Biological Effects of Zosteric Acid in Solution:
Barnacle Behavior and Settlement, Bacterial
Attachment, and Sea Urchin Fertilization
Corita R. Grudzen and Suejin Hwang
Correspondence can be sent to:
Corita Grudzen
Suejin Hwang
ABSTRACT
Zosteric acid (p- sulphooxy cinnamic acid) was studied for its role as a
non-toxic antifouling agent. Cypris larvae of B. amphitrite were used to study
the effects of the compound on larval chemotaxis, mobility, and settlement.
Zosteric acid (ZA) elicited no chemotactic response from the larvae and
therefore it does not appear to inhibit settlement by deterrence. ZA and
sodium acetate, a weak acid with a similar pKa, did both cause larval
immobility, possibly acting as weak acids and changing intracellular pH. Both
pH neutralized (8.0) and unbuffered ZA were shown to inhibit settlement by
up to 30%. Because the presence of microbial films can act as settlement cues
for further fouling by both larval invertebrates and algae, ZA’s effect on
bacterial attachment was also investigated. Both zosteric acid, a sulfated
phenolic ester, and p- coumaric acid, an unsulphated analog of ZA, were
found to suppress bacterial attachment. Therefore, it can be concluded from
this study that the sulphate moiety is not the necessary component for
inhibiting bacterial adherance. The effects of ZA were also assayed on the
attachment of sperm to the egg in the fertilization of the sea urchins
Strongylocentrotus purpuratus and Litichinus pictus. It was found that the
sulphate group on the ZA is necessary for the inhibition of fertilization.
Another fertilization experiment, in which Protease was used to remove the
vitelline layer of the egg, showed that ZA may inhibit fertilization by binding
to the egg receptors on the vitelline layer, thus blocking sperm from
attaching. In conclusion, it should be noted that ZA has a wide variety of
effects and more than one mode of action. Multiple mechanisms seem to be
involved, both individually and synergystically, in producing ZA's
antifouling effect.
INTRODUCTION
Biofouling, the attachment of a wide variety of organisms to à
submerged surface, has been and continues to be a major economic concern.
Both microscopic and macroscopic organisms contribute substantially to drag
and the loss of integrity of ships and other vessels. In the past, the most
effective antifouling compounds have been toxic to marine life prompting
continued research in this area.
The recent identification and synthesis of p-(sulphooxy) cinnamic acid,
commonly known as zosteric acid (ZA), by Dr. Richard Zimmerman is one of
the first natural non-toxic compounds shown to prevent fouling of a wide
range of organisms (Todd, Zimmerman, Crews, and Alberte, 1993). This work
was inspired by the observation that new leaves of Zostera marina, from
which the compound was isolated, were essentially free of fouling organisms
from marine bacteria to barnacles (Harrison, P.G., 1982).
The objective of this study was to determine the mechanism(s) by
which zosteric acid inhibits fouling. The most prevalent form of fouling is
barnacle settlement (Christie, A.O. and Dalley, R., 1987). Therefore, three
Balanus amphitrite bioassays analyzing the effects of ZA on larval
chemotaxis, mobility, and settlement were performed to determine the
compound's mode of action of ZA. The effects of ZA on marine bacteria
were also studied because the presence of microbial films was shown to act as
a settlement cue in recruiting cypris larvae to surfaces (Maki et al., 1990)
Cypris larvae respond to surface associated stimuli during settlement and
metamorphosis. Attached bacteria can present various stimuli by changing
the surface chemistry through their physical presence and or by the
production of extracellular molecules. Therefore, by preventing the
formation of bacterial films, the primary initiator of fouling that leads to the
successive growth of both algae and marine invertebrates, ZA may inhibit
barnacle settlement.
Zosteric acid was also tested in another type of attachment assay, the
attachment of the sperm to the egg in the fertilization of sea urchins.
Gametes from both Strongylocentrotus purpuratus and Litichinus pictus were
used to determine the mode of action of ZA as a possible inhibitor of
attachment.
MATERIALS AND METHODS
The competent cyprids of Balanus amphitrite used in this study were
cultured by James S. Todd at the Marine Sciences Laboratory of Duke
University according to the method described in Rittschof, D., 1992.
Chemotaxis Assay
Chemotactic response was assayed using a 50 cm long trough of PVC
pipe. Barnacle larvae were maintained at a temperature of 6-8° C to prevent
settlement and kept at room temperature during the experiment. Both ends
were closed with silicone gel and rubber stoppers were used to prevent water
flow between the five 10 cm sections. To begin, a rubber stopper was placed at
20 and 30 cm from side #1 and from approximately 250 barnacle larvae were
pipetted along with 8 ml of FSW into this section of the trough. 14 ml of
FSW were then added to the 0-20 cm section and 16 ml to the 30-50 cm
section. At time zero 2 ml of 30 mM ZA were added at 0 cm and the two
rubber stoppers removed allowing the movement of the cyprids and the
diffusion of ZA throughout the trough. At 60 minutes, a rubber stopper was
placed at each 10 cm mark from side f1 (10 cm, 20 cm,...) and the number of
larvae in each of the five 10 cm sections were counted (0-10 cm, 10-20 cm....).
Experiments were disregarded if the number of larvae was less than 100 or if
the two rubber stoppers were not released at the same moment. The side in
which the ZA was added (f1 or f2) was reversed every other experiment. A
control experiment was run exactly as above with one exception¬ 16 ml of
FSW was added to the 0-20 cm section and no ZA was used.
Cyprid Mobility
Plastic Falçon petri dishes were used to evalute the effects of ZA, p
coumaric acid (CA) and sodium acetate on B. amphitrite motility. From 10 to
30 larvae were used in each dish containing 2 ml of solution. 2, 4, 6, 8, and 10
mM concentrations of ZA and 3, 5, and 7 mM of CA were used in both
buffered (pH 8) and unbuffered (pH 3.9 for CA and pH 5.2-5.7 for ZA)
solutions. Sodium acetate was used at 10 mM both buffered with Trisma as
above (pH 8) and unbuffered (pH 5.7). Cyprids were considered motile if they
were noticeably active under the microscope and/or their appendages were
extended after 15 minutes. Two types of control experiments were used: one
contained 2 ml FSW and the other a buffered solution of MES (pH 5.6), both
absent of any of the above weak acids. Two replicate dishes were analyzed for
each concentration, buffered and unbuffered.
Barnacle Settlement Assay
Attachment of cyprids of B. amphitrite was assayed against ZA, CA, and
sodium acetate in plastic dishes (Falcon 43001) containing 20-60 larvae and 5
ml of solution at room temperature. Each experiment was stopped after 24
hours using a few drops of 1 N HCI and the number of attached barnacles
were counted using a dissecting microscope. Varying concentrations of
buffered and unbuffered ZA, CA, and sodium acetate were assayed with 6
replicates for each unbuffered ZA concentration and 4 for each buffered one.
CA and sodium acetate assays were done once. Experiments were disregarded
if there were less than 20 larvae in a petri dish.
Bacterial Attachment
Two different assays were done to test the effects of ZA on bacterial
attachment. The first was a hemagglutination assay done with red blood cells
and bacteria to test for the ability of zosteric acid to prevent agglutination of
red blood cells due to the adherence of bacteria. The experiment was carried
out in eppendorf tubes with an overnight culture of Vibrio cholerae and
human type O- whole blood that was centrifuged at 3000 rpm for five
minutes, washed, and resuspended in phosphate buffered slution (PBS) three
times to arrive at a 5% solution of red blood cells. A 1% solution of fucose,
and 6 mM zosteric acid neutralized to pH 8.0 were additional components.
The first tube contained 15 ul of bacteria and 50 ul of red blood cells in PBS,
the positive control for hemagglutination. The second tube consisted of
bacteria, red blood cells, and 100 ul of fucose (a sugar known to inhibit
hemagglutination). The third tube contained bacteria, red blood cells and 100
ul of 6mM zosteric acid (3mM final concentration in tube). The last two tubes
acted as controls containing red blood cells and either zosteric acid or fucose,
to make sure that these compounds were not affecting the red blood cells.
Appropriate amounts of PBS were added to each tube for a final volume of
200 ul. The tubes were left to sit for 30 min and then samples were removed
and checked under the microscope to observe for bacterial induced
agglutination of the red blood cells.
The second bacterial attachment assay was set up to determine the
relationship between varying doses of zosteric acid in solution and the
density of attached bacteria on a surface. Coumaric acid, an analog of zosteric
acid without the sulphate group, was also tested for the ability to prevent
bacterial attachment to surfaces. Five 50 ml Falcon tubes were set up, each
containing 200 ul of three marine bacterial isolates of the strain Shewanella.
The control tube contained 600 ul of the bacterial strains and SSW, for a total
volume of 30 ml. The four experimental tubes contained bacteria, and three
different concentrations of pH neutralized zosteric acid (0.03mM, O.3mM,
3.OmM) or coumaric acid (3.0 mM), and the appropriate amount of SSW for a
final volume of 30 ml each. A glass slide was then placed in each tube such
that it would float horizontally once the tubes were placed on their sides. The
tubes were placed on a rocker at room temperature for two hours. Each glass
slide was removed and dipped in SSW three times to remove much of the
excess unattached bacteria. The underside of each slide was examined to
ensure that the bacteria were specifically attaching themselves as opposed to
settling due to gravity. The slides were then examined and photographed
under the microscope. The number of bacteria per unit area was counted to
quantify the density of the bacteria attached to the surface of the slide.
Sea Urchin Fertilization
Several experiments were done to determine the effects of zosteric acid
on sea urchin fertilization and the possible mechanism by which it functions.
A dose response experiment for both ZA and CA was performed with eggs
and sperm of Strongylocentrotus purpuratus. Four different concentrations
of ZA and CA were tested: 1mM, 2mM, 3mM, and 4mM. Replicate dishes
were set up for each concentration. Control dishes with just sperm and eggs
were also set up. 1ml of a 2% egg solution in FSW was placed in each dish.
ZA neutralized to pH 7.8 or CA neutralized to pH 7.6 was added to the dishes
in appropriate volumes such that once the final volume of 5 ml was reached
by adding FSW the desired concentration was established. Sperm were then
added to the dishes at a 1/10,000 dilution. The solutions were stirred and the
sperm and eggs were allowed to fertilize. Samples were removed from the
dishes and examined under the microscope. The number of fertilized and
unfertilized eggs were recorded to compare percent fertilization at the varying
concentrations of the two compounds. These dose response experiments for
ZA and CA were done twice with Strongylocentrotus purpuratus and also
once with Litichinus pictus for ZA.
A comparative analysis was done with Litichinus pictus and ZA, CA,
and Heparin, to compare the effects on fertilization of a known compound
such as Heparin with the compounds under study. All dishes were set up in
replicates. Controls were set up with sperm (diluted 1/100,000), eggs (2%
solution in FSW), and FSW for a final volume of 5 ml. The experimental
dishes were set up similiarly except that they contained a final concentration
of 3mM ZA, CA, or Heparin. Once the gametes were allowed to fertilize,
samples were removed from the dishes and examined. The number of
fertilized and unfertilized eggs were recorded to compare the variability in
percent fertilization due to the effects of the different compounds.
One last fertilization experiment was done to determine the
mechanism by which zosteric acid inhibits fertilization. A protease was used
to remove the vitelline layer of the unfertilized eggs to determine whether or
not ZA was inhibiting fertilization by acting on the egg, as opposed to the
sperm. In the control dishes, untreated eggs with intact vitelline layers were
placed in 0 mM, 1mM, and 3 mM concentrations of ZA. The experimental
dishes contained ZA at the same concentrations mentioned above with eg
whose vitelline layers were removed. The vitelline layer of the eggs were
removed by treating dejellied eggs with Protease (100 ug/1 ml egg solution)
for 10 minutes. The eggs were then washed three times to remove excess
Protease. Sperm (1/10,000 dilution) were added to the untreated and treated
eggs. After four hours samples were removed and percent fertilization was
recorded. The Protease treated eggs were checked for fertilization by
observing for cleavage.
RESULTS
Chemotaxis Assay
The chemotactic effects on the cypris larvae did not differ between the
control and ZA experiments (fig. 1). The number of larvae was greatest in the
20-30 cm range (100% at time zero) and then evenly distributed among the 10-
20 cm and 30-40 cm sections and the 0-10 cm and 40-50 cm sections. Despite
the ZA concentration gradient that was established after sixty minutes
between sides #1 and #2 (2.05 mM and .004 mM respectively) the larvae swam
evenly in both directions.
Cyprid Motility
The motility of the cyprids differed greatly between buffered and
unbuffered ZA, CA, and sodium acetate. Regardless of which of the three
compounds was assayed, the larvae were motile at pH 8 (tab. 1). At 8 mM ZA
and above (pH 5.7 and below), the larvae were 100% reversibly immotile with
appendages contracted upon contact. When placed in FSW, there was almost
100% recovery after 10 minutes. At 5 mM CA and above (pH 3.9 and below),
the larvae were 100% irreversibly immotile upon contact, probably dead. In
10 mM sodium acetate (pH 5.7) the larvae were almost 100% reversibly
immotile after 40 minutes. The acidity of the final solution differed between
the same concentrations of the same compound depending on the molarity of
the stock solution from which it was made. The above pH's were calculated
after a 1:1 dilution of ZA or sodium acetate with FSW while CA dishes were
all made from an 8 mM stock solution due to the compound’s low solubility
in sea water. A solution buffered with MES (pH 5.6) or FSW titrated down to
pH 5.2 did not cause larval immotility and in the control experiments (2 ml
FSW) larvae were almost 100% motile.
Barnacle Settlement Assay
Figure 2 shows the dose-effectiveness of pH neutralized and
unbuffered ZA against barnacle settlement. The error bars were determined
by calculating the error (0) using the binomial distribution equation, o
squared=npq, where n equals the total number of replicate experiments, p
equals the fraction of larvae that settled, and q equals the fraction that didn’t.
Four experiments were conducted in which there were more than 20 larvae
in each petri dish. A stock solution of 20 mM ZA (pH 4.2-4.3) was used and
then diluted to 0.05, 0.1, 1, 2, and 3 mM. The pH's of the final solutions were
8.0 in all buffered dishes and 6.5 to 7.6 for 3 mM unbuffered ZA, 7.8 to 7.9 for 2
mM, and 8.0 for 1 mM. pH neutralized and unbuffered percent settlement
were similar at 1, 2, and 3 mM although buffered ZA (pH 8) was a slightly
more powerful inhibitor than unbuffered ZA (pH 6.5 to 7.6) on average and at
each concentration. Neither sodium acetate nor CA significantly inhibited
settlement.
Bacterial Attachment
In the hemagglutination assay, the positive control tube containing
Vibrio cholerae and red blood cells showed significant agglutination due to
the attachment of bacteria (tab. 2). The negative control tube containing
bacteria, red blood cells, and fucose, a known inhibitor of hemagglutination,
showed no agglutination and the red blood cells were free of bacteria. The red
blood cells in the other control wells containing red blood cells and either
hypothetical inhibitor of bacterial attachment, fucose or zosteric acid, without
bacteria were normal. The experimental tube containing bacteria, red blood
cells, and 3mM zosteric acid showed agglutinated red blood cells with bacteria
attached, just as in the positive control.
The second assay showed a difference in the density of bacteria attached
to the glass slides in response to the varying concentrations of zosteric acid in
solution. Bacteria were counted from the photographs taken of the glass
slides immediately after they were washed of excess unattached bacteria in
SW. These numbers were then normalized to the control. Figure 3 shows a
negative correlation between the concentration of zosteric acid and bacterial
density. The lower concentrations (0.03 mM and 0.3 mM) of the zosteric acid
seemed to have a small, yet noticeable effect on the density of attached
bacteria by 18.2% and 24.3% respectively. The highest concentration of
zosteric acid (3 mM) had a more significant effect in preventing bacterial
attachment, decreasing the density of attached barnacles by 36.4%.
Sea Urchin Fertilization
In the dose response experiments with the Strongylocentrotus
purpuratus, ZA decreased percent fertilization while CA did not. The mean
fertilization rates for each concentration were normalized to the percentage of
the control and plotted in Figure 4. The figure shows a negative correlation
between the concentration of ZA and percent fertilization. The data at 2 mM,
3 mM, and 4 mM ZA dishes were statistically different from the control,
having P values of 0.023, 0.006, and 0.00 respectively. The dose response
experiment for ZA on Litichinus pictus showed a similiar negative
correlation between increasing concentrations of ZA and percent fertilization.
Figure 4 also shows the relationship between CA and fertilization in S.
purpuratus. Increasing concentrations of CA have no statistically different
effect on the percentage of eggs fertilized (P value: 0.147) as compared to the
control.
10
The fertilization experiment comparing the effects of CA, Heparin, and
ZA showed a variance in percent fertilization due to the effects of the
different compounds. Figure 5 shows the effects of these compounds at 13
mMl on fertilization of L. pictus. CA decreased the percentage of fertilized
eggs by 14.1% as compared to the control. Heparin decreased fertilization by
36.9% and ZA by 74.8%.
Figure 6 shows the results of the Protease treatment experiment. 96.0%
fertilization was observed in the control eggs with intact vitelline layers and
no ZA. The ZA treated dishes suppressed fertilization by up to 83% as was
seen in the previous dose response curves. In the experimental dishes
containing the protease treated eggs, fertilization was not inhibited by ZA.
DISCUSSION
Although previous investigations (Todd et al., 1993) demonstrated that
p-(sulphooxy) cinnamic acid inhibited settlement of competent cyprids of B.
amphitrite the mechanism was unknown. A bioassay of chemotactic
response to ZA in B. amphitrite was the first step in determining the
compound's mode of action. Analysis of cyprid distribution in the
chemotaxis assay indicated that ZA exhibited no chemotactic response.
Because the larvae did not move in response to a concentration gradient of
ZA, the compound does not appear to be chemorepellent to the cyprids and
therefore does not inhibit settlement by deterrence. During this experiment it
was observed that the larvae in the 0-25 cm range (those exposed to a much
higher concentration of ZA) were noticeably less active than those in the 25-
50 cm range.
A second possible mode of action for the inhibition of settlement by ZA
was therefore hypothesized to be through effects on swimming behavior. If a
11
cyprid can not swim it will never find a suitable substrate on which to settle
and will simply remain in the water column until it recovers or dies. A
necessary distinction must be made between toxic and non-toxic compounds
that cause immobility. Although both cause immobility, a narcotic effect was
differentiated from a toxic one in that the former is reversible and the latter is
not (Rittschof et al., 1992). The results from this assay indicate that at 8 mM
ZA and 10 mM sodium acetate the larvae are 100% reversibly immotile and
therefore inhibited from settling. The CA caused complete irreversible
immotility at 5mM. This is clearly because CA is more acidic (pH 3.9) than
both ZA and sodium acetate and too far from the pH of normal sea water for
the larvae to survive. Because a FSW solution of pH 5.7 did not cause
immobilization, acidic extracellular pH does not appear to be the sole
component in producing this behavioral effect. ZA and sodium acetate may
act as weak acids by partitioning across the cell membrane and changing
intracellular pH. The uncharged species is present in higher concentrations
as the pH decreases in approaching the pKa of a weak acid- 4.0 for ZA and 4.74
for sodium acetate. Therefore, the uncharged species is expected to cross the
cell membrane and in this way could affect intracellular pH possibly resulting
in larval immobility.
Non-toxic antifoulers such as ZA seem to be more variable than toxic
agents in their ability to inhibit settlement (Rittschof et al., 1992). Although
high variability existed among the barnacle inhibition assays, it is apparent
from Figure 2 that both buffered and unbuffered ZA inhibited settlement up
to 30%. Because neither CA nor sodium acetate produced the same inhibitory
effect, the mechanism by which ZA prevents settlement cannot be attributed
to larval immobility in which all three compounds were effective. Therefore,
larval settlement appears unrelated to external pH. However, this does
12
implicate a role for the sulfate moiety of ZA in inhibition of settlement which
may be a key to the compound’s mode of action.
The results of the hemagglutination assay showed no agglutination of
the red blood cells in the negative control with fucose. In the experimental
tube with the zosteric acid, agglutination was not prevented and bacteria
attached to the red blood cells, as in the control. Therefore, the only
conclusion that can be made is that ZA at a 3 mM strength does not prevent
bacterial attachment in this assay. These results do not prove that ZA does
not inhibit bacterial attachment, as can be seen in Figure 3. These results
show that at a 3 mM concentration, ZA can suppress the attachment of
bacteria onto glass surfaces by up to 40%. However, coumaric acid was also
shown to suppress attachment by up to 50%. Because both ZA and CA
seemed effective in suppressing the adherence of bacteria, it can be concluded
that the sulfate group on ZA is not the portion of the compound necessary for
inhibiting bacterial attachment.
In the dose response experiments of ZA and CA with S. purpuratus
and L. pictus, a negative correlation was observed between the concentration
of ZA and percent fertilization. As the concentration of ZA increased, percent
fertilization decreased. CA, however, did not show this effect. Because ZA,
the sulphated phenolic ester, was effective and CA, the unsulfated analog of
ZA, was not, it seems that the sulfate group may play a critical role in
inhibiting fertiliation. The fertilization experiment with Heparin further
supports this hypothesis that the sulfate moiety of ZA is a necessary
component in inhibiting fertilization. Heparin was chosen for this
experiment because it is a highly sulfated polysaccharide, known for its
anticoagulant properties. Figure 5 shows that the fertilization inhibiting
action of Heparin is intermediate to the effects of CA and ZA. CA is
13
unsulfated and has no effect in inhibiting fertilization. Heparin and ZA are
similar in that they both significantly suppppress fertiliation and contain
sulfate groups. There is evidence from the work of DeAngelis and Glabe
(1987), that the egg surface ligands for bindin, a protein secreted from the
acrosomal granule of sea urchin sperm that mediates the sperm’s adhesion to
the egg, have been characterized as containing substantial amounts of
carbohydrate and esterified sulfate. Because zosteric acid is a sulphated
phenolic ester, it may be suppressing fertilization because it is similiar in
structure to the egg surface ligands, thereby tying up sperm and preventing
them from fertilizing.
The Protease treatment experiment further elucidated the mechanism
by which ZA inhibits fertilization. By removing the vitelline layer of the eggs
with Protease, the egg receptors for the sperm were also removed. Figure 6
shows that ZA inhibits fertilization by up to 83.0% when using eggs with
intact vitelline layers. It also showed that ZA lost its inhibitory effect on
fertilization once the vitelline layer was removed. These results suggest that
ZA inhibits fertilization by binding to the egg receptors on the vitelline layer.
Once the vitelline layer is removed, the ZA can no longer bind to the egg
receptors located on this layer and therefore can no longer shield it from
sperm. The sperm can then freely swim to the plasma membrane, where
other egg receptors may also be located, and fertilize. This assay therefore
suggests that the vitelline layer and the receptors on it may be the critical
components affected by ZA in the inhibition of fertilization.
CONCLUSION
The experiments conducted in this study show that zosteric acid
produces a wide variety of effects: on barnacle behavior and settlement,
14
bacterial attachment, and sea urchin fertilization. The results from these
experiments suggest that there may be more than one mode of action.
Zosteric acid not only functions in both its buffered and unbuffered forms, but
also as a sulfated compound and/or simply as a weak acid. Thus, multiple
mechanisms seem to be involved, both individually and synergistically for an
overall antifouling effect.
REFERENCES
Christie, A. O. and Dalley, R., 1987. Barnacle fouling and its prevention.
Barnacle Biology, Crustacean Issues, 5, pp. 419-433.
DeAngelis, P. L. and Glabe, C. G., 1987. Polysaccharide structural features that
are critical for the binding of sulfated fucans to bindin, the adhesive
protein from sea urchin sperm. The Journal of Biological Chemistry,
Vol. 262, No. 29, pp. 13946-13952.
Harrison, P. G., 1982. Control of microbial growth and of amphipod grazing
by water-soluble compounds from leaves of Zostera marina. Marine
Biology, Vol. 67, pp. 225.
Maki, J. S., et. al., 1990. Effect of marine bacteria and their exopolymers on the
attachment of barnacle cypris larvae. Bulletin of Marine Science, Vol.
46, No. 2, pp. 499-511.
Pawlik, J. R., 1992. Chemical ecology of the settlement of benthic marine
invertebrates. Oceanogr. Mar. Biol. Annu. Rev., 30, pp. 273-335.
Rittschof, D., et. al., 1992. Barnacle in vitro assays for biologically active
substances: toxicity and settlement inhibition assays using mass
cultured Balanus amphitrite amphitrite Darwin. Biofouling, Vol. 6,
pp. 115-122.
Todd, J. S., Zimmerman, R. C., Crews, P., and Alberte, R. S., 1993. The
antifouling activity of natural and synthetic phenolic acid sulphate
esters. Phytochemistry, Vol. 34, No. 2, pp. 401-404.
15
gure 1. Lack of ZA Chemotaxis in Barnacle Larvae
Figure 2. ZA Inhibits Barnacle Settlement
Figure 3. ZA Effects Bacterial Attachment to Surface
gure 4. ZA Inhibits Sea Urchin Fertilization
Figure 5. Effects of CA, Heparin, and ZA on Sea Urchin Fertilization
Figure 6. The Effect of the Vitelline Layer on ZA's Inhibition of
Fertilization
Table 1. Effects of ZA, CA, and Sodium Acetate on Cyprid Mobility
Table 2. Hemagglutination Assay: Test for Bacterial Attachment
+ of Larvae
250
200
150-
100
Figure 1.
....
0-10
40-50
2.5
-1.5
ZA Concentration (mM)
0.5



10-20 20-30 30-40
Position in Trough (cm)
ZA concentration (mM)
of Larvae
140
120
-
O 1004
80
O
——
60
O
40
0
88
20
Figure 2.


-4-

2
Concentration (mM)
4 pH 8.0
unbuffered
100
80
.—
0

60
O
40
C
•—
20

Figure 3.


1
3
ZA in mM
1004
75
50
25

8

CA
5
8 ZA
4
Concentration (mM
Figure 4.
+
100
80.0
60.0
400.
20.0-
0.0)+

Treatment (3 mM)
contro
Eca
hep
E Z4
Figure 5.
100.0
80.0
60.0
40.0
20.0
(0.0 +
igure 6.


ZA Concentration

—
protease treated
untreated
Table 1.
Zosteric Acid
Coumaric Acid
Sodium Acetate
Control-FSW
Neutralized (pH 8)
Motile
Motile
Motile
Motile
Unbuffered (pH)
Immotile (5.7)
Dead (3.9)
Immotile (5.7)
Motile (5.7)
Table 2.
Positive Control :
Vibrio strain
Red Blood Cells
Negative Control :
Vibrio strain
Red Blood Cells
Fucose
Experimental :
Vibrio strain
Red Blood Cells
Z4
Agglutination
No
Agglutination
A
lutination