Investigations of the Adhesive Properties of
Phragmatopoma californica Eggs
Annie Chiu
Hopkins Marine Stations, Pacific Grove, CA, 93950
Spring 1993, 17
Investigations of the Adhesive Properties of
Phragmatopoma californica Eggs
Annie Chiu
Hopkins Marine Station, Pacific Grove, CA, 93950
Spring 1993, 175H
Abstract
Phragmatopoma californica is a marine annelid that sheds sticky eggs. The
adhesion of the eggs was found to be dependent on trace levels of calcium.
Transglutaminase inhibitors dansylcadaverine and putrescine prevented or lowered
stickiness, indicating the involvement of tranglutaminase cross-linking in the adhesion
process. Other substrates that prevented stickiness were protease and ethylenediamine.
The egg surface adhered to a polylysine coat, indicating a negatively-charged membrane.
Adhesion does not appear to follow the model system of Mytilus edulis, so P. californica
may present a new system of adhesion in water.
Introduction
Phragmatopoma californica is an intertidal polychaete that sheds eggs that adhere to
many surfaces. Previous investigations of the species have focused on the metamorphosis
(Dales, 1952; Eckelbarger and Chia, 1976; Eckelbarger, 1977), reef-building (Waite, et al,
1992), and settling patterns (Barry, 1989; Eckelbarger, 1976). No mention is made in past
literature describing or studying the adhesive properties of the eggs, with the exception of a
brief description given by Strathmann (1987) for the family Sabellariidae.
Phragmatopoma originally included two species, P. lapidosa and P. californica.
The two species are now considered a single species, called P. lapidosa californica (Barry,
1989), or simply P. californica. The species is reef-dwelling, living in colonies of mixed
sexes (Barry, 1989). Colonies continually increase in size when larvae settle on existing
adult tubes (Eckelbarger, 1976).
P. californica build their sand tubes by cementing grains of sand with organic
mortar containing high levels of 3,4-dihydroxyphenyl-L-alanine (DOPA) (Waite, 1992).
The presence of DOPA in the mortar suggests use of the mussel adhesion system as a
model for the stickiness of the eggs. The mussel Mytilus edulis uses byssus threads
containing DOPA for adhesion (Waite, 1985). The byssus threads contain scleroproteins
which increase the mechanical strength of the threads. These scleroproteins are stabilized
by protein cross-linking. Cross-linking enzymes which may be involved include
transglutaminase, lysyl oxidase, and peroxidase (Waite, 1983).
P. californica was hypothesized to follow the M. edulis adhesion system for the
stickiness of its eggs. Enzyme inhibitors for the above enzymes were tested, and results
suggested the involvement of transglutaminase in egg adhesion. However, other evidence
suggests that the adhesive eggs do not directly conform to the mussel model system.
Materials and Methods
P. californica colonies were collected from the intertidal zone at Boat Works Beach,
Hopkins Marine Station, throughout the time of the study. Eight colonies were used in the
study, with successful spawning in all but one of the colonies. Worm tubes were isolated
from the colony fragment using forceps, then transferred to the desired test solution for
spawning. Worms spawned spontaneously when the tube was broken. In a few cases,
worms began shedding when stressed, before the tube was fully isolated. In these cases,
the whole colony fragment was transferred to the different conditions. Eggs from each
worm were used for multiple tests by transferring the shedding worm to bowls containing
different solutions. Spawning and handling of the worms was carried out as described by
Costello, et al (1957) for the related species Sabellaria vulgaris. The worms were
maintained in a tank with flowing seawater, and colonies were replaced every 2-3 weeks.
Egg adhesiveness was measured using a manometer to monitor water pressure
required to free the eggs from a glass surface (30-ml glass bowl; diameter S cm, height 3.5
cm). An elevated bottle with a bottom spout served as the water source. The bottle was
connected to tubing leading to a manometer (1-ml pipette) and pipette tip. A clamp to
control water pressure was placed on the tubing between the water source and the
manometer. UV-treated, filtered seawater (FSW) was used to measure water pressure in
all solutions.
The experiments were conducted using two methods. The first required spawing
directly into test solutions by placing the shedding worm into the test solution. The test
solutions were FSW, calcium-free seawater (OCaSW), OCaSW with
ethylenediaminetetraacetic acid (EDTA; 5 mM), FSW with protease (0.1%) or borate (10
mM), and FSW with a polylysine surface coat (five microliters of polylysine in 450
microliters of distilled water applied to the bottom of a glass bowl, then blotted off and
allowed to dry). The second method involved spawning into OCaSW-O.02 M
glycylglycine (pH 8) with EDTA as a chelator, then slowly decanting off the OCaSW, and
pouring on a different solution. Enzyme inhibitors for transglutaminase, peroxidase,
trypsin, and lysyl oxidase were tested using this method. See Table 1.
Adhesiveness was also tested on different surfaces, both synthetic and natural.
Eggs were shed directly onto the surfaces, allowed to settle for several minutes, then tested
for their stickiness to each surface. These tests were performed in a petri dish rather than a
glass bowl, so the results do not directly correlate to the tests above. Also, fewer trials
were performed on each surface, resulting in more error in the measurements.
The controls for adhesion were eggs spawned in FSW for stickiness, and
demembranated eggs for unstickiness. Demembranation was carried out using a 1:1
solution of 0.25 M sodium citrate and 1 M sucrose (pH adjusted to 5-6 with hydrochloric
acid) according to Winesdorfer (1967).
The majority of the experiments used unfertilized eggs, although unintended
fertilization was observed in some cases, and may have slightly affected results. Fertilized
eggs were tested for stickiness during various stages of early development. No readily
visible indications of fertilization were apparent prior to cleavage, so testing prior to first
cleavage was not possible.
Results
While spawning was successful in early April, many more eggs were obtained per
worm in late May, indicating that the natural season for spawning begins in late spring (as
also stated by Abbott, et al, 1980). Eggs were shed from each body segment, and emerged
in single-cell columns. The eggs did not appear to stick well to one another, however, as
light agitation disrupted the cell column. Upon reaching a surface such as glass, the eggs
seemed firmly adhered at contact, although the actual time required to cause stickiness is
unknown.
The effects of altering proteins and carbohydrates were measured by spawning into
protease and borate to break down surface proteins and oxidize surface carbohydrates,
respectively. The charge of the egg surface was also measured using a positively-charged
polylysine coat on the glass. Results (Fig. 1) showed reduction of adhesion by protease,
and increase by polylysine. Borate results were inconclusive.
Measurements of adhesion after fertilization showed a slight increase in stickiness
following the early cleavages (Fig. 2). The envelope was successfully removed after
fertilization using the sodium citrate/sucrose solution, and demembranated embryos were
unsticky. This is contrary to Strathmann (1987), which suggests that demembranated eggs
and embryos are sticky. Observed demembranation of unfertilized eggs also eliminated
stickiness, so this condition was used as a control for the stickiness (Fig. 3).
Spawning into various conditions of calcium resulted in different levels of adhesion
(Fig. 4). Calcium-free seawater lowered stickiness only slightly, but addition of EDTA as
a chelator substantially lowered the adhesion. Addition of CaCl crystals and transfer to
FSW restored stickiness.
Enzymes involved in scleroprotein stabilization in the M. edulis model were tested
using several inhibitors (Table 1.) Dansylcadaverine (DC), putrescine (PUT), and
ethylenediamine (EDA) were the only inhibitors that showed a consistent prevention or
decrease of stickiness (Fig. 5). Only sulfanilate (SUL) could be considered uninhibiting to
the adhesion process, while the other inhibitors remain inconclusive. DC was the most
effective inhibitor of stickiness, so a concentration dilution series was tested from 10-4 to
10-8 M (Fig. 6). Effective inhibition was observed up to O.1 uM.
A survey of stickiness was conducted on different surfaces, suggesting a variable
adhesion to different surfaces (Fig. 7). Only extreme values were consistent, showing
Iridea, Macrocystis, and plastic to be very low in adhesiveness, while glass was very high.
No conclusions could be made about the other surfaces, as the standard deviations were
very high.
Discussion
The results of this study indicate that the membrane of P. californica eggs has
properties of other egg outer coats. The strong adhesion to a positively-charged polylysine
coat indicates a negative charge on the egg, a common feature in other species, including
Stronglyocentrotus purpuratus.
Calcium appears to effect adhesion, even at very low levels. The elimination of
calcium and trace metals with a chelator was necessary to lower stickiness substantially.
Calcium may be acting on the egg either directly, as a cofactor for an enzyme or as a
secondary messenger. Direct action may involve a Ca2--dependent system such as that in
cell-cell interactions for keratinocytes (Alberts, et al., 1989). However, the need to remove
all traces of Ca2* (or trace metals) suggests that Ca2+ may be acting as a cofactor or
secondary messenger.
Decreased adhesion in observed solutions of DC and PUT suggest that the cross-
linking enzyme transglutaminase (TGase) may be involved. Although EDA was
consistently low, BAPN results were inconclusive, so the presence of lysyl oxidase is not
conclusive. EDA may have affected the cell in a manner other than a lysyl oxidase
inhibitor. For example, EDA is a weak chelator and may have removed a necessary metal
other than calcium. Furthermore, EDA may serve as an inhibitor for TGase, acting as a
primary amine competitive inhibitor (Lorand, 1979).
DC was the most effective inhibitor of stickiness, and is known as the highest
affinity inhibitor for TGase (Lorand, 1979). It was a potent inhibitor at very low
concentrations, up to 10-7 M (O.1 uM), arguing against the possibility that DC was directly
affecting the egg rather than acting on the enzyme.
Although the results for GEE are inconclusive, the consistent inhibition of
stickiness by DC and PUT suggests that TGase is involved in increasing egg adhesion.
The calcium requirement for adhesion further reinforces the presence of TGase in triggering
egg adhesion, since TGase is calcium dependent.
The ability to inhibit stickiness could increase the usefulness of P. californica as a
model for annelid development. Controlling egg stickiness, by spawning in O.1 uM DC,
would permit experimental manipulations, such as treatments in different solutions without
centrifugation that could be damaging to the eggs.
The role of TGase in causing egg adhesion is unknown. TGase acts by cross-
linking lysine and glutamine residues in peptide bonds in a transamidation reaction (Stryer,
1988). The most studied TGase activity is in the cross-linking of fibrin for blood clotting
TGase activity has also been dicovered in fertilization envelope assembly in
Stronglyocentrotus purpuratus eggs (Battaglia and Shapiro, 1988), endocytosis of yolk
protein in Xenopus laevis eggs (Tucciarone and Lanclos, 1981), and exocytosis of
transferrin receptors in rat reticulocytes (Grasso, et al, 1990).
Several hypotheses for the role of TGase in P. californica egg adhesion are
presented here, but they are merely speculative. One possibility is a conformational change
imposed on surface proteins. Proteins may have regions of stickiness that initially are not
exposed. TGase action may alter the proteins so that the sticky regions are available for
adhesion. Another hypothesis is that sticky proteins may be present in somewhat sparse
distribution around the cell, but upon contact with a surface, the proteins are cross-linked
so they are concentrated at the region of contact. This would result in increased order and
concentration of sticky proteins at the area of contact. Alternately, Ca24 and TGase may be
involved in the exocytosis of vacuoles containing a sticky substance from within the egg,
perhaps upon contact with seawater.
Results of adhesion to different surfaces suggests that only the extreme results are
valid while the others are inconclusive. The results may be highly inconclusive due to the
subjectivity of the test. Water pressure was applied until the majority of the eggs were
freed from the surface, but the definition of majority was subjective for each trial,
especially since the eggs were distributed differently on each surface. Furthermore, the
eggs had a natural variation of stickiness. The variation may not have been evenly
distributed such that a certain area of eggs may have had extremely sticky or unsticky eggs.
These problems had no controls for consistency, so they may have added to the error in
measurements. Because of the subjectivity of the tests, slight differences in water pressure
are not significant.
The variable adhesion to different surfaces poses the question of how the eggs are
attched to the surface. Perhaps two factors, the charge of the egg and the action of TGase
determine the adhesion to any surface. This hypothesis, however, would require that the
less adhesive surfaces are either more negatively charged, or somehow prevent the action
of TGase. The factors affecting stickiness should be investigated further.
The significance of the sticky eggs in nature is also unknown. However, the water
pressure necessary to free the eggs from any tested surface appears to be far less than the
pressure of the waves in the intertidal region. Perhaps the eggs can resist surge or swells,
so they can settle and adhere to the ocean bottom once they are carried past the surf zone.
Adhesion in water is a phenomena with both biological and industrial significance.
Biologically, the significance of egg adhesion may be to increase the fertilization rate of the
eggs (by reducing dilution rate of eggs), or reduce predation by plankton-consuming
animals in the early stages after fertilization (since the eggs would be on the ocean floor
rather than free-floating). P. californica fertilization appears to be very successful, as
larvae are abundant in plankton (Eckelbarger, 1976). This reproductive success may be
attributed in part to the adhesion of the eggs. Industially, the adhesion of P. californica
eggs may serve as a model for making synthetic adhesives for use in marine environments.
Adhesion in water has been difficult to achieve artificially, but adhesives have important
industrial value in the building of bridges and other water structures.
The relation between the M. edulis byssus threads and P. californica adhesion
systems is still unknown. P. californica egg adhesion does seem to use an enzyme,
TGase, that is involved in scleroprotein stability. However, some evidence suggests that
the two systems are not similar. Demembranation was possible in a solution of sodium
citrate and sucrose, whereas the proteins in mussel are insoluble. Spawning in a protease
solution also decreased egg stickiness, while mussel proteins are resistant to protease.
Therefore, and P. californica eggs do not appear to conform directly to the mussel model
system. The P. californica adhesive eggs pose many interesting questions that require
further experimentation.
Literature Cited
Abbott, Donald P., Robert H. Morris, and Eugene C. Haderlie. 1990. Intertidal
invertebrates of California. Stanford: Stanford University Press.
Alberts, Bruce, et al. 1989. Molecular Biology of The Cell (Second Edition). New York:
Garland Publishing, Inc. p. 828.
Barry, James P. 1989. Reproductive response of a marine annelid to winter storms: an
analog to fire adaptation in plants? Mar. Ecol. Prog. Ser. 54:99-107.
Battaglia, David E. and Bennett M. Shapiro. 1988. Hierarchies of Protein Cross-linking
in the Extracellular Matrix: Involvement of an Egg Surface Transglutaminase in Early
Stages of Fertilization Envelope Assembly. J. Cell Biol. 107(6):2447-2454.
Costello, D.P. 1957. Methods for Obtaining and Handling Marine Eggs and Embryos.
Lancaster: Lancaster Press, Inc. pp. 91-97.
Dales, R. Phillips. 1952. The Development and Structure of the Anterior Region of the
Body in the Sabellariidae, with Special reference to Phragmatopoma californica. Quarterly
J. Microscopical Sci. 93(4):435-452.
Eckelbarger, Kevin J. 1976. Larval development and population aspects of the reef-
building polychaete Phragmatopoma lapidosa from the east coast of Florida. Bull. Mar.
Sci. 26(2):117-132.
Eckelbarger, Kevin J. and Fu-Shiang Chia. 1976. Scanning electron microscope
observations of the larval development of the reef-building polychaete Phragmatopoma
lapidosa. Can. J. Zool. 54:2082-2088.
Eckelbarger, Kevin. J. 1977. Larval development of Sabellaria floridensis from Florida
and Phragmatopoma califonica from Southern California (Polychaeta: Sabellariidae), with
a key to the Sabellariid larvae of Florida and a review of development in the family. Bull.
Mar. Sci. 27(2):241-255.
Grasso, J.A., et al. 1990. Calmodulin dependence of transferrin receptor recycling in rat
reticulosytes. Biochem. J. 266(1):261-272.
Lorand, L., et al. 1979. Specificity of Guinea Pig Liver Transglutaminase for Amine
Substrates. Biochemistry. 18(9):1756-1765.
Strathmann, Megumi, F. 1987. Reproduction and Development of Marine Invertibrates of
the Northern Pacific Coast. Seattle: University of Washington Press. pp. 166-169.
Stryer, Lubert. 1988. Biochemistry, Third Edition. New York: W.H. Freeman and
Company. p. 250.
Tucciarone, Linda M. and Kenneth D. Lanclos. 1981. Evidence for the involvement of
transglutaminase in the uptake of vitellogenin by Xenopus laevis oocytes. Biochem.
Biophys. Res. Comm. 99(1):221-227).
Waite, J.H. 1983. Quinone-Tanned Scleroproteins. In The Mollusca (Karl M. Wilbur)
New York: Academic Press. pp. 467-504.
Waite, J.H., Timothy J. Housley, and Marvin L. Tanzer. 1985. Peptide Repeats in a
Mussel Glue Protein: Theme and Variations. Biochemistry. 24(19):5010-5014.
Waite, J.H., Rebecca A. Jensen, and Daniel E. Morse. 1992. Cement Precursor Proteins
of the Reef-Building Polychaete Phragmatopoma californica (Fewkes). Biochemistry.
31(25):5733-5738.
Winesdorfer, John E. 1967. Marine Annelids: Sabellaria. In Methods in developmental
biology (Fred H. Wilt and Norman K. Wessels). New York: Thomas Y. Crowell. pp.
157-162.
10
Table 1. Enzyme Inhibitors
Transglutaminase
Peroxidase
Trypsin
(control)
Lysyl Oxidase
Glycine Ethyl Ester (10 mM)
Dansylcadaverine (1 mM)
Putrescine (1 mM)
Sulfanilate (0.2 mM)
Soybean Trypsin Inhibitor (1 mg/ml)
Bovine Serum Albumin (1 mg/ml)
Ethylenediamine (0.1 mM)
B-aminopropionitrile (0.25 mg/ml)
Figure Legends
Fig. 1 Spawing directly into different solutions to test the effects of altering surface
proteins and carbohydrates, and to test charge of surface. OCa-OCaSW w/EDTA;
FSW-filtered seawater; PRO-protease; BOR-borate; PLY-polylysine coat. Clear region
is standard deviation, grey is mean value. Y-axis is relative stickiness.
Fig. 2 Adhesion before and after fertilization (at 2-4 cell and 32-64 cell stages). All were
spawned directly into normal seawater.
Fig. 3 Membranated and demembranated eggs, in normal seawater. OCaSW with EDTA
is present for comparison. Demembranated eggs are the control for unstickiness.
Fig. 4. Varying conditions of calcium. FSW-filtered seawater; OCa-calcium-free
seawater without chelator; EDTA-OCaSW with EDTA as a chelator; CaCl2-addition of
CaCla crystals to OCaSW w/EDTA; »FSW-transfer from OCaSW w/EDTA to filtered
seawater.
Fig. 5 Spawning into OCaSW w/EDTA then transferring to different enzyme inhibitors.
GEE-glycine ethyl ester; DC-dansylcadaverine; PUT-putrescine; EDA-ethylenediamine
BAPN-B-aminopropionitrile; SUL-sulfanilate; STI-soybean trypsin inhibitor;
BSA=bovine serum albumin. Lowered stickiness (considering standard deviations) was
apparent only for DC, PUT, and EDA.
Fig. 6 Logarithmic concentration dilution curve for dansylcadaverine. Inhibition of
adhesion is apparent until 107 M.
Fig. 7 Spawning onto different surfaces. Surfaces (from left to right) are: Iridaea,
Macrocystis, Gigartina canaliculata, Cystoseira, Gigartina leptorhynchos, rough rock, sand
tube, keyhole limpet shell, smooth rock, plastic, and glass.
12
30

FS
oca
Fig. 1

PR

BO

PLY
30
Fig. 2

SW unfert SW fert (2-4)

SW fert (32-64)
14
whole

dememb.
OCa W/EDTA
Caciz
EDT
calcium conditions
FSV
Fig. 5

A
OCa FSWG DC PUT EDA BAPN SUL STI BSA
a
11•
10 -
Fig
4aaka-
7 -6 -5 -4 -3
-9 -8
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.



Fig. 7