Journal of Experimental Botany, Vol. 52, No. 360, pp. 1409±1415, July 2001
Brefeldin A affects adhesion of zoospores of
the green alga Enteromorpha
Maureen E. Callow1,3, Simon Crawford2, Richard Wetherbee2, Kate Taylor2, John A. Finlay1 and
James A. Callow1
1
2
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
School of Botany, University of Melbourne, Parkville 3052, Victoria, Australia
Received 8 January 2001; Accepted 2 March 2001
Abstract
Primary adhesion of zoospores of the green macroalga Enteromorpha to substrata involves a massive
release of adhesive glycoproteins from Golgi-derived,
membrane-bounded vesicles in the anterior region of
the spore, followed by rapid curing. This process is
sensitive to low concentrations (5±10 mg ml 1) of the
secretion-inhibiting antibiotic, brefeldin A (BFA). The
proportion of cells that settled in BFA was reduced by
approximately 50%, but the effect was fully reversed
by washing in seawater to remove the BFA. Ultrastructural observations showed that BFA caused the
breakdown of Golgi stacks in the majority of cells
examined. When settled cells were subjected to
shear stress, a greater proportion of those settled in
the presence of BFA were detached, compared with
controls, indicating reduced adhesion strength in the
presence of the antibiotic. The most likely reason
for this is that strong adhesion to substrata either
requires the synthesis of extra adhesive materials
beyond those present in the swimming spore, or the
secretion of an additional component required for
adhesive curing. The novel use of atomic force
microscopy in force modulation mode demonstrated that the adhesive secreted by most spores
in the presence of BFA did not undergo the rapid
curing process typical of control spores. However,
some variation between zoospores was observed,
with some cells showing no ultrastructural changes
and normal adhesive curing. These results are
discussed in relation to variations observed in the
propensity and competence of spores to settle, which
may be reflected in differential requirements for de
novo synthesis and secretion of materials needed for
full adhesion.
3
Key words: BFA, adhesive, alga, atomic force microscopy
(AFM), bioadhesion, Enteromorpha, Golgi, secretion.
Introduction
Enteromorpha is a common, green macroalga found
throughout the world in the upper intertidal zone of
shores and as a fouling organism on a variety of manmade structures. Dispersal is achieved mainly through
asexual zoospores; naked (i.e. wall-less), quadri¯agellate
pear-shaped cells, 5±7 mm in length. Colonization of
substrata by these spores involves a variety of surface
selection and cue detection behaviours (Callow et al.,
1997; Callow and Callow, 2000), followed by primary
adhesion which involves the rapid and massive secretion
of adhesive glycoproteins from membrane-bounded,
Golgi-derived vesicles within the apical region of the
motile spore (Evans and Christie, 1970; Callow et al.,
1997; Stanley et al., 1999). The released adhesive undergoes an, as yet, ill-de®ned `curing' process (Callow et al.,
2000a) and later stages in adhesion involve the continued
synthesis and secretion of the same, or related adhesive
glycoproteins into the developing cell wall of the settled
spore (Callow et al., 2000b).
The lipophilic fungal antibiotic, brefeldin A (BFA)
has been widely employed in recent years in studies of
the Golgi apparatus (Ga), vesicle traf®cking and secretory mechanisms in eukaryotic organisms, although there
appear to be differences in the mode of action between
animal and plant cells (Satiat-Jeunemaitre et al., 1996).
Most of the tissues or organisms that have been
investigated, including green algae, display a well-de®ned
secretory pathway. The presence in Enteromorpha
zoospores of an active Ga (Callow and Evans, 1977),
To whom correspondence should be addressed. Fax: q44 121 4145925. E-mail: [email protected]
ß Society for Experimental Biology 2001
1410
Callow et al.
and the extensive involvement of vesicle traf®cking
to release the large quantities of stored adhesive during
settlement, suggests that adhesion of Enteromorpha
zoospores might be sensitive to BFA. The aim of the present study was, therefore, to determine whether treatment
of zoospores with BFA would affect the mechanism of
substratum adhesion anduor the physical properties of
the adhesive, either during settlement, during subsequent
post-settlement consolidation processes, or both. The
results reveal that BFA has a variable, yet distinct effect
on the nature of the secreted adhesive and the adhesion
processes. This variability may be related to the level
of zoospore differentiation relative to its preparedness
to settle. This study is the ®rst to explore the functional
effects of BFA on cell adhesion and adhesives: Enteromorpha, with its relatively discrete, well-characterized
adhesion biology, provides a good model system for such
studies. An understanding of the nature of the zoospore
adhesive and the mechanism of its adhesion may provide
clues for the prevention of the destructive biofouling that
characterizes this organism.
Materials and methods
Plant material and zoospore adhesion assays
Fertile plants of Enteromorpha linza were collected from
Wembury Beach, England (latitude 508189 N; 48029 W) or from
Port Melbourne, Australia (378509 S; 1448559 E). Zoospores
were released and prepared for attachment experiments as
described earlier (Callow et al., 1997). The concentration of
spores used is given for individual experiments.
to give ®nal concentrations of 10 mg ml 1 (35.6 mM) and
5 mg ml 1 (17.8 mM), respectively. Controls contained 1 ml of
ethanol per ml of spore suspension.
The samples, in capped 15 ml centrifuge tubes, were
placed on a rotating wheel (50 rpm) for 1 h, before centrifuging at 2500 rpm for 3 min. The supernatant was removed
and the pellet resuspended in 6 ml seawater containing either
the same concentration of BFA (2 h BFA) or seawater
(1 h BFAq1 h wash). All solutions contained the same concentration of ethanol. The tubes were replaced on the rotating
wheel for 1 h. The control was treated in the same way; the
pelleted spores being resuspended in 6 ml seawater containing
6 ml ethanol. Adhesion assays and cell counts were performed as
described above.
Measurement of the strength of attachment of
BFA-treated zoospores
Thirty ml of the stock BFA solution (10 mg ml 1) were added
to 60 ml of zoospore suspension (1.5 3 106 ml 1), giving a ®nal
concentration of 5 mg ml 1 (17.8 mM). Spores were prevented from settling by placing on a magnetic stirrer. The
control (60 ml ethanol in 60 ml spore suspension), was treated
in the same way. After 1 h, 10 ml aliquots were pipetted
into individual compartments of polystyrene culture dishes
(In Vitro Systems & Services, GmbH), each containing a
standard glass microscope slide (76 3 25 mm). The dishes were
incubated in the dark for 1 h before the slides were washed by
passing backward and forward 10 times through a beaker of
seawater, in order to remove unattached spores.
Three replicate slides from each treatment were ®xed in 2%
glutaraldehyde in seawater and processed as described above.
The remaining three replicates were placed in a ¯ow apparatus
and exposed to a fully-developed turbulent ¯ow as described
previously (Schultz et al., 2000). The three experimental and
three control slides were arranged alternately in the ¯ow
apparatus, which was run for 5 min at a ¯ow of 2.3 l s 1.
After ®xing slides in 2% glutaraldehyde adhered spores were
counted as described above, except counts were taken at 5 mm
intervals along the middle of the long axis. Means and 95%
con®dence limits were calculated as described above. The mean
number of spores remaining attached to the surface after ¯ow
was compared with the mean number before the slides were
subjected to ¯ow.
Time-course of inhibition by BFA
A stock solution contained 10 mg ml 1 BFA (Sigma) in absolute
ethanol. Nine ml of BFA stock were added to 18 ml of a
suspension of zoospores (1.5 3 106 ml 1) giving a ®nal concentration of 5 mg ml 1 (17.8 mM). Six ml aliquots were
pipetted into 3 3 15 ml centrifuge tubes that were capped and
placed on a rotating wheel (50 rpm) for 30, 60 or 90 min.
A control of 18 ml of zoospores containing 9 ml of ethanol was
treated in the same way.
At the end of the period of incubation on the wheel,
spores were allowed to settle and adhere by pipetting 3 3 2 ml
aliquots from both a control and BFA treatment, respectively,
into each of three 3 cm diameter Petri dishes (Sterilin),
containing a 22 mm diameter glass coverslip. The dishes were
incubated in the dark at 20 8C for 1 h. The coverslips were
washed in seawater and ®xed in 2% glutaraldehyde in seawater as described previously (Callow et al., 1997). The
number of spores within an eyepiece grid, viewed using a 3 40
objective, was counted in 10 ®elds of view on each coverslip.
Fields of view were taken at 1 mm intervals across the
middle of each of the three replicate coverslips. The mean of
30 counts"95% con®dence limits was calculated for each
treatment.
Electron microscopy
BFA was added to 2 3 10 ml aliquots of spore suspension containing 1.5 3 106 spores ml 1 to a ®nal concentration
of 5 mg ml 1. A control contained 10 ml spore suspension and
10 ml ethanol. The samples were rotated for 1 h as described
above, centrifuged and resuspended in 10 ml seawater containing either the same concentration of BFA, or seawater with
10 ml ethanol (BFAqwash). The control was resuspended in
10 ml seawater containing 10 ml ethanol. After centrifugation,
the pellet was dispersed and squirted into 2 ml ice-cold ®xative.
Fixation, dehydration and embedding was as described earlier
(Evans and Christie, 1970) using the Epon recipe of
Mollenhauer (Mollenhauer, 1964). Sections were stained with
lead citrate and uranyl acetate and examined in a JEOL
1200EX transmission electron microscope.
Reversal of inhibition caused by BFA
Measurements on adhesive curing by atomic force microscopy
Six ml of BFA stock, or 3 ml BFA stock plus 3 ml of ethanol,
were added to 6 ml spore suspension (2 3 106 spores ml 1),
Released adhesive of Enteromorpha zoospores `cures'
60±80 min after secretion (Callow et al., 2000a). Atomic force
Effect of brefeldin A on Enteromorpha spore adhesion
microscopy was used to determine the effect of BFA on
adhesive curing by measuring the adhesion strengths of the
adhesive glycoprotein, after this curing period. Released
zoospores were preincubated for 1 h on a rotating wheel
(50 rpm) in the presence of 5 mg ml 1 BFA in ®ltered seawater.
Spores were then allowed to settle, in darkness, on 35 mm
diameter polystyrene Petri dishes (Callow et al., 2000a) in the
same solution. After 5 min, unsettled spores were washed
away with six rinses of ®lter sterilized seawater (FSW). Fresh
BFA (5 mg ml 1 in FSW) was added, then the settled spores
were incubated for a further 60 min before mounting the
dish on the stage of a Dimension 3100 AFM (Digital
Instruments, Santa Barbara, California, USA) with a
Nanoscope III controller and a silicon nitride tip of spring constant 0.58 N m 1. Settled spores were located with the optical
objective and manually brought towards the cantilever tip.
The method of `force-curve mapping' was then used to locate
the adhesive pad with the AFM tip, as described earlier
(Callow et al., 2000a). A series of 10 force curves exhibiting
adhesion events was then taken. This procedure takes 10 min,
hence the ®rst spore to be sampled had been settled for a
minimum of 75 min. The procedure was repeated on a further
nine spores over a period of 2 h. Adhesive strength (mN m 1)
was calculated from the retracting portion of force-displacement
curves. Raw output data collected as tip de¯ection (V) versus
piezo displacement (Z, nm) were transformed into force (mN)
versus distance (nm) curves using a value for the tip diameter
of 0.1 mm as described previously (Callow et al., 2000a).
1411
respectively, in the samples incubated with 5 mg ml 1 of
BFA (Fig. 1).
Treatment of zoospores with BFA at 5 and 10 mg ml 1
for 2 h resulted in 39% and 46% fewer cells adhered
than the control (Fig. 2). However, when the spores were
treated with either 5 or 10 mg ml 1 BFA for 1 h followed
by resuspension and incubation in seawater for 1 h,
the inhibitory effect of BFA was reversed and there was
no signi®cant difference in the number of spores adhered
compared to the control.
Effect of BFA on the strength of attachment
Pretreatment of swimming spores with 5 mg ml 1 BFA
for 1 h resulted in 43% fewer cells adhered than in the
control (Fig. 3). When the attached cells were exposed to
shear stress, 20% of the adhered spores detached in the
control compared to 38% for the BFA-treated cells.
Results
Light and video microscopy
Light microscopical observations were made during
zoospore release and for 3 h following release, and the
details of the settlement process recorded by video microscopy (Callow et al., 1997). During the course of these
observations, several features of spore behaviour relevant
to this project were recorded. Firstly, the propensity to
settle following release was different for individual
zoospores, with many zoospores successfully settling
immediately, some failing to settle after initial approaches
to the substratum, and others remaining motile for long
periods prior to settling. Variability in the rate at which
zoospores settle has been noted, but regardless of the
time-frame, the processes involved in settlement and
permanent adhesion always appeared the same as those
described previously (Callow et al., 1997).
Fig. 1. Time-course of zoospore attachment to glass coverslips in the
presence and absence of 5 mg ml 1 BFA. Spore settlement assay was 1 h,
after the period of incubation indicated on the graph. Each point is the
mean of 30 counts; bars show 95% con®dence limits.
The effect of BFA on initial zoospore adhesion
The inclusion of 1 ml ml 1 ethanol in the adhesion assays
had no signi®cant effect on the number of spores that
subsequently attached (data not shown). The time-course
of zoospore attachment in the control was similar to
that reported previously (Callow et al., 1997). BFA
inhibited attachment by 25% after 30 min, but this was
not signi®cantly different to the control. There were 42%
and 47% fewer cells attached after 1 h and 2 h,
Fig. 2. Data showing that the effect of BFA treatment is reversible. All
samples were incubated for 1 h, centrifuged and the pellet resuspended
in either the same medium for a further 1 h (control, 5 and 10 mg ml 1
BFA) or resuspended in seawater (5 and 10 mg ml 1 BFAqwash). Spore
settlement assay was 1 h. Each point is the mean of 30 counts; bars show
95% con®dence limits.
1412
Callow et al.
Fig. 3. Mean number of zoospores attached after 1 h incubation in BFA
followed by a 1 h settlement assay (BFA and control) and after exposure
to turbulent ¯ow in a water channel (BFAq¯ow and controlq¯ow).
Each point is the mean of 30 counts; bars show 95% con®dence limits.
Electron microscopy
The ultrastructure of Enteromorpha zoospores has
been well documented in the classic paper by Evans
and Christie, and control cells had basically the same
morphology (Evans and Christie, 1970). A high concentration of small `adhesive' vesicles characterized by an
electron-opaque core enclosed by a loose undulating
membrane was present at the anterior end of the zoospore
where initial contact and adhesion with the substratum
occurs. Most pro®les were spherical, between 150 and
300 nm in diameter, other pro®les were elongated and
tubular. It has been demonstrated recently that these
vesicles, which appear to be derived from several Golgi
stacks that are always observed in longitudinal sections
through the anterior region (Fig. 4A), contain an adhesive
glycoprotein (Stanley et al., 1999; Callow et al., 2000b).
For cells ®xed following exposure to BFA, a similar
standard of ®xation to the controls was achieved, and
most ®ne structural features of the cells appeared the
same as controls. However, the number and morphology of Golgi stacks varied over a wide range in the
BFA-treated cells. The Golgi apparatus of some cells
appeared unaffected by the BFA (Fig. 4B), and the
number, distribution and ultrastructure of Golgi stacks
appeared the same as in control cells. Approximately
one-third to half of all zoospores sectioned through the
apical region appeared the same as controls, with between
two and four distinct Golgi stacks visible in longitudinal
sections. The majority of the cells displayed a range of
Ga loss or modi®cation, with Fig. 4C and D being
representative of sections where some evidence of Golgi
stacks still existed, other cells showed no ultrastructural
features resembling Ga (not illustrated). Some minor
stacking was occasionally seen in sections (Fig. 4C), as
well as regions characterized by the presence of small
vesicles within a region of ribosome exclusion, a feature
characteristic of the Golgi (Fig. 1D). The appearance of
a higher density of tubular vesicle pro®les in Fig. 4C
compared to the other micrographs is not thought to be
due to BFA treatment since such variation was recorded
earlier (Evans and Christie, 1970). Observation of a large
number of sections of zoospores affected by BFA suggest the disassembly of the Golgi stacks and their
secretory function, a feature also observed in other plant
and animal cells exposed to BFA.
Cells ®xed after the removal of BFA, and following
a recovery period of 60 min in seawater, all appeared
normal when compared with control cells (not illustrated). The Golgi stacks were restored both in number
(typically 2±4 in each longitudinal section) and ultrastructure, and gave the impression of being actively
involved in manufacturing and secreting vesicles into
the anterior region of the cell.
Effect of BFA on adhesive curing
Adhesion strength values for the adhesive pads of a
sample of 10 spores that had settled in the presence
of BFA for a minimal period of 75 min are shown in
Fig. 5. Values for cured adhesive strength ranged from
71 mN m 1 to 793 mN m 1. Two spores had values that
were close to those obtained for control (no BFA)
treatments, but the majority had values considerably
in excess of this indicating that adhesive curing had
been inhibited. The pattern of cured adhesive strengths
was not correlated with the time of sampling over the
2 h period needed to measure 10 spores.
Discussion
The results in this paper demonstrate an effect of BFA
on the secretion processes of Enteromorpha zoospores
that culminate in their primary adhesion to substrata.
The effects were obtained at low BFA concentration
(5 or 10 mg ml 1) and were fully reversible, suggesting a
speci®c mode of action consistent with that reported
previously (Staehelin and Drouich, 1997). Maximum
inhibition of spore attachment was achieved after 1 h
treatment with BFA and a longer period of incubation
(2 h) did not result in a signi®cant increase in inhibition.
There was also no signi®cant difference in the percentage inhibition following treatment with 5 or 10 mg ml 1
BFA. Thus, it appears that only a proportion of spores
(approximately 50%) within the total population are
susceptible to BFA. Higher concentrations (20 mg ml 1)
appeared to be cytotoxic, as motility of the swimming
spores was adversely affected and many spores also lost
their ¯agella. These results were not unexpected since
the same concentration of BFA caused 90% of cells of
the green alga Gonium to de¯agellate within 80 min and
subsequently die (Haller and Fabry, 1998).
Effect of brefeldin A on Enteromorpha spore adhesion
1413
Fig. 4. Thin sections through the apical (adhering) end of an Enteromorpha zoospore, magni®cation 3 40 000. (A) Control cell showing three obvious
Golgi stacks (G) in various orientations plus associated vesicles. Note the electron-opaque vesicles containing adhesive that accumulate in this region.
(B±D) Sections through representative cells exposed to BFA. (B) A section through a cell that shows no obvious effect of the BFA on either the
cytoplasmic morphology or the Golgi stacks (G). (C, D) Sections through cells affected by BFA, which retain some ultrastructural features resembling
Ga. Some stacked cisternae (C, arrow) are observed as well as regions devoid of ribosomes and containing a range of small vesicles (asterisks). The 2± 4
Golgi stacks that are typically present in longitudinal sections of control cells, are absent from these cells.
The EM observations revealed that only a proportion
of the swimming zoospores treated with BFA (approximately 50±70%) exhibited alterations to Golgi structure
compared to the controls. Adhesive vesicles are budded
off the Golgi cisternae (Evans and Christie, 1970),
although their relationship with the Golgi stack is not
fully understood (Callow and Evans, 1977). EM autoradiography indicated that the synthesis of adhesive
1414
Callow et al.
Fig. 5. Distribution of adhesion strength values in a sample of ten spores settled after treatment with 5 mg ml 1 BFA. Spores were pretreated with BFA
for 60 min and settled onto polystyrene for 5 min. Unsettled spores were removed by washing then the settled spores were `aged' in the presence of BFA
for a minimum of 60 min before making AFM measurements on the adhesive pad. Each point is the mean of 10 force curves; bars show 95% con®dence
limits. The range of values for control (minus BFA) cells is shown; mean is shown by the solid line, 95% con®dence limits by the dotted lines.
proteins continued in zoospores after release from the
parent thallus (Callow and Evans, 1974), suggesting that
at least a proportion of the spores may be immature at the
time of release.
Treatment of plant cells with BFA causes a perturbation of vesicular transport in the secretory pathway
(Staehelin and Driouich, 1997), although the effect on
the Golgi varies. Treatment of suspension-cultured
root cells with relatively high concentrations of BFA
(50±200 mg ml 1) caused reversible vesiculation and disassembly of the Golgi stacks (Satiat-Jeunemaitre
and Hawes, 1992a, b). However, treatment of sycamore
suspension-cultured cells at lower concentrations
(2.5±10 mg ml 1) blocked secretion and altered glycosylation patterns of glycoproteins and polysaccharides
but did not cause breakdown of the Golgi stacks
(Driouich et al., 1993). Both types of responses have
been reported in a number of plant systems (SatiatJeunemaitre and Hawes, 1994), which led Staehelin and
Driouich to propose that BFA can block vesicular
transport in at least two sites in the secretory pathway, namely between the ER and the Golgi, and between
the Golgi and the trans-Golgi network (TGN) (Staehelin
and Driouich, 1997). The site at which BFA operates
in Enteromorpha zoospores cannot be established with
certainty from the present study. The Golgi stacks in
Enteromorpha are more variable in appearance and
number than in most of the well-de®ned Golgiusecretory
systems described in the literature. Lower numbers of
spores adhered to the substratum in the presence of
BFA and, of those that did adhere, a greater proportion
were removed by ¯ow and appeared to secrete adhesive
that failed to `cure' compared to the untreated cells.
These data show that there is variability in the response
to BFA within a natural population of spores.
Reduction in the number of cells adhering to glass
slides could be the consequence of two distinct modes of
action of the BFA. Following a surface selection phase
involving perception of surface recognition cues (Callow
and Callow, 2000), the zoospore commits itself to
permanent adhesion which involves the discharge of the
glycoprotein contents of adhesive vesicles to the substratum to form an adhesive pad that anchors the spore
to the surface. BFA might interfere with the transport
of adhesive vesicles to the plasma membrane anduor
fusion of the vesicles with the plasma membrane and
the discharge of their contents. Such an interpretation is
consistent with the results reported earlier (Domozych,
1999) for the alga Closterium. Alternatively, it is now
known from studies with monoclonal antibodies that
the same, or similar glycoprotein contained within the
adhesive vesicles, is still synthesized via the Golgi system
in cells that have already settled (Stanley et al., 1999;
Callow et al., 2000b). It is possible therefore that the
reduced primary adhesion caused by BFA is due to the
effects of this agent on the synthesis or maturation of
components needed for ®rm adhesion, either more
adhesive itself, or components needed for the adhesive
curing and consolidation processes that are known to
take place following primary adhesion (Callow et al.,
2000a). It may be speculated that individual zoospores,
at the time of release, differ in adhesive competence due
to uneven zoosporogenesis. Some spores appear to be
able to settle normally, secreting adhesive together with
Effect of brefeldin A on Enteromorpha spore adhesion
other components from pre-formed materials, and therefore showing no or reduced sensitivity to inhibitors of
Golgi function. Other spores may have suf®cient levels
of pre-formed components to achieve initial adhesion,
but consolidation of adhesion requires continued synthesis and secretion. This group of spores may be
represented by that proportion of the population that
failed to adhere properly and were removed, either
relatively easily by the washing procedure of the adhesion
assay, or with more dif®culty by the higher shear
stresses generated in the ¯ow cell experiment. The AFM
studies that measured the adhesion strength of cured
adhesive, provide strong support for the second hypothesis since BFA reduced adhesive curing, but only in a
proportion of the BFA-treated cells.
The freshly released adhesive glycoprotein of
zoospores that settle normally, i.e. in the absence of BFA,
has a mean adhesive strength of 173 mN m 1 at the earliest
measurable time-point, i.e. 13 min after settlement (Callow
et al., 2000a). Over the next 60 min this value declines to
60 mN m 1 as the adhesive `cures', with relatively little
change over the next 120 min. The aim of the AFM
experiments was therefore to monitor the effect of BFA
on this `cured' value for adhesive strength, the hypothesis
being that BFA might inhibit the secretion of some
component necessary for adhesive curing. The results
support this hypothesis since the majority of spores
displayed adhesive strength values considerably in excess
of the previous maximum value obtained for cured
adhesive. One spore exhibited a value 450% greater than
the highest mean value previously recorded. It is probable,
therefore, that previous values for freshly released adhesive re¯ected a partial curing during the period between
settling and the earliest time point at which measurements
could be made. However, some spores appeared to cure
as normal in the presence of BFA, providing further
evidence of heterogeneity in the spore population.
Wide variation in the behaviour of Enteromorpha
zoospores occurs following their release, with some
zoospores settling immediately and others taking hours
or even days (Callow et al., 1997). The ecological advantages of prolonging settlement are obvious, as the population of zoospores becomes exposed to a wider range
of potential settlement sites. These observations, along
with the evidence presented here, suggests that newlyreleased zoospores display a range of competencies with
respect to settlement and adhesion.
Acknowledgements
JAC wishes to thank the University of Melbourne for support
through the award of the EC Dyason Universitas 21 Visiting
Fellowship, and the Royal Society for additional funds.
Financial support to MEC (award N00014-96-0373) and JAF
(N00014-99-0311) from the US Of®ce of Naval Research is also
acknowledged.
1415
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