Marine Biology 97, 199-206 (1988) Marine

BiOlOgy
..............

@ Springer-Verlag 1988

Inhibition of attachment of larval barnacles, Balanus amphitrite,

by bacterial surface films

J. S. M a k i 1, D . R i t t s c h o f 2, J. D . C o s t l o w 2 and R. M i t c h e l l 1

1 Laboratory of Microbial Ecology, Division of Applied Sciences, Harvard University; Cambridge, Massachusetts 02138, USA
2 Duke University Marine Laboratory; Pivers Island, Beaufort, North Carolina 28516, USA

Abstract (Marshall 1976, Fletcher and Marshall 1982, Mitchell and

Kirchman 1984) which can then be a source of stimuli to
Films of bacteria on solid substrata can positively or nega-
settling larvae (Mitchell and Kirchman 1984). Previous re-
tively influence the attachment of marine invertebrate lar-
ports in the literature have presented conflicting ob-
vae. Effects of marine bacteria on the attachment of cypris
servations on the effect of microbial films on the at-
larvae of the barnacle Balanus amphitrite Darwin were
tachment of barnacle cyprids (e.g. Visscher 1928, Harris
examined in the laboratory. Bacteria, grown to mid-ex-
1946, Crisp and Meadows 1962, Tighe-Ford et al. 1970).
ponential phase and allowed to adsorb irreversibly to poly-
However, larvae from other marine invertebrates including
styrene petri dishes, attached in densities of 107 cells cm -2.
spirorbid polychaetes (Knight-Jones 1951, Meadows and
Assays (22 h) were used to compare the effects of adsorbed
Williams 1963, Kirchman et al. 1982a), several species of
cells of 18 different bacterial species on larval barnacle at-
bryozoa (Mihm et al. 1981, Brancato and Woollacott 1982)
tachment. Most of the adsorbed bacteria either inhibited or
and oysters (Weiner et al. 1985) were shown to respond
had no effect on larval attachment compared to clean sur-
positively to microorganisms on the surface to which they
faces. Experiments testing the effect of larval age on bar-
attach. In some cases a specific bacterium is involved in the
nacle attachment were conducted with six species of bac-
production of the inducer of larval attachment and meta-
teria and showed that older larvae attached in higher per-
morphosis (Muller 1973, Neumann 1979, Kirchman et al.
centages to clean surfaces and that bacterial films generally
1982b, Schmahl 1985, Weiner et al. 1985). The bacterium
inhibited larval attachment. Both the species of bacteria
may only produce the stimulatory substance when at a cer-
and the in situ age of the adsorbed bacteria affected bar-
tain growth stage (e.g. Schmahl 1985, Weiner et al. 1985).
nacle attachment response: older films of Deleya
We hypothesized from preliminary experiments that
(Pseudomonas) marina were more inhibitory. Bacterial extra-
different species of bacteria could elicit various attachment
cellular materials may be involved in the inhibitory process.
responses from cypris larvae and we set out to examine the
following questions: (1) What is the effect of cypris age on
their attachment to films to different species of bacteria?
(2) What is the effect of the age and the nurturing of the
Introduction
bacterial film on cypris attachment? (3) What is the effect
The planktonic larval stages of many sessile marine in- of the density of bacteria on cypris attachment? (4) Can we
vertebrates are recruited to surfaces in response to surface extend results obtained with single species of bacteria to
associated stimuli (Crisp 1984). The barnacle larval stage multispecies situations? We report here the results of
known as the cyprid is non-feeding and specialized for dis- laboratory experiments testing the attachment responses of
persal, settlement, and metamorphosis to the adult. cypris larvae of the barnacle Balanus amphitrite on bacteria
Cyprids possess modified antennules that may serve as tac- irreversibly attached (Marshall et al. 1971) to polystyrene
tile chemical receptors in addition to their role as organs of surfaces.
attachment (Nott and Foster 1969). Physical and chemical
stimuli are important in determining a substratum for bar-
nacle settlement and metamorphosis (Lewis 1978, Crisp Materials and methods
1984).
One consequence of placing solid surfaces in marine All seawater used in the experiments was passed through a
ecosystems is the colonization of those surfaces by bacteria septic, 100 000 Dalton, ultrafiltration system (Millipore)
200 J.S. Maki et al.: Bacterial films and barnacle attachment

and subsequently passed through two sterile 0.2-#m pore USA) and harvested by centrifugation. Bacteria were
size filters (Millipore), placed one on top of the other. washed, centrifuged, and resuspended in seawater (109
Seawater used to dilute the medium in aged film exper- cells ml-1). Petri dishes were filled with 7 ml of the bacte-
iments was also autoclaved. rial solution and incubated at 26 ~ for 2.5 to 3.0 h. Dishes
were rinsed by immersing them ten times in 500 ml of
seawater. Bacteria still attached to the dishes were con-
Bacteria sidered irreversibly adsorbed (Marshall et al. 1971). The
dishes were then filled with 6 ml of seawater.
Initially, Balanus amphitrite Darwin attachment to bacteri- Experiments were performed using either these dishes
al films was tested using 18 cultures of marine bacteria. (for convenience termed Day I) or with dishes in which the
Eight were pure cultures obtained from the American Type bacteria were aged in situ for several days. Aging the at-
Culture Collection (Rockville, MD, USA) (Table 1). Seven tached bacteria in the dishes was accomplished in two
of these eight species were originally isolated directly from ways: every 24 h after the initial filling of the Day I dish
marine or estuarine environments. The eighth species, a with seawater, it was emptied, and either (1) fresh seawater
strain of Vibrio vulnificus, although isolated from blood, is was added, or (2) a 1:10 dilution of Marine Broth 2216 (in
also an estuarine bacterium and requires NaC1 for optimal autoclaved seawater) was used to refill the dish. Aged dish-
growth. The other ten cultures were isolated from rock sur- es were rinsed as above and refilled with 6 ml of seawater
faces and surface waters in the estuarine environment near prior to experiments. To investigate the effect of bacterial
the Duke University Marine Laboratory on Marine Agar density upon cypris attachment, dishes were exposed to the
2216 (Difco, Detroit, MI, USA). These isolates were all bacterial solution for 30, 60, 120, or 180min before the
gram negative, oxidase positive rods, but varied in their dish was emptied and rinsed as above. To examine the ef-
oxidation/fermentation catabolization of glucose (Leifson fect of more natural multispecies films on cypris at-
1963). Further identification is currently in progress. In or- tachment, petri dishes were submerged in a flow-through
der to preserve the bacteria and their respective surface seawater table for 24, 72, or 120 h to allow films to develop
characteristics, cultures upon receipt or isolation were before rinsing and filling the dishes in preparation for the
frozen in vials with glycerol (Gherna 1981) from which experiment.
new stock cultures were periodically established. Prior to, and following the larval attachment exper-
iments, two dishes of each bacterial treatment and control
were fixed with formaldehyde (final concentration 1 to 2%,
Preparation of dishes with attached bacteria v/v) for quantification of attached bacteria using acridine
orange and epifluorescence microscopy (Daley and Hobbie
Bacteria were allowed to attach to polystyrene petri dishes 1975). These dishes did not receive any larvae. At least 300
(Falcon 1006, 5 0 x 9 m m ) following the methods of bacteria were counted per dish and the number of ceils ex-
Fletcher (1977). Cells were grown to mid-exponential pressed as bacteria per cm 2. Prior to fixation, pH and dis-
phase at 26~ in Marine Broth 2216 (Difco, Detroit, MI, solved oxygen (D. O.) were measured in the experimental
dishes using a flat surface electrode (Fisher) combined with
an Orion Research model 801A pH meter, and a probe
Table 1. List of American Type Culture Collection species of bac- combined with a YSI oxygen meter (Yellow Springs Instru-
teria that were used to examine barnacle attachment ment Co., Yellow Springs, OH, USA), respectively.
Species ATCC Source Reference Measurements were compared to levels in the seawater
no. used to fill the dishes. Aged films were streaked on Marine
Agar 2216 to check for contamination.
Alteromonas 27126 Seawater Baumann et al.
macleodii (1972)
Deleya marina 25374 Seawater Cobet et al.
(1970) Preparation of settlement factor
Pseudomonas 19262 Red Algae Yaphe (1957)
atlantica Settlement factor (Crisp and Meadows 1962, Larman et al.
P. perfectomarina 14405 Seawater Zobell and
Upham (1944) 1982), a heat-stable preparation of glycoproteins that pro-
Vibrio algino- 17749 Seawater Sakazaki motes attachment of larval barnacles, was prepared as de-
lytieus a (1968) scribed by Rittschof et al. (1984).
V. campbelli 25920 Seawater Baumann et al.
(1971)
V.fluvialis" 33812 Estuarine Lee et al.
(1981) Test cyprids and the attachment assay
V. vulnifieus a 27562 Blood Reichelt et al.
(1976) Test cyprids were Balanus amphitrite, the most common
balanomorph barnacle found in warm coastal and estuar-
a These three Vibrio spp. were obtained from Dr. R.P. Herwig
(University of Washington, Seattle, USA). All other species were ine waters (Bishop 1950, Crisp and Molesworth 1951, Cost-
obtained directly from the American Type Culture Collection low and Bookhout 1958). Cyprids were mass cultured from
J. S. Maki et al.: Bacterial films and barnacle attachment 201

Stage 1 nauplii on a diet of Skeletonema costatum as pre- Table 2. Balanus amphitrite cypris larvae attachment: preliminary
viously described by Rittschof et al. (1984). The day of experiments with Day I bacterial films. (Species listed according to
percent larvae attached)
metamorphosis from sixth stage nauplius to cyprid was
considered Day 0. Each experimental series used cyprids Species Total no. % total vs
from a single mass spawning from a large number of of Larvae Larvae poly-
adults. (3 replicates) attached styrene
The attachment assay was that previously described by
A a
Branscomb and Rittschof (1984) and Rittschof et al. (1984).
Control polystyrene 264 43 -
Experiments were designed to examine the effects ofcypris Pseudomonas 152 33 NS
age, bacterial film age, and bacterial density on the at- atlantica
tachment of cyprids. The percentage of Balanus amphitrite Vibriofluvialis 163 31 NS
cyprids attaching to polystyrene increases with the age of K alginol/ticus 152 28 S
P. perfectomarina 260 23 S
the larvae due to changes in larval discrimination, with the V. campbeIli 140 18 S
most dramatic change occurring between 2- and 4-d-old K vulnificus 153 8 S
larvae (Rittschof et al. 1984). The sensitivity of the assay is A. macleodii 123 7 S
such that stimulation of cypris attachment is most easily Deleya marina 129 4 S
demonstrated with younger cyprids (i.e. 2-d-old) and inhi- Bb
bition of cypris attachment is most easily demonstrated Control polystyrene 118 66 -
with older larvae (i.e. 4-d-old). With the exception of the DLS1 95 86 S
preliminary experiments, all barnacle attachment assays DLR1 105 74 NS
DLR2 111 69 NS
were conducted using cyprids that were 2- or 4-d-old. Bar- DLR3 92 64 NS
nacle larvae were aged in the dark at 4 ~ to 6 ~ (Rittschof DLS5 124 63 NS
et al. 1984, 1986). Under these conditions, larvae are inac- DLR4 117 63 NS
tive and do not attach to the storage containers. Cyprids DLS4 92 58 NS
DLR5 135 57 NS
were warmed to ambient temperature within 2 h of use.
DLS3 70 54 NS
Controls included seawater with no additions (polystyrene) DLS2 78 42 S
and seawater with 2.5 #g m1-1 settlement factor as a posi-
tive control. Cypris larvae, 15 to 200, were placed in three a Experiment used 2-d-old cyprids. Significance was tested with a
replicate dishes of each treatment (i.e. bacterial films and G statistic from a contingency analysis. A family level of signifi-
cance of a = 0.04 was obtained by using Bonferroni's method for
controls) and incubated for 22 h at 28 ~ At the end of the multiple comparisons with the individual significance level at
incubation interval, dishes were emptied and unattached a/8=0.005 with 1 df, where 8 is the number of comparisons.
cyprids were immobilized by suction filtration and count- NS = not significant. S = significant
ed. Larvae that were not removed by rinsing the dishes b Experiment used 3-d-old cyprids. Significance was tested as
above. A family level of significance of a = 0.05 was obtained as
with distilled water were counted as attached.
above with the individual significance level was a/lO =0.005
Frequencies of attached and not attached individuals with 1 df, where 10 is the number of comparisons. NS = not sig-
combined from the three replicates were compared be- nificant. S = significant
tween treatments by G statistic, corrected for continuity,
generated from a contingency analysis (Zar 1984). The null
hypothesis for the contingency analysis was that there was le/a marina (= Pseudomonas marina: Baumann et al. 1983)
no significant difference between the control (polystyrene) (ATCC 25374), Vibrio campbelli (ATCC 25920), and V. vul-
and any one treatment. The family and individual level of nificus (ATCC27562); two that showed no effect,
significance in each group o f comparisons was determined Pseudomonas atlantiea (ATCC 19263) and isolate DLR1, a
using Bonferroni's method for multiple comparisons (Seber gram negative, oxidative rod-shaped bacterium; and one
1977). that appeared stimulatory, isolate DLS1, a gram negative,
fermentative rod-shaped bacterium.

Results
Preliminary experiments
Preliminary experiments tested cyprid attachment to Day I
films of all 18 species of bacteria (Table 2). The results,
when compared to polystyrene, indicated that seven
species were inhibitory, ten species showed no effect, and
one species was stimulatory (all using G statistic, family
level of significance at least 0.05, with 1 df, Table 2). Based
upon these results, six species of bacteria were chosen for
further experiments: three that appeared inhibitory, De-
Bacterial densities
The adsorption technique used with the pure cultures of
bacteria resulted in densities of l07 attached bacteria per
cm 2 (Tables 3 and 4). Most of the dishes had a high degree
of bacterial confluency, although some bacteria did attach in
small clumps with gaps of 5 to 10 p m between clumps. Sev-
eral of the films, particularly the bacteria aged in situ that
received diluted medium, were multilayered, and thus the
direct counts of the bacteria from these dishes represent a
minimum estimate. Mixed films on dishes placed in flow-
202 J.S. Maki et al.: Bacterial films and barnacle attachment

Table 3. Balanus amphitrite cypris larvae attachment: data from experiments using Day I bacterial films and 4-d-old larvae. A and B: data
from two different experiments

Treatment No. of bacteria a Total no. % larvae G statistic c vs polystyrene

( x 107) cm -2 (_+ SD) of larvae b attached G P

A
Polystyrene nd d 229 69.9
Settlement factor nd 192 79.7 4.83 NS
Pseudomonas atlantiea 4.52 (0.54) 126 34.1 42.28 < 0.001
Deleya marina 3.76 (0.14) 187 28.9 71.06 < 0.001
Vibrio campbelli 2.11 (0.53) 96 42.7 21.37 < 0.001
V. vulnificus 3.81 (0.21) 167 23.9 82.79 < 0.001
DLS 1 1.74 (0.20) 85 49.4 10.18 < 0.005
DLR1 5.84 (0.47) 97 34.0 34.67 < 0.001
B
Polystyrene nd 234 47.0
Settlement factor nd 201 56.2 3.32 NS
P. atlantiea 5.67 (0.05) 250 10.4 82.42 < 0.001
D. marina 1.48 (0.39) 237 22.8 29.81 < 0.001
V. campbelli 3.53 (0.08) 219 29.7 13.45 < 0.001
V. vulnificus 1.62 (0.18) 251 24.3 26.61 < 0.001
DLS1 3.29 (0.28) 195 27.7 16.22 < 0.001
DLR1 4.35 (0.32) 218 13.3 61.61 < 0.001

Mean number of attached bacteria per cm 2 from counts of 4 dishes determined using epifluorescence microscopy after staining with
0.01% (final concentration, wt/vol) acridine orange. Bacteria were grown to mid-exponential phase in Marine Broth 2216 (Difco, De-
troit, MI, USA) at 26 ~ harvested by centrifugation, washed and resuspended to 109 cells m1-1. Dishes were exposed to bacterial solu-
tion for 2.5 to 3.0 h before being rinsed and used in experiments
b Total no. of larvae in 3 replicates
c Using Bonferroni's method of multiple comparisons the family level of significance in each experiment was a = 0.035 with an individual
significance level of a/7 = 0.005 with l df where 7 is the number of comparisons. NS = not significant
d n d = none detected

through seawater tables reached densities o f 106 attached
bacteria per cm 2 after being i m m e r s e d for several days
(Table 5). Attached bacteria were undetected on untreated
polystyrene dishes indicating that the sterile filtration was
fairly effective in removing any bacteria. The densities o f
attached bacteria on in situ aged dishes were sometimes
lower than on D a y I dishes, suggesting that some bacteria
may have desorbed from the surface or lysed. Microscopic
measurements o f the attachment organ o f the cyprid larvae
of Balanus amphitrite used in the experiments indicated
that the organ had an ovoid surface area o f 340/~m 2.
Direct counts of the attached bacteria corrected to this area
were in the 102 range. In all the experiments there was no
change in either p H or D. O. in the dishes.
Barnacle attachment
Larvae attached in similar percentages to polystyrene con-
trols (2-d-old m e a n = 2 3 . 3 % , n = 9 ; 4-d-old m e a n = 4 7 . 9 % ,
n = 8 ) as previously reported (Rittschof et al. 1984). The
m a x i m u m percent attachment and the exact timing when
high percent attachment began to occur on polystyrene
varied between batches o f larvae.
Experiments with cyprids o f different ages
Bacterial films (Day I) of the six species were tested for
their effect upon attachment of 2- and 4-d-old cyprids.
Settlement factor enhanced attachment o f 2-d-old cyprids
(settlement factor = 36 to 49% vs polystyrene = 20%) in two
experiments (P < 0 . 0 5 - < 0.001, G statistic with 1 df). All
bacterial treatments resulted in cyprid attachment at or be-
low polystyrene control levels (family level o f signifi-
cance=0.035, individual level o f significance=0.005, G
statistic with 1 df). Three species o f bacteria, Deleya
marina, Vibrio campbelli, and DLR1, inhibited attachment
of 2-d-old cyprids in at least one of the experiments. No
enhancement o f cypris attachment by DLS1 or any other
bacterium was observed in either experiment.
Experimental results with D a y I bacterial films and
4-d-old cyprids to test treatments for inhibition of at-
tachment showed an increase in overall percentage larval
attachment as c o m p a r e d to 2-d-old cyprids (Table 3). As
expected, settlement factor and polystyrene resulted in
similar cypris attachment. Treatments o f all six species o f
bacteria significantly inhibited cypris attachment when
compared to the polystyrene control (Table 3). Thus,
although higher percentages of older larvae attached to
bacterial films, they did not reach the levels o f attachment
observed in either control.
Film condition experiments
Experiments were conducted with attached bacteria of one
species, Deleya marina, to examine bacteria aged in situ on
the surface and nurturing of bacteria for possible stimula-
J. S. Maki et al.: Bacterial films and barnacle attachment 203

Table 4. Balanus amphitrite cypris larvae attachment: data from experiments using Deleya marina aged in situ with 4-d-old larvae. A and
B: results from two separate experiments

Treatment No. of bacteria" Total no. % larvae G statistic: vs polystyrene

(X 107) cm -2 (• of larvae b attached G P

A
Polystyrene nd d 209 59.8
Day I 6.29 (0.33) 187 22.4 56.74 < 0.001
Day III 4.93 (0.23) 98 12.2 65.19 < 0.001
Day III ( + ) 4.22 (0.12) 111 3.6 111.83 < 0.001
Day V 4.45 (0.74) 152 5.3 127.58 < 0.001
Day V ( + ) 4.18 (0.17) 118 5.1 107.96 < 0.001
B
Polystyrene nd 73 16.4
Day I 1.14 (0.27) 101 9.9 1.09 NS
Day III 2.73 (0.15) 101 3.9 6.46 NS
Day III ( + ) 1.99 (0.02) 97 4.1 6.05 NS
Day V 0.84 (0.21) 98 4.1 6.16 NS
Day V ( + ) 3.36 (0.15) 97 3.1 7.75 <0.01

a Mean number of attached bacteria per cm 2 from counts of 4 dishes determined using epifluorescence microscopy after staining with
0.01% (final concentration, wt/vol) acridine orange. Bacteria were grown to mid-exponential phase in Marine Broth 2216 (Difco, De-
troit, MI) at 26 ~ harvested by centrifugation, washed and resuspended to 109 cells m1-1. Dishes were exposed to bacterial solution for
2.5 to 3.0 h before being rinsed and used for experiments. Day I dishes were prepared the same day of the experiment, while Day III
and Day V dishes were prepared 3 and 5 d prior to the experiment, respectively. Seawater in Day III and Day V dishes was replaced
daily after their preparation while Day III ( + ) and Day V ( + ) dishes were refilled daily with a 1 : 10 dilution of Marine Broth 2216 in
autoclaved seawater after their preparation
b Total no. of larvae in 3 replicates
c Using Bonferroni's method of multiple comparisons the family level of significance in each experiment was a = 0.05 with an individual
significance level of a/5 = 0.01 with 1 dfwhere 5 is the number of comparisons. NS = not significant
nd =none detected

tory or inhibitory activity using 2- and 4-d-old cypris lar-
vae, respectively. Films o f D. marina were aged in situ
either in seawater or in autoclaved seawater s u p p l e m e n t e d
with nutrients.
Deleya marina treatments o f different ages and nutrient
conditions resulted in attachment o f 2-d-old cyprids not
significantly different from polystyrene control levels (fam-
ily level o f significance= 0.05, individual level o f signifi-
cance = 0.01, G statistic with 1 df). Films of D. marina o f all
ages and nutrient conditions inhibited the attachment o f
4-d-old cypris larvae c o m p a r e d to polystyrene controls in
the first experiment (Table 4). In a second experiment with
4-d-old larvae, although the percentages of attached
cyprids were lower in the presence o f bacteria, they were
not always significant (Table 4). Even though estimates o f
the numbers of bacteria per unit surface area did not ap-
pear to increase (Table 4) older films were more inhibitory
to attachment than D a y I films using 4-d-old cyprids (A.
D a y I vs D a y V, G = 20.12, P < 0.001, with 1 df; B. D a y I vs
D a y V ( + ) , G = 2 . 8 2 , P < 0 . 1 0 with 1 dr). Examination o f
the agar plates inoculated with bacteria from the aged
dishes revealed only one colony type.
al solutions for varying amounts of time. These different
exposure times resulted in densities of 106 to 107 cells cm 2.
Frequencies of attached and unattached cypris larvae in
dishes exposed to bacteria for 180 min were c o m p a r e d to
other exposure times. There were no significant differences
in attachment of either 2-d or 4-d-old cyprids between
treatments with varying densities of D. marina in any ex-
periments (family level o f significance=0.075, individual
level of significance = 0.025, G statistic with 1 df).
Multi-species film experiments
Dishes placed in the flow-through seawater tables resulted
in bacterial densities of 106 bacteria cm 2 (Table 5). Exper-
iments with cypris attachment on these dishes yielded simi-
lar results as those with pure cultures o f bacteria, i.e. no
stimulatory effect was observed with 2-d-old larvae (family
level of significance = 0.015, G statistic with 1 df), and 4-d-
old larvae were significantly inhibited by nearly all treat-
ments (Table 5).

Bacterial density experiments
Experiments to determine the effect of the density of ad-
sorbed Deleya marina cells on cypris attachment were con-
ducted using dishes that were exposed to the same bacteri-
Discussion
The irreversible attachment o f bacteria to a substratum is
time d e p e n d e n t and generally involves an extracellular
polymer as an adhesive (Fletcher and Marshall 1982). This
adhesive p o l y m e r m a y be either polysaccharide (Suther-
204 J.S. Maki et al.: Bacterial films and barnacle attachment

Table 5. Balanus amphitrite cypris larvae attachment: data from experiments using mixed films of bacteria of different ages and 4-d-old
larvae. A and B: results from two separate experiments

Treatment No. of bacteria" Total no. % larvae G statistic c polystyrene

(X 107) cm -2 (+__SD) of larvae b attached vs G P

A
Polystyrene - 155 53.5
24 h 0.07 (0.01) 82 36.6 5.57 NS
72 h 0.13 (0.02) 81 32.1 9.15 < 0.005
120 h 0.53 (0.07) 57 19.3 19.62 < 0.001
B
Polystyrene - 167 41.3
24 h 0.09 (0.01) 96 5.2 44.34 < 0.001
72 h 0.84 (0.24) 147 18.4 18.84 < 0.001
120 h 0.64 (0.05) 143 6.9 50.76 < 0.001

a Mean number of attached bacteria per cm2 from counts of 4 dishes determined using epifluorescence microscopy after staining with
0.0170 (final concentration, wt/vol) acridine orange. Polystyrene dishes were allowed to accumulate attached bacteria by placing them
in a flow through seawater table for 24, 72 or 120 h. Dishes were rinsed and used in experiments
b Total no. of larvae in 3 replicates
c Using Bonferroni's method of multiple comparisons the family level of significance in each experiment was a = 0.015 with an individual
significance level of a/3 = 0.005 with 1 df where 3 is the number of comparisons. NS = not significant

land 1983) or protein (Fletcher and Marshall 1982) in
composition. Because exopolymer production by attached
bacteria may be altered both quantitatively and qualita-
tively depending upon growth and environmental con-
ditions (Fletcher and Marshall 1982), the surface chemistry
of a bacterial film being explored by a settling larva can be
quite complex and may not be solely dependent upon the
number of attached bacteria once a certain density of cells
is reached. Substratum surface chemistry has been demon-
strated to be important in the attachment response of bar-
nacle cypris larvae (Crisp and Meadows 1962, 1963, Lewis
1978, Branscomb and Rittschof 1984, Rittschof et al. 1984,
1985, Crisp et al. 1985, Rittschof 1985). In particular, the
cyprid has been shown to be stimulated to attach in the
presence of settlement factor proteins (Crisp and Meadows
1962, 1963) adsorbed onto surfaces but not in solution
(Crisp and Meadows 1962, 1963, Rittschofet al. 1984, 1985,
Rittschof 1985) and to respond to settlement inhibitors ad-
sorbed to surfaces (Rittschofet al. 1985).
Earlier investigations have reported stimulation, inhi-
bition or no effect of bacterial films on the attachment of
barnacle cyprids (Visscher 1928, Harris 1946, Crisp and
Meadows 1962, Tighe-Ford et al. 1970). More recently,
Hudon et al. (1983) observed bacteria at all attachment
sites of the cypris larvae of Balanus crenatus but did not
compare them to uncolonized sites. Our data show that at-
tachment of B. amphitrite cypris larvae on adsorbed bacte-
ria on surfaces can vary with the species of bacteria (e.g.
inhibition, no difference, or possible stimulation). This ob-
servation is similar to data reported by Kirchman et al.
(1982 a) for larval attachment of the spirorbid polychaete
Janua (Dexiospira) brasiliensis and may help explain the
above reports describing a variety of effects of microbial
films on larval attachment. Although we only had one case
of stimulation by a bacterium it is probable that stimula-
tory bacteria exist. The presence or absence of a certain
species of bacteria and its extracellular products in a film
can influence the settlement behavior and subsequent at-
tachment of competent larvae.
Our data also indicate that the age of the Balanus am-
phitrite cypris larvae influenced their attachment response,
supporting the results of Branscomb and Rittschof (1984)
and Rittschof et al. (1984). In the present study, generally
higher percentages of 4-d-old larvae attached to both con-
trols and pure culture bacterial films than did 2-d-old lar-
vae. However, the bacterial films appeared to inhibit at-
tachment, especially o f the older larvae which are less dis-
criminating of the substratum than younger cyprids. This
result was observed in experiments involving both pure
culture and multi-species films.
We also demonstrated that the age of the bacterial film
influenced the attachment response of Balanus amphitrite
cypris larvae. Aged films of Deleya marina became more
inhibitory to larval attachment. Chemical factors in the
exopolymer of the bacteria may be involved, the quantity
and quality of which can be changed by the growth stage
of the bacterium a n d / o r its physiological state. Bacterial
extracellular products have been indicated to stimulate the
larval attachment of a spirorbid polychaete (Kirchman et
al. 1982b) and an oyster (Weiner et al. 1985). Specific
growth stages a n d / o r nutrient conditions have been report-
ed to be necessary for some bacteria to produce stimula-
tory factors for larval metamorphosis (Muller 1973,
Schmahl 1985, Weiner et al. 1985). Even though isolate
DLS1 was stimulatory in only one experiment, its possible
stimulatory nature may also be highly dependent upon
such conditions and further investigations are currently in
progress.
The method of bacterial attachment used in this investi-
gation resulted in 107 attached bacteria per cm 2 for the
pure cultures, which is comparable to previous reports
of bacterial attachment to polystyrene (Fletcher 1977,
J. S. Maki et al.: Bacterial films and barnacle attachment
Fletcher and Loeb 1979). The numbers of attached bacteria
from the pure cultures, although they were achieved in less
time, were about an order o f magnitude higher than those
found on the dishes i m m e r s e d in the seawater table for 5 d,
but were c o m p a r a b l e to the numbers reported by H u d o n et
al. (1983) for sites in the natural environment. Tile data for
Deleya marina suggest that, once bacterial densities are
above 106 cells cm 2, density has no a p p a r e n t effect upon
cyprid attachment.
Barnacle cyprids use their antennules for settlement on
a substratum and for subsequent p e r m a n e n t attachment to
it (Nott and Foster 1969). These antennules secrete a pro-
teinaceous adhesive (Walker and Yule 1984) and adhesion
is stronger to surfaces that have been treated with settle-
ment factor proteins (Yule and Crisp 1983). It has been
suggested that barnacle larvae m a y not settle on or p e r m a -
nently attach to substrata to which their t e m p o r a r y pro-
teinaceous adhesive does not bind strongly (Crisp et al.
1985). This m a y explain the response o f the cypris larvae to
the different bacteria and the multi-species films in the
present study. I f the cypris antennular cement cannot bind
strongly to the exopolymer o f a bacterium, the larvae m a y
not settle or attach a n d leave to search for a suitable sub-
stratum for metamorphosis. On the other hand, if an exo-
polymer allows strong binding with the antennular cement,
the larvae m a y settle and p e r m a n e n t l y attach in greater
numbers. We are currently attempting to better define the
nature o f both the inhibitory and possible stimulatory fac-
tors involved in the attachment o f these cyprid larvae on
bacterial films. The characterization of both types of factors
would be useful in ecological and applied research.
Acknowledgements. Special thanks are given to the excellent
technical assistance of S. Wolff and P. Sullivan at Harvard and E.
S. Branscomb and A. Schmidt at Duke. We also thank Drs. T. E.
Ford, J. F. Manning, J. D. Oliver, and H. Spencer for their helpful
comments and criticisms of an earlier draft of this manuscript. This
work was supported in part by ONR contracts ~N00014-78-
C-0294 and #N00014-86-K-0261 (Duke University), ONR con-
tract :~N00014-85-K-0061 (Harvard University), and NOAA Sea
Grant No. NA79AA-D-00091 (Harvard University).
Literature cited
Baumann, P., Baumann, L., Mandel, M. (1971). Taxonomy of ma-
rine bacteria: the genus Beneckea. J. Bact. 107:268-294
Baumann, L., Baumann, P., Mandel, M., Allen, R. D. (1972). Tax-
onomy of aerobic marine eubacteria. J. Bact. 110:402-429
Baumann, L., Bowditch, R. D., Baumann, P. (1983). Description of
Deleya gen. nov. created to accomodate the marine species
Acaligenes aestus, A. pacificus, A. cupidus, A. venustus, and
Pseudomonas marina. Int. J. Syst Bact. 33:793-802
Bishop, M. W. H. (1950). Distribution of Balanus amphitrite Dar-
win var. denticulata Broch. Nature, Lond. 165:409-410
Brancato, M. S., Woollacott, R. M. (1982). Effect of microbial films
on settlement of bryozoan larvae (Bugula simplex, B.
stolonifera, and B. turrita). Mar. Biol. 71:51-56
Branscomb, E. S., Rittschof, D. (1984). An investigation of low fre-
quency sound waves as a means of inhibiting barnacle settle-
ment. J. exp. mar. Biol. Ecol. 79:149-154
Cobet, A. B., Wirsen, C. Jr., Jones, G. E. (1970). The effect of nick-
el on a marine bacterium, Arthrobacter marinus sp. nov. J. gen.
Microbiol. 62:159-169
205
Costlow, J. D. Jr., Bookhout, C. G. (1958). Larval development of
Balanus amphitrite var. denticulata Broch reared in the labora-
tory. Biol. Bull. mar. biol. Lab., Woods Hole 114:284-295
Crisp, D. J. (1984). Overview of research on marine invertebrate
larvae, 1940-1980. In: Costlow, J. D., Tipper, R. C. (eds.) Ma-
rine biodeterioration: an interdisciplinary study. Naval Insti-
tute Press, Annapolis, p. 103-126
Crisp, D. J., Meadows, P. S. (1962). The chemical basis of gregari-
ousness in cirripedes. Proc. R. Soc. Lond. Ser. B 156:500-520
Crisp, D. J., Meadows, P. S. (1963). Adsorbed layers: the stimulus
to settlement in barnacles. Proc. R. Soc. Lond. Set. B 158:
364-387
Crisp, D. J., Molesworth, A. H. N. (1951). Habitat of Balanus am-
phitrite var. denticulata in Britain. Nature, Lond. 167:489-490
Crisp, D. J., Walker, G., Young, G. A., Yule, A. B. (1985). Adhe-
sion and substrate choice in mussels and barnacles. J. Coll. In-
terface Sci. 104:40-50
Daley, R. J., Hobbie, J. E. (1975). Direct counts of aquatic bacteria
by a modified epifluorescence technique. Limnol. Oceanogr.
20:875-882
Fletclaer, M. (1977). The effects of culture concentration and age,
time, and temperature on bacterial attachment to polystyrene.
Can. J. Microbiol. 23:1-6
Fletcher, M., Loeb, G. I. (1979). Influence of substratum charac-
teristics on the attachment of a marine pseudomonas to solid
surfaces. Appl. envir. Mircobiol, 37:67-72
Fletcher, M., Marshall, K. C. (1982). Are solid surfaces of ecologi-
cal significance to aquatic bacteria? Adv. microb. Ecol. 6:
199-236
Gherna, R. L. (1981). Preservation. In: Gerhard, P., Murray,
R. G. E., Costilow, R. N., Nester, E. W., Wood, W. A., Krieg, N.
R., Phillips, G. B. (eds.) Manual of methods for general micro-
biology. Washington, D.C., Am. Soc. Microbiol. p. 208-217
Harris, J. E. (1946). Report on anti-fouling research, 1942-1944. J.
Iron Steel Inst. 154:297-334
Hudon, C., Bourget, E., Legendre, P. (1983). An integrated study
of the factors influencing the choice of the settling site of
BaIanus crenatus cyprid larvae. Can. J. Fish. aquat. Sci. 40:
1186-1194
Kirchman, D., Graham, S., Reish, D., Mitchell, R. (1982 a). Bacte-
ria induce settlement and metamorphosis of Janua (Dexio-
spira) brasiliensis Grube (Polychaeta: Spirorbidae). J. exp. mar.
Biol. Ecol. 56:153-163
Kirchman, D., Graham, S., Reish, D., Mitchell, R. (1982b). Lectins
may mediate in the settlement and metamorphosis of Janua
(Dexiospira) brasiliensis Grube (Polychaeta: Spirorbidae). Mar.
Biol. Lett. 3:131-142
Knight-Jones, E. W. (1951). Gregariousness and some other as-
pects of the setting behaviour of Spirorbis. J. mar. biol. Ass.
U. K. 30:201-222
Larman, V. N., Gabbott, P. A., East, J. (1982). Physico-chemical
properties of the settlement factor proteins from the barnacle
Balanus balanoides. Comp. Biochem. Physiol. 72 B: 329-338
Lee, J. V., Shread, P., Furniss, A. L., Bryant, T. N. (1981). Tax-
onomy and description of Vibrio fluvialis sp. nov. (Synonym
Group F Vibrios, Group EF6). J. appl. Bact. 50:73-94
Leifson, E. (1963). Determination of carbohydrate metabolism of
marine bacteria. J. Bact. 85:1183-1184
Lewis, C. A. (1978). A review of substratum selection in free-living
and symbiotic cirripeds. In: Chia, F. S., Rice, M. E. (eds.)
Settlement and metamorphosis of marine invertebrate larvae.
Elsevier, New York, p. 207-218
Marshall, K. C. (1976). Interfaces in microbial ecology. Harvard
University Press, Cambridge, 156 pp.
Marshall, K. C., Stout, R., Mitchell, R. (1971). Mechanism of the
initial events in the sorption of marine bacteria to surfaces. J.
gen. Microbiol. 68:337-348
Meadows, P. S., Williams, G. B. (1963). Settlement of Spirorbis
borealis Daudin larvae on surfaces bearing microorganisms.
Nature, Lond. 198:610-611
206
Mihm, J. W., Banta, W. C., Loeb, G. I. (1981). Effects of adsorbed
organic and primary fouling films on bryozoan settlement. J.
exp. mar. Biol. Ecol. 54:167-179
Mitchell, R., Kirchman, D. (1984). The microbial ecology of ma-
rine surfaces. In: Costlow, J. D., Tipper, R. C. (ed.) Marine bio-
deterioration: an interdisciplinary study. Naval Institute Press,
Annapolis, p. 49-56
Muller, W. A. (1973). Induction of metamorphosis by bacteria and
ions in the planulae of Hydraetinia echinata; an approach to
the mode of action. In: Tokioka, T., Nishimura, S. (ed.) Proc.
2nd Int. Syrup. on Cnidaria (Recent trends in coelenterate
biology). PuN. Seto Mar. Biol. Lab., no. 20, pp. 195-208
Neumann, R. (1979). Bacterial induction of settlement and meta-
morphosis in the planula larvae of Cassiopea andromeda (Cni-
daria: Scyphozoa, Rhizostomeae). Mar. Ecol. Prog. Ser. h
21-28
Nott, J. A., Foster, B. A. (1969). On the structure of the antennular
attachment organ of the cypris larva of Balanus balanoides (L.).
Philas. Trans. R. Soc. Lond. Ser. B 256:115-134
Reichelt, J. L., Baumann, P., Baumann, L. (1976). Study of genetic
relationships among marine species of the genera Beneckea
and Photobacterium by means of in vitro DNA/DNA hybridi-
zation. Arch. Microbiol. 110:101-120
Rittschof, D. (1985). Oyster drills and the frontiers of chemical
ecology: unsettling ideas. Am. malacol. Bull., Spec. Ed. No. 1:
111-116
Rittschof, D., Branscomb, E. S., Costlow, J. D. (1984). Settlement
and behavior in relation to flow and surface in larval bar-
nacles, Balanus amphitrite Darwin, J. exp. mar. Biol. Ecol. 82:
131-146
Rittschof, D., Hooper, I. R., Branscomb, E. S., Costlow, J. D.
(1985). Inhibition of barnacle settlement and behavior by natu-
ral products from whip corals, Leptogorgia virgulata (Lamarck
1815). J. Chem. Ecol. 11:551-563
Rittschof, D., Maki, J., Mitchell, R., Costlow, J. D. (1986). Ion and
neuropharmacological studies of barnacle settlement. Neth. J.
Sea Res, 20:269-275
J.S. Maki et al.: Bacterial films and barnacle attachment
Sakazaki, R. (1968). Proposal of Vibrio alginolyticus for the bio-
type 2 of Vibrio parahaemolytieus. Japan. J. Med. Sci. Biol. 21:
359-362
Schmahl, G. (1985). Bacterially induced stolon settlement in the
Scyphopolyps of A ureIia aurita (Cnidaria, Scyphozoa). Hel-
gol~inder Meeresunters. 39:33-42
Seber, G. A. F. (1977). Linear regression analysis. John Wiley and
Sons, New York
Sutherland, I. W. (1983). Microbial exopolysaccharides - their
role in microbial adhesion in sequeous system. CRC Crit. Rev.
Microbiol. 10:173-201
Tighe-Ford, D. J., Power, M. J. D., Vaile, D. C. (1970). Laboratory
rearing of barnacle larvae for antifouling research. Hel-
gol~inder wiss. Meeresunters. 20:393-405
Visscher, J. P. (1928). Nature and extent of fouling of ships' bot-
toms. Bull. U.S. Bur. Fish. 43:193-252
Walker, G., Yule, A. B. (1984). Temporary adhesion of the bar-
nacle cyprid: the existence of an antennular adhesive secretion.
J. mar. biol. Ass. U. K. 64:679-686
Weiner, R. M., Segall, A. M., Colwell, R. R. (1985). Characteri-
zation of a marine bacterium associated with Crassostrea vir-
ginica (the Eastern Oyster). Appl. Envir. Microbiol. 49:83-90
Yaphe, W. (1957). The use of agarase from Pseudomonas atlantiea
in the identification of agar in marine algae (Rhodophyceae).
Can. J. Microbiol. 3:987-993
Yule, .4. B., Crisp, D. J. (1983). Adhesion of cypris larvae of the
barnacle, Balanus balanoides, to clean and arthropodin treated
surfaces. J. mar. biol. Ass. U. K. 63:261-271
Zar, J. H. (1984). Biostatistical analysis, 2nd ed., 718 pp. Prentice-
Hall, Inc., Englewood Cliffs
Zobell, C. E., Upham, H. C. (1944). A list of marine bacteria in-
cluding descriptions of sixty new species. Bull. Scripps Inst.
Oceanog. 5:239-292
Date of final manuscript acceptance: September 28, 1987.
Communicated by J. M. Lawrence, Tampa