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IntroductionSessile marine invertebrates, such as barnacles, mussels and

tube worms, that populate wave-swept shallow waters attachthemselves to almost any kind of surface to colonize. Thesetype of animals use various unique strategies to ‘glue’themselves to hard surfaces found in these waters, includingman-made marine installations and ships’ hulls. An estimated2500–3000 species of marine organisms are known to causesignificant biofouling worldwide. This type of marinebiofouling is a tremendous economic burden on marineindustries such as shipping and oil-drilling structures. This isespecially true of the many species of barnacles found in theoceans globally, and thus they are exceptional models toinvestigate the nature of the adhesive and the biology of thesecretory process that support settling of these animals.

The life cycle of Balanus improvisus has seven planktoniclarval stages before it metamorphoses into a sessile organism.Between the sixth and seventh transitions, the larva transformsfrom a nauplii larva into a cyprid larva. The cyprid larvasearches for a suitable surface to attach itself to and tometamorphose into a sessile reproducing animal. Theprerequisite for settling and metamorphosis is the ability to

produce, store and secrete the adhesive proteins once thecyprid identifies an appropriate surface to adhere. Theadhesive-secreting cells are located within a pair of cementglands, which are connected by cement ducts that widen intomuscular cement sacs, the presumed temporary storagelocation during cement secretion. Cement ducts connect thesacs to an antenna, which is composed of four segments. Thecement duct extends into the third segment, the adhesive discs.The adhesive is secreted through the discs, and the cyprid larvais able to attach itself to the surface and begin metamorphosis(Harrison and Sandeman, 1999; Nott and Foster, 1969).

Our approach is to understand the biology of barnaclecement secretion in detail so that new techniques could bedeveloped to control their settling on to man-made marinesurfaces. Current methods of control mainly use biocide dopedpaints on surfaces, and such biocides leach in significantquantities to cause serious toxicity to the marine environment.New approaches are necessary to devise more environmentallybenign modes of control of barnacle-induced biofouling (deNys and Steinberg, 2002; Omae, 2003). Understanding theneural control of cement secretion is expected to provide thelead to develop novel substances that inhibit cement secretion.

Barnacles, like many marine invertebrates, causeserious biofouling to marine industrial constructions andhulls of vessels as they attach themselves to such surfaces.Precise biochemical understanding of the underwateradhesion to surfaces requires a detailed characterizationof the biology of the control of barnacle cement secretionand the proteins that make up the cement. In this study,we have investigated cement secretion by cyprid larvae ofBalanus improvisus (D.) and the morphology of theircement glands. We studied the cement proteinorganization within cement granules and categorized thegranules into four different types according to their sizeand morphology, before and after stimulation of secretion.In addition, we followed the exocytotic process of cement

secretion in vivo and discovered that granules undergo adramatic swelling during secretion. Such swelling mightbe due to an increased osmotic activity of granulecontents, following a process of hydration. We hypothesizethat this hydration is essential for exocytotic secretion andconclude that cement protein exocytosis is a more complexprocess than previously thought and is similar toexocytotic secretion in vertebrate systems, such ashistamine secretion from mast cells and exocrine secretionin the salivary gland and the pancreas.

Key words: barnacle cyprid, cement gland, cement secretion,exocytosis, dense core granules, granule swelling.

Summary

The Journal of Experimental Biology 209, 956-964Published by The Company of Biologists 2006doi:10.1242/jeb.02031

An in vivo study of exocytosis of cement proteins from barnacle Balanusimprovisus (D.) cyprid larva

Kristin Ödling1, Christian Albertsson1, James T. Russell2 and Lena G. E. Mårtensson1,*1Göteborg University, Department of Zoology, Zoophysiology, Medicinaregatan 18 SE-413 90 Göteborg, Sweden and

2Section on Cell Biology and Signal Transduction, NICHD, NIH, Building 49, Room 5A-78, 22 Convent Drive,MSC 4480, Bethesda, MD 20892-4480, USA

*Author for correspondence (e-mail: lena.martensson@zool.gu.se)

Accepted 12 December 2005

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In the present study, we have focused on the morphology ofthe cyprid cement glands and the physiology of adhesivesecretion.

Materials and methodsRearing of barnacle cyprid larvae

Cyprid larvae of Balanus improvisus (Darwin 1854) werereared in a laboratory culture as described by (Berntsson etal., 2000). In short, adult barnacles were allowed to settle onPlexiglas panels placed in the sea off the west coast of Swedenin the vicinity of Tjärnö Biological Laboratory (58°53� N,11°8� E). The panels were brought to the laboratory andplaced in buckets with running seawater. These animals wereused as brood stock in all laboratory experiments. Adult B.improvisus will spawn throughout the year when regularly fedwith nauplii of Artemia sp. and the prymnesiophyte Isocrysisgalbana. When kept at 27–28°C, the development to cypridlarvae takes 6–7·days, and the newly moulted cyprid larvaewere filtered through a stack of sieves and washed to removealgae and detritus. The cyprids were then transported in25·cm3 flasks to the laboratory, stored at 4°C and usually usedwithin a week.

All experiments were performed in filtered seawater (FSW,0.22·�m), and drugs used were purchased from Sigma. Thedrugs were diluted in FSW to desired concentrations.

Preparation for light microscopy and transmission electronmicroscopy (TEM)

The cyprids were placed in FSW containing no dopamine,or 1·mmol·l–1 dopamine for a time series of 2, 4 and 10·mintreatment. The cyprids were thereafter immediately transferredover to the fixative.

The cyprids were fixed according to Harrison andSandeman (Harrison and Sandeman 1999). Cyprids werechilled to 4°C for 30·min and transferred to fixation solutioncontaining 2.5% glutaraldehyde and 2% formalin in FSW,pH·8.2. The cyprids were then microwaved in an ice bath upto 37°C and thereafter placed in fixative at 4°C overnight.Several washings were then performed in FSW over 1·h,followed by treatment with 2% osmium tetroxide for 30·minand then 2% uranyl acetate for 20·min. Dehydration wasthereafter performed in an ethanol series of 50%, 70% and90%. Finally, propylenoxide and infiltration of Agar 100 resin(Epon) was performed overnight in 4°C. Samples wereembedded in gelatine capsules in Agar 100 and polymerizedat 60°C for 2 days.

For light microscopy, the blocks were sectioned into 2·�m-thick sections and stained with 1% Toluidine Blue and PyroninG for 1·min at 60°C. The sections were then mounted in Pertexand examined in a Nikon Optiphot microscope connected to aNikon DXM 1200 digital camera. Images were acquired usingthe ACT-1 software (Nikon Microscopes, Europe BV,Badhoevedorp, The Netherlands). Contrast levels were thenadjusted in Adobe Photoshop CS (Adobe Systems, Inc.,Mountain View, CA, USA).

Confocal microscopy

Cyprid larvae are transparent animals and thus are easilyobserved under a confocal microscope to study the secretoryprocess in situ. We found that Acridine Orange accumulateswithin secretory vesicles in the cement glands of cyprids andcould be used as a flourophore to study the exocytoticsecretion. This is similar to secretory granules of pancreatic �-cells due to the acidic intragranular environment with respectto the cytosol (Pace and Sachs, 1982). The excitationwavelength was 490·nm and fluorescence emission wascollected at 519·nm. We incubated cyprid larvae in FSWcontaining 10·mmol·l–1 of Acridine Orange for 1·h and washedthem several times in FSW prior to microscopy. The cypridsseemed unaffected by the Acridine Orange loading.

The larvae were mounted on a cover glass using Kwik Silsilicon glue (World Precision Instruments, Herts, UK) at thecaudal end, while the anterior end remained free. They werekept in FSW in darkness for 1·h. A perfusion chamber was builtup by using Tack It™ (Faber-Castell, Germany) on a glassslide, and the cover glass with the cyprid was inverted andattached to Tack It, thereby creating a perfusion chamber.Larvae were imaged in a Bio-Rad MRC 1024 laser scanningconfocal microscope equipped with a Krypton/Argon laser.Images were acquired using Lasersharp 2000 (Bio-Rad,Hercules, CA, USA) at a magnification of 800� and wereanalyzed using Adobe Photoshop CS.

Differential interference contrast (DIC) microscopy

Cyprid larvae were immobilized in 1% low gellingtemperature agarose, type VII (Sigma A-9045, gellingtemperature below 25°C) dissolved in FSW. Agar was pouredover the larvae and they were placed inside a refrigerator for5·min for polymerization. Animals were then examined underan inverted microscope and imaged. Images were acquiredusing a CCD camera (Pulnix Corp., San Jose, CA, USA), andthe microscope and camera systems were controlled bySynapse (Synergy Research Inc., Silver Spring, MD, USA) asdescribed earlier (Simpson et al., 1997). Cyprids immobilizedin agarose appeared unharmed and tried to move within the gelduring microscopic observation.

Estimation of secretory granule number and size

In order to measure the changes in secretory granule typeand number during the secretory event, we stimulated cementsecretion in intact larvae using dopamine (1·mmol·l–1). Larvaewere exposed to dopamine for 0, 2, 4 or 10·min and wereimmediately fixed as described earlier. Sections were cut andwere stained with Toluidine Blue for light microscopicobservation and granule counting. Statistical comparison wascarried out using analysis of variance (ANOVA, P

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measured. All granules of type 2, 3 and 4 within a section weremeasured. Due to the presence of large numbers of type 1granules, the cross-sectional area of all granules within 2–3cells was measured. The result was evaluated byKruskall–Wallis test (P

959Barnacle cyprid cement exocytosis

10·min with dopamine. The most abundant are the dense, darkgranules stained blue (type 1). The granules that appear lightblue represent type 2 vesicles. Granules that appear to be ‘motheaten’ are type 3 granules. The fourth type of granules are

partially or completely empty and appear as vacuoles. Fig.·3summarizes the differences between the size of the differentgranule types. Overall, type 1 granules were the smallest, anddopamine stimulation seemed to induce granule swellingfollowed by varying degrees of emptying of granule contents,finally resulting in completely empty vesicles that appear asvacuoles. Control, unstimulated glands contained mostly type1 granules, and only occasionally were type 2 and 3 vesiclesfound in some cells. This observation supports our view thatthe loss of granule contents and vacuole formation were notexperimental artefacts caused by fixatives.

Secretory granule ultrastructure was examined using TEM incontrols (Fig.·4A) and in animals stimulated with dopamine for10·min (Fig.·4B). Cement glands of unstimulated control cypridshad mostly type 1 granules, which were small and their interiorappeared dense with a variegated crystal-like structure. Type 1granules in the stimulated glands were identical in structure tothose in unstimulated glands. Stimulated glands containedgranules whose contents showed varying morphologiescategorized into the four different types (see Fig.·2 forcomparison). Fig.·5A shows dense type 1 granules with anintragranular pattern of an organized electron-dense core. Thisappearance might indicate that the cement proteins are stored invery high concentrations within the vesicles. Most endocrine andneurosecretory vesicles in mammalian cell systems are knownto contain secretory material in concentrations as high as0.2·mol·l–1 (Dreifuss, 1975). Fig.·5B shows type 2 granules thatappear swollen, filled with smooth, amorphous contents thatdiffer in appearance from the organized regular pattern seen indense-core type 1 granules. Type 3 granules appeared partiallyempty, containing empty holes that gave a moth-eatenappearance (Fig.·5C) reflecting their appearance under the lightmicroscope. Closer examination shows that the empty holescontain disorganized proteinaceous material with a filamentousstructure interspersed within the smooth amorphous materialsimilar to type 2 granules. Finally, we classified the emptyvacuole-like granules as post-exocytotic type 4 granules devoidof contents. Fig.·5D shows an example of a vacuole, and in thisexample it is seen together with type 1 granules. Thus, differenttypes of granules occasionally occur within the same cell. Type4 granules (vacuoles) are membrane-bound areas where thecement proteins have been almost completely lost, and onlysome remnants are retained. These structures appear similar todegranulation sacs seen in histamine-secreting mast cellsfollowing maximal stimulation (Cho et al., 2002b; Crivellato etal., 2002a). Overall, these morphological characteristics ofsecretory granules in the cement gland of cyprids appear to besimilar to secretory vesicles in other secretory systems duringexocytosis such as the entero-endocrine cells (Crivellato et al.,2002b), the different cells of the immune system (Crivellato etal., 2003) and the pancreatic acinar cells (Raraty et al., 2000)(for a review, see Jena, 2005).

Visualization of secretion in vivo

Acridine Orange accumulated into cement granules in livecyprids served as a fluorescent marker for following cement

Fig.·4. Electron microscopy of cement glands in (A) control,unstimulated cyprids and (B) cyprids stimulated with 1·mmol·l–1

dopamine for 10·min. The different types of granules are labelled inB and can be compared with Fig.·2. In control, unstimulated animals,most of the granules are type 1, but all the four types of granules arevisible in stimulated cement glands (B). Note also that the differenttypes of granules are within the same cell in B. Scale bar: 3.8·�m inA; 2.5·�m in B.

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secretion-dependent changes in granule morphology and lossof contents. Stimulation of cyprids immobilized in Kwik Silwith dopamine resulted in a gradual loss of fluorescence invesicles and appearance of large dark vacuoles. Fig.·6demonstrates this observation (Fig.·6A, control; Fig.·6B, after15·min in 1·mmol·l–1 dopamine). Dopamine at concentrationsbetween 100·�mol·l–1 and 1·mmol·l–1 induced massive vacuoleformation in stimulated cyprids. Noradrenaline (1·mmol·l–1)induced vacuole formation about half of the time (50% oftrials), similar to previously published observations (Okano etal., 1996). We tested a number of biogenic amines using thistechnique, namely serotonin, histamine, octopamine, tyramineand melatonin, all at a concentration of 1·mmol·l–1 (data notshown). Unlike dopamine and noradrenaline, none of the otheramines tested stimulated cement secretion (vacuole formation).In control experiments, we tested spontaneous vacuoleformation in the absence of stimulation. Ten animals wereincubated in filtered seawater for 2·h and none of them showedany signs of vacuole formation during that time. Thus,vacuoles do not form spontaneously in the absence ofstimulation in cement glands of cyprids kept in FSW. The lossof fluorescence and vacuole formation is probably due to adecrease in concentration of Acridine Orange within vesiclesas they swell and a loss of dye as secretion of vesicle contentsproceeds.

In another set of experiments, we investigated dopamine-induced cement secretion in intact live cyprids immobilized inlow-melting-point agarose using DIC microscopy. Similar toour observation of loss of Acridine Orange fluorescence, wewere able to monitor changes in interference contrast followedby vacuole formation within cement glands in all the animals

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that we examined (Fig.·7). We counted the appearance of thedifferent types of granules at various times during dopamine(1·mmol·l–1) stimulation. Fig.·8 shows the change in numberof different types of granules during dopamine stimulation overtime. Similar to the results from Toluidine Blue-stainedsections, dense-core, type 1 granules reduced in number asstimulation proceeded, and the number of vacuoles increased.Thus, a number of different microscopy techniques – light andelectron microscopy of fixed and stained sections, confocalmicroscopy of Acridine Orange-loaded cyprids and DICmicroscopy of live cyprids – all show stimulation-dependentchanges in granule morphology and granule emptying. Whilethe loss of intragranular material is easily observed, themechanism through which such loss occurs is not revealed bythese experiments. The morphological data, however, suggestthat sequential exocytosis might account for the loss of granulecontents (see Figs·2,·9). The emergence of vacuoles is seen asearly as 2·min after onset of stimulation, and significant lossof granules is observed after 10·min, when approximately halfof the dense-core granules have been secreted. Throughout theduration of stimulation, there is no difference in the number oftype 2 and type 3 granules. This observation might suggest thatthe granules undergo swelling upon stimulation andsequentially undergo exocytosis.

In some early experiments, Walker suggested that there aretwo types of cells within the cement gland (Walker, 1971), �and �-cells, based on histological criteria and themorphological appearance of the cement granules. In ourhands, within B. improvisus cyprid cement glands, differenttypes of granules could be observed within the same cell(Figs·2,·4B). While the possibility exists that different types of

Fig.·5. Electron microscopy ofsecretory vesicle types. (A)Unstimulated cement gland wheremost of the cement granules appeardensely packed with secretorymaterial. Granule contents appear tohave a distinct organization. Scalebar, 0.6·�m. (B) Type 2 granulesappear larger and their contentsappear amorphous and lack theorganization observed in the dense-core vesicles seen in A. Scale bar,0.6·�m. (C) Type 3 granules appearsimilar to type 2, except have a ‘moth-eaten’ appearance with clear spaces orhydration channels due to partial lossof contents. Scale bar, 0.25·�m. (D)Type 4 vesicles appear like vacuoleswith a reticulated appearance. Notethat the reticulated granules appearwithin the same cell as the dense-coretype 1 granules. Scale bar, 0.4·�m.

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cells might exist within the cement gland, it appears thatdifferent types of granules occur within the same cell. Weconclude that the difference in appearance of the granules ismore likely linked to the secretory process.

DiscussionIn this study, we used several microscopy techniques to

visualize the secretory process in the cement glands of B.improvisus cyprids. This analysis allowed us to characterizesecretory vesicle morphology and dynamics during stimulatedsecretion. In addition, the data are consistent with anexocytotic secretory process for cement release by the cyprids.Upon stimulation of cement secretion in intact cyprids,secretory vesicles undergo a series of changes, starting withan initial swelling to complete emptying of contents. DIC

microscopy allowed direct visualization of exocytoticsecretion in vivo.

The observation that secretory granules undergo a series ofmorphological changes upon stimulation of secretion issignificant and may be somewhat similar to two previousreports on barnacle cement secretion (Okano et al., 1996;Walker, 1971). Walker described cement gland morphology inboth free-swimming as well as settled Balanus amphitritecyprids. He discovered that the cement gland in non-settledanimals contains a large number of dense granules with adiameter of approximately 3–5·�m, inside columnar cells, andcalled these cells �-cells. He also described another type ofcell, �-cells, with granules that had a reticulated appearance.In addition, Walker observed vacuoles in the apical part of �-cells, where material had been discharged within glands fromsettled cyprids. We found that in unstimulated cyprids, thecement glands contain small, dense-core secretory granules,which begin to swell within minutes of stimulation withdopamine. Swollen granules gradually lose their contents,possibly due to secretion, to finally become empty vacuole-likeorganelles. The appearance of vacuoles indeed occurred nearthe median central groove of the cement glands, similar to thefinding by Walker. In addition, like Walker, we occasionallyfound granules with type 2 or type 3 appearance in control,unstimulated cyprid cement glands. In cement glands fromdopamine-stimulated cyprids, we always found the threedifferent types of granules as well as vacuoles within the samecells (Figs·2,·4B,·9B,C).

This finding might question the previous assertion thatdifferent types of cells exist in cyprid cement glands containingdifferent types of granules possibly differing in their proteincompositions (Walker, 1971). The fact that Walker foundvacuoles and reticulated granules in unsettled cyprids mightindicate that the cyprid larvae, as well as the recently settledcyprids, might have undergone partial secretion of cement,associated with larval settlement. Vacuole formation suggestssecretion has occurred, possibly in an attempt at settlement bythe cyprid (see later). Similar vacuole-like structures have beenseen in other secretory systems where secretory cells containdense-core secretory vesicles, e.g. pancreatic acinar cells (Choet al., 2002b; Raraty et al., 2000), gastrointestinal epithelia(Crivellato et al., 2002b; Kuver et al., 2000) and mast cells(Crivellato et al., 2002a). Similar observations of exocytosisand membrane retrieval in invertebrate neurosecretion weremade in the crab sinus gland over two decades ago (May andGolding, 1983; Morris and Nordmann, 1980).

The current dogma in the case of cyprid settling is thatcement secretion occurs in an all-or-none fashion. Theobservation of granules with partial loss of contents in normalunstimulated cyprid cement glands might suggest that somesecretion can occur in the absence of settling in a form ofpiecemeal degranulation (Aravanis et al., 2003; Crivellato etal., 2003). Secondly, it is possible that only some of thecement-secreting cells participate in secretion during a settlingattempt, sparing other cells in order to provide the cyprid withthe possibility of multiple attempts to settle. Finally, the

Fig.·6. Confocal microscopy of an Acridine Orange-stained livingcyprid cement gland. The larva was immobilized on a cover slip usingKwik Sil and imaged. (A) Unstimulated cement gland within theliving cyprid. (B) The cyprid was stimulated with dopamine(1·mmol·l–1) and imaged 15·min later. Note the brightly stainedsecretory vesicles in the control gland (A) and their loss and theappearance of large vacuoles after stimulation.

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conclusion that the different types of granules found instimulated cement glands indeed represent granules that haveundergone varying degrees of loss of contents calls intoquestion the idea that vesicles with different intragranularcomposition might exist in order to support cement curing.While it is entirely possible that several different proteins makeup the secreted cement, it is not clear if they are derived frommultiple types of secretory granules. Not enough is known ofthe composition of the vesicle contents, nor the cement, toconclude if the barnacle adhesive is a single- or multi-component glue. Dense-core secretory vesicles in general areknown to contain multiple protein and other components thatform a complex mixture that is held at extremely highconcentrations within the vesicles (Lagercrantz, 1976; Uvnäset al., 1970; Winkler, 1976). It is quite likely that all thenecessary components that make up the barnacle adhesive arestored within the vesicles such that, upon secretion, interactionwith seawater or solid surfaces with the right chemical featuresresults in glue hardening. More detailed information of thechemistry of the cement or the granule contents is necessary inorder to precisely understand the adhesive curing mechanism.

We confirm previously published observations thatdopamine and, to a lesser extent, noradrenaline stimulatecement secretion from cyprid larvae (Okano et al., 1996). Themajor difference between the previously published work and

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the present study is that Okano’s experiments were carried outusing isolated cement gland preparations in vitro while ourstudy is performed in intact cyprids in vivo. Perhaps, for that

1 2 3 4

5 6 7 8

9 10 11 12

Fig.·7. Visualization of cyprid cement secretion under differential interference contrast (DIC) optics. A cyprid larva was immobilized in agaroseand observed in a coverslip chamber using an inverted microscope using DIC optics. The montage shows a series of images separated by 10·sintervals. Images are arranged starting at the top and going left to right. Note the appearance of a vacuole that seems to grow larger with time(arrow). Note the increase in size of the cement sac between frames 1 and 12. See supplemental material for a movie sequence of the originaldata at http://vivaldi.zool.gu.se/film/Movie-2-sm.mov and http://vivaldi.zool.gu.se/film/Movie-2-sm.avi.

0

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175 Control 2 min4 min10 min

***

Granules

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1 432 1 4321 4321 432

Fig.·8. Dopamine stimulation causes secretory vesicle loss andappearance of vacuoles. Cyprids were fixed at different times duringexposure to dopamine (1·mmol·l–1) and sectioned. Sections werestained with Toluidine Blue, and the different types of vesicles in thestained sections were counted under the microscope. Note that dense-core granules (type 1) reduce in number over time, with aproportionate increase in the number of vacuoles (type 4) after 10·minof dopamine treatment.

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reason, our experiments required a higher concentration ofdopamine to achieve stimulation. The higher concentration ofdopamine might induce a massive cement secretion and,consequently, more granule swelling and vacuole formation(Kelly et al., 2004). Another difference could be that theextracellular medium used in Okano’s studies did not supportgranule swelling like the extracellular fluid in the living cypridkept in seawater. Granule swelling is known to be dependenton medium pH, Ca2+ (Espinosa et al., 2002) and ionic strength(Finkelstein et al., 1986; Nanavati and Fernandez, 1993),which may differ in the two experimental conditions. Inzymogen granules of the pancreas, the mechanism of granuleswelling during exocytosis was found to be regulated by aGTP-mediated process involving G�i3 and aquaporin (Cho etal., 2002a).

As in most secretory systems, the cement proteins arestored within the vesicles at extremely high concentrations ina colloidal complex with numerous components such that theosmotic pressure inside vesicles is low (Kreuger et al., 1989).The hydration forces on the granule will be expected to becontrolled by the colloid osmotic pressure within the vesiclesand would be modulated by changes in the osmolality of thesurrounding medium (Whitaker and Zimmerberg, 1987). Theintragranular complex needs to dissociate during exocytosisfor secretion to occur. In some granules, a phase boundarybetween compact electron-dense material and less-compactamorphous material within vesicles has been observed,together with loss of material (see Walker, 1971 forcomparison). Granule swelling following hydration of thegranule contents might therefore be essential for secretion tooccur. The structure within the type 3 granules, appearingas hydration channels, might be due to such changes, andsimilar structures have been observed in other dense-coregranules such as the sea urchin egg cortical granules and mastcell granules (Dvorak and Morgan, 2000; Whalley et al.,2000).

Exocytosis is an ubiquitous event in biology and severalhypotheses have been suggested as possible molecularmechanisms. It seems likely that the detailed molecularmechanisms of exocytosis might differ in different secretorysystems depending upon the physiologically required speed ofthe exocytotic secretory event. It seems possible that in somecellular systems a single granule might undergo multiplefusion–secretion cycles (for reviews, see Burgoyne and Morgan,2003; Lindau and Alvarez de Toledo, 2003), leading to partialsecretion of vesicle contents or a pulsatile form of secretion.Similar ‘kiss-and-run’ type partial exocytosis has been observedin synaptic transmitter release (Aravanis et al., 2003). Duringexocytosis, the granule membrane transiently becomes part ofthe cell membrane and then is selectively retrieved, and it ispossible that rapid cycling between a fusion state and a non-fusion state may occur (Burgoyne and Morgan, 2003; Schneider,2001). The duration and diameter of the fusion pore opening willregulate how much granular material is secreted for eachsecretion cycle (Tsuboi and Rutter, 2003; Tabares et al., 2003).Thus, it is possible that the empty vacuoles, like the type 4

Fig.·9. Light microscopy of dopamine-stimulated cement glands.Cement glands were fixed at various times during dopamine exposure,and sections were cut. Toluidine Blue-stained sections were examinedunder the microscope. Note the absence of vacuoles in the control gland(A) and the appearance of vacuoles after 4·min exposure to dopamine(B), which increase in number after 10·min exposure (C).

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granules we observe, could result from multiple rounds of partialexocytosis, or complete exocytosis and total emptying.

In conclusion, the data we present here support thesuggestion that the dense-core secretory vesicles within cementglands in cyprid larvae are secreted through a process ofexocytosis. Exocytotic secretion of barnacle cyprid cementresembles the secretory event observed in many mammaliancell systems, including pulsatile or partial secretion of granulecontents. In addition, we observed four different types ofcement granules in dopamine-stimulated cyprids. With theexception of type 1 granules, all others appear swollen withpartial or complete loss of granule contents and might representvesicles that have undergone partial or complete loss ofcontents. Thus, the cement gland in barnacles appears to be aprecisely regulated exocrine organ that is more complex in itsorganization and regulation than previously thought.

We would like to acknowledge Ulla Svedin and ElisabethNorström for their outstanding technical skills. Financialsupport was provided by the MISTRA program Marine Paint,Stiftelsen Konung Carl XVI Gustafs 50-årsfond and CarlTryggers Foundation.

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