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First published online March 31, 2007
Journal of Experimental Biology 210, 1481-1488 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02759
The role of calcium and magnesium in the concrete tubes of the sandcastle worm
1 Molecular, Cellular and Developmental Biology, University of California at
Santa Barbara, Santa Barbara, CA 93106, USA
2 Physics Department, University of California at Santa Barbara, Santa
Barbara, CA 93106, USA
3 Marine Science Institute and Chemistry and Biochemistry Department,
University of California at Santa Barbara, Santa Barbara, CA 93106,
USA
* Authors for correspondence (e-mail: sun{at}lifesci.ucsb.edu; waite{at}lifesci.ucsb.edu)
Accepted 20 February 2007
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Key words: Phragmatopoma californica, tube worm, protein cement, calcium, magnesium, biomechanics
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; Simmons et al.,
2005). In collaboration with other worms, each sandcastle worm
collects, inspects and cements wave-borne particles into the walls at the
growing end of its home, a long concrete tube that is fused with the tubes of
many other worms (Vovelle,
1965). The concretions made by a worm colony resemble honeycombed
mounds that can be several meters in diameter
(Vovelle, 1965;
Jensen and Morse, 1988).
The particles that make up the walls of each tube are held together by
droplets of cement that are sparingly dispensed by the building organ in the
thorax of the worm. Each worm is thus continuously engaged in the manufacture
of a composite material typically defined as a dispersion of stiff filler
particles in a viscoelastic matrix
(Wainwright et al., 1982). The
cement, which serves as the matrix of the tube, was the subject of a recent
extensive protein analysis. Three major protein families were identified
(Waite et al., 1992;
Zhao et al., 2005), two of
which were basic and contain almost 10 mol% 3,4-dihydroxyphenylalanine (DOPA),
whereas the third was acidic with very high levels of O-phosphoserine
(Stewart et al., 2004). The
proteins are deposited onto the sand surface as a colloidal emulsion rich in
calcium (Ca) and magnesium (Mg) (Stewart
et al., 2004) that sets within seconds. Cysteinyl-DOPA cross-links
are detectable in the cement and may reinforce the setting process
(Zhao et al., 2005). The role
of Ca2+ and Mg2+ ions in the cement is not understood,
but recent atomic force microscopy (AFM) studies of bone suggest that
Ca2+-mediated interactions between bone mineral, collagen and
phosphoproteins contribute significantly to the cohesiveness and fracture
toughness of the bone structure (Fantner et
al., 2005).
The sandcastle worm is not an important fouler of commercial surfaces, but
presents useful features for studying wet adhesion. Its attachment strategy
has much to teach about engineering durable adhesive bonds to wet mineral and
metal surfaces, a feat that eludes most synthetic polymers
(Brockmann, 1983). Indeed,
environmental moisture is the leading cause of adhesive failure in
manufactured products, including silica-filled composites (Comyn,
1982).
The aim of the present study was to explore the contribution of the divalent ions to the mechanical performance of the Phragmatopoma californica cement. Three hypotheses were tested: (1) Ca and Mg contribute to the compressive strength of worm tubes, (2) Ca and Mg provide cemented sand grains with resistance to pull-out forces, and (3) Ca and Mg are crucial for adhesion of the cement. Our results indicate that divalent ions Ca2+ and Mg2+ play crucial roles in the structural integrity and cohesive strength of the Phragmatopoma cement.
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Materials and methods |
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).
Briefly, colonies of P. californica collected from the intertidal
zone near Campus Point, Santa Barbara, CA, USA were transferred to shallow
laboratory tanks circulated with seawater at 
12°C. Single worms
residing in only 1–2 cm of their original tube length were spaced out on
a bed of clean sand or glass beads. Commercial sand (grain size range
400–600 µm) was obtained from Sigma and placed in a large Petri dish
maintained under flowing filtered and aerated seawater. The worms extended the
anterior tube ends with the available clean sand. A batch of the newly built
tube increments was harvested regularly and washed with either filtered
seawater or deionized water followed by five washes with Milli-Q water. A
batch here refers to all of the tubes made under the same condition by a newly
collected cohort of worms during a given period in captivity. Depending on the
experiment, the tubes were then incubated in different treatment
solutions. Special conditions were required for construction of tubes used for pull-out tests. The worms were placed on a single layer of sand or glass beads in a 13-cm diameter Petri dish, with the bottom of the dish covered with small coverslips (18 mmx18 mm, 0.13–0.16 mm thick) affixed to the Petri dish with double-sided tape. Newly built portions of the tubes on coverslips usually resembled tunnels because the worm used the coverslip surface as one facet of the tube. The newly built tubes were harvested with the attached coverslip and subjected to further treatment and mechanical analysis.
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).
Pull-out force test
The pull-out or detachment force of sand grains was measured using the worm
tubes attached to coverslips, the latter providing an adequate amount of flat
surface to be secured to the spring arm by a pair of magnets. The pull-out
test system is based on a simple spring gauge, as shown in
Fig. 1. The spring was a flat
strip of type 302 steel (width 10 mm and thickness 0.127 mm), which was fixed
in a vise at one end and bent 90° at the other, 56 mm from the vise mount.
The bent overhang was 18 mm long, which was adequate to enable a pair of
magnets to secure a coverslip with attached tube. Sand grains were pulled from
the end of a worm tube one at a time with fine-tipped forceps (Dumont type #3)
in a trajectory perpendicular to the long axis of the spring and at a pulling
speed of approximately 0.5 mm s–1 based on calculations from
video-recordings. Pull-outs were monitored under a light microscope. Spring
deflection, which was measured by ruler with an accuracy of 0.5 mm, gauged the
pull-out force and was converted to Newtons by comparison with a standard
calibration curve determined by using a microbalance (Mettler Toledo, 0.1 mg
readability). The spring was calibrated by measuring deflections for a range
of known metric loads. The force at each deflection was calculated according
to force=massxacceleration of gravity.
A total of 81 and 134 sand grains were pulled from a batch of approximately 10 EDTA-treated and untreated tubes, respectively. The sample number for EDTA treatment is lower only because many of the tubes did not survive the EDTA treatment. The tubes we used came from different worms. However, each tube from the same worm was divided into at least two sections (depending on how many pieces of the coverslips the tube resided on) and were treated with seawater and EDTA, respectively, to minimize sample variation. The Kolmogorov–Smirnov test was used for statistics.
Mechanical compression
Compressive loading of tubes was performed with a mechanical tester (Bionix
200; MTS Systems, Cary, NC, USA) equipped with a 10-N load cell set at a
cross-head speed of 1 mm min–1. Worm tubes were placed on a
flat stainless-steel mount and secured at both ends by tape. Force and
deformation were recorded while the end of a motor-driven steel plate with a
2.5 mmx40 mm cross-section was pushed at right angles against the long
axis of the tube. All samples were wet-tested (capillarity was adequate to
completely wet the tube) with the appropriate treatment buffer. Each tube was
tested to compressive failure, which was defined as the point at which visible
structural collapse occurred. A batch of 18 tubes was tested. Each worm tube
was divided in two, one half subjected to EDTA treatment and the other half
subjected to seawater treatment. Change in the peak load was expressed as the
NEDTA/Nseawater. In order to look into the effect of
Milli-Q water wash, we also tested another 16 tubes with half of each tube
rinsed and tested in Milli-Q water and the other half of each tube rinsed and
tested in seawater. Results were also expressed as NMilli-Q
water/Nseawater for inter-sample comparison.
SEM imaging and energy dispersive spectrometry (EDS)
For SEM imaging, tubes built from glass beads were preferred on account of
the uniformity of the beads and cement deposits. Following each treatment,
glass beads were extensively rinsed with Milli-Q water to eliminate residual
salt, followed by freeze-drying and mounting on SEM posts. The samples were
sputter-coated with gold for 60 s for imaging and 15 s for in situ
elemental analysis using a Denton Vacuum DESK II coater (45 mA, 6.666 Pa)
(Moorestown, NJ, USA), and examined with a Tescan Vega TS 5130MM thermionic
emission scanning electron microscope equipped with an IXRF Systems energy
dispersive spectrometer (Tescan, Houston, TX, USA). Sample pore size and
trabecular (distance between the pores) thickness were measured by using the
measurement function that comes with the Tescan software.
AFM test on cement in seawater and EDTA
To measure the adhesion of Phragmatopoma cement under cyclic
stress–strain conditions, AFM experiments were performed using cement
deposits or `glue prints' on coverslips. Because of the nonhomogeneity of the
cement patches, a 50 µm glass bead was glued to the end of an AFM
cantilever (CSC21; Mikromash USA, Wilsonville, OR, USA) using 2-ton epoxy
(Devcon, Danvers, MA, USA). The beaded cantilever was positioned over a cement
patch using the OMV optical microscopy system of a Multimode Picoforce system
(Veeco, Santa Barbara, CA, USA). The bead tip was held on the surface with a
force of 10 nN for 3 s for each pull. Pulling experiments were performed at a
rate of 0.33 Hz. Sufficient protein adhesion to the bead tip during a
`pick-up' is provided by multiple noncovalent interactions
(Rief et al., 1997). Pulling
curves were first performed in seawater (several-hundred pulls), after which
the sample chamber was flushed with EDTA (500 mmol l–1, pH
8.0) in which another several-hundred pulls were performed at the same
position. Water-rinse effect after seawater was also investigated. The pulling
curves were analyzed for energy dissipation, maximum adhesion force and
pulling length using custom software written in LabView 7.1 (National
Instruments, Austin, TX, USA). Statistical analysis reflecting the change in
adhesion from seawater to EDTA was performed in Origin 7 (OriginLab,
Northhampton, MA, USA) using an independent Student's t-test at a
significance level of 0.05. Because of the nature of the testing, only one out
of many glue spots on each coverslip could be tested with a set of
experiments. In all, five different cement prints from five different worms
were tested and analyzed.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). The
trabecular structure of each pore consisted of a thin pore wall (30–50
nm thickness) and a connecting matrix approximately 166 nm thick (range
42–599 nm with an s.d. of 60 nm, N=1724 from three images).
Adhesive failure revealed a deeply dimpled interfacial `footprint'. Curiously,
although a thin skin covered both the exposed and interfacial portions of the
cement, only the skin of the interfacial portion appeared to be sufficiently
compliant to dimple.
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).
Binding force test results on normal and EDTA-treated tube
Because of the technical difficulty of testing singly bonded pairs of sand
grains, a miniature force gauge was designed to measure the pull-out strength
per sand grain in tubes made by worms maintained in laboratory tanks. For the
untreated tubes tested in seawater, approximately 70% of the sand grains
exhibited a pull-out force greater than 21 mN
(Fig. 3). With regard to the
EDTA-treated tubes, over 80% of the sand grains had a pull-out force of 9 mN
or less (Fig. 3A). It must be
emphasized that no pull-out measurements could be made for many of the sand
grains (40% of total) because, as noted earlier, cement contacts were
frequently dislodged by EDTA treatment. On average, the untreated sand grains
required approximately three times more pull-out force than the surviving
EDTA-treated ones. The Kolmogorov–Smirnov test showed this difference
between EDTA-treated and untreated grains to be highly significant
(Fig. 3B).
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AFM test results of EDTA-treated cement (with and without Ca and Mg)
AFM was used to investigate the nanoscale adhesive properties of the tube
cement. A cantilever with an attached glass bead (see
Fig. 5A) was pressed onto the
edge of a cement deposit on a coverslip to prevent picking up too much cement.
During cantilever retraction, adhesion resulted in force–distance
pulling curves like those shown in Fig.
5B, which are representative for pulls in seawater and EDTA,
respectively. The approach-retraction cycle was repeated 500 times at the same
spot in seawater and then another 500 times after flushing with EDTA. For each
trial, the averages of total pulling length, maximum force and total energy
dissipation were calculated (one trial shown in
Fig. 5C). Because of the
inhomogeneous nature of the cement deposits, the absolute values of the pulls
varied considerably. These variations could have been caused by the
heterogeneity in the cement deposits, by a slight drift in the pulling
position or by distortion of the cement deposit by the cantilever. To make
comparisons between samples easier, the absolute values were normalized to the
seawater value for each trial. For all five trials, all of the changes in
measured adhesion parameters from seawater to EDTA were statistically
significant at P=0.05. Fig.
5D shows the time dependence of the adhesion for the trial shown
in Fig. 5C, with each point
representing one pull. The approach-retraction cycle was repeated 500 times at
the same spot in seawater and then another 500 times after flushing with EDTA.
To ensure that the reduction in adhesion by EDTA was not just an effect from
rinsing, we also flushed the sample chamber with seawater after the first 250
pulls and found no significant effect (P=0.05). In addition, tests in
Milli-Q water after seawater did not show significant difference between the
two. The adhesion loss following EDTA treatment was not recoverable by
extensive flushing with seawater (data not shown).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). At one
time the metals were thought to be present as minerals, possibly
calcium/magnesium phosphate (Gruet et al.,
1987), but evidence for this has never materialized
(Stewart et al., 2004).
Indeed, the phosphate appears to be entirely associated with the
phosphorylated Pc-3 proteins in the cement
(Zhao et al., 2005). The aim
of this research was to assess the contribution of Ca/Mg to the mechanical
properties of the worm tubes. This was done by measuring the effects of
divalent ion removal using EDTA, a divalent ion chelator. EDTA is not specific
for Ca/Mg, but chelates a variety of ions, including some such as
Cu2+ with extremely high affinity (log
Ks=18.8). Compositional analysis of the cement by EDS before
and after EDTA treatment, however, showed convincingly that Ca/Mg removal was
the only detectable chemical effect of the treatment
(Stewart et al., 2004)
(Fig. 2i). The mechanical effects of Ca/Mg removal were explored at several levels ranging from the macro- to nanoscale. The compressive strengths of whole tubes made under laboratory conditions with commercial sand exhibited only a quarter of the strength of tubes collected from the wild, but were at least twice as strong as the EDTA-treated laboratory tubes. The wild-type tubes were not subjected to EDTA treatment because they included a high proportion of calcareous particles, which would have been dissolved by EDTA. By contrast, the laboratory-made tubes were built using only silica-based sand, which could confidently be assumed to be inert towards EDTA. The superior strength of wild-type tubes should be subjected to closer scrutiny in future analyses. At present we attribute the better wild-type performance to three factors: use of a wider range of particle sizes, more irregular particles and particle packing with fewer (if any) gaps. Additional factors are of course also possible.
To investigate microscale mechanics, the pull-out force of sand grains from laboratory-made tubes was measured. Sand grains from the untreated tubes required two to three times more pull-out force than the EDTA-treated ones. The magnitude of this effect was somewhat different than that in the compression tests, but not unexpected because the two tests were measuring different properties. In pull-outs, the mechanical properties most directly tested were the tensile and shear strengths of the cement and thus would be closely linked to the structural integrity of the cement itself. EDTA treatment caused the rigid, porous cement structure to collapse (Fig. 2E-H). Judging by the number of stretched trabeculae and distorted pore shapes, Ca/Mg depletion appeared to soften the cement. It is possible that EDTA removed components in addition to Ca/Mg, but this remains to be determined.
Nanomechanical analysis of cement deposits was performed with a modified AFM cantilever. EDTA treatment greatly diminished both the maximum observed adhesive force and energy dissipation of cement; extensibility as approximated by pull-out length also decreased by approximately 50% but there was much variability. For the untreated cement tested in seawater, the approach-retract cycles were largely reversible. The energy dissipation, maximum adhesion force and the pulling length were not significantly diminished after 500 AFM pulls in seawater. This suggests that the cement depends on noncovalent interactions that can reform with re-established contact and which would enable considerable `rehealing' following moderate deformation. After EDTA treatment, however, this `self-healing' ability was lost even after reintroduction of Ca/Mg.
The emerging picture reveals a cement that is fairly robust and with a
striking dependence on Ca/Mg. An approximate adhesive strength can be
calculated from the mean sand grain pull-out force of 25 mN. Given that each
sand grain or glass bead is typically held in place by four to five spot
`welds' of the cement, approximately 5–6 mN would be required to break
each spot. Taking 6 mN as the breaking force for one spot with a diameter of
150 µm gives an estimated adhesive strength of 350 kPa. This is consistent
with other marine adhesives such as mussel byssus, which on glass exhibited an
adhesive strength of 320 kPa in the winter and 750 kPa in summer
(Young and Crisp, 1982). Two
caveats, however, are worth mentioning for this comparison: (1) our pull-out
tests involved a mixed mode of loading (tensile and shear) that may not be
comparable to the mechanical tests used in other studies; and (2) given the
mechanical superiority of field specimens, it is possible that laboratory
conditions (including the building material, silica sand) are not optimal for
cement maturation. In view of these considerations, our estimate of strength
should be treated as a minimum.
The dependence of cement performance on Ca/Mg is intriguing because it
superficially resembles Ca-dependent cell–cell adhesion. Adhesion
between cells is mediated by cadherins, which are modular proteins extending
out from the cell surface. Although the interactions between cadherins from
different cells are not directly Ca-mediated, cadherin conformation and
rigidity are (Leckband and Sivasankar,
2000; Nagar et al.,
1996; Prakasam et al.,
2006); thus, with Ca depletion, cadherins unravel, and the
three-dimensional conformation-dependent adhesive contact surfaces lose their
identity.
Is a similar mechanistic understanding of adhesion in
Phragmatopoma cement possible at this point? Practical adhesion is
governed by two overriding factors – the strength and number of
interfacial interactions between the cement and a surface and the cohesive
strength or `cure' of the cement (Fig.
6). Is the Ca/Mg dependence of cement a reflection of interfacial
activity or curing? Wholesale dislodgement of cement discs from sand and glass
beads following EDTA treatment suggests interfacial involvement, as does the
decreased adhesive force in AFM. The repertory of building materials for
sabellariids, however, is larger than silica so the proposed model
(Fig. 6B) may not apply to all
surfaces. Indeed, silica surfaces are negatively charged at seawater pH
(Neihof and Loeb, 1972),
providing a perfect setting for ion bridging by Ca and Mg. However, the
structural collapse in the cement, evidence of increased stretching and
bending in the trabecular network, as well as the lower slopes in the
compression plots (Fig. 4) are
more suggestive of a structural cohesive role for Ca/Mg as proposed for Ca in
the frustule of diatoms (Kröger et
al., 1994) and in the PEVK region of titin
(Kellermayer and Bustamante,
1997; Labeit et al.,
2003). At this stage, given the narrow scope of this study, we can
conclude only that the role of Ca/Mg in Phragmatopoma cement is
crucial and multifunctional.
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The defining significance of Ca/Mg in the performance of
Phragmatopoma cement seems to mystify the role of DOPA, an amino acid
commonly present in marine adhesives
(Waite et al., 2005). Indeed,
two of the cement precursors, Pc-1 and Pc-2, each contain almost 10 mol% DOPA
(Waite et al., 1992). The
detection of cysteinyl-DOPA cross-links in the cement suggests DOPA plays a
role in the curing of the structure (Zhao
et al., 2005). However, such covalent cross-links would not have
been labile to EDTA treatment. In addition, the interfacial coordination
complexes formed between peptidyl-DOPA and surface oxides
(Lee et al., 2006) are
considerably more stable than the corresponding ones with EDTA. Additional
adhesive mechanisms and interactions will undoubtedly emerge as particulates
other than silica are tested under conditions more akin to those occurring in
the wild.
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Acknowledgments |
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