved in settlement.
wettable, so the substrata chosen for the field studies
were borosilicate glass, baked at 500?C for 4 h (GL),
Methods
and polycarbonate, rinsed with 70% (v/v) ethanol in
Sampling distilled water (PC). The initial surface wettability af-
ter being baked or rinsed was determined using the
Field studies were performed in the Greenfield Slip
Standard Harmonic Mean (SHM) method (Gerhart et
of the inner harbor at Milwaukee, WI, which is located
al. 1992). This technique assigns a numerical value (0-
next to the University of Wisconsin Great Lakes WA-
to a surface based on drop-spread measurements
TER Institute (Fig. 1). The Kinnickinnic River flows 100)
of varying concentrations of aqueous methanol solu-
north past the Greenfield Slip (Fig. 1). In general, the
tions, which estimates the polar components of surface
water in the slip is still, so the possible contributions
The higher the numerical value is, the
of the river to the sediment load and nutrient levels wettability.
more wettable the substratum.
would be minimal. The slip is -five meters deep.
Dreissena bugensis, or the quagga mussel, has to our
Mesh method
knowledge not been reported in the Milwaukee harbor
(New York Sea Grant 1999b). The adult mussels that The role of biofilms in the attachment of postveli-
were observed in the Greenfield Slip over the duration gers of D. polymorpha was tested using a method sim-
of the study had the zebra mussel's characteristic shell ilar to the technique utilized by Todd & Keough (1994)

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140
and Keough & Raimondi (1995, 1996) in their inves-
tigations of settlement of marine invertebrate larvae.
In their studies, biofilms were developed on substrata
in the field with minimal invertebrate attachment by
covering the substrata with a plankton mesh, which
made it difficult for larval invertebrates to attach to the
substrata. After exposing the "meshed" substrata in
the field and allowing a biofilm to develop, the plank-
ton mesh was removed. The filmed substrata were then
exposed to the macroorganisms in the field. The tech-
nique allows investigators to develop biofilms in the
field and study their effects, if any, on invertebrate
attachment.
In our studies, a 100-pim mesh was used. Biofilms
were developed on the two different types of substrata
previously described in the Greenfield Slip. After ex-
posure in the slip, the mesh was removed and the
filmed substrata were exposed to settling postveligers,
along with initially clean surfaces. Attachment to the
various substrata was then compared.
Mesh effects on biofilm development
Before sampling for zebra mussel attachment in the
field, the effects of the 100-p[m mesh on biofilm de-
velopment were assessed on both substrata over time.
Three aluminum-hinged sampling devices (21 X 28 X
1 cm), each holding 20 substrata in two rows of 10
substrata, were constructed. They could be opened and
closed underwater during deployment and retrieval,
which limited the exposure of the substrata to the or-
ganic and microbially rich air-water interface. Substra-
ta (70 X 54 mm), covered or not covered by the 100-
btm mesh, were positioned randomly (using a random
number table) on the devices in a vertical orientation.
The devices were suspended from the southern side of
the Greenfield Slip at -2 m depth in an area always
shaded, and biofilms were allowed to develop for 1-,
2-, or 3-week periods.
Quantitative analysis of total bacteria attached to the
substrata was performed by acridine orange direct
counts (AODC) with epifluorescent microscopy at
1250X magnification (Maki et al. 1990a; Murray et al.
1994). The number of attached bacteria was compared
between the mesh treatments (covered or uncovered,
two substrata per treatment) to determine if the mesh
had any quantitative effect on biofilm development.
The number of attached bacteria was also compared
between GL substrata and PC substrata (covered or
uncovered, two substrata per type) to determine if
there were any quantitative differences between sur-
faces.
Semi-quantitative analysis was also performed by
comparing the number of colony-forming units (CFU)
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Kavouras & Maki
from substrata covered by the mesh or left uncovered
(two surfaces per treatment). After exposure in the har-
bor, substrata were placed in sterile Whirlpak bags
(Nasco) with sterile phosphate-buffered saline (PBS,
pH 7.6; Smibert & Krieg 1994) and scrubbed with
sterile toothbrushes to remove attached bacteria
(Burton et al. 1982). Serial dilutions of the cell sus-
pensions were prepared using PBS and used to inoc-
ulate triplicate plates of R2A (Reasoner & Geldreich
1985) agar. The plates were incubated in the dark at
room temperature (-22?C) for 2 weeks after which the
CFU were counted. Comparisons of the numbers of
CFU were made between meshed treatments (covered
and uncovered, two substrata per treatment) and be-
tween GL and PC substrata (covered or uncovered,
two substrata per type). Data were collected and ana-
lyzed over five consecutive seasons (Summer 1997
through Summer 1998).
Postveliger attachment in the field
Two sampling devices (84 X 70 X 3 cm), which
carried a maximum of 36 substrata (80 X 100 mm) in
six rows by six columns, were constructed to investi-
gate postveliger attachment in the field. The devices
were also designed with hinged doors, which were
opened after the devices were submerged and closed
before retrieval from the water, thus limiting the ex-
posure of the test substrata to the organically and mi-
crobially enriched air-water interface. These experi-
ments consisted of three periods: Prefilming, Period 1
and Period 2 (Fig. 2). Prefilming was always two
weeks in length, while Periods 1 & 2 were approxi-
mately the same length of time, ranging from 1-3
weeks each, depending on the trial. There were three
replicates of each substratum per treatment (Fig. 2).
Six substrata, three of each type and covered with
a mesh, were positioned randomly (using a random
number table) on the device in a vertical orientation.
The device was submerged in the same area of the
Greenfield Slip as described above for two weeks (Pre-
filming). After Prefilming, the device was retrieved,
the mesh was removed from the six substrata (Treat-
ment F, Fig. 2), the remaining 30 substrata (Treatments
A-E, Fig. 2) were positioned randomly on the device
according to treatment, and the device was re-sub-
merged (Period 1). After Period 1, the device was re-
trieved, all adjustments to Treatments C, D, and E
were made (Fig. 2), and the device was re-submerged
for a third time (Period 2). At the end of Period 2, the
device was retrieved, all substrata were removed, and
the attached postveligers on each surface were counted
with a dissecting microscope at 40X magnification.
Postveligers were identified based on morphology and
Biofilms and zebra mussel settlement
Treatment Prefilming Period Period
Code Period 1 2
A I I
B
B E E *
C
E li
E1 E1 pni
E2
F L1i -1
size (Conn et al. 1993). Results were expressed
number of postveligers per m2.
Treatments and planned comparisons
Various experimental treatments were used in the
postveliger attachment studies (Fig. 2). Treatments A
and D provided initially clean surfaces for exposure to
settling postveligers. Treatments B and E provided
controls for the effectiveness of the mesh on blocking
postveliger attachment. Treatments C and F provided
filmed surfaces for exposure to settling postveligers.
Four planned comparisons were created to examine if
biofilms had an effect on postveliger attachment in the
field and if postveliger conspecific cues were used. The
last three comparisons are similar to those used by
Todd & Keough (1994).
1. A vs. F. This comparison examined biofilm in-
fluence on postveliger attachment by comparing at-
tachment on Treatment A, initially clean substrata ex-
posed to zebra mussels for the entire experiment
(Periods 1 and 2), to attachment on Treatment F, filmed
substrata exposed to zebra mussels for the entire ex-
periment (Fig. 2). For example, if the biofilm enhanced
postveliger attachment, then the result should be num-
ber of mussels on Treatment F > number of mussels
on Treatment A.
2. C vs. E1 + D2. This comparison also examined
the influence of biofilms by comparing the number of
postveligers present on Treatment C (initially clean
substrata covered by a mesh during Period 1 only and
exposed to postveligers during Period 2) to the number
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141
End of
Study
--
=
Fig. 2. Diagram illustrating experi-
mental treatments used during the
field studies. Hatched boxes repre-
sent substrata covered by a mesh
and clear boxes represent uncovered
substrata. Arrows indicate length of
immersion. For Treatments C and F,
the mesh is removed, but the sub-
strata are not replaced. For Treat-
Efl_^A
ments D and E, the substrata are re-
placed. Only Treatment F substrata
were present during the Prefilming
Period.
as present on the sum of Treatments E1 and D2 (initially
clean, substrata covered by a mesh and initially clean,
substrata without a mesh, respectively) (Fig. 2). Again,
if the biofilm enhanced attachment, the result should
be the number of mussels on Treatment C > number
of mussels on Treatments E, + D2. (Note: The data
for El + D2 was derived by adding the mean of E1 to
each value of D2.)
3. A vs. D. This comparison examined the ability
of initial zebra mussel colonizers to attract secondary
colonizers (i.e., the presence of conspecific cues). At-
tachment on Treatment A (initially clean substrata ex-
posed for both Periods 1 & 2) was compared to at-
tachment on Treatment D (initially clean substrata
exposed to zebra mussels during Period 1 [D1] that was
replaced with new, initially clean substrata [D2] for
Period 2) (Fig. 2). If conspecific cues were used, then
the result should be number of mussels on Treatment
A > number of mussels on Treatment D (i.e., D1 +
D2). (Note: The data for D was derived by adding the
mean of D1 to each value of D2.)
4. A vs. (C - E1 + D1). This comparison also ex-
amined the presence of conspecific cues. Attachment
on Treatment A was compared to the attachment ob-
served on Treatment C minus the attachment on Treat-
ment El, plus the attachment on Treatment D1 (Fig. 2).
If conspecific cues were used, the result should be
number of mussels on Treatment A > number of mus-
sels on Treatments C - El + Dl. (Note: The data for
C - E1 + D1 was derived by subtracting the mean of
E1 from each value of C and adding the mean of D1.)
142
Table 1. Postveligerattachmentsamplingdates duringthe
1998, 1999, and 2000 field seasons.
Trialno. Year Dates of exposurea
1 1998 August 20-September 30
2 1999 July 12-August 13
3 1999 August 30-October 20
4 1999 September3-October 29
5 2000 June 30-August 11
6 2000 August 1-September14
a
Prefilmingperiod included.
Postveliger attachment on biofilms
Based on the results of the postveliger attachment
data collected in 1998-2000, three additional field
studies were performed in 2000-2001 with only PC to
determine the length of time necessary for develop-
ment of an influential biofilm on this substratum. Sub-
strata were covered with a 100-pxm mesh (4-6 sub-
strata) and were randomly loaded onto the sampling
device as previously described. Biofilms were allowed
to develop for three weeks, two weeks, one week, and
one day. After these substrata were filmed for the ap-
propriate length of time, the mesh was removed and
4-6 initially clean PC substrata were loaded onto the
device. All the substrata were exposed to postveligers
in the field for two weeks. After two weeks, the device
was retrieved, all substrata were removed, and the at-
tached postveligers on each surface were counted as
described with a dissecting microscope at 40X mag-
nification. During the Prefilming period of this exper-
iment, extra PC substrata were loaded on to the device
in order to examine whether postveligers attached to
the substrata covered by the mesh. The purpose of add-
ing extra PC substrata covered with a mesh was to
determine if a correction factor was necessary for these
studies (See Results).
Statistical analyses
The comparisons of bacterial direct counts (AODC)
and colony-forming units (CFU) were performed using
a three-factor analysis of variance (ANOVA). The fac-
tors were treatment (covered GL, uncovered GL, cov-
ered PC, and uncovered PC), length of biofilm devel-
opment (1, 2, and 3 weeks), and time of year (June
1997, July 1997, October 1997, December 1997, April
1998, and July 1998). Treatment and length of biofilm
development were treated as fixed factors, while time
of year was treated as a random factor. These statistical
tests were carried out using SPSS 11.0 for Windows.
A probability level of p-.05 was considered signifi-
cant. The Tukey test was utilized to determine differ-
ences among the groups.
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Kavouras & Maki
The postveliger attachment data from 1998-2000
were analyzed by three-factor ANOVA. The factors
were planned comparison (A, C, D, E1 + D2, C - E1
+ DI, and F), substratum (GL and PC), and Trial (Ta-
ble 1). Planned comparison and substratum were treat-
ed as fixed factors, while trial was treated as a random
factor. These statistical tests were carried out using
SPSS 10.0 for Windows. A probability level of p-.05
was considered significant. The Tukey test was utilized
to determine differences among the groups. If no sig-
nificant interaction was observed between planned
comparison and substratum, then each comparison was
analyzed by single-factor ANOVA within each trial.
These statistical tests were carried out using SigmaStat
2.0.3. A probability level of p<.05 was considered sig-
nificant. The Tukey test was utilized to determine dif-
ferences between the groups.
The data collected from the biofilm development ex-
periment were analyzed using single-factor ANOVA.
These statistical tests were carried out using SigmaStat
2.0.3. A probability level of p<.05 was considered sig-
nificant. The Tukey test was utilized to determine dif-
ferences among the groups.
Results
Substratum wettability
The initial substratum wettability of the test sub-
strata, before exposure in the field, showed that PC
had a SHM value of 22.3 ?+ 1.7 (n=5), indicating that
is was a low-wettable substratum. In contrast, GL had
a SHM value of 97.3 + 4.9 (n=5), indicating that it
was a wettable substratum.
Mesh effects on biofilm development
The effect of the mesh on the quantitative devel-
opment of biofilms over time was examined by com-
paring the number of attached bacteria on substrata
covered and not covered by a mesh using AODC and
CFU on R2A. A quantitative assessment of the bio-
films on GL and PC substrata was also made. There
were no differences detected among the treatments
(Tables 2-3, Fig. 3). There were also no interactions
detected between treatments and the length of biofilm
development (Tables 2-3, Fig. 3), indicating that the
mesh did not have an effect on the quantitative devel-
opment of biofilms after 1 week, 2 weeks, and 3 weeks
and that there were no quantitative differences between
the biofilms on these surfaces after 1 week, 2 weeks,
and 3 weeks. It was decided, based on the data, that 2
weeks was a sufficient amount of time for develop-
ment of a biofilm on the substrata, which could be
used in the postveliger attachment studies. Therefore,
Biofilms and zebra mussel settlement 143

Table 2. Three-factor ANOVA of AODC from substrata covered and not covered by the mesh.

Source of variation DF SS MS F p
Intercept 1 2159674.397 2159674.397 11.565 0.027
Treatment 3 28824.488 9608.163 0.829 0.503
Length of biofilm development 2 20628.461 10314.230 0.530 0.608
Time of year 4 753280.723 188320.181 8.509 0.006
Treatment X Length of biofilm development 6 14520.891 2420.148 0.264 0.948
Treatment X Time of year 12 139763.559 11646.963 1.267 0.301
Length of biofilm development X Time of year 8 157160.525 19645.066 2.147 0.072
Treatment X Length of biofilm development X Time of year 23 212076.575 9220.721 2.599 0.002

all Treatment F surfaces (Fig. 2) were filmed for 2
weeks (See below).
Postveliger attachment in the field
Field studies examining the role of biofilms in post-
veliger attachment to GL and PC substrata were per-
formed between May and October 1998, 1999, and
2000. Sampling began in May, but postveliger attach-
ment was not observed on any test surfaces until July.
The data presented here are from trials in which post-
veliger attachment to the test substrata was observed.
Postveligers were observed most of the time on
Treatments B, substrata covered by a mesh during the
entire experiment, and E, substrata covered by a mesh
during Period 1 and replaced with new substrata cov-
ered by a mesh for Period 2 (Fig. 2). There was no
difference between the total numbers of postveligers
present on Treatments B and E when they were com-
pared using analysis of variance (data not shown). If
postveligers were present on these surfaces, then it was
possible that postveligers may have attached to Treat-
ment F (Fig. 2) during the Prefilming period, which
was prior to the experiment. The number of postveli-
gers on Treatment F, substrata filmed prior to the ex-
periment and then exposed to postveligers for the en-
tire experiment, was adjusted for possible postveliger
attachment during the prefilming period by subtracting
the average number of postveligers found attached to
substrata covered by a mesh over two weeks, using the
data from Treatment B. The numbers subtracted from
Treatment F surfaces were generally <5% of the mean
number of postveligers present on these filmed surfac-
es, but never exceeded 15.5% of the mean on PC and
11.6% of the mean on GL (data not shown). However,
based on the 2000-01 postveliger attachment data, the
adjustment may not have been necessary (see last par-
agraph of this subsection).
The data analyzed by three-factor ANOVA indicat-
ed that there was no significant interaction between the
factors planned comparison and treatment (Table 4,
p>.05). Therefore, the data was analyzed by single-
factor ANOVA within each trial.
To test whether natural biofilms had an effect on
zebra mussel attachment, two sets of planned compar-
isons were made. The first set compared the number
of attached postveligers between initially clean surfac-
es (Treatment A) and filmed surfaces (Treatment F),
both exposed to postveligers for the entire experiment
(Fig. 2). The results of this comparison showed there
was a consistent difference in the number of postve-
ligers attached to these two treatments on PC substrata
(five out of six trials, Fig. 4a). In each case the filmed
Treatment F had significantly more postveliger attach-
ment than the initially clean Treatment A. Alternative-
ly, on GL substrata, only one of the six trials showed
that postveliger attachment to Treatment F was signif-
icantly greater than to Treatment A (Fig. 4b).

Table 3. Three-factor ANOVA of CFU from substrata covered and not covered by the mesh.

Source of variation DF SS MS F p
Intercept 1 3.4 X 1012 3.4 X 1012 2.353 0.186
Treatment 3 5.2 X 1011 1.7 X 1011 0.549 0.656
Length of biofilm development 2 1.15 X 1012 5.75 X 1011 0.846 0.458
Time of year 5 7.2 X 1012 1.44 X 1012 3.9 0.209
Treatment X Length of biofilm development 6 3.16 X 1012 5.26 X 1011 0.841 0.549
Treatment X Time of year 15 4.76 X 1012 3.18 X 1011 0.507 0.917
Length of biofilm development X Time of year 10 6.8 X 1012 6.8 X 1011 1.086 0.403
Treatment X Length of biofilm development X Time of year 30 1.88 X 1012 6.26 X 1011 6.122 <0.001

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144 Kavouras & Maki

The second set compared the number of attached

postveligers between a substratum covered by a mesh
1e+8
a and filmed for Period 1 that was exposed to postveli-
April 1998
gers during Period 2 (Treatment C) and a substratum
1e+7 .L TI covered by a mesh and filmed for Period 1 only (Treat-
0
L ment E1) plus an exposed substratum for Period 2 only
I-I (Treatment D2; i.e., Treatment E1 + Treatment D2)
O)
le+6
(Fig. 2). The filming with the mesh present and the
U _.
0 exposure time for this set of comparisons was about
le+5 half of that in the first set, A vs. F (Fig. 2). The results
showed that on PC, there were two of the five trials
where the filmed and exposed Treatment C had sig-
1e+4
nificantly greater postveliger attachment than the sum
1 2 3 of the filmed only Treatment E1 and the exposed only
Treatment D2 (Fig. 4c). The dates of these two trials
Time (weeks)
correspond to times where a difference was also de-
tected for A vs. F (Fig. 4a). On GL substrata, no dif-
ferences were detected (Fig. 4d). Overall, the data
from both sets of these planned comparisons showed
le+7 that postveliger attachment was enhanced by natural
biofilms on PC substrata but not on GL.
le+6
Two sets of planned comparisons were also made to
Ie+5 determine if zebra mussel postveligers used conspe-
E
0
le+4 cific cues. The first compared postveliger attachment
UL
between initially clean Treatment A, exposed to post-
U Ie+3 veligers for the entire experiment, and Treatments D1
le+2 + D2, initially clean substrata exposed to postveligers
during Period 1 (D1) or Period 2 (D2) only (Fig. 2).
le+l One difference was detected out of six trials for this
le+O set of comparisons for both PC and GL substrata and
1 2 3 they occurred at the same time (Figs. 5a, 5b). For both
substrata, the number of attached postveligers on
Time(weeks) Treatment A was significantly greater than on Treat-
ments DI + D2. The second set compared postveliger
attachment between initially clean Treatment A, ex-
posed to postveligers for the entire experiment, to ini-
CoveredPC tially clean Treatment C, filmed with a mesh for Period
'^^' UncoveredPC 1 and exposed for Period 2, minus the attachment of
CoveredGL Treatment El, covered by a mesh for Period 1 only,
, l Uncovered GL plus the attachment on Treatment on D1, exposed for
Period 1 only (Fig. 2). No differences in the second
set of comparisons were detected for either PC or GL
Fig. 3. Representative results of the effects of mesh and
initial substratum wettability on quantitative biofilm devel- substrata (Figs. 5c, 5d). Overall, the data for both sets
opment, from April 1998. Data were also collected in June, of comparisons did not reveal a consistent difference
July, October, and December 1997 and July 1998. a. Direct that could be interpreted as the use of conspecific cues.
counts of bacteria attached to PC and GL substrata that were To determine the length of time necessary to devel-
covered or not covered by a mesh. b. Colony-forming units
op an influential biofilm on PC substrata, filmed PC
(CFU) on R2A from PC and GL substrata that were covered substrata were compared to initially, clean PC substra-
or not covered by a mesh. No differences were detected
ta. There was no difference among the number of post-
among the treatments and there were no interactions detected
between treatments and the length of biofilm development veligers present on PC substrata filmed for one week,
for AODC and CFU on R2A (Tables 2-3). Similar results one day, and initially clean (Fig. 6). The number of
were obtained during the other times of the year. postveligers attached to PC substrata filmed for two

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All use subject to JSTOR Terms and Conditions
Biofilms and zebra mussel settlement 145

Table 4. Three-factor ANOVA of postveliger attachment (1998-2000).

Source of variation DF SS MS F p
Intercept 1 24032.671 24032.671 6.529 0.051
Planned comparison 5 4884.993 976.999 4.021 0.010
Substratum 1 1263.322 1263.322 11.503 0.019
Trial 5 18895.728 3779.146 12.635 0.000
Trial X Planned comparison 22 5410.161 245.916 4.306 0.001
Trial X Substratum 5 556.737 111.347 1.952 0.126
Planned Comparison X Substratum 5 666.780 133.356 2.341 0.075
Trial X Planned comparison X Substratum 22 1256.480 57.113 1.227 0.241

and three weeks was different from the number at-
tached to initially clean PC substrata (Fig. 6). Thus,
prefilming substrata for two weeks seemed to be
enough time to develop an influential biofilm. No post-
veligers were found attached to PC substrata covered
by a mesh present during the Prefilming period, so a
correction factor was not applied.
Discussion
The adaptation of the mesh method, to allow bio-
films to form while preventing larval attachment, to
the freshwater environment worked well for studying
zebra mussels. The presence of the 100-pim mesh did
not have any adverse quantitative effect on the devel-

12000 12000
10000 10000

8000 0 8000
n. S.
ce
6000 6000
o,
,....
&. 4000 a:
OA
o
0 4000
O4
2000 2000
0 0
1 2 3 4 5 6
1 2 3 4 5 6
TrialNo.
TrialNo.
6000 2500
d
m- C (GL)
5000
2000 - E + D (GL)
" 4000 a)
c) 1500 -
I 3000 0.
-
1000 -
c 2000 '"a

1000 500

0
2 3 4 5 6
0 I 2 3 4 5 6
TrialNo. TrialNo.
Fig. 4. The influence of biofilms on postveliger attachment. Comparison (A vs. F) of larval zebra mussel attachment
between initially clean substrata (A) and filmed substrata (F) in order detect the influence of biofilms on a. PC substrata
and b. GL substrata. Comparison (C vs. El + D2) of larval zebra mussel attachment between substrata covered by a mesh
during Period 1 and without a mesh during Period 2 (C), and the sum of attachment to substrata covered by a mesh present
during Period 1 only (El) plus the attachment to substrata without a mesh present during Period 2 only (D2), in order to
detect the influence of biofilms on c. PC substrata and d. GL substrata. Error bars are one standard deviation of the mean.
Asterisks indicate level of significant difference detected by ANOVA: *, p'.05; ** p'.Ol; ***, p<.OOl.

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All use subject to JSTOR Terms and Conditions
146 Kavouras & Maki

7000 5000
6000
4000
5000 E
v
8-
4000 - 3000
.)

0
3000 t 2000
2000 0
1000
1000
0 0
1 2 3 4 5 6 1 2 3 4 5 6
Trial No. Trial No.
4000 3000

2500
'4
3000
a) s- 2000
0.

| 2000

i 1000
0
X 1000
500

0 0
2 3 4 5 6 2 3 4 5 6

Trial No. Trial No.

Fig. 5. The influence of postveliger conspecific cues on postveliger attachment. Comparison (A vs. D) of larval zebra
mussel attachment between initially clean substrata (A), and the sum of attachment to substrata without a mesh present
during Periods 1 and 2 exclusively (DI and D2, respectively) in order to detect the presence of larval conspecific cues on
a. PC substrata and b. GL substrata. Comparison (A vs. C - E1 + D1) of larval zebra mussel attachment between initially
clean substrata (A), and substrata covered by a mesh during Period 1 and then without a mesh during Period 2 (C), minus
the attachment to substrata covered by a mesh present during Period 1 only (E1), plus the attachment to substrata without
a mesh present during Period 1 only (D1), in order to detect the presence of larval conspecific cues on c. PC substrata and
d. GL substrata. Error bars are one standard deviation of the mean. ** indicate a difference detected by ANOVA, p<.Ol.

opment of biofilms as determined using bacterial direct
counts (AODC) and colony-forming units (CFU) on
R2A agar (Tables 2-3, Fig. 3). However, the mesh
could have affected the diversity of microorganisms
that attached, resulting in a qualitative difference be-
tween biofilms on covered and uncovered substrata, as
well as on GL and PC substrata. Also, a quantitative
difference was not found between biofilms on wettable
GL and low-wettable PC (Tables 2-3, Fig. 3). How-
ever, surface chemistry (e.g., wettability) can affect
bacterial attachment and biofilm development (Shea et
al. 1991; Dalton et al. 1994; Busscher & van der Mei
1997; Wimpenny et al. 2000), which means that the
diversity of the microorganisms attaching to GL and
PC could have been different. Thus, the microbial
composition of the biofilms present on the substrata
could be affected by the mesh and/or initial surface
chemistry.
The 100-(pm mesh employed in these studies was
not completely successful at blocking the appearance
of postveligers on substrata covered by the mesh in
the initial postveliger attachment studies, but it was
very effective in the biofilm development experiments.
The presence of invertebrates on covered substrata was
found in other studies (Todd & Keough 1994). The
100-tJm mesh should have prevented attachment of
any postveligers, which have an average length of
>220 tpm (Conn et al. 1993). One explanation is that
zebra mussel eggs (40-50 ,um in size; Claudi & Mack-
ie 1994) or veligers (lengths 100 !Lm,Neumann et al.
1993) passed through the mesh and developed into
postveligers. This is supported by observations that ze-

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All use subject to JSTOR Terms and Conditions
Biofilms and zebra mussel settlement 147

bra mussel fertilized eggs can become trapped in shel- 6000

tered microsites, where the larvae remain and develop X 5000
(Clarke 1952; Yankovich & Haffner 1993). The cor-
rection factor employed did not eliminate any possible ?? 4000
differences between Treatments A and F (Figs. 4a,b),
so its use was deemed acceptable. Although the 100- o 3000
pxmmesh did prove effective in the biofilm develop- Q 2000
ment experiments, in future studies the use of a smaller
size mesh, such as 40 |pm, should prevent penetration E 1000
N 0
of mussel eggs and veligers. However, the effect of the O 2
smaller size on biofilm development would need to be
determined.
*^\ / 1
zo ^1*
Densities of attached postveligers observed in this
study (Figs. 4-6) were similar to densities of zebra
mussels reported in other field studies (Walz 1975; " 2500
Kilgour & Mackie 1993; Yankovich & Haffner 1993).
The purpose of the planned comparisons was to de- C, 2000
0
termine whether associative (e.g., biofilms) or conspe-
cific cues could be used by postveligers during the , 1500
C,
settling stage of their life cycle. The comparisons of 0
0.
A vs. F and C vs. E1 + D2 examined the possibility - 1000
of associative cues, while A vs. D and A vs. (C - E1
+ D1) examined the possibility of conspecific cues. E 500
The results indicate that postveligers may use associa- ,0
N 0
tive cues to identify attachment sites, but not on all
surfaces.
The data indicated that biofilms on filmed PC sub-
strata enhanced postveliger attachment. While A vs. F
consistently showed that filmed PC (Treatment F) had Eg 1200
greater densities of zebra mussels than clean PC c y,z
(Treatment A, Fig. 4a) the comparison of C vs. E1 + Q 1000
D2 detected only an occasional difference (Fig. 4a, 4c). .? 800
Because biofilms on Treatment C have less time to
develop than on Treatment F, the data suggest that the 0600-
x ,y
length of time for biofilm development on PC was a
critical component to biofilm influence on zebra mus- 0 400
sel attachment. The experiments conducted to deter- E
200
mine the length of time it takes for biofilms on PC |
substrata to become influential supported the idea that N 0 VF''t;: x21141d;i7-
i........i !..2................ I........ ...
a-z.Y;.A :x.fe.>5 :?r?r:?

exposure time was critical, indicating that postveliger IN

attachment to PC substrata filmed for -two weeks was I & 1
different than attachment to initially clean PC substrata
o^ p I

(Fig. 6). On the other hand, GL substrata showed sim-

ilar results in postveliger attachment with and without
films (Fig. 4b,d). Lewandowski (1982) filmed glass
slides in an aquarium before exposing them to zebra
mussels in the field, because he believed the film was
necessary for zebra mussel attachment. Our observa-
tions indicate that it is not necessary to film glass sur-
faces treated in the manner described before exposing
them to zebra mussels in the field. As previously men-
tioned, the GL and PC substrata used in these studies
represent extremes of initial substratum wettability, so
it is not known how far the stimulatory effects on post-
Fig. 6. Attachmentof postveligersto PC substratawith dif-
ferent levels of biofilm development. The experimentwas
performedin a. September2000, b. August 2001, and c.
September2001. PC substratawere filmed for 3 weeks, 2
weeks, 1 week, and 1 day. Attachmentto filmed PC was
comparedto initially clean PC substrataafter exposure in
the field for two weeks. Errorbarsare one standarddeviation
of the mean. The letters above each bar denote similarities
among the groups.

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148
veliger attachment would extend on substrata of inter-
mediate wettability.
A change in surface wettability is one possible ex-
planation for the ability of filmed PC substrata to en-
courage postveliger attachment. For example, Crisp et
al. (1985) showed that larvae of the marine mussel
Mytilus edulis attached in greater numbers to surfaces
with higher critical surface energies, i.e., a wettable
surface. In one study, low-wettable substrata exposed
to fresh waters became medium-wettable after 13 days,
but had a lower density of bacteria than wettable sub-
strata after 10-20 days of exposure (Meyer et al.
1988). Our data indicated there were no quantitative
differences in bacterial densities between low-wettable
PC and wettable GL over 1 week, 2 weeks, and 3
weeks (Tables 2-3, Fig. 3), so it is unlikely that biofilm
density was the influential factor on postveliger at-
tachment. If wettability alone made PC substrata with
a developed biofilm more attractive to zebra mussel
attachment than PC without a film, then it should fol-
low that the number of postveligers on initially clean
and wettable GL should have been greater than the
number on initially clean PC. However, no difference
in the number of mussels attached to these two sub-
strata could be detected (p >.05, ANOVA, Fig. 4a,
4b).
Mihm et al. (1981), studying the attachment of
bryozoan larvae to glass and polystyrene substrata,
concluded that biofilm influence on the attachment of
these larval invertebrates was more than a mere change
in wettability. Other field studies in the marine envi-
ronment have demonstrated that initial substratum wet-
tability can influence colonization by invertebrates
(Roberts et al. 1991), but these effects are not long-
term (Holm et al. 1997). Although a change in wet-
tability caused by biofilms may play some role in the
initial attachment of postveligers to a surface similar
to the conclusions of Mihm et al. (1981), it is unlikely
that it is the only cause of the observed effects of
biofilms on PC substrata.
There are testable alternatives to wettability being a
predominant factor in zebra mussel attachment. One is
that the biofilm that developed on PC was influential
due to qualitative differences. The microbial diversity
of the biofilms on PC and GL could be different, be-
cause the surfaces would have different effects on the
bioadhesion of microorganisms (Baier 1980; Shea et
al. 1991; Dalton et al. 1994; Busscher & van der Mei
1997). If this were the case, because of their different
composition, biofilms on PC substrata may be produc-
ing different extracellular products than biofilms on
GL substrata. The extracellular products from PC bio-
films would then act as a positive chemical cue to set-
tling postveligers.
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Kavouras & Maki
A second alternative is that the biofilms on GL and
PC are composed of the same populations, but the na-
ture of the substratum influences the microbial extra-
cellular products available for interactions with the
postveligers. In this case, the microbial products avail-
able to act as associative cues at the biofilm surface
are what separate the observed biofilm effects on GL
and PC substrata. This latter explanation has been
demonstrated in experiments studying the attachment
of barnacle cypris larvae, where biofilms composed of
a single bacterial species adsorbed on different sub-
strata elicited different attachment responses from the
larvae (Maki et al. 1990b; Maki et al. 1994; O'Connor
& Richardson 1996, 1998; Maki et al. 2000). Further
investigations into the diversity of the biofilms on their
respective surfaces will be necessary to begin to re-
solve this problem.
Initial postveliger colonizers did not attract more
postveligers to the surface. Comparisons of both A vs.
D and A vs. (C - E1 + D1) did not detect reproducible
differences that would clearly indicate the presence of
postveliger conspecific cues (Fig. 5). Also, in many of
the trials, it was observed that D and (C - E1 + D1)
> A (Fig. 5), which is opposite to what should have
been observed if postveliger conspecific cues were
used. These experiments examined only the ability of
zebra mussel postveligers to attract other postveligers
to a surface. Thus, the data do not rule out the possi-
bility of conspecific cues playing some role in zebra
mussel recruitment.
Some of the previous reports that examined zebra
mussel attachment suggested that exposure of artificial
substrata to an aqueous environment and/or coloniza-
tion of the surfaces with algae and bacteria (i.e., bio-
films) may have been a factor in stimulating postve-
ligers to attach to surfaces (Walz 1975; Lewandowski
1982; van Diepen & Davids 1986). Another more de-
finitive study investigating attachment to natural sur-
faces found that biofilms stimulated dreissenid (i.e.,
quagga mussel, D. bugensis and/or zebra mussel, D.
polymorpha) attachment to the three natural substrata
(mussels, shells, stones) they examined (Wainman et
al. 1996). These authors scrubbed the substrata under
study to remove biofilms and compared larval attach-
ment between scrubbed and filmed surfaces. They
found that the scrubbed substrata had 10-20% lower
mussel attachment than filmed substrata (Wainman et
al. 1996). The data reported here using artificial sub-
strata indicate that biofilms do not stimulate zebra
mussel attachment to all surfaces on which they form.
In conclusion, the mesh method for studying the ef-
fect of biofilms on postveliger attachment in the ma-
rine environment worked well for investigating zebra
mussel attachment in fresh waters. Although only GL
Biofilms and zebra mussel settlement
and PC substrata were used in the present study, the
method is readily adaptable for using a variety of nat-
ural and artificial substrata. The presence of the mesh
did not adversely affect the quantitative development
of biofilms on either substratum. However, any quali-
tative effects of the mesh were not ascertained. Field
studies demonstrated a positive role for biofilms in ze-
bra mussel attachment to polycarbonate, but not glass.
Based on our results, plastics that are submerged in
zebra mussel infested waters can be very attractive to
settling postveligers when biofilms have formed upon
them, which may explain observations previously
made by others (Walz 1975; van Diepen & Davids
1986). The results presented here may also have im-
plications for other studies using plastics as substrata
for zebra mussel attachment (Walz 1973; Kilgour &
Mackie 1993; Marsden & Lansky 2000). The eluci-
dation of the responsible regulatory signals required in
recruitment, which appears to be more than just a
change in wettability and may involve chemical cues,
is necessary in order to understand the exact role of
biofilms in zebra mussel attachment.
Acknowledgments. Thanks to Nick Herrider for construct-
ing the sampling devices, Naveen Bansal for his assistance
with the statistical analyses, and the staff of the University
of Wisconsin Great Lakes W.A.T.E.R. Institute for their ac-
commodations. Work was funded in part by the University
of Wisconsin Sea Grant Institute under grants from the Na-
tional Sea Grant College Program, National Oceanic and At-
mospheric Administration, U.S. Department of Commerce,
and from the State of Wisconsin. Federal grant number
NA46RG0481, project number R/BT-9.
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