Dissertation

DISSERTATION

Titel der Dissertation „Characterization of the cement of the stalked barnacle Dosima fascicularis (Crustacea, Cirripedia Thoracica): Morphology, biochemistry and mechanical properties“

verfasst von Mag.rer.nat. Vanessa Zheden

angestrebter akademischer Grad Doktorin der Naturwissenschaften (Dr.rer.nat.)

Wien, 2014

Studienkennzahl lt. Studienblatt: A 091 439 Dissertationsgebiet lt. Studienblatt: Zoologie Betreut von: Ao. Univ.-Prof. i.R. Dr. Waltraud Klepal

“If you can dream it, you can do it.”

Walt Disney

Table of contents

I. Introduction 1

II. Manuscript 1 9

Zheden V, von Byern J, Kerbl A, Leisch N, Staedler Y, Grunwald I, Power AM, Klepal W. 2012. Morphology of the cement apparatus and the cement of the buoy barnacle Dosima fascicularis (Crustacea, Cirripedia, Thoracica, Lepadidae). Biol Bull. 223:192-204.

III. Manuscript 2 23

Zheden V, Klepal W, von Byern J, Bogner FR, Thiel K, Kowalik T, Grunwald I. 2014. Biochemical analyses of the cement float of the goose barnacle Dosima fascicularis - a preliminary study. Biofouling. 30:949-963.

IV. Manuscript 3 39

Zheden V, Klepal W, Gorb SN, Kovalev A. Mechanical properties of the cement of the stalked barnacle Dosima fascicularis (Cirripedia, Crustacea). Interface Focus. Submitted

V. Manuscript 4 63

Zheden V, Kovalev A, Gorb SN, Klepal W. Characterization of cement float buoyancy in the stalked barnacle Dosima fascicularis (Crustacea, Cirripedia). Interface Focus. Submitted

VI. Discussion 81

VII. Abstract 87

VIII. Zusammenfassung 88

IX. Acknowledgements 89

X. Curriculum Vitae 90

“Characterization of the cement of the stalked barnacle Dosima fascicularis (Crustacea, Cirripedia Thoracica): Morphology, biochemistry and mechanical properties“ I. Introduction

Barnacles (Cirripedia Thoracica) are sessile, marine Crustacea, which comprise acorn

(Sessilia) and stalked barnacles (Pedunculata). As adult, all of them attach to the substratum with an adhesive (cement). An overview of the mechanism of adhesion was given in a preliminary study (Power et al. 2010). Barnacles have six free-swimming nauplius stages and one cypris larva. Prior to permanent settlement, the cyprid seeks a suitable place for attachment. During the exploration process (“walking” over surfaces with the antennules), it needs a temporary adhesive produced by unicellular antennular glands (Nott & Foster 1969;

Phang et al. 2008). Once an appropriate site is selected, the larva secretes the permanent cyprid cement formed in paired cement glands within the cyprid carapace (Walker 1971;

Phang et al. 2006). After metamorphosis to the subadult, cells of the cyprid cement glands first dedifferentiate and then contribute to the formation of the new cement apparatus. From now on the permanent cement of the adult is secreted (Walker 1973; Yule & Walker 1987).

The cement produced in cement gland cells (in acorn barnacles located in the basal mantle, in stalked barnacles in the peduncle) is transported through a complex canal system. In many species this canal system consists of intracellular, collector, secondary and principal canals, in some species e.g. Balanus tintinnabulum and B. hameri the intracellular canal is lacking

(Lacombe & Liguori 1969; Walker 1970). The cement is secreted to the outside through pores in the attachment disk on the third antennular podomere (Nott & Foster 1969). Cement production continues throughout life time of the animal in accordance with the moulting cycle

(Fyhn & Costlow 1976; Kamino 2006). Most studies about cement synthesis were conducted on acorn barnacles. They secrete a thin, solid or reticulate layer of cement through a branching network of canals which lead to the outside with a number of pores at the base of the shell (Lacombe 1970; Walker 1973; Sangeetha et al. 2010). In contrast, pedunculate barnacles have the characteristic stalk (peduncle), an expansion of the forehead (Anderson

1994). The peduncle is attached to the substratum with the basal attachment disc of the

1 antennules (Lacombe & Liguori 1969; Walker 1974; Walker & Youngson 1975). From there a thicker cement layer is secreted than in the acorn barnacles.

Barnacles are among the most troublesome and dominant fouling organisms, because of their gregarious settlement on any hard substratum, whether it is natural or artificial (Knight-Jones

& Crisp 1953; Wiegemann 2005; Kamino 2010, 2013). Accordingly, attention focused on fouling-release coatings, which prevent firm attachment of the barnacle cyprids and other encrusters (Schultz et al. 1999; Yebra et al. 2004; Aldred & Clare 2008; Scardino et al. 2008;

Callow & Callow 2011). The adhesion strength of these organisms is reduced on low free energy surfaces, whereas higher free energy surfaces promote a much stronger bond between the attachment site and the surface (Yule & Walker 1987; Sangeetha & Kumar 2011). By using elastomeric coatings, the detachment stress can be lowered and barnacles or other encrusters can be removed easily from the substratum (Berglin & Gatenholm 1999; Kavanagh et al. 2003; Holm et al. 2009).

During the last decades the biochemical composition of the barnacle cement was in the focus of interest. The cement consists of approximately 90% proteins and is a multi-protein complex (Walker 1972; Kamino et al. 1996). More than ten cement proteins, of which five are unique among underwater adhesives, are identified in the Megabalanus rosa cement (Kamino

2006). Each of these proteins has a special characteristic and it is assumed that it fulfils either surface or bulk function in the underwater attachment process (Kamino 2010). The cement of barnacles is cured and remains durable underwater like the adhesives of other marine organisms e.g. mussels and tube-building polychaetes (Khandeparker & Anil 2007; Sangeetha

& Kumar 2011; Stewart et al. 2011). There are, however, differences between the adhesives of these marine animals: The amino acid compositions and the primary structures of the barnacle cement proteins are highly complex and not as simple as those found in mussel byssal proteins and polychaete cement proteins (Kamino 2012). The adhesives of the mussels and polychaetes contain significant amounts of the amino acid L-3,4-dihydroxyphenyalanine

2 (L-DOPA), a posttranslational modification of tyrosine, which plays an important role in interfacial adhesion (Nicklisch & Waite 2012; Wang & Stewart 2013). In contrast, no indication of L-DOPA (Jonker et al. 2012) nor of any other protein modification have been found in the holdfast system of barnacles with the exception of the glycosylated 52 kDa- protein found in the cement of Megabalanus rosa (Kamino et al. 2012).

Most pelagic stalked barnacles of the family Lepadidae are typical rafting organisms (Thiel &

Gutow 2005) settling mainly on floating objects (Minchin 1996; Hinojosa et al. 2006). One of these species, Dosima (Lepas) fascicularis (Ellis and Solander, 1786), has developed a special floating device. It secretes a large amount of foam-like cement with gas-filled bubbles enclosed (Newman & Abbott 1980). The cypris larva of D. fascicularis attaches mainly on small, floating objects like feathers or parts of ruptured macroalgae (Cheng & Lewin 1976).

After metamorphosis to the adult, the amount of cement increases and a voluminous cement ball may overgrow the substratum. The so formed float gives buoyancy to the animal and enables it to drift autonomously at the water surface (Thiel & Gutow 2005). Consequently, D. fascicularis is a cosmopolitan species, found in tropical and temperate seas, carried along by ocean currents (Young 1990).

Most studies about the buoy barnacle Dosima fascicularis refer to the ecology and distribution of this drifting animal and its substratum preferences (Cheng & Lewin 1976; Minchin 1996;

Alvarez & Celis 2004; Hinojosa et al. 2006). The present thesis is an attempt to give an overall picture of the special adhesive of D. fascicularis.

Emphasis of the first manuscript (Zheden et al. 2012) is the morphology of the cement apparatus and the cement of this animal. The origin, formation and structure of the cement as well as its way through a complex canal system to the outside are presented. Also a hypothesis about the possible origin and the kind of gas inside the bubbles in the cement is

3 proposed. The methods used in this study are x-ray microtomography, light microscopy, transmission and scanning electron microscopy.

In our second manuscript (Zheden et al. 2014) interest focuses on the biochemistry of the

Dosima cement, particularly on its protein and carbohydrate content. We analysed the amino acids and chemical elements. Our results are compared with the earlier investigations on the cement of Dosima made by Barnes and Blackstock (1974, 1976), of other barnacles (Kamino

2006; Barlow et al. 2010; Lin et al. 2014) and of the adhesives of other marine animals

(Stewart et al. 2004; Flammang 2006; Silverman & Roberto 2007). The main methods applied in this study are scanning electron microscopy with energy dispersive X-ray microanalysis,

1D- and 2D-gel electrophoresis, mass spectrometry, FTIR- and Raman spectroscopy.

The third manuscript (Zheden et al. submitted-a) describes the mechanical properties of the cement of Dosima fascicularis considering differences between the outer and inner regions of the float. Using series of micro-indentations and tensile experiments, the hardness, the elastic modulus and the tensile stress of the cement float are investigated. These mechanical properties are compared with those of the rigid cement of acorn barnacles (Ramsay et al.

2008; Sullan et al. 2009; Sangeetha & Kumar 2011).

The fourth manuscript (Zheden et al. submitted-b) focuses on the buoyancy of this float. We adapted a force transducer apparatus for measuring the buoyant force of the cement. Further the water content, the gas volume inside the float and the correlation of the buoyancy and the gas volume inside the float are investigated. With our pressure experiments we could answer the question whether the foam-like cement float is an open or closed porous system.

References

Aldred N, Clare AS. 2008. The adhesive strategies of cyprids and development of barnacle- resistant marine coatings. Biofouling. 24:351-363.

Alvarez F, Celis A. 2004. On the occurrence of Conchoderma virgatum and Dosima fascicularis (Cirripedia, Thoracica) on the sea snake, Pelamis platurus (Reptilia, Serpentes) in Jalisco, Mexico. Crustaceana. 77:761-764.

4 Anderson DT. 1994. Barnacles: Structure, function, development and evolution. London: Chapman & Hall.

Barlow DE, Dickinson GH, Orihuela B, Kulp JL, Rittschof D, Wahl KJ. 2010. Characterization of the adhesive plaque of the barnacle Balanus amphitrite: amyloid-like nanofibrils are a major component. Langmuir. 26:6549-6556.

Barnes H, Blackstock J. 1974. The biochemical composition of the cement of a pedunculate cirripede. J Exp Mar Biol Ecol. 16:87-91.

Barnes H, Blackstock J. 1976. Further observations on biochemical composition of the cement of Lepas fascicularis Ellis and Solander; electrophoretic examination of the protein moieties under various conditions. J Exp Mar Biol Ecol. 25:263-271.

Berglin M, Gatenholm P. 1999. The nature of bioadhesive bonding between barnacles and fouling-release silicone coatings. J Adhes Sci Technol. 13:713-727.

Callow JA, Callow ME. 2011. Trends in the development of environmentally friendly fouling-resistant marine coatings. Nat Commun. 2:244.

Cheng L, Lewin RA. 1976. Goose barnacles (Cirripedia: Thoracica) on flotsam beached at La Jolla, California. Fish Bull. 74:212-217.

Flammang P. 2006. Adhesive secretions in echinoderms: an overview. In: Smith AM, Callow JA, editors. Biological adhesives. Berlin: Springer; p. 183-206.

Fyhn UEH, Costlow JD. 1976. A histochemical study of cement secretion during intermolt cycle in barnacles. Biol Bull. 150:47-56.

Hinojosa I, Boltaña S, Lancellotti D, Macaya E, Ugalde P, Valdivia N, Vásquez N, Newman WA, Thiel M. 2006. Geographic distribution and description of four pelagic barnacles along the south east Pacific coast of Chile - a zoogeographical approximation. Rev Chil Hist Nat. 79:13-27.

Holm ER, Kavanagh CJ, Orihuela B, Rittschof D. 2009. Phenotypic variation for adhesive tenacity in the barnacle Balanus amphitrite. J Exp Mar Biol Ecol. 380:61-67.

Jonker J-L, von Byern J, Flammang P, Klepal W, Power AM. 2012. Unusual adhesive production system in the barnacle Lepas anatifera: an ultrastructural and histochemical investigation. J Morphol. 273:1377-1391.

Kamino K. 2006. Barnacle underwater attachment. In: Smith AM, Callow JA, editors. Biological Adhesives. Berlin: Springer; p. 145-166.

Kamino K. 2010. Molecular design of barnacle cement in comparison with those of mussel and tubeworm. J Adhes. 86:96-110.

5 Kamino K. 2012. Diversified material designs in biological underwater adhesives. In: Thomopoulos S, Birman V, Genin GM, editors. Structural interfaces and attachments in biology. New York: Springer; p. 175-199.

Kamino K. 2013. Mini-review: Barnacle adhesives and adhesion. Biofouling. 29:735-749.

Kamino K, Nakano M, Kanai S. 2012. Significance of the conformation of building blocks in curing of barnacle underwater adhesive. Febs J. 279:1750-1760.

Kamino K, Odo S, Maruyama T. 1996. Cement proteins of the acorn barnacle, Megabalanus rosa. Biol Bull. 190:403-409.

Kavanagh CJ, Swain GW, Kovach BS, Stein J, Darkangelo-Wood C, Truby K, Holm E, Montemarano J, Meyer A, Wiebe D. 2003. The effects of silicone fluid additives and silicone elastomer matrices on barnacle adhesion strength. Biofouling. 19:381-390.

Khandeparker L, Anil AC. 2007. Underwater adhesion: The barnacle way. Int J Adhes Adhes. 27:165-172.

Knight-Jones EW, Crisp DJ. 1953. Gregariousness in barnacles in relation to the fouling of ships and to anti-fouling research. Nature. 171:1109-1110.

Lacombe D. 1970. A comparative study of the cement glands in some balanid barnacles (Cirripedia, Balanidae). Biol Bull. 139:164-179.

Lacombe D, Liguori VR. 1969. Comparative histological studies of the cement apparatus of Lepas anatifera and Balanus tintinnabulum. Biol Bull. 137:170-180.

Lin HC, Wong YH, Tsang LM, Chu KH, Qian PY, Chan BKK. 2014. First study on gene expression of cement proteins and potential adhesion-related genes of a membranous-based barnacle as revealed from Next-Generation Sequencing technology. Biofouling. 30:169-181.

Minchin D. 1996. Tar pellets and plastics as attachment surfaces for lepadid cirripedes in the North Atlantic Ocean. Mar Pollut Bull. 32:855-859.

Newman WA, Abbott DP. 1980. Cirripedia: the Barnacles. In: Morris RH, Abbott DP, Haderlie EC, editors. Intertidal Invertebrates of California. Stanford, CA: Stanford University Press; p. 504-535.

Nicklisch SCT, Waite JH. 2012. Mini-review: The role of redox in Dopa-mediated marine adhesion. Biofouling. 28:865-877.

Nott JA, Foster BA. 1969. On the structure of the antennular attachement organ of the cypris larva of Balanus balanoides (L.). Philos Trans R Soc Lond Ser B-Biol Sci. 256:115-133.

Phang IY, Aldred N, Clare AS, Callow JA, Vancso GJ. 2006. An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM). Biofouling. 22:245-250.

6 Phang IY, Aldred N, Clare AS, Vancso GJ. 2008. Towards a nanomechanical basis for temporary adhesion in barnacle cyprids (Semibalanus balanoides). J R Soc Interface. 5:397- 401.

Power AM, Klepal W, Zheden V, Jonker J, McEvilly P, von Byern J. 2010. Mechanisms of adhesion in adult barnacles. In: von Byern J, Grunwald I, editors. Biological adhesive systems: From nature to technical and medical application. Vienna: Springer; p. 153-168.

Ramsay DB, Dickinson GH, Orihuela B, Rittschof D, Wahl KJ. 2008. Base plate mechanics of the barnacle Balanus amphitrite (=Amphibalanus amphitrite). Biofouling. 24:109-118.

Sangeetha R, Kumar R. 2011. Interfacial morphology and nanomechanics of cement of the barnacle, Amphibalanus reticulatus on metallic and non-metallic substrata. Biofouling. 27:569-577.

Sangeetha R, Kumar R, Venkatesan R, Doble M, Vedaprakash L, Kruparatnam, Lakshmi K, Dineshram. 2010. Understanding the structure of the adhesive plaque of Amphibalanus reticulatus. Mater Sci Eng C. 30:112-119.

Scardino AJ, Guenther J, de Nys R. 2008. Attachment point theory revisited: the fouling response to a microtextured matrix. Biofouling. 24:45-53.

Schultz MP, Kavanagh CJ, Swain GW. 1999. Hydrodynamic forces on barnacles: Implications on detachment from fouling-release surfaces. Biofouling. 13:323-335.

Silverman HG, Roberto FF. 2007. Understanding marine mussel adhesion. Mar Biotechnol. 9:661-681.

Stewart RJ, Ransom TC, Hlady V. 2011. Natural underwater adhesives. J Polym Sci Pol Phys. 49:757-771.

Stewart RJ, Weaver JC, Morse DE, Waite JH. 2004. The tube cement of Phragmatopoma californica: a solid foam. J Exp Biol. 207:4727-4734.

Sullan RMA, Gunari N, Tanur AE, Chan Y, Dickinson GH, Orihuela B, Rittschof D, Walker GC. 2009. Nanoscale structures and mechanics of barnacle cement. Biofouling. 25:263-275.

Thiel M, Gutow L. 2005. The ecology of rafting in the marine environment. II. The rafting organisms and community. Oceanogr Mar Biol Ann Rev. 43:279-418.

Walker G. 1970. The histology, histochemistry and ultrastructure of the cement apparatus of three adult sessile barnacles, Elminius modestus, Balanus balanoides and Balanus hameri. Mar Biol. 7:239-248.

Walker G. 1971. A study of the cement apparatus of the cypris larva of the barnacle Balanus balanoides. Mar Biol. 9:205-212.

Walker G. 1972. The biochemical composition of the cement of two barnacle species, Balanus hameri and Balanus crenatus. J Mar Biol Assoc UK. 52:429-435.

7 Walker G. 1973. The early development of the cement apparatus in the barnacle, Balanus balanoides (L.) (Crustacea: Cirripedia). J Exp Mar Biol Ecol. 12:305-314.

Walker G. 1974. The occurrence, distribution and attachment of the pedunculate barnacle Octolasmis mülleri (Coker) on the gills of crabs, particularly the blue crab, Callinectes sapidus Rathbun. Biol Bull. 147:678-689.

Walker G, Youngson A. 1975. The biochemical composition of Lepas anatifera (L.) cement (Crustacea: Cirripedia). J Mar Biol Assoc UK. 55:703-707.

Wang CS, Stewart RJ. 2013. Multipart copolyelectrolyte adhesive of the sandcastle worm, Phragmatopoma californica (Fewkes): catechol oxidase catalyzed curing through peptidyl- DOPA. Biomacromolecules. 14:1607-1617.

Wiegemann M. 2005. Adhesion in blue mussels (Mytilus edulis) and barnacles (genus Balanus): Mechanisms and technical applications. Aquat Sci. 67:166-176.

Yebra DM, Kiil S, Dam-Johansen K. 2004. Antifouling technology - past, present and future steps towards efficient and environmentally friendly antifouling coatings. Prog Org Coat. 50:75-104.

Young PS. 1990. Lepadomorph cirripeds from the Brazilian coast. I.-Families Lepadidae, Poecilasmatidae and Heteralepadidae. Bull Mar Sci. 47:641-655.

Yule AB, Walker G. 1987. Adhesion in barnacles. In: Southward AJ, editors. Crustacean Issues 5: Barnacle Biology. Rotterdam: Balkema; p. 389-402.

Zheden V, Klepal W, Gorb S, Kovalev A. submitted-a. Mechanical properties of the cement of the stalked barnacle Dosima fascicularis (Cirripedia, Crustacea). Interface Focus.

Zheden V, Kovalev A, Gorb S, Klepal W. submitted-b. Characterization of cement float buoyancy in the stalked barnacle Dosima fascicularis (Crustacea, Cirripedia). Interface Focus.

II. Manuscript 1

Morphology of the cement apparatus and the cement of the buoy barnacle

Dosima fascicularis (Crustacea, Cirripedia, Thoracica, Lepadidae)

Authors: Zheden V, von Byern J, Kerbl A, Leisch N, Staedler Y, Grunwald I, Power AM,

Klepal W

Publication status: published 2012 in Biological Bulletin 223:192-204

Personal contributions of Vanessa Zheden:

Collection of material

Sample preparation for EM and LM

Cutting and staining of ultra- and semi-thin sections

EM and LM imaging and documentation

Performing statistical analyses

Manuscript preparation

Morphology of the Cement Apparatus and the Cement of the Buoy Barnacle Dosima fascicularis (Crustacea, Cirripedia, Thoracica, Lepadidae)

VANESSA ZHEDEN1,*, JANEK VON BYERN2, ALEXANDRA KERBL1,3, NIKOLAUS LEISCH1, YANNICK STAEDLER4, INGO GRUNWALD5, ANNE MARIE POWER6, AND WALTRAUD KLEPAL1 1University of Vienna, Faculty of Life Sciences, Core Facility of Cell Imaging and Ultrastructure Research, Austria; 2University of Vienna, Medical University of Vienna, University of Veterinary Medicine Vienna, Center for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories,Austria; 3University of Vienna, Faculty of Life Sciences, Department of Integrative Zoology, Austria; 4University of Vienna, Faculty Center of Biodiversity, Department of Structural and Functional Botany, Austria; 5Fraunhofer Institute for Manufacturing Technology and Advanced Materials IFAM, Bremen, Germany; and 6Ryan Institute for Environmental, Marine and Energy Research, School of Natural Sciences (Zoology), National University of Ireland, Galway, Ireland

Abstract. Barnacles produce a proteinaceous adhesive hemolymph into the cement canal system and from there called cement to attach permanently to rocks or to other into the cement. hard substrata. The stalked barnacle Dosima fascicularis is of special interest as it produces a large amount of foam-like Introduction cement that can be used as a float. The morphology of the cement apparatus and of the polymerized cement of this Many sedentary marine organisms such as mussels, tube- species is almost unknown. The current study aims at filling worms, and acorn and stalked barnacles attach to surfaces these gaps in our knowledge using light and electron mi- with proteinaceous underwater adhesives (Kamino, 2008; croscopy as well as x-ray microtomography. The shape of Stewart et al., 2011). These substances have increasingly the cement gland cells changes from round to ovoid during become a focus of interest in connection with research on barnacle development. The cytoplasm of the gland cells, antifouling (Schultz et al., 2011) and on biomedical and unlike that of some other barnacles, does not have distinct biomaterial applications as potential biological adhesives secretory and storage regions. The cement canals, which (Khandeparker and Anil, 2007; Lee et al., 2007; Ang et al., transport the cement from the gland cells to the base of the 2011; Stewart, 2011; Yang et al., 2011). Publication of stalk, end at different positions in juvenile and mature comprehensive books on plant and animal adhesion (Smith animals. With increasing size of the cement float, the exit of and Callow, 2006; von Byern and Grunwald, 2010) further the cement canals shift from the centrally positioned attach- highlights the current interest in, and growing importance ment disk of the vestigial antennules to more lateral posi- of, this field. tions on the stalk. The bubbles enclosed in the foam-like Barnacles (Cirripedia, Thoracica) are among the most troublesome and dominant fouling organisms due to their float are most likely filled with CO2 that diffuses from the abundance and gregarious settlement (Knight-Jones and Crisp, 1953). Ships may lose up to 86% of their cruising Received 15 March 2012; accepted 29 June 2012. speed because of heavy fouling (Schultz, 2007). Therefore, * To whom correspondence should be addressed. E-mail: [email protected] since the Second World War, attempts have been made Abbreviations: ␮CT, x-ray microtomography; ESEM, environmental to prevent the attachment of barnacles by developing resis- scanning electron microscope; RER, rough endoplasmic reticulum. tant coatings for ships (Saroyan et al., 1970b; Yebra et al.,

192

10 CEMENT FLOAT OF A BARNACLE 193

2004; Aldred and Clare, 2008; Xie et al., 2011), especially the highly unusual cement of Dosima fascicularis. Early using low-energy surface materials (Crisp et al., 1985; biochemical analyses of the cement exist (Barnes and Yule and Walker, 1987). These investigations have often Blackstock, 1974, 1976), and a preliminary study to eluci- been carried out in parallel with intensive studies of date the mechanism of adhesion of this barnacle was carried barnacles—their larval development, morphological out (Power et al., 2010). Our current interest is focused on changes during metamorphosis from cyprid to juvenile, and the morphology of the cement apparatus and on the forma- adult anatomy (Walker, 1970, 1971, 1973; Cheung and tion and morphological structure of the cement float of Nigrelli, 1975). D. fascicularis. This knowledge is necessary for the under- The last larval stage, the free-swimming cyprid, settles standing of the chemical and physical (mechanical) proper- and attaches to the substratum using the attachment disk on ties of the cement. its third antennular podomere (segment) (Lagersson and Høeg, 2002). The adhesive, called cement, is extruded at the Materials and Methods attachment disk and cured underwater. Cement production continues in the adult during the growth period and in Juvenile specimens of Dosima fascicularis (Ellis and accordance with the molting cycle (Fyhn and Costlow, Solander, 1786) with a capitulum length of 2 to 6 mm and 1976; Walker, 1978; Kamino, 2006). This adhesive is pro- a width of 1 to 3 mm were collected on the western coast of duced in cement gland cells and conducted through a canal Ireland. Adult, mature animals with a capitulum length of system to the area of attachment. 10 to 25 mm and a width of 8 to 19 mm were collected on Most studies of the cement apparatus (cement gland cells the northwestern coast of Denmark and on the western coast and canals) were carried out on Sessilia (acorn barnacles) of Ireland. Only animals that had been washed ashore were in the 1970s. Cyprids (Walker, 1971, 1973; Cheung and collected. The peduncles of 5 juvenile and 10 mature ani- Nigrelli, 1975) and adult acorn barnacles (Lacombe and mals were cut into small pieces, fixed in 2.5% glutaralde- Liguori, 1969; Lacombe, 1970; Walker, 1970, 1978; Fyhn hyde, and post-fixed in 1% osmium tetroxide, both at pH 7.3 and Costlow, 1976) were investigated using histological and in 0.1 mol lϪ1 sodium cacodylate buffer with 10% (w/v) histochemical methods. These barnacles secrete a thin layer sucrose, for 2 h. The samples were dehydrated in a graded of cement, which is either solid or reticulate, between the ethanol series, and acetonitrile was used as intermediate base of the shell and the substratum (Walker and Youngson, before embedding in Agar low viscosity resin. All these 1975; Kamino, 2010). Pedunculata (stalked, or goose, bar- steps were carried out at room temperature. Sections of nacles) often extrude an excess of tough and solid adhesive 60 nm were cut on a Leica Ultracut-S microtome and (Walker and Youngson, 1975) by which they anchor the stained with 0.5% uranyl acetate and 2% lead citrate. The peduncle firmly in fissures of the substratum (pers. obs.). sections were viewed in a Zeiss EM 902 transmission elec- The buoy barnacle Dosima fascicularis (Ellis and Solander, tron microscope (80 kV). Semithin sections (1 ␮m) were 1786) of the family Lepadidae is unique among the Cirripe- stained with toluidine blue and examined with a Nikon dia (Thoracica) in producing a bulge of foam-like cement Eclipse E800 light microscope. around the basal part of the peduncle by which it attaches to For scanning electron microscopy (SEM), the polymer- any floating substrata it encounters. D. fascicularis is found ized cement of adult specimens of D. fascicularis was fixed on driftwood, feathers, seaweed, macroalgae (Boe¨tius, in 2.5% glutaraldehyde in 0.1 mol lϪ1 sodium cacodylate 1952-53; Cheng and Lewin, 1976; Hinojosa et al., 2006), buffer and rinsed with distilled water. The wet cement was and inorganic material such as tar pellets and plastics examined in a Philips XL 30 environmental scanning elec- (Minchin, 1996). As the barnacle grows, the substratum tron microscope (ESEM) (30 kV). The air-dried cement was may become enclosed in the cement ball (Boe¨tius, 1952– coated with gold by an Agar B7340 sputter coater and 53), thereby allowing the animal to float independently just examined in a Philips XL 20 scanning electron microscope below the water surface. (15 kV). Little is known about the cement apparatus in Peduncu- For x-ray microtomography (␮CT), the glutaraldehyde- lata. In juvenile stalked barnacles the cement glands are at fixed stalk of a mature animal with the attached cement was the apical region of the peduncle just beneath the capitulum. rinsed in 0.1 mol lϪ1 sodium cacodylate buffer and dehy- In adult individuals the glands may extend over most of the drated in a graded series of ethanol. Subsequently, the length of the peduncle, embedded between the ovaries sample was treated with a 70% alcoholic solution of 1% (Lacombe and Liguori, 1969; Walker, 1974). The canal (w/v) phosphotungstic acid for one week and afterward system leading from the cement glands to the base of the rinsed with distilled water. The sample was imaged at low stalk can be differentiated by location and function into four resolution for an overview (9.6-␮m voxelsize) and at high canal sections: the intracellular, the collector, the secondary, resolution for a close-up (2.4-␮m voxelsize) with an Xradia and the principal (Lacombe and Liguori, 1969). MicroXCT-200 scanner while immersed in distilled water. Little is known about the production and the structure of This system uses a 90-keV/8 W tungsten x-ray source, a

11 194 V. ZHEDEN ET AL.

Figure 1. Habitus of Dosima fascicularis with special emphasis on the peduncle. (A) Macroscopic image of the animal. (B) Transmission electron micrograph of prismatic hypodermal cells underlying the cuticle. (C) Virtual longitudinal section through the peduncle (volume rendering based on an x-ray microtomography (␮CT) image stack) focusing on the three muscle layers. (D) Virtual longitudinal ␮CT section of the peduncle of a mature animal. The wide hemolymph canal extends between the paired ovaries and the cement gland cells. (E) Semithin cross section of the peduncle, including the hemolymph canal. Its wall is formed by elongate connective tissue cells. Inset: Magnified wall cell of the hemolymph canal. c ϭ cement, ca ϭ capitulum, ci ϭ cirri, cm ϭ circular muscles, cu ϭ cuticle of the peduncle, gc ϭ cement gland cell, h ϭ hemolymph canal, hc ϭ hypodermal cells, lm ϭ longitudinal muscles, n ϭ nucleus, o ϭ ovaries, om ϭ oblique muscles, pe ϭ peduncle. Scale bars: A ϭ 2 cm; B ϭ 5 ␮m; C, D ϭ 500 ␮m; E ϭ 0.1 mm, Inset ϭ 12.5 ␮m. cooled 2 k ϫ 2 k CCD camera, and switchable scintillator- Results objective lens units. The schemata were created in Photo- General morphology of the peduncle shop CS5 (Adobe Systems Inc., San Jose, CA, USA) based on the virtual longitudinal sections obtained by ␮CT. The peduncle of Dosima fascicularis (Fig. 1A) tapers For statistical analysis, the structured-walk technique toward the base and becomes embedded in the cement as the known as the Richardson plot (Mandelbrot, 1967) was used float increases in size. The peduncle is covered by unar- to compare the ruggedness of the nuclear membrane of mored cuticle with an average thickness of 40 ␮m in juve- the young (n ϭ 10) and the mature (n ϭ 10) cement gland niles and 100 ␮m in adults. The external thin epicuticle is 52 cells. On sections, the circumference of the nucleus was nm Ϯ 31 (n ϭ 20) thick. This and all confidence intervals measured along its margin by using rods of different length. represent standard deviations (SD). Within the internal pro- The fractal dimension is determined by plotting the log of cuticle, layers of electron-dense and electron-lucent mate- the nuclear margin versus the log of the rod length (see rial alternate. Exo- and endocuticle cannot be distinguished. Table 1). The prismatic hypodermal cells (Fig.1B) underlying the

12 CEMENT FLOAT OF A BARNACLE 195

Figure 2. Illustration of longitudinal section through the peduncle and the cement ﬂoat. (A) In juvenile animals, young cement gland cells are situated close to the longitudinal muscles and connected to the canal system. The branched ends of the principal canals lead to pores at the attachment disk of the vestigial antennules. (B) In adult animals, mature cement gland cells are intermingled with the ovaries. The branched ends of the principal canals lead to lateral pores on the peduncle. Distally to the latter, previously used pores are visible in the cuticle of the peduncle. These pores are covered by layers of cement and have no connection to the cement canals. A large amount of cement is formed. a ϭ alga, arrowhead ϭ indicated position of hemolymph canal, cl ϭ cement layers, cs ϭ cement canal system, cu ϭ cuticle of the peduncle, m ϭ different muscle layers, mgc ϭ mature cement gland cells, nc ϭ newly secreted cement, o ϭ ovaries, p ϭ pores, pp ϭ pores used previously, ygc ϭ young cement gland cells. Scale bars: A, B ϭ 500 ␮m.

cuticle are 9.9 ␮m Ϯ 1.9 (n ϭ 60) high, and their oval Young cement glands nucleus is located in the center of the cell. Beneath the hypodermis is an outer layer of circular and a middle layer Young cement gland cells (Fig. 3A) occur only in small, of oblique muscles, followed by the innermost layer with juvenile animals. The cells are initially round, then acquire three to four rows of longitudinal muscles (Fig. 1C). A pair an oval shape. They are then 22.2 ␮m Ϯ 3.1 long and of antennulary nerves passes laterally through the peduncle. 16.6 ␮m Ϯ 3.3 (n ϭ 30) wide. Around these cells is a thin The wide hemolymph canal extends from the rostral sinus layer of extracellular matrix (0.1 ␮m Ϯ 0.02 wide, n ϭ 20). of the capitulum into the base of the peduncle, and its thin The initially globular nucleus (fractal dimension D ϭ 1.06, wall is formed by serially orientated connective tissue cells Table 1) is 10.7 ␮m Ϯ 2.7 (n ϭ 20) in diameter and rich in (Fig. 1D, E, Inset). Unicellular cement glands are in the euchromatin. In the nuclear membrane are a high number of upper part of the peduncle beneath the capitulum. In juve- pores (13 Ϯ 2 pores per 10 ␮m of membrane length, n ϭ nile animals, two sets of cement glands are embedded in the 10) (Fig. 3B). One electron-dense globular nucleolus is reticulate connective tissue at the periphery of the peduncle. located centrally in the nucleus, measuring on average 5 ␮m From there they extend toward the center of the peduncle in diameter. In very young animals the nucleolus has a (Fig. 2A). In mature animals, ovaries ﬁll almost the whole sharply contoured interface with the nucleoplasm. The cy- peduncle and the cement glands are intermingled with them toplasm is full of vesicular rough endoplasmic reticulum (Fig. 2B). A canal system leads from the cement glands to (RER) and free ribosomes (Fig. 3B). Other organelles are the base of the peduncle where the cement is extruded and few: Golgi bodies (consisting of two to three cisternae hardens upon contact with the substratum and the seawater. only), mitochondria, and vesicles (mostly lysosomes and Different sections of the canal system can be distinguished multivesicular bodies). Some vesicles enclose membrane mainly by the appearance of the apical surface of their wall fragments (Fig. 3C). In young cement gland cells the intra- cells (see below). cellular canal has begun to form. The lumen of this canal is

13 196 V. ZHEDEN ET AL.

Figure 3. Transmission electron micrographs of the cement apparatus of a juvenile of Dosima fascicularis. (A) Overview of a young cement gland cell with globular nucleus and one centrally located nucleolus. An extracellular matrix surrounds the cell. (B) The nuclear membrane has a large number of pores (arrowheads). The cytoplasm is full of vesicular rough endoplasmic reticulum and free ribosomes. (C) Transition between gland cell (with intracellular canal) and collector canal. Near the transition are vesicles with membrane fragments. cw ϭ collector canal wall, ex ϭ extracellular matrix, gc ϭ cement gland cell, ic ϭ intracellular canal, n ϭ nucleus, nu ϭ nucleolus, RER ϭ rough endoplasmic reticulum, ri ϭ ribosomes, vm ϭ vesicle with membrane fragments. Scale bars: A ϭ 2 ␮m; B, C ϭ 1 ␮m. usually very narrow, and few cell processes (0.08 ␮m in 140 ␮m long and 40 to 50 ␮m (n ϭ 35) wide (Fig. 4A). The diameter) project into it. The gland cell is connected to the layer of extracellular matrix around these cells is 0.21 ␮m Ϯ cells of the collector canal (Fig. 3C) and thus to the extra- 0.16 (n ϭ 30) wide. Unlike the continuous smooth surface cellular canal system. of the young gland cell, the surface of the mature gland has some narrow indentations, 0.08 ␮m Ϯ 0.02 (n ϭ 30) wide Mature cement glands (Fig. 4B). The large oblong nucleus (37.2 ␮m Ϯ 9.9 long, ϭ Mature cement gland cells occur in animals that are in the n 20) of the mature gland cell is lobed (fractal dimension ϭ reproductive stage. The gland cells are large, ovoid, 50 to D 1.42, Table 1, Fig. 4A) and contains up to 20 nucleoli embedded in heterochromatin. The nuclear membrane has 11 Ϯ 1 pores per 10 ␮m of membrane length (n ϭ 10). Table 1 RER, free ribosomes, mitochondria, and Golgi bodies are distributed throughout the cytoplasm of the cell. The cister- Comparison of the ruggedness of the nuclear membrane of the young and the mature cement gland cells with Richardson Plot (Mandelbrot, nae of the RER are round to oval (with a diameter of 0.13 1967) ␮m Ϯ 0.03, n ϭ 40), or elongate (0.09 ␮m Ϯ 0.02 wide, n ϭ 40) (Fig. 4C). The elongate cisternae are often arranged Nuclear membrane Juvenile Mature parallel to each other, forming stacks (Fig. 4D). The Golgi animals animals bodies consist, on average, of three cisternae. Depending on Sample size (gland cells) n 10 10 the plane of the section, the shape of the Golgi bodies differs Mean value of fractal dimension D 1.06 1.42 greatly: some are ﬂat, others have a C- or S-shape, and some Standard deviation 0.03 0.17 are almost round. On the trans-side of the Golgi bodies are

14 CEMENT FLOAT OF A BARNACLE 197

Figure 4. Transmission electron micrographs of the cement apparatus of a mature adult of Dosima fascicularis. (A) Overview of a mature cement gland cell with prominent intracellular canal and cross section of collector canal. The gland cell has a lobed nucleus with several nucleoli. The cell and the collector canal are surrounded by extracellular matrix. (B) Indentations of the gland cell membrane. (C) Elongate cisternae of the rough endoplasmic reticulum. (D) Golgi bodies and stacks of rough endoplasmic reticulum are present in the cytoplasm. (E) Vesicle containing crystals. (F) Autophagic vacuoles are in the cytoplasm in proximity to the intracellular canal (cut several times in the section). av ϭ autophagic vacuoles, cc ϭ collector canal, cw ϭ collector canal wall, cy ϭ crystals, ex ϭ extracellular matrix, G ϭ Golgi body, gc ϭ cement gland cell, i ϭ indentations, ic ϭ intracellular canal, n ϭ nucleus, nu ϭ nucleoli, RER ϭ rough endoplasmic reticulum. Scale bars: A ϭ 10 ␮m; B ϭ 1 ␮m; C ϭ 200 nm; D ϭ 1 ␮m; E ϭ 200 nm; F ϭ 1 ␮m.

round vesicles (diameter from 0.2 to 0.5 ␮m) with fuzzy Extracellular canal system contents. Apart from these, many lysosomes and multi- Depending on the distension of the peduncle, the cells vesicular and residual bodies are in the cytoplasm. In some that constitute the monoepithelial wall of the extracellular such vesicles, crystals occupy up to a quarter of the canal sections (collector, secondary, and principal canals) vesicle volume (Fig. 4E). The intracellular canal with its appear cuboidal or elongate, forming a squamous epithe- lumen is prominent in mature gland cells. In the cyto- lium. Their round-to-oval nuclei lie mostly in the center of plasm around the intracellular canal there is an especially the cells. Like the gland cells, the canal is surrounded by an high number of autophagic vacuoles (Fig. 4F). The cell extracellular matrix (in the latter, 0.25 ␮m Ϯ 0.11 wide, n ϭ processes, resembling microvilli (0.08 ␮m in diameter 30). The canal sections could not be distinguished by their and 0.45 ␮m long), project into the lumen and are some- epithelial height in adult animals (4 ␮m Ϯ 1.8, n ϭ 30). times branched (Fig. 5A). Several vesicles (0.3 to 0.6 ␮m in Slight differences could be observed between the collector diameter) with fuzzy content are found within the lumen. canal and the other two canal sections in juvenile animals The intracellular canal is connected to the extracellular (collector canal: 4.3 ␮m Ϯ 1.3, n ϭ 20; secondary and cement canal system. principal canals: 6.1 ␮m Ϯ 2.7, n ϭ 40). The cells within

15 198 V. ZHEDEN ET AL.

Figure 5. Transmission electron micrographs of the intracellular and collector canals. (A) Branched microvilli-like cell processes facing the lumen of the intracellular canal. Inset: Septate junction connecting cement gland cell with wall cell of the collector canal. (B) Vesicles with fuzzy contents in the lumen of the collector canal. (C) Unlike the apical surface of the intracellular canal, the surface of the collector canal wall cells is almost smooth. cc ϭ collector canal, cw ϭ collector canal wall, ex ϭ extracellular matrix, gc ϭ cement gland cell, ic ϭ intracellular canal, n ϭ nucleus, mv ϭ microvilli-like cell processes, sj ϭ septate junction, v ϭ vesicle. Scale bars: A ϭ 500 nm, Inset ϭ 100 nm; B ϭ 1 ␮m; C ϭ 2 ␮m. one canal section and between two consecutive sections are and the principal canals branch laterally at the base of the connected by an adhesion complex, parts of which are stalk. The branches of the principal canal lead through the septate junctions (Fig. 5A, Inset). muscle layers and form new pores through the cuticle (Fig. The collector canal, the first section of the extracellular 6B, Inset, C). When these are covered by the cement layer system, is continuous with the intracellular canal of the secreted last, new pores are opened more proximally on the gland cell and the secondary canal. Its wall consists of three stalk. The lumen of the principal canals is lined with a to five cells. In juvenile animals, the lumen of the canal dense, probably chitinous, cuticle without any structure. sections is at times very narrow and sometimes not even The cuticle stains strongly with toluidine blue (Fig. 6C). In visible. A few vesicles with fuzzy contents are present in the juvenile animals this cuticle is 0.18 ␮m Ϯ 0.04 (n ϭ 40); in lumen of the collector canal of mature animals (Fig. 5B). mature animals it is 0.84 ␮m Ϯ 0.22 (n ϭ 40) thick. Within These vesicles are comparable to those within the gland cell the lumen are round vesicles (Fig. 6C), electron-dense and in the intracellular canal. The apical surface of the amorphous traces, or both, probably cement (Fig. 6D). collector canal cells is almost smooth (Fig. 5C), apart from a few microvilli-like cell processes that have the same Cement dimension as those in the intracellular canal. The collector canals of several gland cells lead to a single secondary The polymerized cement usually forms an oval, whitish- canal, which in turn ends in two principal canals. In cross beige foam-like float. Animals washed ashore carry bacte- section, a single layer of three to five cells forms the wall of ria, diatoms, and sand grains on the surface of the cement the secondary canal, as in the collector canal. The wall ball. Bacteria can also be found inside the cement float and surface facing the lumen of the secondary canal is smooth. in some bubbles. Small, cement-covered flies were found in The principal canals are opposite to each other laterally at the outer region of the float. the base of the peduncle, close to the longitudinal muscles. Young animals possess only a small amount of cement They extend over two-thirds of the peduncle length. In that hardly extends beyond the diameter of the peduncle. juvenile animals they end in pores between the villi of the The adult animals (with a peduncle diameter of 4 to 5 mm vestigial antennulesЈ attachment disk (Fig. 6A). As the at its basal end) have cement floats that are mostly oval with animal grows and the float enlarges, the antennules are lost diameters between 8 and 15 mm. The polymerized cement

16 CEMENT FLOAT OF A BARNACLE 199

Figure 6. Principal canal and its pores in juvenile and mature individuals of Dosima fascicularis. (A) Semithin section of the attachment disk on the third antennular podomere of a juvenile. The principal canal ends in pores between the villi of the attachment disk. (B) Virtual longitudinal x-ray microtomography (␮CT) section of the peduncle of a mature animal. Several pores (arrows) are found laterally at the base of the peduncle. Inset: Higher magnification of the pores (arrows). (C) Semithin section of the principal canal of a mature animal leading to a pore in the cuticle of the peduncle. The cuticle lining of the principal canal is prominent. Some round vesicles are present inside the lumen. (D) Transmission electron micrograph of the principal canal formed by cuboidal cells. The lumen is lined by a thin, dense cuticle layer, and remnants of cement are present inside the canal. III and IV ϭ third and fourth antennular podomere, a ϭ alga, ad ϭ attachment disk, c ϭ cement, cu ϭ cuticle of the peduncle, l ϭ cuticle lining, o ϭ ovaries, p ϭ pores, pc ϭ principal canal, pw ϭ principal canal wall, v ϭ vesicles, vi ϭ villi. Scale bars: A ϭ 50 ␮m; B, Inset ϭ 500 ␮m; C ϭ 10 ␮m; D ϭ 5 ␮m. of animals attached to a large, smooth, and stiff surface firm, but it is still elastic after fixation. The float consists of (e.g., a plastic box) is flat and only about 5-mm thick. Its layers arranged concentrically around the stalk and the volume is about 1000 mm3. In animals attached to small and object to which it is attached. The layers are secreted from soft surfaces (e.g., algae), the cement is globular and often pores in the cuticle of the stalk, which are between 0.3- and surrounds the substratum completely. In these cases, the 0.6-mm apart in mature animals. This distance corresponds cement float is 7- to 10-mm thick and its volume is up to to the thickness of the layers within the polymerized ce- 2000 mm3. The foam-like cement contains bubbles of dif- ment. Observed under the stereo microscope in wet condi- ferent sizes (Fig. 7A). These bubbles are gas-filled and tion, the surface of the cement float appears gelatinous confer buoyancy to the animal. The cement float appears without any structure (Fig. 7A). Under the scanning electron

17 200 V. ZHEDEN ET AL.

Figure 7. Stereo and electron microscopic images of the cement float of Dosima fascicularis. (A) Stereo microscopic image of a cross section through a cement float. The inner region is filled with bubbles of different sizes. The outer region, formed by the most proximal peduncular pores, appears as a gelatinous rind. (B) Scan- ning electron micrograph of the rind of the float consisting of narrow layers with small bubbles (arrows). (C) Environmental scanning electron microscope (ESEM) micrograph of the cement float around the peduncle. In the cement around the stalk, many small round bubbles are present, followed by larger elongate bubbles with widened distal ends. (D) ESEM micrograph of a bubble with a reticulated wall. (E) Transmission electron micrograph of the fibrous cement forming condensed zones. (F) Transmission electron micrograph of the frame of a bubble. a ϭ alga, b ϭ bubble, lb ϭ large elongate bubble, pe ϭ peduncle, r ϭ rind, sb ϭ small round bubbles. Scale bars: A ϭ 5 mm; B ϭ 200 ␮m; C ϭ 1 mm; D ϭ 200 ␮m; E, F ϭ 5 ␮m. microscope the layers near the surface of the float are ␮m Ϯ 7.65 in diameter, n ϭ 20). Next to these are large narrower than in the center, giving the appearance of a rind and elongate bubbles (470 ␮m to 2460 ␮m in length) with (Fig. 7B). Within this region bubbles are closely packed, widened distal ends radiating to the periphery (Fig. 7C). The rounded, and very small (11.36 ␮m Ϯ 2.93 in diameter, bubbles have a reticulated wall (Fig. 7D). In ultrathin sec- n ϭ 50). Some elongate bubbles, compressed in parallel tions the cement appears fibrous, sometimes forming con- to the surface of the float, are also present. In the cement densed zones (Fig. 7E) that are often found framing the around the stalk are many small rounded bubbles (25.42 bubbles (Fig. 7F).

18 CEMENT FLOAT OF A BARNACLE 201

Discussion line segment is almost the same. The nucleus of the mature gland cell is up to 4 times bigger than the rounded nucleus The morphology of the cement apparatus of Dosima of the young glands, and its surface is considerably larger fascicularis is similar to that of Lepas anatifera (Lacombe due to its elongate and highly lobed form. In the young and Liguori, 1969; Jonker et al., 2012). Both species belong gland cells of D. fascicularis, the RER is vesicular and to the family Lepadidae, whose members are early diverg- appears swollen. Swollen RER was also described in the ing barnacles considered to have retained ancestral features cement gland cells of the cyprid of Semibalanus balanoides (Newman, 1982, 1987; Walker, 1992; Kugele and Yule, (Walker, 1973), in the cement cells of adult Balanus hameri 2000). One of these ancestral features may be the absence of and Chelonibia testudinaria, and especially in the synthesis differentiation within the glandular cells—that is, the pres- region of Elminius modestus (Walker, 1970, 1978). This is ence of large unicellular cement gland cells with uniform generally interpreted as a sign of synthetic activity (Han and distribution of organelles in the cytoplasm (Lacombe and Bordereau, 1982), but it is also known in the context of Liguori, 1969). In the more derived acorn barnacles (e.g., programmed cell death (Klepal et al., 2008). In our samples Balanus tintinnabulum, Elminius modestus, and Chelonibia of D. fascicularis, it cannot be ruled out that swelling of the testudinaria), a secretory (synthesis) and a storage (aggre- RER (and of some mitochondria) could be due to the gation) region can be distinguished in the cytoplasm of the suboptimal condition of the animals, which had been gland cell (Lacombe and Liguori, 1969; Lacombe, 1970; washed ashore. In the mature cement glands are many Walker, 1970, 1978). Another indication that D. fascicularis residual bodies, some with crystalline inclusions. Like Che- retains ancestral character states is that the maturation of the lonibia testudinaria and Octolasmis mu¨lleri, D. fascicularis gland cells proceeds with that of the animal, as in the produces a large volume of cement, and residual bodies are primitive balanid Semibalanus balanoides (Lacombe, a result of the high synthetic activity of the gland cells 1970). It has been suggested that maturation of the animal (Walker, 1978). The high production of proteins or the and of the cement gland cells is synchronized with the surplus of unused proteins may lead to the accumulation of molting cycle in the primitive, but not in the derived, these proteins as crystals in the residual bodies of D. fas- barnacles (e.g., Balanus psittacus) (Lacombe, 1970). In the cicularis. latter and in Lepas anatifera (Lacombe and Liguori, 1969), The formation of the intracellular canal in cement glands all stages of gland cell development can be observed in one is only possible via the partial degeneration of the cyto- animal at the same time. A more recent study (Jonker et al., plasm and some organelles. This explains the many auto- 2012) has not shown this in L. anatifera, nor does it apply phagic vacuoles, lysosomes, and multivesicular bodies near to Dosima fascicularis. Juvenile animals have only young the intracellular canal. cement cells; mature individuals only mature cells. The cement gland cells release cement either by apocrine During maturation, the gland cells and their nucleus en- secretion as in Lepas anatifera (Lacombe and Liguori, large, the cells change their shape, and the number of 1969; Jonker et al., 2012) or as vesicles by exocytosis as in nucleoli increases. The change of appearance of the gland many acorn barnacles, for example, in Balanus improvisus cells during their development was also documented for or Megabalanus rosa (Okano et al., 1996; O¨ dling et al., Lepas anatifera, Balanus nubilis, B. kondakovi, and Semi- 2006). In our samples of D. fascicularis, the mode of balanus balanoides (Lacombe and Liguori, 1969; Lacombe, secretion into the intracellular canal could not be observed. 1970; Karande and Gaonkar, 1977). The great number of It has been reported that the cement glands arise by nucleoli is a hallmark for highly active cells. In addition to differentiation of the wall cells of the secondary canal and the cement gland cells of barnacles, other gland cells are that the cement canals originate from the invagination of the known to possess nuclei with many nucleoli (Lommelen et hypodermal cells of the exterior mantle wall (Lacombe and al., 2002), as also do oocytes in, for example, amphibians Liguori, 1969; Lacombe, 1970). Other authors suggest that and fish (Shankar and Kulkarni, 2006; Mali and Bulog, the cement cells are derived from the collecting canal cells 2010). in the cyprid (Cheung and Nigrelli, 1975). So far we cannot The cement gland cells of D. fascicularis are specialized confirm either suggestion about the origin of the cement for the synthesis and secretion of the mainly proteinaceous gland cells because the available animals had developed too cement and are thus very active (Barnes and Blackstock, far to detect these processes. For such analyses newly set- 1974, 1976; Walker and Youngson, 1975). The high activity tled cyprids and stages of their metamorphosis to the juve- of the cement gland cells is also indicated by the presence of nile are necessary, which is made difficult by the fact that a great number of nuclear pores, the numerous well-devel- the lepadid barnacle D. fasciularis is a fast-growing species oped Golgi bodies, many free ribosomes, and the appear- (Anderson, 1994). ance of the rough endoplasmic reticulum (RER). Although Intracellular, collector, secondary, and principal canals the dimensions of the nuclei in young and mature glands are distinguished mainly by the appearance of the apical differ greatly, the number of nuclear pores along a defined surface of their wall cells. In D. fascicularis the cell pro-

19 202 V. ZHEDEN ET AL. cesses projecting into the lumen decrease in number from The cement in the principal canal is structureless, but the the intracellular canal to the secondary canal. The principal cement ﬂoat has a speciﬁc foam-like appearance (Boe¨tius, canal is lined by a structureless chitinous-like cuticle as 1952–53). The nature of the gas inside the bubbles is not described for other barnacles (Lacombe and Liguori, 1969; known, and it is still not clear how it penetrates into the

Walker, 1970, 1974). In juvenile animals the principal canal cement. It is most likely that CO2, a byproduct of metabo- leads into the attachment disk of the vestigial antennules, lism, is transported by the hemolymph to the collector and where the cement is extruded through several pores. As the the secondary canals, where it enters their lumen. Our animal grows the antennules are lost and new pores are preliminary observations indicate that the pH of the cement formed through the cuticle at the distal part of the peduncle. ﬂoat is 3 to 4. It is known that in an acid environment

From there the newly secreted cement is released, forming (below pH 4) more than 90% of CO2 is gaseous. Together a new layer around the existing cement ﬂoat. The new layer with the cement, CO2 would be transported from the col- covers the pores that have been used for the previous lector and secondary canals through the principal canal to cement excretion. When growth is continued, again new the outside. The lipid bilayer of cell membranes is perme- pores break through the cuticle more proximally on the able to CO2 and proteins lower CO2 permeability (Gut- stalk, and the next layer of cement is formed on top of the knecht et al., 1977; Missner et al., 2008). When the cement previous one. In this way the peduncle and its pores get and CO2 are transported through the principal canal, the more and more embedded in the cement ﬂoat as the latter penetration of CO2 through the proteinaceous cuticular lin- increases in size. The formation of new pores along with ing of the canal may be negligible. In addition, increased proceeding growth is also known in other Cirripedia Tho- hemolymph pressure within the peduncle may aid CO2 racica, for example, Semibalanus balanoides, Balanus cre- diffusion into the collector and the secondary canals, thus natus, and Lepas anatifera (Bocquet-Ve´drine, 1970; preventing its diffusion back into the peduncular plexus. An

Walker, 1973; Kugele and Yule, 2000). In connection with excretion of CO2 through the cement canal system could not active relocation, new pores are formed in the Pedunculata only cause the formation of the gas bubbles but also con- Capitulum mitella and Pollicipes pollicipes. In these species tribute to the pH regulation of the hemolymph. the pores and canals of the trailing edge are lost through Two forces act on the cement ﬂoat in opposite directions: sloughing, and their antennules cannot be detected anymore gravity, which exerts traction downward due to the weight (Kugele and Yule, 1993, 2000). In the parasitic barnacle of the animal; and buoyancy, which exerts a pressure up-

Clistosaccus paguri (Rhizocephalia), pores supposedly de- ward that is mostly caused by the light gaseous CO2. This is velop by chemical dissolution of the host and parasite seen in the elongate outline of the large bubbles radiating cuticles around the distal end of the cyprid antennules from the peduncle (caused by traction) and in their widened

(Høeg, 1985, 1990). The authors suggested that their ce- distal ends (caused by pressure of CO2). A relationship ment contains lytic enzymes. It is possible that such en- between the size of the cement float and the buoyancy of the zymes are together with the cement also present in the substratum on which the cyprids of D. fascicularis settled principal canal of D. fascicularis. They could penetrate the could not be determined in the available adult animals. cuticle of the stalk to form the pores. However, the shape of the cement float seems to correlate The mechanism of extrusion of the cement differs in the with the surface properties of the substratum. various species of Cirripedia Thoracica. A kind of flushing The cement floats of D. fascicularis individuals that have fluid as described for some Balanidae may keep the ducts been washed ashore often seem to be surrounded by a clear of any cement residues (Saroyan et al., 1970a). The “gelatinous rind.” This is newly secreted cement of low vesicles seen in the principal canal of D. fascicularis may viscosity so that it can be transported through the cement contain such a fluid. Cement secretion is confined to the canal and through the cuticular pores, as described for Lepas short intermolt period (Walker, 1971, 1978; Fyhn and Cost- anatifera (Walker and Youngson, 1975). The cement layers low, 1976; Khandeparker and Anil, 2007), suggesting that in this rind are narrower than in the center, which can be cement synthesis and accumulation in the gland cell occur explained by the increase in size of the float. Under the around the postecdysial stage. As the animal grows, the assumption that the animal can secrete only a certain quan- marginal area of the attachment region enlarges and must be tity of cement at a time, the amount of newly produced continually fixed to the substratum (Kamino, 2006). The cement available is only enough for the formation of layers extrusion of cement in D. fascicularis is hypothesized to be that become narrower toward the periphery of the float. regulated by hemolymph pressure (Power et al., 2010). In The mechanism by which the newly secreted cement is Lepas anatifera, oblique muscles between the cement cells transported from the pores in the peduncle over the entire aid the extrusion of secretion from the cement cells (Walker, surface of the float against gravity seems to depend on the 1974). In acorn barnacles, contraction of the striated basal Archimedes force on the cement (which has a lower density muscles of the mantle layer is supposed to have the same than seawater, further lowered by the bubbles within it), and function (Lacombe, 1970). the adhesive power of the cement and the gas within it.

20 CEMENT FLOAT OF A BARNACLE 203

When cement is excreted from the pores on the peduncle cement gland in different developmental stages of the barnacle Balanus just below the cement float, adhesive forces cause the newly eburneus. Mar. Biol. 32: 99–103. formed cement to adhere to that formed previously. This Crisp, D. J., G. Walker, G. A. Young, and A. B. Yule. 1985. Adhesion and substrate choice in mussels and barnacles. J. Colloid Interface Sci. may also be aided by the pressure of the surrounding water. 104: 40–50. The low density of the cement causes the animal to rise to Fyhn, U. E. H., and J. D. Costlow. 1976. A histochemical study of the water surface. The light gaseous CO2 within the cement cement secretion during intermolt cycle in barnacles. Biol. Bull. 150: may produce an additional uplift, allowing the newly 47–56. formed cement to spread all over the surface of the float. Gutknecht, J., M. A. Bisson, and F. C. Tosteson. 1977. Diffusion of carbon dioxide through lipid bilayer membranes. Effects of carbonic Flies stuck to and partly embedded within the cement prove anhydrase, bicarbonate, and unstirred layers. J. Gen. Physiol. 69: that the cement float of stranded individuals of Dosima 779–794. fascicularis was still very soft and sticky on its surface. This Han, S. H., and C. Bordereau. 1982. Ultrastructure of the fat body of could either mean that the D. fascicularis cement takes a the reproductive pair in higher termites. J. Morphol. 172: 313–322. relatively long time to harden or that cement secretion in Hinojosa, I., S. Boltanã, D. Lancellotti, E. Macaya, P. Ugalde, N. Valdivia, N. Va´squez, W. A. Newman, and M. Thiel. 2006. Geo- this particular individual was going on while the animal was graphic distribution and description of four pelagic barnacles along the washed ashore. south east Pacific coast of Chile—a zoogeographical approximation. Rev. Chil. Hist. Nat. 79: 13–27. Acknowledgments Høeg, J. T. 1985. Male cypris settlement in Clistosaccus paguri Lillje- borg (Crustacea: Cirripedia: Rhizocephala). J. Exp. Mar. Biol. Ecol. We thank D. Stuebing (Fraunhofer Institute IFAM, Bre- 89: 221–235. Høeg, J. T. 1990. “Akentrogonid” host invasion and an entirely new men, Germany) and J. L. Jonker and P. McEvilly (Ryan type of life cycle in the rhizocephalan parasite Clistosaccus paguri Institute for Environmental, Marine and Energy Research, (Thecostraca: Cirripedia). J. Crustac. Biol. 10: 37–52. National University of Ireland, Galway, Ireland) for their Jonker, J. L., J. v. Byern, P. Flammang, W. Klepal, and A. M. Power. help in collecting the animals. We greatly appreciate the 2012. Unusual adhesive production system in the barnacle Lepas help of H. L. Nemeschkal with the statistics. We thank D. anatifera: an ultrastructural and histochemical investigation. J. Mor- phol. doi: 10.1002/jmor.20067.9. Gruber and N. Cyran for interesting discussions. The com- Kamino, K. 2006. Barnacle underwater attachment. Pp. 145–166 in ments of T. Valentincic, J. Suppan, and two anonymous Biological Adhesives, A. M. Smith and J. A. Callow, eds. Springer, referees helped to improve the manuscript. This research Berlin. was carried out with the financial support of the Austrian Kamino, K. 2008. Underwater adhesive of marine organisms as the vital Science Fund (FWF) P21767-B17. One of us (A. M. Power) link between biological science and material science. Mar. Biotechnol. was funded by the Beaufort Marine Biodiscovery Pro- 10: 111–121. Kamino, K. 2010. Molecular design of barnacle cement in comparison gramme, Irish Marine Institute and Science Foundation with those of mussel and tubeworm. J. Adhes. 86: 96–110. Ireland (09RFPMTR2311). Karande, A. A., and S. N. Gaonkar. 1977. Histology and histochemistry of cement glands of Balanus kondakovi. Proc. Indian Acad. Sci. 86 B: 409–416. Literature Cited Khandeparker, L., and A. C. Anil. 2007. Underwater adhesion: the Aldred, N., and A. S. Clare. 2008. The adhesive strategies of cyprids barnacle way. Int. J. Adhesion Adhes. 27: 165–172. and development of barnacle-resistant marine coatings. Biofouling 24: Klepal, W., D. Gruber, and B. Pflugfelder. 2008. Natural cyclic 351–363. degeneration by a sequence of programmed cell death modes in Semi- Anderson, D. T. 1994. Barnacles: Structure, Function, Development balanus balanoides (Linnaeus, 1767) (Crustacea, Cirripedia Tho- and Evolution. Chapman & Hall, London. racica). Zoomorphology 127: 49–58. Ang, Q. J., J. X. C. Lee, and J. H. T. Sng. 2011. Potential of barnacle Knight-Jones, E. W., and D. J. Crisp. 1953. Gregariousness in barna- cement in dentistry. Innovation 10: 14–19. cles in relation to the fouling of ships and to anti-fouling research. Barnes, H., and J. Blackstock. 1974. The biochemical composition of Nature 171: 1109–1110. the cement of a pedunculate cirripede. J. Exp. Mar. Biol. Ecol. 16: 87– Kugele, M., and A. B. Yule. 1993. Mobility in lepadomorph barnacles. 91. J. Mar. Biol. Assoc. UK 73: 719–722. Barnes, H., and J. Blackstock. 1976. Further observations on biochem- Kugele, M., and A. B. Yule. 2000. Active relocation in lepadomorph ical composition of the cement of Lepas fascicularis Ellis and Solan- barnacles. J. Mar. Biol. Assoc. UK 80: 103–111. der: electrophoretic examination of the protein moieties under various Lacombe, D. 1970. A comparative study of the cement glands in some conditions. J. Exp. Mar. Biol. Ecol. 25: 263–271. balanid barnacles (Cirripedia, Balanidae). Biol. Bull. 139: 164–179. Bocquet-Ve´drine, J. 1970. Contribution a`l’e´tude du syste`me ce´men- Lacombe, D., and V. R. Liguori. 1969. Comparative histological taire chez Balanus crenatus Brug. (Crustace´ Cirripe`de thoracique). studies of the cement apparatus of Lepas anatifera and Balanus tintin- Arch. Zool. Exp. Gen. 111: 521–557. nabulum. Biol. Bull. 137: 170–180. Boe¨tius, J. 1952–53. Some notes on the relation to the substratum of Lagersson, N. C., and J. T. Høeg. 2002. Settlement behavior and Lepas anatifera L. and Lepas fasciculars E. et S. Oikos 4: 112–117. antennulary biomechanics in cypris larvae of Balanus amphitrite (Crus- Cheng, L., and R. A. Lewin. 1976. Goose barnacles (Cirripedia: Tho- tacea: Thecostraca: Cirripedia). Mar. Biol. 141: 513–526. racica) on floatsam beached at La Jolla, California. Fish. Bull. 74: Lee, H., B. P. Lee, and P. B. Messersmith. 2007. A reversible wet/dry 212–217. adhesive inspired by mussels and geckos. Nature 448: 338–341. Cheung, P. J., and R. F. Nigrelli. 1975. Secretory activity of the Lommelen, E., E. Schoeters, and J. Billen. 2002. Ultrastructure of the

21 204 V. ZHEDEN ET AL.

labial gland in the ant Pachycondyla obscuricornis (Hymenoptera, Smith, A. M., and J. A. Callow. 2006. Biological Adhesives. Springer, Formicidae). Neth. J. Zool. 52: 61–68. Berlin. Mali, L. B., and B. Bulog. 2010. Ultrastructure of previtellogene Stewart, R. J. 2011. Protein-based underwater adhesives and the pros- oocytes in the neotenic cave salamander Proteus anguinus anguinus pects for their biotechnological production. Appl. Microbiol. Biotech- (Amphibia, Urodela, Proteidae). Protoplasma 246: 33–39. nol. 89: 27–33. Mandelbrot, B. 1967. How long is the coast of Britain? Statistical Stewart, R. J., T. C. Ransom, and V. Hlady. 2011. Natural underwater self-similarity and fractional dimension. Science 156: 636–638. adhesives. J. Polym. Sci. B Polym. Phys. 49: 757–771. Minchin, D. 1996. Tar pellets and plastics as attachment surfaces for von Byern, J., and I. Grunwald. 2010. Biological Adhesive Systems: lepadid cirripedes in the North Atlantic Ocean. Mar. Pollut. Bull. 32: From Nature to Technical and Medical Application. Springer, Vienna. 855–859. Walker, G. 1970. The histology, histochemistry and ultrastructure of the Missner, A., P. Ku¨gler, S. M. Saparov, K. Sommer, J. C. Mathai, M. L. cement apparatus of three adult sessile barnacles, Elminius modestus, Zeidel, and P. Pohl. 2008. Carbon dioxide transport through mem- Balanus balanoides and Balanus hameri. Mar. Biol. 7: 239–248. branes. J. Biol. Chem. 283: 25340–25347. Walker, G. 1971. A study of the cement apparatus of the cypris larva of Newman, W. A. 1982. Evolution within the Crustacea. Pt. 3: Cirripedia. the barnacle Balanus balanoides. Mar. Biol. 9: 205–212. Pp. 197–221 in The Biology of Crustacea: Systematics, the Fossil Walker, G. 1973. The early development of the cement apparatus in the Record, and Biogeography, L. G. Abele, ed. Academic Press, New barnacle, Balanus balanoides (L.) (Crustacea: Cirripedia). J. Exp. Mar. York. Biol. Ecol. 12: 305–314. Newman, W. A. 1987. Evolution of cirripedes and their major groups. Walker, G. 1974. The occurrence, distribution and attachment of the Pp. 3–42 in Crustacean Issues 5: Barnacle Biology, A. J. Southward, pedunculate barnacle Octolasmis mu¨lleri (Coker) on the gills of crabs, ed. Balkema, Rotterdam. particularly the blue crab, Callinectes sapidus Rathbun. Biol. Bull. 147: O¨ dling, K., C. Albertsson, J. T. Russell, and L. G. E. Mårtensson. 2006. 678–689. An in vivo study of exocytosis of cement proteins from barnacle Walker, G. 1978. A cytological study of the cement apparatus of the Balanus improvisus (D.) cyprid larva. J. Exp. Biol. 209: 956–964. barnacle, Chelonibia testudinaria Linnaeus, an epizoite on turtles. Bull. Okano, K., K. Shimizu, C. G. Satuito, and N. Fusetani. 1996. Visu- Mar. Sci. 28: 205–209. alization of cement exocytosis in the cypris cement gland of the Walker, G. 1992. Cirripedia. Pp. 249–311 in Microscopic Anatomy of barnacle Megabalanus rosa. J. Exp. Biol. 199: 2131–2137. Invertebrates: Crustacea Power, A. M., W. Klepal, V. Zheden, J. Jonker, P. McEvilly, and J. von , F. W. Harrison and A. G. Humes, eds. Byern. 2010. Mechanisms of adhesion in adult barnacles. Pp. 153– Wiley-Liss, New York. 168 in Biological Adhesive Systems: From Nature to Technical and Walker, G., and A. Youngson. 1975. The biochemical composition of Medical Application, J. von Byern and I. Grunwald, eds. Springer, Lepas anatifera (L.) cement (Crustacea: Cirripedia). J. Mar. Biol. Vienna. Assoc. UK 55: 703–707. Saroyan, J. R., E. Lindner, and C. A. Dooley. 1970a. Repair and Xie, L., F. Hong, C. He, C. Ma, J. Liu, G. Zhang, and C. Wu. 2011. reattachment in the Balanidae as related to their cementing mechanism. Coatings with a self-generating hydrogel surface for antifouling. Poly- Biol. Bull. 139: 333–350. mer 52: 3738–3744. Saroyan, J. R., E. Lindner, C. A. Dooley, and H. R. Bleile. 1970b. Yang, W. J., T. Cai, K. G. Neoh, E. T. Kang, G. H. Dickinson, S. L. M. Barnacle cement. Key to second generation antifouling coatings. In- Teo, and D. Rittschof. 2011. Biomimetic anchors for antifouling dustrial & Engineering Chemistry Product Research and Development and antibacterial polymer brushes on stainless steel. Langmuir 27: 9: 122–133. 7065–7076. Schultz, M. P. 2007. Effects of coating roughness and biofouling on Yebra, D. M., S. Kiil, and K. Dam-Johansen. 2004. Antifouling ship resistance and powering. Biofouling 23: 331–341. technology—past, present and future steps towards efﬁcient and envi- Schultz, M. P., J. A. Bendick, E. R. Holm, and W. M. Hertel. 2011. ronmentally friendly antifouling coatings. Prog. Org. Coatings 50: Economic impact of biofouling on a naval surface ship. Biofouling 27: 75–104. 87–98. Yule, A. B., and G. Walker. 1987. Adhesion in barnacles. Pp. 389–402 Shankar, D. S., and R. S. Kulkarni. 2006. Effect of cortisol on female in Crustacean Issues. 5: Barnacle Biology, A. J. Southward, ed. freshwater ﬁsh Notopterus notopterus. J. Environ. Biol. 27: 727–731. Balkema, Rotterdam.

III. Manuscript 2

Biochemical analyses of the cement float of the goose barnacle

Dosima fascicularis - a preliminary study

Authors: Zheden V, Klepal W, von Byern J, Bogner FR, Thiel K, Kowalik T, Grunwald I

Publication status: published 2014 in Biofouling 30:949-963

Personal contributions of Vanessa Zheden:

Collection of material

Sample preparation for SEM, EDX and LM

Staining for LM

SEM, LM imaging, EDX analyses and documentation

Sample preparation for and performance of 1D- and 2D-gel electrophoresis

Manuscript preparation

23 Biofouling, 2014 Vol. 30, No. 8, 949–963, http://dx.doi.org/10.1080/08927014.2014.954557

Biochemical analyses of the cement ﬂoat of the goose barnacle Dosima fascicularis – a preliminary study Vanessa Zhedena, Waltraud Klepala, Janek von Byernb,c, Fabian Robert Bognerd, Karsten Thield, Thomas Kowalikd and Ingo Grunwaldd* aUniversity of Vienna, Faculty of Life Sciences, Core Facility Cell Imaging and Ultrastructure Research, Vienna, Austria; bLudwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria; cUniversity of Kiel, Zoological Institute, Functional Morphology and Biomechanics, Kiel, Germany; dFraunhofer Institute for Manufacturing Technology and Advanced Materials IFAM, Department Adhesives and Chemistry of Polymers, Bremen, Germany (Received 14 May 2013; accepted 11 August 2014)

The goose barnacle Dosima fascicularis produces an excessive amount of adhesive (cement), which has a double function, being used for attachment to various substrata and also as a ﬂoat (buoy). This paper focuses on the chemical composition of the cement, which has a water content of 92%. Scanning electron microscopy with EDX was used to measure the organic elements C, O and N in the foam-like cement. Vibrational spectroscopy (FTIR, Raman) provided further information about the overall secondary structure, which tended towards a β-sheet. Disulphide bonds could not be detected by Raman spectroscopy. The cystine, methionine, histidine and tryptophan contents were each below 1% in the cement. Analyses of the cement revealed a protein content of 84% and a total carbohydrate content of 1.5% in the dry cement. The amino acid composition, 1D/2D-PAGE and MS/MS sequence analysis revealed a de novo set of peptides/proteins with low homologies with other proteins such as the barnacle cement proteins, largely with an acidic pI between 3.5 and 6.0. The biochemical composition of the cement of D. fascicularis is similar to that of other barnacles, but it shows interesting variations. Keywords: barnacle adhesive/cement; 1D/2D PAGE; FTIR/Raman spectroscopy; protein primary/secondary structure; hydrogel; carbohydrate

Introduction Kamino et al. 1996; Kamino 2010; Nicklisch & Waite Proteinaceous underwater adhesives are secreted for 2012). Barnacle cement is produced by unicellular locomotion by the sea star and sea urchin (Flammang glands throughout the lifetime of the animal in accor- et al. 1998; Santos et al. 2009), for defence by the sea dance with its moulting cycle (Saroyan et al. 1970; Fyhn cucumber (DeMoor et al. 2003), for building protective & Costlow 1976; Jonker et al. 2012; Zheden et al. tubes by sabellariid polychaetes (Stewart et al. 2004) and 2012). Two types of adhesive can be distinguished: the for attachment to the substratum by mussels and primary cement, produced while the animal is attached barnacles (Kamino et al. 1996; Waite 2002). The best to a substratum, and the secondary cement, secreted characterised marine bioadhesive systems are those of when the animal is injured or detached (Saroyan et al. the sandcastle worm Phragmatopoma californica 1970, 1970; Kamino et al. 1996). The two kinds of Downloaded by [Vienna University Library] at 02:06 19 September 2014 (Stewart et al. 2004), the blue mussel Mytilus edulis cement are very similar in their amino acid composition (Silverman & Roberto 2007) and the acorn barnacle (Naldrett 1993) and consist of ~90% protein (Walker Megabalanus rosa (Kamino 2008). The adhesives of the 1972; Kamino et al. 1996). Usually the secondary sandcastle worm (Wang et al. 2010) and mytilid mussels cement is used for biochemical analyses, because it is (Waite 2002) contain significant amounts of the amino easier to collect. acid L-3,4-dihydroxyphenylalanine (L-DOPA). L-DOPA Most studies on the cement proteins of cirripedes can complex with metal ions and oxides (Sever et al. have been conducted on acorn barnacles, especially 2004), as well as with semimetals, such as silica, there- Megabalanus rosa. Its cement is composed of more than fore allowing adherence to rocks and glass (Silverman & 10 proteins, of which five are novel in their primary struc- Roberto 2007). The holdfast system of the barnacle dif- ture compared with other underwater adhesives (Kamino fers from that of mytilid mussels and sabellariid poly- 2006). Each protein has a unique characteristic and it is chaetes by the lack of a detectable L-DOPA system and assumed that it fulfils either a surface or bulk function in the slow curing process of the adhesive (Naldrett 1993; the multifunctional process of underwater attachment

*Corresponding author. Email: [email protected]

24 950 V. Zheden et al.

(Kamino 2010). Only a few studies exist on the biochemis- General cement analysis fi try of the cement of goose or stalked barnacles. The rst The cement was removed from the animal and any investigations of the biochemical composition of the adherent substratum. Pieces of cement were washed in cement of the buoy barnacle Dosima (Lepas) fascicularis distilled water to remove debris and salt before freezing (Ellis & Solander 1786) were by Barnes and Blackstock at –20°C or freeze-drying (Christ Alpha1–4, Christ (1974, 1976). They determined the carbohydrate, lipid and GmbH, Osterode, Germany). To estimate the water amino acid composition as well as the electrophoretic prop- content of the cement, four samples of three different erties of the cement. Walker and Youngson (1975) focused individuals of Dosima were weighed before and after on the related goose barnacle Lepas anatifera and assumed freeze-drying. For the protein analyses, the freeze-dried that the cement of acorn and goose barnacles had a similar samples were powdered in a small porcelain mortar. biochemical composition, but that it may act in a different way. Goose barnacles normally secrete a thick layer of primary cement in contrast to the thin film produced by acorn Total carbohydrate analysis of the cement barnacles (Walker & Youngson 1975). Most pelagic goose Carbohydrate analysis was performed using a kit from barnacles of the family Lepadidae are, like acorn barnacles, BioVision (Catalogue # K645–100. Milpitas, CA, USA). known to be major fouling organisms. They often cover This is based on the phenol-sulphuric acid method and floating objects such as ships, buoys, tar pellets, plastic, can detect most forms of carbohydrates, including simple driftwood, feathers and seaweed (Boëtius 1952–53; Cheng and complex saccharides, glycans, glycoproteins and & Lewin 1976; Minchin 1996; Hinojosa et al. 2006). glycolipids. The experiments were performed according Dosima fascicularis does not usually cover large surfaces. to the supplier’s manual. Freeze-dried cement and glu- It clusters either on organic or inorganic substrata of differ- cose for the standard curves (supplied in the kit) were ent size or on the cement of conspecifics. Its stalk is flexible completely dissolved in 150 μl of concentrated H2SO4 and the plates are fairly soft. It is unique among the barna- (98%) and incubated at 90°C for 15 min. Thereafter cles due to the amount and morphological structure of the 30 μl of developer were added and the mixture was cement it produces. It secretes layer by layer an excess mixed on a shaker for 5 min. Control analysis took place amount of foam-like cement (Zheden et al. 2012) (Figure 1 without the developer. The samples were detected spec- and Supplementary Figure S1). [Supplementary material is trophotometrically at 490 nm with a multimode reader available via a multimedia link on the online article web- Mithras LB940 (Berthold Technologies, Bad Wildbad, page.] The bubbles in the foam-like cement are probably Germany). The total carbohydrate content of the freeze- filled with CO2, a metabolic product of the haemolymph dried material was calculated on the basis of the glucose (Zheden et al. 2012). As the animal grows, small substrata standard curve via linear regression analysis. such as feathers or pieces of seaweed can be surrounded and sometimes enclosed by the enlarging cement, which then forms a float (Figure S1a, b). As a result, Staining methods D. fascicularis can drift on the surface of the water (Boëtius The periodic acid-Schiff (PAS) method (McManus & 1952–53; Young 1990). Mowry 1960) was used to detect the presence of neutral In the present study some of the biochemical compo- hexose sugar units in the cement. The negative control nents of the cement of D. fascicularis were investigated. did not contain periodic acid. The slime of the slug The large amount of primary cement is easily accessible Arion rufus served as a positive control. To test for the

Downloaded by [Vienna University Library] at 02:06 19 September 2014 and therefore ideal for analysis. Although there is overall possible presence of L-DOPA in the cement, pieces were agreement on the composition of the cement of stained according to the protocol of Arnow (1937). D. fascicularis compared with that of other barnacles, Samples from the tube-dwelling polychaete Sabellaria some differences are noticeable, including the acidic alveolata were used as a positive control (Becker et al. composition of the adhesive proteins, their structural 2012). architecture, the absence of Raman detectable disulphide bonds and the very low amounts of cystines. Scanning electron microscopy with energy dispersive X-ray microanalysis (EDX) The cement ﬂoat was cut into pieces, washed with dis- Materials and methods tilled water overnight and air-dried. The elemental com- Dosima fascicularis was collected during the summer position was determined without any carbon coating on months of 2012 and 2013 on the west coast of Denmark the samples using a Philips XL 20 scanning electron (56°57’37.63” N, 8°21’37.60” E) after having been microscope (SEM) (Eindhoven, The Netherlands) washed ashore. Only primary cement was used for the equipped with an energy dispersive X-ray spectrometer analyses. with a lithium-drifted silicon (SiLi) detector crystal.

25 Biofouling 951

Analyses were performed using EDAX Genesis 5.11 Protein Dual Colour Standards (Bio-Rad) were used as software (Mahwah, NJ, USA). Spectra of the elements markers. Gels were run at 100 V, 50 mA for 90 min in detected in the cement were collected over a time of 100 SDS PAGE running buffer 1x (25 mM Tris, 192 mM s and a ~30% dead time. Line scans with a distance of glycine, 0.1% (w/v) SDS at pH 8.3). The proteins were 140 μm and dot mappings (resolution 256 x 200, 22 visualised by staining with Page Blue™, Protein Staining frames) of the elements were also acquired. Operating Solution (Thermo Fisher Scientific Inc., Waltham, MA, parameters for all these analyses were: accelerating USA). For the two-dimensional polyacrylamide gel elec- voltage 20 kV, acquisition at 15° take-off angle and a trophoresis (2D-PAGE), the final soluble fraction was working distance of 12 mm. For high-resolution images, precipitated with 20% trichloroacetic acid (TCA) and some samples were coated with gold using an Agar 0.2% DTT in ice-cold acetone. IEF rehydration solution B7340 sputter coater (Stansted, Essex, UK) and exam- (2 M thiourea, 6 M urea, 1% (w/v) CHAPS, 1% (w/v) ined in the SEM at an accelerating voltage of 15 kV. DTT, 0.5% (w/v) IPG buffer pH 3–4) was added to the fractions and incubated at 37°C for 20 min with stirring followed by consecutive centrifugation at 21,000 x g for Amino acid analysis 5 min. The protein solution (~150 μg of protein) was The amino acid composition of the cement was deter- applied to a ReadyStrip™ IPG Strip (pH 3–10, 11 cm) mined by the Institut Kuhlmann GmbH (Ludwigshafen, (Bio-Rad). Isoelectric focusing (IEF) was performed with Germany). A small piece of the cement was washed sev- an Ettan IPGphor3 unit (GE Healthcare, Sweden) for eral times with ultra pure water and then freeze-dried. 13,000 Vh at 50 μA strip−1. The strips were equilibrated Three samples were hydrolysed and diluted in buffer in equilibration buffer 1 (50 mM Tris-HCl pH 8.8, 6 M solution. The amino acid composition was then deter- urea, 30% glycerol, 2% SDS, 1% (w/v) DTT) and equili- mined using an S443 analyser (Sykam GmbH, Fuersten- bration buffer 2 (50 mM Tris-HCl pH 8.8, 6 M urea, feldbruck, Germany). After chromatographic separation 30% glycerol, 2% SDS, 4% (w/v) idoacetamide) with of the amino acids they were post-column derivatised gentle agitation for 15 min each. 2D-PAGE for each strip with ninhydrin and detected at 570 nm (prolin 440 nm). was performed at 200 V, 50 mA for 50 min in precast For cystine and methionine analysis the samples were 10–20% polyacrylamide ready gels for IPG strips treated with performic acid for 15 h and then hydrolysed (Bio-Rad). Proteins were visualised by overnight staining for 24 h with 6 N HCl at 120°C. The amino acid trypto- in Page Blue™ and destained in distilled water phan was analysed after alkaline hydrolysis for 24 h with (Grunwald et al. 2007). 5.6 N NaOH at 110°C. All other amino acids were hydrolysed under acidic conditions with 6 N HCl at 120°C for 24 h. Sequence analysis Gel slices from 1D-PAGE gels of protein bands at a size of 85.2, 68.4, 63.5 and 60.5 kDa were digested by Solubilisation experiments trypsin/chymotrypsin according to the company’s protein The freeze-dried cement was solubilised in thiourea/urea digestion instructions for In-Gel samples (protocol lysis buffer (2 M thiourea, 7 M urea, 2% (w/v) CHAPS), and performance by Proteome Factory AG, Berlin, 2% (w/v) and alternately 5% (w/v) dithiothreitol (DTT)) Germany). The acidified peptides were applied to na- by incubation at 37°C overnight with stirring (Rabilloud noLC-ESI-MS (LTQ-FT, Thermo Finnigan) analyses

Downloaded by [Vienna University Library] at 02:06 19 September 2014 1998). Afterwards the samples were centrifuged at using a 35 min nanoLC gradient (Agilent 1,100 nanoLC 21,000 x g for 15 min. The supernatant was removed system) with solvent A (0.1% formic acid, 5% acetoni- and stored (–20°C). The remaining pellets were dis- trile, 94.9% ddH2O) and solvent B (0.1% formic acid, solved in SDS sample solubilisation buffer (1% SDS, 99.9% acetonitrile). The mass accuracy was better than 100 mM Tris-HCl pH 9.5) and incubated at 37°C for 3 h 5 ppm for MS data and ± 0.5 Da for MS/MS data. MS/ with stirring. After centrifugation the final soluble MS was subjected to de novo sequencing. The induced fraction was stored (–20°C). modification propionamide adducts (at cysteine) and common sample handling modifications were allowed during the MS/MS data search. To minimise false posi- Electrophoretic analyses tive hits, common laboratory proteins (ie trypsin, keratin) Samples for one-dimensional polyacrylamide gel electro- were taken into account and their peptide signals were phoresis (1D-PAGE) were incubated at 95°C for 15 min disregarded. Each candidate sequence was used for a in Laemmli sample buffer (Bio-Rad, Munich, Germany). BLAST alignment search to further exclude contaminants. The samples were run on a 1D precast 10–20% poly- The de novo sequencing of the MS/MS spectra was per- acrylamide ready gel (Bio-Rad) with two different formed by using the PEAKS® program (Bioinformatics amounts of protein (10 and 20 μg). Precision Plus Solutions Inc., Waterloo, ON, Canada). This software

26 952 V. Zheden et al.

computed the best peptide sequences based on the fragment Results ions collected from the peaks in the MS/MS spectrum SEM with EDX (Zhang et al. 2012). The SPIDER program (Han et al. 2005) The excess amount of foam-like cement produced by was used in conjunction with the PEAKS tool to perform D. fascicularis (Figure 1a, Figure S1) consisted of con- database searches for the MS/MS generated sequence tags. centrically arranged layers with gas-filled bubbles of dif- The SPIDER tool is able to cope with common sequence ferent sizes (Figure 1b, c). Near the surface of the substitutions such as I/L, N/GG, SAT/TAS in the de novo cement, the layers were closer packed, giving the appear- generated sequence tags. The ALC score was used as ance of a rind containing small bubbles (Figure 1b, c). selection parameter for the MS/MS sequence tags for the EDX analyses gave a rough overview of the elemen- resulting Dosima peptide sequence (see Table 2); only the tal composition of the primary cement. The elements sequences with the highest score are given here. (De novo carbon (C), oxygen (O) and nitrogen (N) had the highest sequencing by MS/MS is less reliable than chemical counts in the spectrum, followed by sulphur (S) and methods such as the EDMAN degradation and therefore magnesium (Mg). Calcium (Ca), phosphorus (P) and makes scoring of the tags necessary. The selection parameter potassium (K) were present only in smaller quantities for the candidates was their ALC score for the given (Figure 2a). EDX line scan spectra and dot mappings of sequence and only de novo peptide sequences with the very the elements showed that C, O and N were distributed highest score were given.) evenly throughout the cement (Figure 2b, c). S, Mg, Ca, The identified peptide sequences were additionally P and K seen in the spectra were not detected by line analysed by a NCBI blastp search in non-redundant data- scans and dot mappings, supposedly because the base. The blastp search algorithm automatically adjusted amounts of these elements were below the detection limit the parameter to find optimal matches to short peptides. of the system. The search was made first without limitations in the taxa settings, and second only with the search option barnacles (taxid:6,676). The first two results of each peptide Water, carbohydrate and amino acid analysis were displayed and a selection of other identified The cement had a high water content of 92% ± 2.7 sequences is shown in Table S1. (mean ± SD; n = 4), estimated by comparing the fresh and freeze-dried samples. The cement itself had the con- sistency of a stable gel, which could not be broken down FTIR and Raman spectroscopy by the fingers. The dried cement was found to contain Protein samples were measured (a) as freeze-dried 1.5% ± 0.08 (mean ± SD; n = 4) carbohydrates, as was powder without any further procedure and (b) after confirmed by PAS staining (Figure S2a). dissolution in common D2O (Sigma Aldrich, Munich, The amino acid analysis showed that the total protein Germany). For D2O exchange a fresh piece of cement content of the cement was at least 84%. Eighteen amino was washed five times with ultra pure water (0.05 μS acids were identified by hydrolysis (Table 1). The amino −1 cm ) for 1 h. After this initial washing step, the cement acids AsX (asparagine or aspartic acid) and GlX (gluta- piece was immersed in 2 ml of D2O for 7 days (the D2O mine or glutamic acid) were most prominent, each at solution was changed daily). about 10% (Table 1). The methionine, histidine, cystine For interpretation of the results normal peak picking and tryptophane amino acids were only found in small was used. The Fourier transform infrared (FTIR) mea- quantities (<1%). The proteins contained slightly more

Downloaded by [Vienna University Library] at 02:06 19 September 2014 surements were performed on a Bruker Equinox 55 polar (51%) than non-polar (49%) amino acids. High instrument (Bruker Optics GmbH, Ettlingen, Germany) amounts of amino acids with hydrophobic side chains with a Harrick Golden Gate ATR inlet (diamond crystal, (about 43%) and only small amounts of basic amino one reflection). Measurements were taken with a resolu- acids (about 11%) were detected in the cement. −1 tion of 4 cm and 32 scans. Background measurements In summary, the cement was composed of (w/w) were taken against air. The FT-Raman measurements 92% water, 6.7% proteins (84% of dry cement), 0.12% were performed on a Bruker Vertex 80 with RAM II carbohydrates (1.5% of dry cement) and 1.2% undefined module (Bruker Optics). A 1,064 nm Nd-Yg laser was substances. Part of the latter could be salts from the used. Measurements were taken with 250 scans at a reso- seawater or lipids. lution of 4 cm−1. The spectra and images were evaluated using the Bruker software package OPUS 6.5 and the Solubility and electrophoretic properties CONFOCHECK system (Bruker Optics), which is a The cement could not be fully solubilised with a thio- dedicated FTIR system for the investigation of proteins. urea/urea lysis buffer and some unresolved debris Identified peaks were aligned manually and plotted on remained. At least 10 proteins were identified using 1D- the spectra. and 2D-PAGE stained with coomassie dye Page Blue™.

27 Biofouling 953

Figure 1. Overview of the cement ﬂoat of D. fascicularis. (a) D. fascicularis attached to driftwood with an excess of cement (c). Downloaded by [Vienna University Library] at 02:06 19 September 2014 The stalk or peduncle (p) (blue) is partly embedded in the cement. The cirri (ci), the thoracopods of the animal, are seen between the opened plates of the capitulum (ca). (b) Stereo microscopic image of a piece of cement showing bubbles (*) arranged in layers; r: rind. (c) SEM image of a cross-section of the cement containing small and large bubbles (*); r: rind. Scale bar: a: 1 cm, b: 2.5 mm, c: 200 μm.

Their molecular mass ranged from 47.4 to 205.0 kDa adopted by Kamino et al. (2000 ) the D. fascicularis (Figure 3a). The apparent molecular masses of the prom- cement proteins were named according to their molecular inent proteins were 60.5, 63.5, 68.4, 85.2, 111.4, 149.3 masses (Dfcp-60, -63, -68 and -85). and 205.0 kDa. The 2D-PAGE showed three additional protein bands the molecular mass of which could not be conﬁdently determined due to the lack of marker bands Sequence analysis above 250.0 kDa. All the detected proteins were acidic The sequence tags in the four different protein fragments (or neutral) with an isoelectric point (pI) ranging from obtained from the 1D-PAGE and following PEAKS 3.5 to 6.0 (Figure 3b). The prominent proteins in the 2D (Zhang et al. 2012) and SPIDER search (Han et al. gel had molecular masses of 47.4, 60.5, 63.5, 68.4, 2005) indicated a variety of possible proteins. Each of 135.4, 149.3 and 205.0 kDa. Following the nomenclature the identiﬁed 20 peptides (Table 2) was further analysed

28 954 V. Zheden et al. Downloaded by [Vienna University Library] at 02:06 19 September 2014

Figure 2. SEM images and EDX spectra of the cement of D. fascicularis. (a) EDX spectrum of the elemental composition of the cement ﬂoat (left). The mean values in weight per cent (Wt %) and the internal error (Error) are shown on the right side. (b) SEM image with the position of the line scan (left). The associated EDX line scan spectrum (right) shows high counts of carbon (C), oxygen (O) and nitrogen (N). Other elements such as sulphur (S) are not detected with this method. (c) SEM image (left) and corresponding dot mappings of the elements C, O, N and S (right). Only C, O and N give fair signals and an even distribution of the elements; S gives a random distribution of noise signals. Scale bar: b, c: 50 μm.

by NCBI blastp database search (Altschul et al. 1997). assignment of the sequences of the bands with the In a ﬁrst approach the peptides were searched against the molecular masses 60.5, 63.5, 68.4 or 85.2 kDa to a non-redundant protein sequence (nr) entries without speciﬁc protein and/or to a related barnacle species was taxa limitations. Within these results, no conclusive possible. In a second approach the search was limited to

29 Biofouling 955

Table 1. Comparison of the amino acid composition (values in residues per hundred) of the cement of D. fascicularis* with previous data for D. fascicularis (Barnes & Blackstock 1974), Lepas anatifera (Walker & Youngson 1975), Megabalanus rosa (Kamino et al. 1996), Balanus hameri and B. crenatus (Walker 1972).

Goose barnacles Acorn barnacles Amino acid D. fascicularis* D. fascicularis L. anatifera M. rosa B. hameri B. crenatus AsX 9.30 10.12 8.75 9.066 7.81 8.28 Thr 4.80 6.35 5.25 7.049 6.56 6.23 Ser 6.58 9.88 8.75 9.905 11.36 7.69 GlX 10.30 11.41 9.10 9.149 9.05 8.63 Gly 4.21 8.71 9.57 7.920 8.27 8.59 Ala 5.60 9.88 8.99 7.473 6.87 6.47 Val 6.05 7.76 6.65 7.251 2.74 2.19 Met 0.49 0.59 0.56 1.600 0.72 0.67 Ile 5.72 6.82 5.91 5.301 4.43 5.34 Leu 8.13 9.76 9.79 8.276 8.78 8.11 Tyr 2.65 0.12 3.69 4.184 4.92 5.38 Phe 5.06 4.94 3.69 3.709 3.67 3.98 His 0.43 0.47 2.70 1.329 2.28 2.16 Lys 2.25 2.47 3.85 5.666 5.47 6.79 Arg 6.42 6.12 6.07 5.602 5.85 6.13 Pro 4.32 4.35 4.99 4.916 8.39 6.06 Cys/2 0.67 0.24 1.10 1.603 6.81 7.28 Trp 0.05 – 0.56 – – – Protein content 84% 75.9% 96% >90% 85.9% 84.5% Downloaded by [Vienna University Library] at 02:06 19 September 2014

Figure 3. 1D-PAGE and 2D-PAGE of the solubilised D. fascicularis cement proteins. (a) 1D-PAGE. Excised gel bands at 85.2 (Dfcp-85), 68.4 (Dfcp-68), 63.5 (Dfcp-63), 60.5 kDa (Dfcp-60) of the coomassie-stained gel after 1D-PAGE of the ﬁnal soluble fraction of the cement of D. fascicularis. 10 μg and 20 μg of protein were applied to the 1D gel. (b) 2D-PAGE. Corresponding positions of the excised protein bands in the coomassie-stained gel after 2D-PAGE of the ﬁnal soluble fraction after TCA acetone precipitation of the cement.

30 Downloaded by [Vienna University Library] at 02:06 19 September 2014 956 .Zheden V. tal et . Table 2. De novo peptide sequences and PEAKS® results gained from sequence analysis of protein bands obtained from PAGE of the cement of D. fascicularis by MS/MS analysis.

Dfcp Scan Sequence TLC ALC (%) m/z z ppm Local conﬁdence (%) Dfcp-85 1,212 HPLLSSLNGLVSR 10.8 83 696.8984 2 −4.6 78 77 88 96 96 91 86 72 87 89 94 95 21 Dfcp-85 994 FNVAFDELAATR 10.2 85 677.3482 2 7.0 62 56 80 82 88 89 92 95 95 93 93 96 Dfcp-85 1,279 RRGLVLSHLAQPK 10.6 82 737.9545 2 3.6 80 83 80 78 86 92 93 92 84 84 70 87 51 Dfcp-85 1,330 SSVSPSLLELSDAEPEK 12.8 75 894.4531 2 4.5 87 90 90 49 42 61 79 90 83 86 93 94 89 84 70 63 21 Dfcp-85 822 TPPAAMEELATDK 10.4 80 687.3303 2 −5.9 21 86 86 92 90 84 85 89 88 90 84 85 54 Dfcp-68 1,847 FASADELDDLLDTVK 12.2 81 826.4038 2 −3.5 45 49 70 88 91 95 96 96 94 94 92 91 81 83 49 Dfcp-68 2,911 RLAAVVLSR 7.8 86 492.8160 2 −6.6 79 84 91 93 93 93 86 88 66 Dfcp-68 3,169 LGSGYVDFLR 8.1 81 563.8023 2 7.4 78 78 89 89 89 90 92 90 90 24 Dfcp-68 1,797 LSVTQLPR 6.6 82 457.2794 2 2.0 86 87 93 92 80 93 96 24 Dfcp-68 2,245 RSGEVTSAWSSKVR 11.0 78 775.4098 2 2.8 76 78 87 88 94 93 84 84 85 88 87 56 57 30 Dfcp-63 4,320 WVTSAWSSKAR 9.0 82 639.8329 2 −0.3 78 76 89 90 89 91 93 92 75 76 41 Dfcp-63 5,370 FEDFLVNNLNAFSR 11.4 81 843.4136 2 −4.8 54 56 77 91 96 97 88 80 86 84 93 87 87 54 Dfcp-63 5,066 ELYGGLLTADELTK 10.6 76 761.9024 2 −1.4 25 81 68 90 87 91 91 91 92 88 83 74 72 25 Dfcp-63 4,765 RLEQLAGGKR 8.5 85 564.3314 2 −7.7 90 89 90 65 88 89 85 82 83 86 Dfcp-63 4,626 GPDYEVELQR 8.1 81 603.2953 2 3.1 73 84 84 91 74 91 92 80 80 59 Dfcp-60 6,857 SFLADVLGR 7.4 82 489.2779 2 7.4 48 53 91 94 92 93 94 94 73 Dfcp-60 6,795 KFAALEDFAVSR 9.8 82 677.3605 2 −1.6 69 69 91 93 92 93 91 90 87 86 90 26 Dfcp-60 6,023 MMSVSNKVR 7.4 82 526.2734 2 0.8 75 82 82 86 85 75 85 90 70 Dfcp-60 6,453 FGALGLSR 6.7 84 410.7344 2 −7.4 91 90 92 85 83 85 88 57 Dfcp-60 6,697 TPLSLESVTR 8.4 84 551.8099 2 2.0 90 92 91 87 87 91 88 80 77 49

Dfcp: D. fascicularis cement protein (followed by the molecular mass). ‘Local confidence is the confidence that a particular amino acid is present in the de novo peptide at a particular position. It is presented as a percentage. Total local confidence (TLC) is the sum of the local confidence scores (0 to 1) from each amino acid in the peptide sequence. Average local confidence (ALC) is the average of the TLC. It is TLC divided by the number of amino acids in the peptide sequence. ppm = precursor mass error, calculated as 106 × (precursor mass – peptide mass) / peptide mass. m/z = precursor mass-to-charge ratio. z = precursor charge.’ Source: PEAKS® user manual (http://www.bioinfor.com/peaks/support/). 31 Biofouling 957

the taxon barnacles. Table S1 shows the summarised Severcan & Haris 2012) and mostly buried tyrosine resi- results of over 2,000 hits. The first two identified dues (855 and 832 cm−1) (Severcan & Haris 2012). Bands proteins of each Dfcp peptide with the lowest E-value for L-DOPA (735–730 cm−1) (Ooka & Garrell 2000) and were displayed. Other results with higher E-values disulphide bridges (550–500 cm−1) (Severcan & Haris (= less reliable) are exemplarily shown to display 2012) were not found by Raman spectroscopy indicating an possible homologue sequences in other barnacle proteins. absence or an amount below the sensitivity of the method It must be remembered that, due to the short peptide (Figure 4b). L-DOPA was also not detected on staining the sequences, false positive results can occur. cement with the Arnow method (Figure S2b). Within the identified proteins the barnacle cement proteins cp-100, cp-20 and cp-19 were found, but never with the lowest E-value. In addition, the cement proteins were Discussion spread over the Dfcps like following: Dfcp-60 with 1x D. fascicularis is the only barnacle which produces cp100; Dfcp-63 with 7x cp100 and 1x cp19; Dfcp-68 with gas-filled cement, thereby allowing the animal to float. The 2x cp100, 1x cp20 and 1x cp19; Dfcp-85 with 4x cp100 animals occur either individually or in clusters attached to and 1x cp19 matches. E-values <1 were found only for the flotsam or to the cement-buoys of conspecifics. The cement Dfcp-60 (TPLSLESVTR) matching ‘neurofibromin’, consisted of 8% dry matter and had a high water content of Dfcp-63 (WVTSAWSSKAR) matching ‘lectin BRA-2’, 92% in total, which fulfilled the definition of a hydrogel Dfcp-68 (FASADELDDLLDTVK) matching ‘clathrin (Zavan et al. 2009). Other adhesives have also been classi- heavy chain’ and Dfcp-85 (RRGLVLSHLAQPK) match- fied as hydrogels, for example the gastropod adhesive gels ing ‘settlement inducing protein complex’ (Table S1). (Smith 2006), the adhesive skin exudates of the Australian Furthermore, >20 times sequences from the barnacle frog Notaden bennetti (Graham et al. 2005), the prey protein ‘MULTIFUNCin’ were identified. capture glue of the velvet worm Euperipatoides sp. (Graham et al. 2013) and the egg attachment glue of the moth Opodiphthera sp. (Li et al. 2008), but none of these FTIR spectroscopy have a foam-like structure as found in Dosima. The analysis showed significant peaks corresponding to In agreement with the organic nature of the cement proteins containing a β-sheet structure (amide I between and the results of the adhesives of other barnacle species 1,640 and 1,620 cm−1) and/or amyloid-like structure (Berglin & Gatenholm 2003; Sullan et al. 2009) the (amide I between 1,630 and 1,610 cm−1) (Barlow & Wahl EDX spectra of the cement float of D. fascicularis 2012). Distinct peaks at 1,517 cm−1 and 1,446 cm−1 in showed high counts of carbon (C), oxygen (O) and nitro- the spectrum of the dried and D2O equilibrated sample gen (N). However, in D. fascicularis the elements were respectively indicated amide II vibration in agreement distributed evenly, unlike the cement of Amphibalanus with the results of Omoike & Chorover (2004) and amphitrite where high amounts of C, N and O were con- Barlow and Wahl (2012). The proteinaceous cement that centrated in rod-shaped structures (Sullan et al. 2009). was analysed did not contain detectable amounts of phos- Sulphur (S), magnesium (Mg) and small amounts of cal- phate (1,253 cm−1) (Gremlich & Yan 2001). Multiple cium (Ca), phosphorus (P) and potassium (K), presum- peaks between 1,236 cm−1 and 950 cm−1 corresponded to ably originating from seawater, were detected in the amide III vibration (1,234 cm−1) (Cai & Singh 2004). In EDX spectra of the cement, but not in the line scans and addition, vibrations of polysaccharides (1,150– dot mappings. This indicated that the quantities of these −1 −1

Downloaded by [Vienna University Library] at 02:06 19 September 2014 1,000 cm ) and phosphodiester bonds (1,230–950 cm ) elements were near the detection limit of the EDX-sys- (Omoike & Chorover 2004) were also found. Due to the tem, which was 0.1 wt %. Interestingly, NaCl was not lack of detectable bands in the region between 1,000 and seen in the EDX spectra. The reason could be that the 700 cm−1 characteristic for phosphate-sugar backbone cement was rinsed in fresh water before it was used for vibrations (Socrates 2001), the peaks at 1,076 and EDX investigations and NaCl was washed away. 1,055 cm−1 were assigned to polysaccharide vibrations High amounts of Ca have been found in the cement rather than phosphodiester bonds (Figure 4a, Figure S3). of balanoid barnacles with a calcareous base (Walker 1972; Sangeetha et al. 2010). It is possible that Ca in the cement of these species came from the basal plate and/or Raman spectroscopy the edge of the calcareous shell (Sangeetha & Kumar The results confirmed the proteinaceous nature of the 2011). This kind of distortion could be ruled out in D. cement containing a significantly higher content of β-sheet fascicularis because this goose barnacle has a membra- structure than β-turn, α-helix and random coil (1,680– nous attachment disc and its calcareous plates do not 1,665 cm−1) (Jiskoot & Crommelin 2005). The proteins come into contact with the cement. contained a significant amount of phenylalanine Although sulphur was identified in the EDX spectra (1,003 cm−1 and 1,606 cm−1) (Jiskoot & Crommelin 2005; of the cement of D. fascicularis no disulphide bridges

32 958 V. Zheden et al. Downloaded by [Vienna University Library] at 02:06 19 September 2014

Figure 4. FTIR and Raman spectra of dry and wet D. fascicularis cement. (a) FTIR spectrum of the cement. The region between −1 4,000 and 500 cm is magniﬁed. Blue: sample equilibrated in D2O; Red: dry sample. (b) Raman spectrum of the cement. The region −1 between 4,000 and 480 cm is magniﬁed. Blue: sample equilibrated in D2O; Red: dry sample.

33 Biofouling 959

could be detected with Raman spectroscopy. In acorn cement of the closely related goose barnacle Lepas barnacle cement, disulphide bonds are known to stabilise anatifera (Walker & Youngson 1975) and that of the the protein complex and they contribute to its insolubility acorn barnacle Megabalanus rosa (Kamino et al. 1996) (Naldrett 1993; Kamino et al. 2000). Disulphide bonds had a higher protein content (>90%) than D. fascicularis. are also commonly found in the adhesives of echino- According to Barlow et al. (2010), barnacle cement derms, mussels, snails and sabellariid polychaetes is largely composed of fibrillar proteinaceous material. (Benedict & Waite 1986; Flammang et al. 1998; Smith Fibrous structures were identified by microscopic analy- et al. 1999; DeMoor et al. 2003; Zhao et al. 2005). ses in the cement of acorn barnacles (Wiegemann & Sulphur can also occur as sulphated polysaccharides as Watermann 2003; Dickinson et al. 2009; Sullan et al. seen in the adhesive of the sandcastle worm Phragmatop- 2009) and the goose barnacle D. fascicularis (Zheden oma californica (Wang & Stewart 2013). A small distinct et al. 2012). One fibrillar proteinaceous structure associ- peak at 1,055 cm−1 in the FTIR spectrum of the D. ated with bioadhesion in barnacles is amyloid (Kamino fascicularis cement equilibrated in D2O corresponds to 2008; Sullan et al. 2009; Barlow et al. 2010). In polysaccharide vibration, which suggests that some D. fascicularis, amyloid was detected by histochemical glycosylated proteins occur in the cement. This was con- methods in the cement glands and in the cement firmed by the PAS method and the total carbohydrate (McEvilly 2011) but it was not found in the closely analysis. related barnacle Lepas anatifera (Jonker et al. 2012). With the EDX system, phosphorus was detected in The FTIR spectrum of D. fascicularis cement indi- small amounts in the cement of D. fascicularis, which cated the probability of a mixture of cross, parallel and has only been reported for other barnacle cement by antiparallel β-sheets due to major overlap of the relevant Walker (1972). Phosphorus is required as an activation areas for amyloid-like and β-sheet structures (1,610– constituent for post-translational modification to form 1,630 cm−1 vs 1,620–1,640 cm−1), which could not be phosphoserine, which plays an important role in the for- further specified by processing the amide I region via mation of a strong adhesive. This component was also deconvolution (Barlow & Wahl 2012) (not shown). The found in the adhesives of caddis flies, mytilid mussels, β-sheet structures were previously found to be a feature sandcastle worms and the Cuvierian tubules of the sea of barnacle adhesives (Sullan et al. 2009; Burden et al. cucumber as well as in kelp spore adhesive (Waite & 2012). Furthermore, the FTIR spectra of wet and dry Qin 2001; Zhao et al. 2005; Flammang et al. 2009; cement differed in the amide I band shift (Figure S3), Stewart & Wang 2010; Petrone et al. 2011). However, which was most likely due to refolding of the secondary phosphoserines were not found in the adhesive system of structure caused by dehydration (Hédoux et al. 2012; barnacles (Kamino 2010) and could not be chemically Hartwig et al. 2013). verified in D. fascicularis. Also L-DOPA, a post-transla- The amino acid composition of the cement of tional modification of tyrosine, has never been identified D. fascicularis differed from earlier data by Barnes and in barnacle cement (Naldrett 1993; Kamino et al. 1996). Blackstock (1974, 1976), mainly in the smaller quantities This result was confirmed by the Arnow staining and the of alanine, glycine and serine and the larger quantity of FTIR and the Raman spectra of the cement of tyrosine. Tyrosine ring vibration is also indicated at D. fascicularis. 1,517 cm−1 in the FTIR spectra (Barth & Zscherp 2002) The adhesives of barnacles, mussels and sandcastle and as doublets at 833 and 855 cm−1 in the Raman spec- worms consist mostly of proteins (Walker & Youngson tra (Severcan & Haris 2012). Comparing the Dosima

Downloaded by [Vienna University Library] at 02:06 19 September 2014 1975; Kamino et al. 1996; Zhao et al. 2005; Silverman cement with that of L. anatifera, M. rosa, B. hameri and & Roberto 2007; Stewart et al. 2011), while the tempo- B. crenatus (Walker 1972; Walker & Youngson 1975; rary adhesives of echinoderms and gastropods are made Kamino et al. 1996), the main differences are the smaller up of proteins and carbohydrates (Flammang 2006; amounts of alanine, glycine, histidine, lysine and serine. Smith 2010). Distinct amide I, II and III bands in the The values of phenylalanine in the Dosima cement were FTIR spectrum and an amide I band in the Raman spec- higher, which was confirmed by Raman spectroscopy. trum indicated a proteinaceous cement in D. fascicularis, Comparing the Dosima cement with that of B. hameri as confirmed by the elemental analysis. The protein con- and B. crenatus (Walker 1972), the amount of valine tent of 84% was higher than reported previously (75.9%, was considerably higher and that of cystine and proline Barnes & Blackstock 1974). This may be due to smaller (see Table 1). The high amounts of hydrophobic improved methods or to some individual variation. It is amino acids found in the cement of D. fascicularis interesting that the protein content of the cement varies agree with the findings in the cement of B. crenatus, between acorn and goose barnacles and also in the B. perforatus and M. rosa. Naldrett & Kaplan (1997), different species of these two groups. Almost the same Kamino (2010) and Kamino et al. (2012) stated that protein content as in D. fascicularis was found in hydrophobic interactions rendered the cement matrix Balanus hameri and B. crenatus (Walker 1972). The insoluble and made it resistant to decomposition by

34 960 V. Zheden et al.

marine bacteria (Naldrett 1993). Both qualities are partic- The poor coverage of the database searches used ularly important for Dosima cement because the cement could indicate unexpected post-translational modifica- needs stability against mechanical impact while drifting tions or peptide mutations (Ma & Johnson 2012), but it in the sea (Zheden et al. unpublished). Because, unlike could also imply that in addition to the known cement other barnacles, an excess amount of cement is produced proteins some unknown proteins are part of the cement. by the animal, most of the cement surface is exposed to The applied MS/MS techniques can be used as the basis the environment and thus also to bacteria. Kamino for understanding the Dosima cement as a natural under- (2008) reported that the major bulk proteins of barnacle water adhesive. cement are hydrophobic, whereas proteins for surface Understanding more about the Dosima cement could functions are hydrophilic. Barlow et al. (2009) indicated lead to the production of non-toxic artificial glues for that the cement of live barnacles was moderately medical and technical purposes (Smith & Callow 2006; hydrated and therefore hydrophilic in its natural state. von Byern & Grunwald 2010). In a follow-up study, it is The isoelectric point of the observed cement proteins of planned to use peptide sequence tags to identify the full- D. fascicularis ranged from pH 3.5 to 6.0, indicating that length proteins by genome or transcriptome sequencing, mainly acidic proteins could be solubilised. or by RT-PCR methods (Hennebert et al. 2012; Lin et al. Indistinct protein bands with a molecular weight 2014). Furthermore, the peptides characterised in this from <10.0 to 90.0 kDa were found in the cement of D. study will be used to identify possible inorganic surface fascicularis by Barnes & Blackstock (1976). In the pres- binding sequences in a combined computational and ent study protein bands with a molecular weight between experimental approach as described by Steckbeck et al. 47.4 and 250.0 kDa were identified. N-terminal sequence (2014). The peptides can also be the basis for designing analysis by EDMAN degradation failed due to blocked fluorescent-labelled nucleic acids for in situ hybridisation termini of the proteins (data not shown). The reason for probes to localise RNA sequences (Wang & Stewart the N-terminal blocking can be by natural processes as 2012). In addition, anti-peptide antibodies will be raised protection for example against bacterial proteolytic to localise the protein using immuno histochemistry enzymes or because of an undesirable effect of the sam- approaches. ple preparation (eg by impurities of solubilisation or PAGE chemicals) (Wellner et al. 1990). De novo sequencing was originally performed mainly Acknowledgements manually with the help of database search, but is now The authors thank Cornelius Luetz and Stuart Fegan for criti- commonly carried out by computational systems using cally reading the manuscript. They also thank Sascha Thies, Paul Hagedorn und Thomas Montforts for their help with the algorithms to reduce time and costs (Ma & Johnson protein and peptide analyses and helpful discussions. They are 2012). In the present study a set of 20 peptide sequences grateful to Dr Patrick Flammang who provided histological was identified (five of each analysed Dcfp). A NCBI sections of Sabellaria alveolata as positive controls in the blastp search of each of the peptides showed no results L-DOPA test. The authors would also like to thank the anony- that allowed definite conclusions to be drawn about the mous reviewers for their constructive and helpful comments. This research was carried out with the financial support of the analysed Dfcp or the underlying mechanisms of the Austrian Science Fund (FWF) [P21767-B17 to Waltraud adhesion process. In addition, it must be taken into Klepal] and of the Fraunhofer-Gesellschaft zur Foerderung der account that only some of the solubilised proteins were angewandten Forschung e.V., Germany. A scholarship was analysed. Some of the identified peptides showed simi- granted to Vanessa Zheden from the German Academic Exchange Service (DAAD). This work was also supported by Downloaded by [Vienna University Library] at 02:06 19 September 2014 larities to the barnacle cement proteins (cp) (Kamino COST action TD0906 ‘Biological Adhesives: from Biology to 2013). At least 14 sequence hits were found for cp100 k, Biomimetics’. three hits for cp19 k and two for cp20 k and no hit for cp68 k or cp52 k. Markedly, the most abundant cement protein cp100 k (Kamino 2013; Lin et al. 2014) was also References found in the present study with most matches (but not Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, with the lowest E-values). Miller W, Lipman DJ. 1997. Gapped BLAST and PSI- The NCBI blastp showed more than 20 matches for BLAST: a new generation of protein database search pro- – the protein MULTIFUNCin. This ~ 1,500 amino acid grams. Nucleic Acids Res. 25:3389 3402. Arnow LE. 1937. Colorimetric determination of the compo- large glycoprotein (accession no: AFY13480) functions nents of 3,4-dihydroxyphenylalanine tyrosine mixtures. J within the biomineralisation of barnacle shell and as Biol Chem. 118:531–537. attractant for the larvae of the barnacle. The latter applies Barlow DE, Dickinson GH, Orihuela B, Kulp JL, Rittschof D, also to Dosima whose larvae settle on the cement of con- Wahl KJ. 2010. Characterization of the adhesive plaque of fi specifics. However, MULTIFUNCin was not mentioned the barnacle Balanus amphitrite: amyloid-like nano brils are a major component. Langmuir. 26:6549–6556. in the context of cement formation (Ferrier 2010).

35 Biofouling 961

Barlow DE, Dickinson GH, Orihuela B, Rittschof D, Wahl KJ. JA, et al. 2005. Characterization of a protein-based 2009. In situ ATR-FTIR characterization of primary cement adhesive elastomer secreted by the Australian frog Notaden interfaces of the barnacle Balanus amphitrite. Biofouling. bennetti. Biomacromolecules. 6:3300–3312. 25:359–366. Graham LD, Glattauer V, Li D, Tyler MJ, Ramshaw JAM. 2013. Barlow DE, Wahl KJ. 2012. Optical spectroscopy of marine The adhesive skin exudate of Notaden bennetti frogs (Anura: bioadhesive interfaces. Annu Rev Anal Chem. 5:229–251. Limnodynastidae) has similarities to the prey capture glue Barnes H, Blackstock J. 1974. The biochemical composition of of Euperipatoides sp. velvet worms (Onychophora: the cement of a pedunculate cirripede. J Exp Mar Biol Peripatopsidae). Comp Biochem Phys B. 165:250–259. Ecol. 16:87–91. Gremlich HU, Yan B. 2001. Infrared and Raman spectroscopy Barnes H, Blackstock J. 1976. Further observations on bio- of biological materials. New York, NY: Marcel Dekker. chemical composition of the cement of Lepas fascicularis Grunwald I, Heinig I, Thole HH, Neumann D, Kahmann U, Ellis and Solander; electrophoretic examination of the Kloppstech K, Gau AE. 2007. Purification and characterisa- protein moieties under various conditions. J Exp Mar Biol tion of a jacalin-related, coleoptile specific lectin from Ecol. 25:263–271. Hordeum vulgare. Planta. 226:225–234. Barth A, Zscherp C. 2002. What vibrations tell us about Han Y, Ma B, Zhang K. 2005. SPIDER: software for protein proteins. Q Rev Biophys. 35:369–430. identification from sequence tags with de novo sequencing Becker PT, Lambert A, Lejeune A, Lanterbecq D, Flammang P. error. J Bioinform Comput Biol. 3:697–716. 2012. Identification, characterization, and expression levels Hartwig A, Meissner R, Merten C, Schiffels P, Wand P, of putative adhesive proteins from the tube-dwelling poly- Grunwald I. 2013. Mutual influence between adhesion and chaete Sabellaria alveolata. Biol Bull. 223:217–225. molecular conformation: molecular geometry is a key issue Benedict CV, Waite JH. 1986. Composition and ultrastructure in interphase formation. J Adhes. 89:77–95. of the byssus of Mytilus edulis. J Morphol. 189:261–270. Hédoux A, Paccou L, Achir S, Guinet Y. 2012. In situ monitor- Berglin M, Gatenholm P. 2003. The barnacle adhesive plaque: ing of proteins during lyophilization using micro-Raman morphological and chemical differences as a response to sub- spectroscopy: a description of structural changes induced strate properties. Colloids Surf B Biointerfaces. 28:107–117. by dehydration. J Pharm Sci. 101:2316–2326. Boëtius J. 1952-53. Some notes on the relation to the substra- Hennebert E, Wattiez R, Waite JH, Flammang P. 2012. Charac- tum of Lepas anatifera L. and Lepas fasciculars E. et S. terization of the protein fraction of the temporary adhesive Oikos. 4:112–117. secreted by the tube feet of the sea star Asterias rubens. Burden DK, Barlow DE, Spillmann CM, Orihuela B, Rittschof Biofouling. 28:289–303. D, Everett RK, Wahl KJ. 2012. Barnacle Balanus amphi- Hinojosa I, Boltaña S, Lancellotti D, Macaya E, Ugalde P, trite adheres by a stepwise cementing process. Langmuir. Valdivia N, Vásquez N, Newman WA, Thiel M. 2006. 28:13364–13372. Geographic distribution and description of four pelagic bar- Cai S, Singh BR. 2004. A distinct utility of the amide III infra- nacles along the south east Pacific coast of Chile – a zoo- red band for secondary structure estimation of aqueous pro- geographical approximation. Rev Chil Hist Nat. 79:13–27. tein solutions using partial least squares methods. Jiskoot W, Crommelin DJA. 2005. Methods for structural anal- Biochemistry. 43:2541–2549. ysis of protein pharmaceuticals. Arlington (VA): American Cheng L, Lewin RA. 1976. Goose barnacles (Cirripedia: Association of Pharmaceutical Scientists. Thoracica) on floatsam beached at La-Jolla, California. Fish Jonker J-L, von Byern J, Flammang P, Klepal W, Power AM. Bull. 74:212–217. 2012. Unusual adhesive production system in the barnacle DeMoor S, Waite JH, Jangoux M, Flammang P. 2003. Characteriza- Lepas anatifera: an ultrastructural and histochemical inves- tion of the adhesive from Cuvierian tubules of the sea cucumber tigation. J Morphol. 273:1377–1391. Holothuria forskali (Echinodermata, Holothuroidea). Mar Kamino K. 2006. Barnacle underwater attachment. In: Smith Biotechnol. 5:45–57. AM, Callow JA, editors. Biological adhesives. Berlin: Dickinson GH, Vega IE, Wahl KJ, Orihuela B, Beyley V, Springer; p. 145–166. Rodriguez EN, Everett RK, Bonaventura J, Rittschof D. Kamino K. 2008. Underwater adhesive of marine organisms as 2009. Barnacle cement: a polymerization model based on the vital link between biological science and material sci- evolutionary concepts. J Exp Biol. 212:3499–3510. ence. Mar Biotechnol. 10:111–121.

Downloaded by [Vienna University Library] at 02:06 19 September 2014 Ferrier GA. 2010. The sensory basis for ecological paradigms Kamino K. 2010. Molecular design of barnacle cement in com- on wave-swept shores [dissertation]. Los Angeles (CA): parison with those of mussel and tubeworm. J Adhes. University of California. 86:96–110. Flammang P. 2006. Adhesive secretions in echinoderms: an Kamino K. 2013. Mini-review: barnacle adhesives and adhe- overview. In: Smith AM, Callow JA, editors. Biological sion. Biofouling. 29:735–749. adhesives. Berlin: Springer; p. 183–206. Kamino K, Inoue K, Maruyama T, Takamatsu N, Harayama S, Flammang P, Lambert A, Bailly P, Hennebert E. 2009. Poly- Shizuri Y. 2000. Barnacle cement proteins – importance of phosphoprotein-containing marine adhesives. J Adhes. disulﬁde bonds in their insolubility. J Biol Chem. 85:447–464. 275:27360–27365. Flammang P, Michel A, Van Cauwenberge A, Alexandre H, Kamino K, Nakano M, Kanai S. 2012. Signiﬁcance of the con- Jangoux M. 1998. A study of the temporary adhesion of the formation of building blocks in curing of barnacle under- podia in the sea star Asterias rubens (Echinodermata, Aster- water adhesive. FEBS J. 279:1750–1760. oidea) through their footprints. J Exp Biol. 201:2383–2395. Kamino K, Odo S, Maruyama T. 1996. Cement proteins of the Fyhn UEH, Costlow JD. 1976. A histochemical study of acorn barnacle, Megabalanus rosa. Biol Bull. 190:403–409. cement secretion during intermolt cycle in barnacles. Biol Li DM, Huson MG, Graham LD. 2008. Proteinaceous adhesive Bull. 150:47–56. secretions from insects, and in particular the egg attachment Graham LD, Glattauer V, Huson MG, Maxwell JM, Knott RB, glue of Opodiphthera sp. moths. Arch Insect Biochem. White JW, Vaughan PR, Peng Y, Tyler MJ, Werkmeister 69:85–105.

36 962 V. Zheden et al.

Lin HC, Wong YH, Tsang LM, Chu KH, Qian PY, Chan BKK. Smith AM. 2006. The biochemistry and mechanics of gastro- 2014. First study on gene expression of cement proteins pod adhesive gels. In: Smith AM, Callow JA, editors. and potential adhesion-related genes of a membranous- Biological adhesives. Berlin: Spinger; p. 167–182. based barnacle as revealed from next-generation sequencing Smith AM. 2010. Gastropod secretory glands and adhesive technology. Biofouling. 30:169–181. gels. In: von Byern J, Grunwald I, editors. Biological Ma B, Johnson R. 2012. De novo sequencing and homology adhesive systems: from nature to technical and medical searching. Mol Cell Proteomics. 11.O111: 014902. application. Vienna: Springer; p. 41–51. McEvilly P. 2011. Components of the adhesive of the peduncu- Smith AM, Callow JA. 2006. Biological adhesives. Berlin: late barnacles Dosima fascicularis, Lepas anatifera, Concho- Springer. derma auritum, Lepas pectinata and Pollicipes pollicipes Smith AM, Quick TJ, Peter RLS. 1999. Differences in the [Masters thesis]. Galway: National University of Ireland. composition of adhesive and non-adhesive mucus from the McManus JFA, Mowry RW. 1960. Staining methods: histologi- limpet Lottia limatula. Biol Bull. 196:34–44. cal and histochemical. New York, NY: Paul B. Hoeber Inc. Socrates G. 2001. Infrared and Raman characteristics group Minchin D. 1996. Tar pellets and plastics as attachment sur- frequencies: tables and charts. Chichester: Wiley. faces for lepadid cirripedes in the North Atlantic Ocean. Steckbeck S, Schneider J, Wittig L, Rischka K, Grunwald I, Mar Pollut Bull. 32:855–859. Ciacchi LC. 2014. Identification of materials’ binding pep- Naldrett MJ. 1993. The importance of sulphur cross-links and tide sequences guided by a MALDI-ToF MS depletion hydrophobic interactions in the polymerization of barnacle assay. Anal Methods. 6:1501–1509. cement. J Mar Biol Assoc UK. 73:689–702. Stewart RJ, Wang CS. 2010. Adaptation of caddisfly larval Naldrett MJ, Kaplan DL. 1997. Characterization of barnacle silks to aquatic habitats by phosphorylation of H-fibroin (Balanus eburneus and B. cenatus) adhesive proteins. Mar serines. Biomacromolecules. 11:969–974. Biol. 127:629–635. Stewart RJ, Weaver JC, Morse DE, Waite JH. 2004. The tube Nicklisch SCT, Waite JH. 2012. Mini-review: the role of redox cement of Phragmatopoma californica: a solid foam. J Exp in Dopa-mediated marine adhesion. Biofouling. 28:865–877. Biol. 207:4727–4734. Omoike A, Chorover J. 2004. Spectroscopic study of extracel- Stewart RJ, Ransom TC, Hlady V. 2011. Natural underwater lular polymeric substances from Bacillus subtilis: aqueous adhesives. J Polym Sci B Polym Phys. 49:757–771. chemistry and adsorption effects. Biomacromolecules. Sullan RMA, Gunari N, Tanur AE, Chan Y, Dickinson GH, 5:1219–1230. Orihuela B, Rittschof D, Walker GC. 2009. Nanoscale Ooka AA, Garrell RL. 2000. Surface-enhanced Raman spec- structures and mechanics of barnacle cement. Biofouling. troscopy of DOPA-containing peptides related to adhesive 25:263–275. protein of marine mussel, Mytilus edulis. Biopolymers. von Byern J, Grunwald I. 2010. Biological adhesive systems: 57:92–102. from nature to technical and medical application. Vienna: Petrone L, Easingwood R, Barker MF, McQuillan AJ. 2011. In Springer. situ ATR-IR spectroscopic and electron microscopic analy- Waite JH. 2002. Adhesion à la moule [Adhesion like a mussel]. ses of settlement secretions of Undaria pinnatifida kelp Integr Comp Biol. 42:1172–1180. spores. J R Soc Interface. 8:410–422. Waite JH, Qin XX. 2001. Polyphosphoprotein from the adhe- Rabilloud T. 1998. Use of thiourea to increase the solubility of sive pads of Mytilus edulis. Biochemistry. 40:2887–2893. membrane proteins in two-dimensional electrophoresis. Walker G. 1972. The biochemical composition of the cement Electrophoresis. 19:758–760. of two barnacle species, Balanus hameri and Balanus cren- Sangeetha R, Kumar R, Venkatesan R, Doble M, Vedaprakash atus. J Mar Biol Assoc UK. 52:429–435. L, Kruparatnam, Lakshmi K, Dineshram. 2010. Walker G, Youngson A. 1975. The biochemical composition of Understanding the structure of the adhesive plaque of Lepas anatifera (L.) cement (Crustacea: Cirripedia). J Mar Amphibalanus reticulatus. Mater Sci Eng C. 30:112–119. Biol Assoc UK. 55:703–707. Sangeetha R, Kumar R. 2011. Interfacial morphology and Wang CS, Stewart RJ. 2012. Localization of the bioadhesive nanomechanics of cement of the barnacle, Amphibalanus precursors of the sandcastle worm, Phragmatopoma califor- reticulatus on metallic and non-metallic substrata. nica (Fewkes). J Exp Biol. 215:351–361. Biofouling. 27:569–577. Wang CS, Stewart RJ. 2013. Multipart copolyelectrolyte adhe-

Downloaded by [Vienna University Library] at 02:06 19 September 2014 Santos R, da Costa G, Franco C, Gomes-Alves P, Flammang P, sive of the sandcastle worm, Phragmatopoma californica Coelho AV. 2009. First insights into the biochemistry of tube (Fewkes): catechol oxidase catalyzed curing through pepti- foot adhesive from the sea urchin Paracentrotus lividus dyl-DOPA. Biomacromolecules. 14:1607–1617. (Echinoidea, Echinodermata). Mar Biotechnol. 11:686–698. Wang CS, Svendsen KK, Stewart RJ. 2010. Morphology of the Saroyan JR, Lindner E, Dooley CA. 1970. Repair and reattach- adhesive system in the sandcastle worm, Phragmatopoma ment in the Balanidae as related to their cementing mecha- californica. In: von Byern J, Grunwald I, editors. Biologi- nism. Biol Bull. 139:333–350. cal adhesive systems: from nature to technical and medical Saroyan JR, Lindner E, Dooley CA, Bleile HR. 1970. Barnacle application. Vienna: Springer; p. 169–179. cement - key to second generation antifouling coatings. Ind Wellner D, Panneerselvam C, Horecker BL. 1990. Sequencing of Eng Chem Prod Res Dev. 9:122–133. peptides and proteins with blocked N-terminal amino acids: Sever MJ, Weisser JT, Monahan J, Srinivasan S, Wilker JJ. 2004. N-acetylserine or N-acetylthreonine. PNAS. 87:1947–1949. Metal-mediated cross-linking in the generation of a Wiegemann M, Watermann B. 2003. Peculiarities of barnacle marine-mussel adhesive. Angew Chem Int Edit. 43:448–450. adhesive cured on non-stick surfaces. J Adhes Sci Technol. Severcan F, Haris PI. 2012. Vibrational spectroscopy in 17:1957–1977. diagnosis and screening. Amsterdam: IOS Press. Young PS. 1990. Lepadomorph cirripeds from the brazilian Silverman HG, Roberto FF. 2007. Understanding marine coast. I.-Families Lepadidae, Poecilasmatidae and Hetera- mussel adhesion. Mar Biotechnol. 9:661–681. lepadidae. Bull Mar Sci. 47:641–655.

37 Biofouling 963

Zavan B, Cortivo R, Abatangelo G. 2009. Hydrogels and tissue Zhao H, Sun CJ, Stewart RJ, Waite JH. 2005. Cement proteins engineering. In: Barbucci R, editor. Hydrogels: biological of the tube-building polychaete Phragmatopoma californica. properties and applications. Milan: Springer; p. 1–8. J Biol Chem. 280:42938–42944. Zhang J, Xin L, Shan B, Chen W, Xie M, Yuen D, Zhang W, Zheden V, von Byern J, Kerbl A, Leisch N, Staedler Y, Zhang Z, Lajoie GA, Ma B. 2012. PEAKS DB: de novo Grunwald I, Power AM, Klepal W. 2012. Morphology of sequencing assisted database search for sensitive and accu- the cement apparatus and the cement of the buoy barnacle rate peptide identiﬁcation. Mol Cell Proteomics. 11:M111. Dosima fascicularis (Crustacea, Cirripedia, Thoracica, 010587. Lepadidae). Biol Bull. 223:192–204. Downloaded by [Vienna University Library] at 02:06 19 September 2014

IV. Manuscript 3

Mechanical properties of the cement of the stalked barnacle

Dosima fascicularis (Cirripedia, Crustacea)

Authors: Zheden V, Klepal W, Gorb SN and Kovalev A

Publication status: submitted to Interface Focus

Personal contributions of Vanessa Zheden:

Collection of the material

Sample preparation for TEM and SEM

Cutting and staining of ultra-thin sections

TEM and SEM imaging and documentation

Performing micro-indentation experiments and tensile tests

Creation of graphs, box plots and performance of statistical analyses

Manuscript preparation

39 Mechanical properties of the cement of the stalked barnacle Dosima fascicularis (Cirripedia, Crustacea)

Vanessa Zheden1, Waltraud Klepal1, Stanislav N. Gorb2 and Alexander Kovalev2

1 University of Vienna, Faculty of Life Sciences, Core Facility Cell Imaging and

Ultrastructure Research, Vienna, Austria

2 Kiel University, Zoological Institute: Functional Morphology and Biomechanics, Kiel,

Germany

Corresponding author: [email protected]

Abstract

The stalked barnacle Dosima fascicularis secretes foam-like cement, whose amount usually exceeds that produced by other barnacles. When Dosima settles on small objects, this adhesive is additionally used as a float which gives buoyancy to the animal. The dual use of the cement by D. fascicularis requires mechanical properties different from those of other barnacle species. In the float, two regions with different morphological structure and mechanical properties can be distinguished. The outer compact zone with small gas bubbles is harder than the interior one, and forms a protective rind presumably against mechanical damage. The inner region with large, gas-filled bubbles is soft. This study demonstrates that

D. fascicularis cement is soft and visco-elastic. We show that the values of the elastic modulus, hardness and tensile stress are considerably lower than in the rigid cement of other barnacles.

Key words: adhesive, elastic modulus, hardness, tensile stress

40 Introduction

Sessile marine organisms secrete adhesives which are cured underwater and which remain durable in the water [1, 2]. The best-studied animals, producing a strong adhesive, are the invertebrates, such as barnacles, mussels and tubeworms [3].

Barnacles are among the most troublesome and dominant fouling organisms [4]. They settle as cypris larvae on any hard substratum, whether it is man-made like ships and bridges or organisms like crabs and turtles. After metamorphosis from the cyprid to the juvenile both acorn and stalked barnacles usually produce only a thin layer of permanent adhesive, the so- called cement, by which they adhere to the substratum. The stalked barnacle Dosima fascicularis (Ellis and Solander, 1786) is exceptional, secreting a large amount of foam-like proteinaceous cement which is produced by the cement glands and led through the stalk in a complex canal system. At the base of the stalk the cement is extruded through pores in the cuticle. The bubbles enclosed in the cement contain gas [5-7] whose nature is not known so far. It is assumed that it may be CO2, a byproduct of metabolism, which is transported by the hemolymph and diffuses through the lining cells of the cement canals into the lumen of the ducts. The excretion of CO2 together with the cement is doubly advantageous for the animal: it causes the formation of gas bubbles in the cement and contributes to the pH regulation in the hemolymph [8].

D. fascicularis mainly attaches to floating objects such as feathers, driftwood, seaweed and tar pellets [5, 7, 9, 10]. As the animal grows, the amount of cement increases and can subsequently enclose any small substratum to which it adheres. With this so formed float D. fascicularis drifts passively in the neuston [11]. Other animals, like marine snails of the family Janithidae, are also rafting in the water but their float is of different origin and consists of mucus bubbles [12, 13]. It is assumed that the float is derived from an egg mass which is modified for buoyancy [12]. The precise composition of this material has not yet been analysed. Apart from the use for locomotion foam-like materials serve for attachment and

41 protection. Marine mussels like Mytilus californianus and M. edulis adhere to the substratum by means of the proteinaceous byssus. The adhesive plaque of the byssus resembles a solid foam with a spongy inner structure and a fibrous surface matrix [14-16]. According to

Grayson [17] a solid foam is defined as material “the apparent density of which is decreased substantially by the presence of numerous cells disposed throughout its mass”. The marine sandcastle worm Phragmatopoma californica builds protective tubes of solid foam [18] by gluing sand grains and parts of shells together with a proteinaceous cement [19, 20].

In addition, biofoams are frequently used by animals for the protection of brood. Examples are the nymphs of spittlebugs which secrete a froth, consisting of a proteoglycan and glycoprotein complex. The froth surrounds their body and thus protects them from e.g. desiccation [21]. Fish like the armoured catfish protect their eggs in floating mucus foam nests [22, 23]. Tropical frogs produce proteinaceous foams to protect their eggs and embryos against environmental challenges [24].

Little to nothing is known about the mechanical properties of the biofoams [e.g. 25, 26] and so far nothing is known about the mechanical qualities of the cement of stalked barnacles.

After the detailed documentation of the morphology of the cement apparatus and the cement

[8] and the study of the biochemical composition of the cement of D. fascicularis [27], it is the aim of this study to investigate the mechanical properties of this adhesive. Studies about the acorn barnacle cement show that its structure differs between different species and also within the same species depending on the substratum to which the animal is attached [2, 28].

The adhesive may be fibrous, globular or sponge-like [1, 2, 29, 30]. Accordingly mechanical properties of the cement differ. Among the best investigated qualities are adhesive tenacity, elastic modulus, and hardness. On hard substratum the adhesive tenacity or removal stress of the temporary adhesive of the cyprid (which is searching for a suitable place for attachment) and of the permanent cement of the juvenile is almost the same (around 0.2 MPa).

Considerably higher tenacity values (around 0.9 MPa) were found in the permanent adhesive

42 of a settled cyprid and the cement of the adult [31, 32]. By using elastomeric coatings, the removal stress can be lowered and barnacles can easily be detached [30, 33, 34]. The substratum also influences the elastic modulus and hardness of the cement. These values are higher on non-metallic than on metallic substrata. In previous experiments, it was shown that acorn barnacles secrete more cement on low energy, polymeric surfaces to adhere firmly than on high energy surfaces like metal [2]. Compared to the cement of acorn barnacles the

Dosima cement has a different structure [8] and according to the definition by Grayson [17] it is a solid foam. Its main function is to give buoyancy to the animal [9], apart from providing a reliable bond to the substratum. Therefore, it is to be expected that its mechanical properties differ from those of other barnacles whose only function is to adhere strongly to the substratum. In contrast to the thin and firm cement of acorn barnacles, the cement float of D. fascicularis appears soft and elastic.

In the present study, both the elastic modulus and hardness of the cement were measured using micro-indentation. Furthermore, the cement float was pulled apart in a tensile test until it ruptured, and the tensile stress was compared with the stress measured in acorn barnacles at the pull off from the substratum. The mechanical properties of the cement of D. fascicularis are interesting in comparison with those of acorn barnacles and in the context of the possible use of this adhesive in medicine and technology.

Material and methods

Dosima fascicularis which had been washed ashore was collected on the North-West coast of

Denmark. The cement was removed from the animals and stored in sea water with a 2% antibiotic antimycotic solution (Sigma-Aldrich, Vienna, Austria) prior to the analyses.

43 Electron microscopy

The cement was fixed in 2.5% glutaraldehyde in 0.1 mol l-1 sodium cacodylate buffer with

10% (w/v) sucrose at pH 7.3 for 2 h. For scanning electron microscopy, cross sections of the pre-fixed cement floats were rinsed in distilled water, air-dried and coated with gold by an

Agar B7340 sputter coater (Agar Scientific Ltd., Stansted, Essex, U.K.). The samples were examined in a Philips XL 30 scanning electron microscope (FEI and Philips, Eindhoven, NL) at 15 kV.

For transmission electron microscopy small pieces of pre-fixed cement were post-fixed in 1% osmium tetroxide in 0.1 mol l-1 sodium cacodylate buffer for 2 h. The samples were dehydrated in a graded ethanol series, and acetonitrile was used as intermediate medium before embedding in Agar Low Viscosity Resin. 60 nm sections were cut on a Reichert

Ultracut-S microtome (Leica Microsystems, Vienna, Austria), stained with 0.5% uranyl acetate and 2% lead citrate. The sections were viewed in a Zeiss EM 902 transmission electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) at 80 kV.

Micro-indentation experiments

The elastic (Young’s) modulus and the hardness of the D. fascicularis cement were measured by micro-indentation. The cement floats were cut into halves and the almost smooth surface and the inner foam-like region were measured in sea- and distilled water at room temperature.

Also the surface of dried cement was measured. On each of these samples three indentations were performed on different spots. The samples were fixed in a metal frame, so that they could not drift away. In our experiments, the Basalt 01 microtribometer (Tetra GmbH,

Germany) [35-37] was used. Indentations were performed using a glass sphere (3 mm diameter) fixed to a metal spring (figure 1a). The spring constant was calculated experimentally from the slope of the force-distance curve obtained on hard substratum

(aluminum block). The effective Young’s modulus E and the hardness H of the cement

44 samples were defined from the fit of unloading part of the force-indentation depth curves according to the Hertz equation [38] (figures 3a, b):

4 = . and = , 3 1 퐸√푅 1 5 퐹푚푎푥 2 퐹 훿 퐻 푚푎푥 R is the radius of the glass sphere, ν −is 휈the Poisson ratio,휋푅 δ is훿 the indentation depth caused by the applied force F, Fmax and δmax are the maximal values of the applied force and indentation depth respectively. The software MATLAB 7.12.0 (The MathWorks, Inc., Natick, MA, USA) was used for the fit. Graphs, box plots and statistical tests were created in Sigma Plot 11.0

(Systat Software Inc, Bangalore, India and San Jose, CA, USA). Statistical analysis was done with the nonparametric Kruskal-Wallis one way analysis of variance on Ranks with Dunn’s multiple comparison procedure (significant set at P < 0.05). Data were fitted by linear regression and tested for normal distribution using a Shapiro-Wilk’s test.

Tensile test

In this experiment whole cement floats as well as parts of the outer region of the cement float were used. These parts were cut into cubes of about 10 x 10 x 0.2 mm. The samples were then fixed by two clamps (figure 1b), one clamp was fixed on a force transducer FORT 100 (World

Precision Instruments, Sarasota, FL, USA) which pulled the sample with a constant speed of

200 µm/s. The cement was pulled in 5 mm steps, each followed by a resting period of around

8 s until the sample ruptured. The pulling force and distance were measured. Additionally, the strain at rupture was defined. Knowing the area (width multiplied by height) and the length of the cement samples, a stress–strain curve could be generated. The value of the stress was obtained from the applied force F divided by the cement cross-section area A ( = ) an휎d the 퐹 휎 퐴 strain from the extension δ divided by the length l of the cement sample ( = ) . The 훿 graphs휀 were created in Sigma Plot 11.0. The data for the relaxation (figure 8b) were휀 fitted푙 by a single exponential function and tested for normal distribution utilising Shapiro-Wilk’s test.

45 Results

Cement morphology

Dosima fascicularis occurred individually or in groups (figure 2a), producing a large amount of cement. The foam-like cement enclosed gas-filled bubbles of different size (figure 2b), which gave buoyancy to the animal. The cement was secreted in concentric layers around the stalk. The outer layers, forming a kind of rind, were narrow and contained small bubbles

(figure 2c). Inside the float were mainly large bubbles. In the scanning electron microscope, the cement had a rough structure (figure 2d), in the transmission electron microscope it appeared fibrous with condensed zones, where the fibres aggregated (figures 2e, f). Some of these zones formed the frames of the bubbles.

Micro-indentation test

Typical curves of loading force versus indentation depth of the wet and the dry cement were shown in figures 3a and 3b. The force – indentation depth curves of two consecutive indentations at the same place of the float provided information about visco-elastic-plastic deformation of the cement during the loading/unloading process (figure 4). The plastic deformation is seen as a shift of the second indentation curve (gray) relative to the first indentation curve (black). The large difference in loading and unloading parts of the indentation curves demonstrates the pronounced viscose properties of the float. However, the main mechanical response of the float on the indentation is elastic.

The elastic modulus of the cement surface measured in sea water was 16.4 kPa ± 8.8, whereas in distilled water it was 11.6 kPa ± 5.3. In the inner region the modulus was 9.3 kPa ± 5.3 measured in sea water and 8.5 kPa ± 3.6 in distilled water. Statistically there was a significant difference between the different regions of the cement in the two media (P < 0.001, Kruskal-

Wallis test). However, the pairwise comparison revealed that the inner regions were not

46 significantly different. The dry cement had a much higher Young’s modulus (0.76 MPa ±

0.87) than the cement under wet conditions (figure 5a).

The hardness of the cement surface measured in sea water was 2.5 kPa ± 1.2, whereas in distilled water it was 1.7 kPa ± 0.7. In sea water the inner region had a hardness of 1.5 kPa ±

0.9, in distilled water 1.3 kPa ± 0.4. As for the elastic modulus, the hardness differed significantly (P < 0.001, Kruskal-Wallis test), but the pairwise comparison showed that there was no significant difference between the inner regions in sea- and distilled water. The dry cement had the highest hardness values (39.0 kPa ± 23.2) (figure 5b).

The elastic modulus and the hardness were higher at the surface than in the inner zone of the wet cement. With an increasing indentation depth, the elastic modulus (R2 = 0.22, Shapiro-

Wilk’s test) and the hardness (R2 = 0.06, Shapiro-Wilk’s test) decreased (figures 6a, b). In the inner region of the cement float no correlation between the indentation depth and the elastic modulus (R2 = 0.0002, Shapiro-Wilk’s test) or hardness (R2 = 0.006, Shapiro-Wilk’s test) was observed (figures 6c, d). In the dry cement the values of the elastic modulus (R2 = 0.47,

Shapiro-Wilk’s test) and hardness (R2 = 0.43, Shapiro-Wilk’s test) decreased significantly with an increasing indentation depth (figures 6e, f).

Tensile test

During pulling, the cement was elastically extensible to the point of rupture (figure 7). The tensile stress of the cement was lying below 0.2 MPa (figure 8a). During the resting period, the force decreased, indicating slow relaxation of the material (figures 8a, b). This showed the visco-elastic properties of the cement.

Discussion

Many organisms use adhesives for a variety of purposes e.g. for attachment, defence or protection [18, 39, 40]. In marine animals the attachment can be permanent as in mussels,

47 transitory as in turbellarians or temporary as in echinoderms [41]. Barnacles use both temporary and permanent adhesion during their life cycle. The last larval stage, the cyprid, uses temporary adhesion to explore the substratum before settlement and the adult is permanently attached [32]. According to Yule and Walker [31] the cyprid used a low bond strength cement in comparison to the higher bond strength cement of the adult.

Normally barnacles deposit a thin layer (a few µm thick) of firm adhesive on high energy surfaces. Only when they adhere firmly on polymeric substrata with low energy they produce a thicker cement layer with sponge-like structure. Interestingly this spongy cement was harder and had a higher elastic modulus than the firm cement [2]. In contrast to all other barnacles

Dosima fascicularis produces a large amount of foam-like cement which contains gas-filled bubbles. The cement is secreted in concentric layers around the stalk and the attached substratum [8]. It is known that the layered structure of barnacle cement is the result of cyclic secretion during the growth of the animal [30, 42, 43]. Sun et al. [30] described the multilayered structure of the adhesive plaque of Balanus eburneus and B. variagatus. In these species the elastic modulus, in the range of 0.01-100 MPa, increased from the outer to the inner layer. In contrast to the Balanus cement, the surface of the Dosima cement had a higher elastic modulus and was harder than the inner region (figure 6). A reason for this may be that the salt in the sea water hardens the surface of the cement. In experiments we could show that the elastic modulus and hardness of the cement surface were higher in sea water than in distilled water. In addition, the outer narrow layers formed a rind, containing only small bubbles (about 11 µm in diameter), in comparison to the inner region where the elongate bubbles (up to 2460 µm in length) predominated [8]. This structure will give greater mechanical stability and stronger protection to the surface region. The rind, which acts as interface towards the environment, is prone to any mechanical- or UV-damage and possible dehydration. Similar structural and mechanical characteristics were also observed in the cement of Phragmatopoma californica, where the smallest bubbles were at the interface to the

48 surrounding water [18]. These authors suggested that the highest elastic modulus would be at the interface. Also the inner spongy plaque matrix of the mussel Mytilus edulis became increasingly dense toward the outside [14]. This seems to be a common phenomenon in the adhesives of aquatic animals.

The cement of barnacles is generally fibrous [8, 29, 44-46] and visco-elastic [2, 30]. Visco- elasticity is known to be a property of many, if not all, biological materials [47]. It was described of natural fibrous composites, like the cuticle of the attachment pads of Orthoptera

[35, 37]. Also non fibrous materials like those of echinoderm tube foot discs [48] and the adhesive gels of gastropods [49] had visco-elastic properties. Visco-elasticity of the cement of

D. fascicularis may be necessary to protect the gas-filled bubbles inside the float from rupture by any fast and strong mechanical impact e.g. caused by water current and waves.

Most values of the elastic modulus of the wet cement of D. fascicularis were in the range of

5-20 kPa. Investigations of the cement of the acorn barnacle Amphibalanus spp. showed that the elastic modulus of the cement was higher than that of D. fascicularis. The modulus of elasticity of the cement of A. amphitrite measured by Ramsay et al. [50] with the punch test apparatus was between 2.9 GPa and 6.5 GPa. Sullan et al. [1] performed AFM- nanoindentations on different structures of the cement of the same species and got lower values of the elastic modulus ranging from 0.2 to 90 MPa. The difference of these results may be due to the different methods used. Sangeetha and Kumar [2] analysed the cement of A. reticulatus, growing on metallic and non-metallic substrata, using a nanomechanical testing system. The hardness and the elastic modulus of the cement were higher on non-metallic (H =

52.6 MPa and E = 1.2 GPa) than on metallic substrata (H = 8.7 MPa and E = 0.4 GPa) and again lower than those of the Dosima cement. These authors reported that barnacles needed more cement to adhere firmly to non-metallic substrata than to metal and that detachment of barnacles from metallic surfaces was generally more difficult than from non-metallic ones.

49 The acorn barnacles described above, had a calcareous base. It cannot be ruled out that parts of the hard calcareous base were included in the measurements. In our experiments, only the pure cement, free of any animal tissue and free of any substratum, was investigated. In the indentation experiments, we selected indentation depths at least 10-fold lower than the sample thickness, in order to prevent contribution of the stiff support to the results of our measurements.

Our results revealed that the D. fascicularis cement is a soft biological material. Its elastic modulus was in the same range as that of other animal adhesive structures such as the tube foot discs of echinoderms (3-140 kPa) [48], the adhesive pads of ensiferan insects (25-100 kPa) [37] and the adhesive secreted by the Australian frog Notaden bennetti (170-1035 kPa)

[51]. Very hard marine biomaterials were the adhesive secreted by the serpulid Hydroides dianthus (about 3 GPa [52]) and the byssal threads of Mytilus mussels. The protective outer cuticle of the byssal threads of Mytilus californianus and M. galloprovincialis had a modulus of about 1.7 GPa and a hardness of around 0.1 GPa. The elastic modulus and the hardness of the cuticle were 4-6 times greater than that of the inner collagen core of the byssal threads

[25].

The dry cement of D. fascicularis had an elastic modulus of approximately 0.8 MPa, and was thus much harder than the wet cement. It is known that dehydration hardened the originally soft barnacle adhesive [30]. Desiccation also hardened biological materials, like insect cuticle or bones [53, 54].

The adhesive strength or removal stress of acorn barnacles was, like the elastic modulus and the hardness, influenced by the substratum. Unlike the elastic modulus and hardness, the tenacity (adhesive strength) was higher on “natural”, hard surfaces (around 0.9 MPa) than on polymeric substrata (< 0.1 MPa) [32, 33, 55]. D. fascicularis mostly settles on small, organic floating objects, which may be gradually enclosed in the cement float. Therefore, the cement could not be removed from the substratum and the removal stress could not be measured. In

50 order to compare the acorn barnacle cement with that of D. fascicularis, the Dosima cement was pulled apart until rupture. The tensile stress determined in such an experiment was below

0.2 MPa. Accordingly, the tensile stress of the Dosima cement was lower than the removal stress of the cement of acorn barnacles attached to natural stiff substratum, but it was slightly higher than that of barnacles attached to polymeric substrata.

Conclusions

The cement of Dosima fascicularis is a soft biological material and like that of other barnacles fibrous and visco-elastic. The values of elastic modulus, hardness and tensile stress were much lower than in the rigid cement of acorn barnacles investigated so far. A physical explanation for these differences is the foam-like structure of the Dosima cement caused by the gas-filled bubbles. An ecological explanation could be the differing living conditions of acorn barnacles and Dosima with the partly different use of the adhesive. In contrast to the gregariously settling acorn barnacles, which are firmly attached to the substratum, D. fascicularis is either singly or in small numbers attached to floating objects or drifting through the sea autonomously with a cement float. For this lifestyle the Dosima cement has to be resilient to withstand mechanical impact in the water, not hard as in other barnacles and – very important – the float must not be permeable for water and gas. The great elasticity enhanced by the foam-like structure gives the cement damping properties. In addition, this special structure of the cement is more economical for the animal than a solid structure. It saves material and thus energy.

The shock-absorbing properties combined with the expected biocompatibility of the Dosima cement make it interesting for possible applications in orthopaedics. Its structure and the assumed biodegradability make it like other biofoams [56, 57] perfectly suitable as three- dimensional scaffolds for tissue growth and wound healing. Besides the possible application in medicine the Dosima cement could also be used in technology. Its foam-like weight saving

51 structure in conjunction with the fact that the cement cures and is durable underwater could make it an appropriate material for construction works in wet environment.

Acknowledgements

We thank H. L. Nemeschkal for help with the statistics. This research was carried out with the financial support of the Austrian Science Fund (FWF) P21767-B17 to Waltraud Klepal.

Vanessa Zheden was granted a “Short-Term Scientific Mission” from the COST-action

TD0906 to perform experiments at the Kiel University.

References

[1] Sullan, R.M.A., Gunari, N., Tanur, A.E., Chan, Y., Dickinson, G.H., Orihuela, B., Rittschof, D. Walker, G.C. 2009 Nanoscale structures and mechanics of barnacle cement. Biofouling 25, 263-275. (doi:10.1080/08927010802688095).

[2] Sangeetha, R. Kumar, R. 2011 Interfacial morphology and nanomechanics of cement of the barnacle, Amphibalanus reticulatus on metallic and non-metallic substrata. Biofouling 27, 569-577. (doi:10.1080/08927014.2011.589027).

[3] Stewart, R.J., Ransom, T.C. Hlady, V. 2011 Natural underwater adhesives. J. Polym. Sci. Pol. Phys. 49, 757-771. (doi:10.1002/polb.22256).

[4] Kamino, K. 2013 Mini-review: Barnacle adhesives and adhesion. Biofouling 29, 735-749. (doi:10.1080/08927014.2013.800863).

[5] Hinojosa, I., Boltaña, S., Lancellotti, D., Macaya, E., Ugalde, P., Valdivia, N., Vásquez, N., Newman, W.A. Thiel, M. 2006 Geographic distribution and description of four pelagic barnacles along the south east Pacific coast of Chile - a zoogeographical approximation. Rev. Chil. Hist. Nat. 79, 13-27.

[6] Newman, W.A. Abbott, D.P. 1980 Cirripedia: the Barnacles. In Intertidal Invertebrates of California (eds. R.H. Morris, D.P. Abbott & E.C. Haderlie), pp. 504-535. Stanford, CA: Stanford University Press.

[7] Cheng, L. Lewin, R.A. 1976 Goose barnacles (Cirripedia: Thoracica) on flotsam beached at La Jolla, California. Fish. Bull. 74, 212-217.

[8] Zheden, V., von Byern, J., Kerbl, A., Leisch, N., Staedler, Y., Grunwald, I., Power, A.M. Klepal, W. 2012 Morphology of the cement apparatus and the cement of the buoy barnacle Dosima fascicularis (Crustacea, Cirripedia, Thoracica, Lepadidae). Biol. Bull. 223, 192-204.

52 [9] Boëtius, J. 1952-53 Some notes on the relation to the substratum of Lepas anatifera L. and Lepas fascicularis E. et S. Oikos 4, 112-117. (doi:10.2307/3564807).

[10] Minchin, D. 1996 Tar pellets and plastics as attachment surfaces for lepadid cirripedes in the North Atlantic Ocean. Mar. Pollut. Bull. 32, 855-859. (doi:10.1016/S0025- 326X(96)00045-8 ).

[11] Thiel, M. Gutow, L. 2005 The ecology of rafting in the marine environment. II. The rafting organisms and community. Oceanogr. Mar. Biol. Ann. Rev. 43, 279-418.

[12] Churchill, C.K.C., Foighil, D.O., Strong, E.E. Gittenberger, A. 2011 Females floated first in bubble-rafting snails. Curr. Biol. 21, R802-R803. (doi:10.1016/j.cub.2011.08.011).

[13] Churchill, C.K.C., Strong, E.E. Foighil, D.O. 2011 Hitchhiking juveniles in the rare neustonic gastropod Recluzia cf. Jehennei (Janthinidae). J. Mollus. Stud. 77, 441-444. (doi:10.1093/mollus/eyr020).

[14] Benedict, C.V. Waite, J.H. 1986 Composition and ultrastructure of the byssus of Mytilus edulis. J. Morphol. 189, 261-270. (doi:10.1002/jmor.1051890305 ).

[15] Waite, J.H. 1986 Mussel glue from Mytilus californianus Conrad: a comparative study. J. Comp. Physiol. B 156, 491-496. (doi:10.1007/bf00691034).

[16] Silverman, H.G. Roberto, F.F. 2007 Understanding marine mussel adhesion. Mar. Biotechnol. 9, 661-681. (doi:10.1007/s10126-007-9053-x).

[17] Grayson, M. 1983 Encyclopaedia of composite materials and components, 1st edn. New York: Wiley & Sons.

[18] Stewart, R.J., Weaver, J.C., Morse, D.E. Waite, J.H. 2004 The tube cement of Phragmatopoma californica: a solid foam. J. Exp. Biol. 207, 4727-4734. (doi:10.1242/jeb.03110).

[19] Jensen, R.A. Morse, D.E. 1988 The bioadhesive of Phragmatopoma californica tubes: a silk-like cement containing L-DOPA. J. Comp. Physiol. B. 158, 317-324. (doi:10.1007/bf00695330).

[20] Zhao, H., Sun, C.J., Stewart, R.J. Waite, J.H. 2005 Cement proteins of the tube-building polychaete Phragmatopoma californica. J. Biol. Chem. 280, 42938-42944. (doi:10.1074/jbc.M508457200 ).

[21] Mello, M.L.S., Pimentel, E.R., Yamada, A.T. Storopoli-Neto, A. 1987 Composition and structure of the froth of the spittlebug, Deois sp. Insect. Biochem. 17, 493-502. (doi:10.1016/0020-1790(87)90009-6).

[22] Andrade, D.V. Abe, A.S. 1997 Foam nest production in the armoured catfish. J. Fish. Biol. 50, 665-667. (doi:10.1006/jfbi.1996.0318).

[23] Hostache, G. Mol, J.H. 1998 Reproductive biology of the neotropical armoured catfish Hoplosternum littorale (Siluriformes - Callichthyidae): a synthesis stressing the role of the

53 floating bubble nest. Aquat. Living. Resour. 11, 173-185. (doi:10.1016/s0990-7440(98)80114- 9).

[24] Cooper, A., Kennedy, M.W., Fleming, R.I., Wilson, E.H., Videler, H., Wokosin, D.L., Su, T.J., Green, R.J. Lu, J.R. 2005 Adsorption of frog foam nest proteins at the air-water interface. Biophys. J. 88, 2114-2125. (doi:10.1529/biophysj.104.046268).

[25] Holten-Andersen, N., Zhao, H. Waite, J.H. 2009 Stiff coatings on compliant biofibers: the cuticle of Mytilus californianus byssal threads. Biochemistry 48, 2752-2759. (doi:10.1021/bi900018m).

[26] Bell, E.C. Gosline, J.M. 1996 Mechanical design of mussel byssus: material yield enhances attachment strength. J. Exp. Biol. 199, 1005-1017.

[27] Zheden, V., Klepal, W., von Byern, J., Bogner, F.R., Thiel, K., Kowalik, T. Grunwald, I. 2014 Biochemical analyses of the cement float of the goose barnacle Dosima fascicularis - a preliminary study. Biofouling 30, 949-963. (doi:10.1080/08927014.2014.954557).

[28] Murosaki, T., Noguchi, T., Hashimoto, K., Kakugo, A., Kurokawa, T., Saito, J., Chen, Y.M., Furukawa, H. Gong, J.P. 2009 Antifouling properties of tough gels against barnacles in a long-term marine environment experiment. Biofouling 25, 657-666. (doi:10.1080/08927010903082628).

[29] Dickinson, G.H., Vega, I.E., Wahl, K.J., Orihuela, B., Beyley, V., Rodriguez, E.N., Everett, R.K., Bonaventura, J. Rittschof, D. 2009 Barnacle cement: a polymerization model based on evolutionary concepts. J. Exp. Biol. 212, 3499-3510. (doi:10.1242/jeb.029884).

[30] Sun, Y.J., Guo, S.L., Walker, G.C., Kavanagh, C.J. Swain, G.W. 2004 Surface elastic modulus of barnacle adhesive and release characteristics from silicone surfaces. Biofouling 20, 279-289. (doi:10.1080/08927010400026383).

[31] Yule, A.B. Walker, G. 1984 The adhesion of the barnacle, Balanus balanoides, to slate surfaces. J. Mar. Biol. Assoc. U.K. 64, 147-156.

[32] Yule, A.B. Walker, G. 1987 Adhesion in barnacles. In Crustacean Issues 5: Barnacle Biology (ed. A.J. Southward), pp. 389-402. Rotterdam: Balkema.

[33] Wendt, D.E., Kowalke, G.L., Kim, J. Singer, I.L. 2006 Factors that influence elastomeric coating performance: the effect of coating thickness on basal plate morphology, growth and critical removal stress of the barnacle Balanus amphitrite. Biofouling 22, 1-9. (doi:10.1080/08927010500499563).

[34] Berglin, M. Gatenholm, P. 1999 The nature of bioadhesive bonding between barnacles and fouling-release silicone coatings. J. Adhes. Sci. Technol. 13, 713-727. (doi:10.1163/156856199x00956).

[35] Gorb, S., Jiao, Y.K. Scherge, M. 2000 Ultrastructural architecture and mechanical properties of attachment pads in Tettigonia viridissima (Orthoptera Tettigoniidae). J. Comp. Physiol. A 186, 821-831. (doi:10.1007/s003590000135).

54 [36] Jiao, Y.K., Gorb, S. Scherge, M. 2000 Adhesion measured on the attachment pads of Tettigonia viridissima (Orthoptera, Insecta). J. Exp. Biol. 203, 1887-1895.

[37] Perez Goodwyn, P., Peressadko, A., Schwarz, H., Kastner, V. Gorb, S. 2006 Material structure, stiffness, and adhesion: why attachment pads of the grasshopper (Tettigonia viridissima) adhere more strongly than those of the locust (Locusta migratoria) (Insecta: Orthoptera). J. Comp. Physiol. A 192, 1233-1243. (doi:10.1007/s00359-006-0156-z).

[38] Hertz, H. 1881 Über den Kontakt elastischer Körper. J. Reine Angew. Math. 92, 156-171.

[39] DeMoor, S., Waite, J.H., Jangoux, M. Flammang, P. 2003 Characterization of the adhesive from Cuvierian tubules of the sea cucumber Holothuria forskali (Echinodermata, Holothuroidea). Mar. Biotechnol. 5, 45-57. (doi:10.1007/s10126-002-0049-2).

[40] Kamino, K. 2010 Molecular design of barnacle cement in comparison with those of mussel and tubeworm. J. Adhes. 86, 96-110. (doi:10.1080/00218460903418139).

[41] Flammang, P. 2006 Adhesive secretions in echinoderms: an overview. In Biological adhesives (eds. A.M. Smith & J.A. Callow), pp. 183-206. Berlin: Springer.

[42] Walker, G. 1978 A cytological study of the cement apparatus of the barnacle, Chelonibia testudinaria Linnaeus, an epizoite on turtles. Bull. Mar. Sci. 28, 205-209.

[43] Fyhn, U.E.H. Costlow, J.D. 1976 A histochemical study of cement secretion during intermolt cycle in barnacles. Biol. Bull. 150, 47-56.

[44] Barlow, D.E., Dickinson, G.H., Orihuela, B., Kulp, J.L., Rittschof, D. Wahl, K.J. 2010 Characterization of the adhesive plaque of the barnacle Balanus amphitrite: amyloid-like nanofibrils are a major component. Langmuir 26, 6549-6556. (doi:10.1021/la9041309).

[45] Barlow, D.E., Dickinson, G.H., Orihuela, B., Rittschof, D. Wahl, K.J. 2009 In situ ATR- FTIR characterization of primary cement interfaces of the barnacle Balanus amphitrite. Biofouling 25, 359-366. (doi:10.1080/08927010902812009).

[46] Wiegemann, M. Watermann, B. 2003 Peculiarities of barnacle adhesive cured on non- stick surfaces. J. Adhes. Sci. Technol. 17, 1957-1977. (doi:10.1163/156856103770572070).

[47] Vincent, J. 2012 Structural biomaterials, 3rd edn. Princeton, NJ: Princeton University Press.

[48] Santos, R., Gorb, S., Jamar, V. Flammang, P. 2005 Adhesion of echinoderm tube feet to rough surfaces. J. Exp. Biol. 208, 2555-2567. (doi:10.1242/jeb.01683).

[49] Smith, A.M. 2006 The biochemistry and mechanics of gastropod adhesive gels. In Biological adhesives (eds. A.M. Smith & J.A. Callow), pp. 167-182. Berlin: Springer.

[50] Ramsay, D.B., Dickinson, G.H., Orihuela, B., Rittschof, D. Wahl, K.J. 2008 Base plate mechanics of the barnacle Balanus amphitrite (=Amphibalanus amphitrite). Biofouling 24, 109-118. (doi:10.1080/08927010701882112).

55 [51] Graham, L.D., Glattauer, V., Huson, M.G., Maxwell, J.M., Knott, R.B., White, J.W., Vaughan, P.R., Peng, Y., Tyler, M.J., Werkmeister, J.A., et al. 2005 Characterization of a protein-based adhesive elastomer secreted by the Australian frog Notaden bennetti. Biomacromolecules 6, 3300-3312. (doi:10.1021/bm050335e).

[52] Tanur, A.E., Gunari, N., Sullan, R.M.A., Kavanagh, C.J. Walker, G.C. 2010 Insights into the composition, morphology, and formation of the calcareous shell of the serpulid Hydroides dianthus. J. Struct. Biol. 169, 145-160. (doi:10.1016/j.jsb.2009.09.008).

[53] Enders, S., Barbakadse, N., Gorb, S.N. Arzt, E. 2004 Exploring biological surfaces by nanoindentation. J. Mater. Res. 19, 880-887. (doi:10.1557/jmr.2004.19.3.880).

[54] Hengsberger, S., Kulik, A. Zysset, P. 2002 Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. Bone 30, 178-184. (doi:10.1016/s8756-3282(01)00624-x).

[55] Watermann, B., Berger, H.-D., Sönnichsen, H. Willemsen, P. 1997 Performance and effectiveness of non-stick coatings in seawater. Biofouling 11, 101-118. (doi:10.1016/S0040- 6090(97)00337-4 ).

[56] Cooper, A. Kennedy, M.W. 2010 Biofoams and natural protein surfactants. Biophys. Chem. 151, 96-104. (doi:10.1016/j.bpc.2010.06.006).

[57] Kennedy, M.W. Cooper, A. 2013 Surfactant proteins and natural biofoams. In Proteins in solution and at interfaces: methods and applications in biotechnology and materials science (eds. J.M. Ruso & A. Pineiro), pp. 365-378. Hoboken, NJ: Wiley & Sons.

56 Figure captions

Figure 1. (a) Schematic drawing of the indentation experiment setup. The glass sphere was attached to a spring, which was driven by a motor and indented the cement. (b) For the tensile experiment, a part of the outer region of a cement float (c) was fixed between two clamps.

The upper clamp attached to the force transducer (ft) was pulling the sample with a constant speed while the lower clamp was fixed.

Figure 2. (a) Stranded aggregation of Dosima fascicularis connected by a single cement float

(c). (b-d) Scanning electron micrographs. (b) Cross section of a cement float. The outer zone forming a rind (r) contained mainly small bubbles, whereas in the inner region of the float large bubbles (b) were dominant. (c) Higher magnification of the rind with narrow layers

(arrows). (d) On the layers and the inner lining of the bubbles the cement showed some roughness. (e-f) Transmission electron micrographs. (e) The cement had a fibrous structure.

58 (f) In the region, where the fibres were condensed, irregular lines were seen. Scale bars: (a) 1 cm; (b) 500 µm; (c) 100 µm; (d) 5 µm; (e) 2 µm, (f) 5 µm.

Figure 3. Typical force versus indentation depth curves of the cement of Dosima fascicularis.

(a) The cement surface measured in sea water. (b) The surface of the dry cement. The solid line indicates the fit of the indentation data with the Hertz theory. (E = elastic modulus)

Figure 4. Two consecutive indentations, applied with the same force, measured on the same place at the surface of a wet cement float of Dosima fascicularis. The second indentation

(grey) demonstrated a visco-elastic-plastic deformation when compared to the first indentation (black).

Figure 5. The elastic modulus (a) and the hardness (b) of the surface and inner region of the cement float of Dosima fascicularis measured in sea water and distilled water as well as of the surface of the dry cement. Box plots show the median value (line), the ends of the boxes define the 25th and 75th percentiles and the error bars the 10th and 90th percentiles. The outlines are illustrated as black dots. The difference between the wet and dry cement was obvious, therefore only the differences between the different regions under wet conditions were analysed using Kruskal-Wallis one way analysis of variance on Ranks. (a, b) P < 0.001.

Figure 6. Correlation between the indentation depth and the elastic modulus (a, c, e).

Correlation between the indentation depth and the hardness (b, d, f). (a, b) Surface of the wet cement (in sea water, n = 116). (c, d) Inner region of the wet cement (in sea water, n = 46). (e, f) Surface of the dry cement (n = 40). The data were fitted by linear regression and tested for normal distribution using a Shapiro-Wilk’s test. (a) y = 37.85 – 0.11x, R2 = 0.22, P < 0.0001;

(b) y = 4.002 – 0.007x, R2 = 0.06, P < 0.0001; (c) y = 9.77 – 0.002x, R2 = 0.0002, P < 0.0001;

61 (d) y = 1.063 + 0.002x, R2 = 0.006, P < 0.0001; (e) y = 2.69 – 02x, R2 = 0.47, P = 0.07; (f) y =

94.18 – 46x, R2 = 0.43, P = 0.1 (R2 = coefficient of determination).

Figure 7. The curve shows a force – distance dependence during pulling the cement (in this case, a parallelepiped-shaped sample of the outer region of the float was used) until it ruptured.

Figure 8. (a) The stress-strain curve obtained in the pulling experiment shows relaxation (see b) of the cement, when the pulling was stopped for around 8 s. (b) Relaxation (decrease in the interacting force) of the cement, lasting for several seconds, was observed during the resting period. The relaxation process was strongly pronounced during deformation of the cement. It was represented by a single exponential function and tested for normal distribution utilising

Shapiro-Wilk’s test. f = 0.6 – 0.2e – 0.4x, R2 = 0.88, P < 0.0001 (R2 = coefficient of determination).

V. Manuscript 4

Characterization of cement float buoyancy in the stalked barnacle

Dosima fascicularis (Crustacea, Cirripedia)

Authors: Zheden V, Kovalev A, Gorb SN and Klepal W

Publication status: submitted to Interface Focus

Personal contributions of Vanessa Zheden:

Collection of the material

Sample preparation for SEM and LM

SEM and LM imaging and documentation

Performance of all experiments

Creation of graphs, histograms and performance of statistical analyses

Manuscript preparation

63 Characterization of cement float buoyancy in the stalked barnacle

Dosima fascicularis (Crustacea, Cirripedia)

Vanessa Zheden1, Alexander Kovalev2, Stanislav N. Gorb2 and Waltraud Klepal1

1 University of Vienna, Faculty of Life Sciences, Core Facility Cell Imaging and

Ultrastructure Research, Vienna, Austria

2 Kiel University, Zoological Institute: Functional Morphology and Biomechanics, Kiel,

Germany

Corresponding author: [email protected]

Abstract

Dosima fascicularis is the only barnacle which can drift autonomously at the water surface.

The animal secretes a large amount of foam-like cement which often overgrows the substratum to which it is attached. When several individuals share one float, their size and not their number are crucial for the production of both volume and mass of the float. The gas content within the cells (bubbles) of the foam gives buoyancy to the whole float. The higher the gas volume, the greater is the positive buoyancy. The cement consists of more than 90% water and the gas volume is on average 18.5%. Our experiments demonstrate that the intact foam-like cement float is sealed from the surrounding water. The effective density of the cement floats without gas is greater than that of the sea water.

Key words: adhesive, buoyancy, foam, biological material, Arthropoda

64 Introduction

Barnacles (Cirripedia Thoracica) are sessile, marine Crustacea with free-swimming larval stages. The adult barnacle attaches permanently with its proteinaceous adhesive, the so-called cement, to stationary hard materials like rocks or to natural and man-made floating objects.

Lepadomorph stalked barnacles are typical rafting organisms [1]. Their pelagic larvae colonise any floating objects like driftwood, macroalgae, plastic or tar pellets [2-4]. They even settle on living animals, e.g. sea snakes [5, 6], turtles [7], birds [8], and seals [9]. With this strategy, barnacles can extend their habitat. The normally sedentary animals turn into

“hitchhikers” which raft through the sea [9, 10]. Generally lepadomorph barnacles secrete a thin layer of cement for attachment. One of them, Dosima (Lepas) fascicularis (Ellis and

Solander, 1786), has evolved special adaptations. This animal produces a large amount of foam-like cement with enclosed gas-filled bubbles [11]. The gas is presumably CO2, a metabolic product [12].

The cypris larva of D. fascicularis settles mainly on small floating objects, like bird feathers or algae. After metamorphosis of the cyprid and as the animal grows, the amount of cement increases. It may overgrow the substratum and thus form a float [13, 14] which gives

1buoyancy to the animal and enables it to drift autonomously just at the water surface [1]. The cement is produced by cement glands in the upper part of the stalk, transported together with the gas through the cement canal system and secreted through pores on the stalk base [12].

The phenomenon of positive buoyancy is known of other marine organisms too like some snails, cnidarians and brown algae. They also produce their own floating devices with which they drift through the water. The snails of the family Janthinidae secrete a float of mucus bubbles [15, 16]. With that float they drift and hunt in the neuston in contrast to the related benthic species. Neustonic cnidarians like Velella velella and Physalia physalis have one gas- filled bladder which acts as a sail allowing them to float on the water surface [17, 18]. Brown algae like Fucus vesiculosus have gas-filled floats, called pneumatocysts, which give positive

65 buoyancy to the blades [19, 20]. By that they are exposed to more sunlight which improves their photosynthetic activity.

Most studies on such biological floats deal with the ecology of drifting or rafting organisms

[1, 10, 21], but to the best of our knowledge nothing is known about positive buoyant properties of these structures. After having dealt with some mechanical properties [22], the aim of this study was to investigate the unusual function of the cement of Dosima fascicularis with special emphasis on the positive buoyancy of the cement float, the gas- and the water content as well as the volume-weight correlation.

Material and methods

Individuals of the stalked barnacle Dosima fascicularis, which had been washed ashore, were collected on the north-west coast of Denmark. The animals occurred individually (figure 1a), in groups on the same cement float (figure 1b) or attached to small floating objects (e.g. feathers). For our investigations we used colonies of two to seven animals attached to one float. All animals of one colony had a capitulum length between 2 and 3 cm. For the experiments only the cement floats were used. Prior to the analyses the cement was separated from the animals and stored in sea water with a 2% antibiotic antimycotic solution (Sigma-

Aldrich, Vienna Austria).

Microscopy

Razor blade sections through whole cement floats were photographed with a Lumix DMC-

GH1 camera (Panasonic, Hamburg, D) mounted on a MZ3000 stereo microscope (Micros

Austria, St. Veit/Glan, A). For scanning electron microscopy, cross sections of the float were fixed in 2.5% glutaraldehyde in 0.1 mol l-1 sodium cacodylate buffer with 10% (w/v) sucrose at pH 7.3 for 2 h and rinsed in distilled water. After air-drying the cement samples were coated with gold by an Agar B7340 sputter coater (Agar Scientific Ltd., Stansted, Essex,

66 U.K.) and examined in a Philips XL 30 scanning electron microscope (FEI and Philips,

Eindhoven, NL) at 15 kV.

Mass, volume and water content

Ten cement floats were weighed under wet conditions and their volumes were determined by water displacement. For measuring the water content of the cement the floats were wiped with tissues to remove any excess water. Two floats were then air dried for two days, in addition three floats were dried in the oven at 65°C overnight. Afterwards the dried cement floats were weighed again. The water content was calculated by subtracting the dry mass from the wet mass. Some salts of sea water may still have been on the surface of the dried floats.

Buoyant force and gas volume

Ten cement floats were placed one after the other in a 100 ml beaker filled with sea water.

Each float was totally submerged to a depth of 1 cm below the water surface by a thin wire- frame attached to a force transducer using a motorised micromanipulator (FORT-100 and

DC3001R, World Precision Instruments, Sarasota, FL, USA) (figure 1c). The floats were kept underwater for about 20 s and their buoyant force was measured. The data were collected with the software AcqKnowledge 3.7.0 (Biopac Systems, Inc., Goleta, CA, USA). Afterwards, volume, mass and buoyant force of the cement floats were determined. The volume of the gas

Vg inside the cement float was calculated using the following equation

( ) = 퐹 휌푤 . 푔+푀 1− 휌푐 푉푔 휌푤−휌푔 F was the positive buoyant force, g the gravitational acceleration equals 9.81 m/s2 and M was

3 the mass of the dried cement float. The density of the sea water ρw (1018.65 kg/m ) was calculated as mass divided by volume. Because of the irregular internal structure of the cement float (due to the greatly differing size of the gas bubbles) and the resulting uncertainty

67 in determining the density of the cement we used for our calculations the standard value ρc =

3 1350 kg/m for the density of proteins [23, 24]. For the density of CO2 within the bubbles

3 [12] we used the standard value ρg = 1.977 kg/m .

Reduced pressure experiments

In a vacuum-desiccator cement floats were placed in a beaker filled with sea water. Two of them were exposed to a pressure of 700 mbar and two others to 30 mbar. 30 mbar were reached after about 20 min. During pressure reduction the floats were observed to detect any structural changes and any escaping gas bubbles. After achievement of the desired pressure the floats were kept at this pressure for five minutes. The buoyant force of the floats at atmospheric pressure (996 mbar) was measured before and after the reduced pressure treatment.

Statistical analyses

The regression calculations were done via a random bootstrap approach [25, 26] (with 10,000 iterations) by the program routine MUREG from the software package of ‘computer intensive statistics’ [27]. Graphs and histograms for all experiments were made in Sigma Plot 11.0

(Systat Software Inc, Bangalore, India and San Jose, CA, USA).

Results

Cement morphology

The cement was secreted in concentric layers around the stalk and the attachment site (figure

2a). Large gas-filled bubbles were mainly found in the inner region of the cement float.

Small, round bubbles arose throughout the float, but dominated near the surface of the float forming a kind of rind (figure 2b).

68 Mass, volume and water content

There was a significant correlation between the volume of the cement float of Dosima fascicularis and its wet mass (R2 = 0.96, P < 0.001). The greater the volume, the heavier was the float (figure 3). The number of animals attached to the same cement float did not influence mass and volume of the float. Only the size of the animals was crucial. For example, the float with four animals attached was heavier and bigger than the floats with seven animals (table

1).

The water content of the cement floats was over 90% (w/w), no matter whether they had been air dried or oven dried (figure 4a). When the dried cement was immersed in sea water again, it was still floating. Within a few minutes the cement float got heavier because of water uptake.

After three days in water the air dried cement floats had more than the threefold dry mass, but they did not return to the original wet mass. Interestingly, the oven dried floats did not take up as much water as the air dried ones (figure 4b).

Buoyant force and gas volume

The volume of the gas was linearly dependent on the volume of the float (R2 = 0.65, P =

0.018) (figure 5a). In essence, the bigger the float, the higher was the gas volume. The gas volume fraction in a float was on average 18.5% ± 6.7 (n = 10). A typical graph of the measured positive buoyant force is shown in figure 5b. In this figure the force required to hold the cement float underwater was 9.05 mN. The volume of the gas inside the float and the buoyant force were significantly correlated (R2 = 0.99, P < 0.001) (figure 5c). Clearly, the higher the gas content in the float, the more positive buoyancy it had.

Reduced pressure experiments

For the pressure experiments two cement floats, kept in sea water, were exposed to 700 mbar, two others to 30 mbar ambient pressure. There was almost no difference between the buoyant

69 force, measured before and after treatment with 700 mbar underpressure (figure 6a: floats 1 and 2). During pressure reduction gas bubbles were observed leaking out and accumulating at the surface of the float around 400 mbar (figure 6b arrows). At that pressure the cement float started to crack. Below 250 mbar most bubbles in the cement had burst and water filled the voids. Immediately after ambient pressure reduction to 30 mbar, the cement floats had still some positive buoyancy, but in comparison with the floats exposed to 700 mbar underpressure, the buoyant force was much lower (figure 6a: floats 3 and 4). As a consequence, the floats, which were exposed to 30 mbar, sank a few hours after the experiment. Due to the water uptake the mass of the floats increased. After five days the cement was >25% heavier than measured immediately after the underpressure experiment.

Discussion

The stalked barnacle Dosima fascicularis is unique among the barnacles being able to secrete its own float. It can even form colonies attached to a central float [28]. Interestingly, volume and mass of the float are primarily correlated with the size of the attached animals and not with their number. The colonies investigated in this study contained barnacles of similar size.

This is presumably the result of several cypris larvae having settled on the same substratum during the same reproductive period. The increasing cement of the adult eventually formed one joint float. This was also previously suggested by Ryan and Branch [28].

Water content

The original wet cement of D. fascicularis had a water content of more than 90% [see also

29]. Interestingly, after air drying the cement took up more water than when it was oven dried.

A possible explanation is that the protein structure of the cement changes due to irreversible protein denaturation during heating up to 65°C. Another reason for the difference in water uptake could be that the process of air drying is slower and thus gentler than oven drying.

70 Therefore the air dried material did not shrink that much and water could get more easily into it again. The reason that the dried floats did not get their original mass after rehydration might be that the immersion time was too short.

Buoyancy

Floating is a typical feature of lepadomorph barnacles. Lepas anatifera or L. testudinata attach with a thin layer of cement to large and highly buoyant objects like kelp or plastic [30].

In contrast D. fascicularis normally settles on small floating objects, which are often overgrown by the cement. By developing its own foam-like cement full of gas bubbles, the D. fascicularis cement maintains positive buoyancy in accordance with the growth of the animal throughout its life time [2, 28].

For the estimation of the cement density we used the value 1350 kg/m3 of protein density [23,

24]. According to this value the density of the proteinaceous cement was higher than that of sea water (1018.65 kg/m3). Consequently the cement without gas bubbles would sink. In principle, the greater the volume of the float the bigger was the gas volume, but the gas content of the floats varied between the D. fascicularis colonies (see table 1). As to be expected the floats with less gas content had lower positive buoyancy than floats with higher gas content. The gas volume of the float and thus its positive buoyancy could also depend on the object to which the float is attached. The use of floating objects, such as feathers, as attachment sites lead automatically to the increase of buoyancy. In any case the total positive buoyancy of the float must compensate the weight of the attached animals.

Reduced pressure experiments

At atmospheric pressure and at underpressure of 700 mbar, the float buoyancy was unaffected. This means that the walls of the bubbles in the cement could sustain at least 700 mbar. It was observed that at an ambient pressure below 400 mbar the cement burst and the

71 gas escaped from the bubbles. The float was now open to the outside, water filled the voids and the floats began to sink. The fact that the floats sank due to structural failure of the cement is an indirect proof that the intact cement float is a porous system closed to the surrounding water.

Conclusions

The prerequisite for the use of the Dosima fascicularis cement as a float is its positive buoyancy depending on the balance between the volume of the float and the volume of the gas within the bubbles. The mass and the volume of the float depend on the size of the attached animals and not on their number. The water content of the float is >90% and the gas volume is on average 18.5%. Although the float is porous, it is crucial that it is sealed from the surrounding water. In addition, a kind of rind gives mechanical stability to the float and forms a barrier to the environment.

All lepadomorph barnacles are floating and the function of their cement is adhesion as in other barnacles. In D. fascicularis the cement has a double function: Initially it is adhesion and in addition it is floating independent of any floating substratum. The cement of D. fascicularis is an interesting example of how the adhesive material can change its function due to a slight modification of the structure. The use of the cement as a float at the water surface allows the essentially sessile adult animal secondary mobility. By that Dosima can extend its habitat and occupy new ecological niches.

Acknowledgements

We thank H. L. Nemeschkal for help with the statistics. This research was carried out with the financial support of the Austrian Science Fund (FWF) P21767-B17 to Waltraud Klepal. We also thank Ingo Grunwald (Fraunhofer Institute IFAM, Bremen) for financial support.

Vanessa Zheden was granted a “Short-Term Scientific Mission” from the COST-action

72 TD0906 to perform experiments at the Kiel University and a PhD Completion Grant from the

Faculty of Life Sciences of the University of Vienna. Stanislav Gorb was supported by the

German Science Foundation Excellence Cluster “Future Ocean, Phase 2” at the Kiel

University.

References

[1] Thiel, M. Gutow, L. 2005 The ecology of rafting in the marine environment. II. The rafting organisms and community. Oceanogr. Mar. Biol. Ann. Rev. 43, 279-418.

[2] Minchin, D. 1996 Tar pellets and plastics as attachment surfaces for lepadid cirripedes in the North Atlantic Ocean. Mar. Pollut. Bull. 32, 855-859. (doi:10.1016/S0025- 326X(96)00045-8 ).

[3] Cheng, L. Lewin, R.A. 1976 Goose barnacles (Cirripedia: Thoracica) on flotsam beached at La Jolla, California. Fish. Bull. 74, 212-217.

[4] Hinojosa, I., Boltaña, S., Lancellotti, D., Macaya, E., Ugalde, P., Valdivia, N., Vásquez, N., Newman, W.A. Thiel, M. 2006 Geographic distribution and description of four pelagic barnacles along the south east Pacific coast of Chile - a zoogeographical approximation. Rev. Chil. Hist. Nat. 79, 13-27.

[5] Alvarez, F. Celis, A. 2004 On the occurrence of Conchoderma virgatum and Dosima fascicularis (Cirripedia, Thoracica) on the sea snake, Pelamis platurus (Reptilia, Serpentes) in Jalisco, Mexico. Crustaceana 77, 761-764. (doi:10.1163/1568540041958536).

[6] Yamato, S., Yusa, Y. Tanase, H. 1996 Distribution of two species of Conchoderma (Cirripedia: Thoracica) over the body of a sea snake, Laticauda semifasciata (Reinwardt), from the Kii Peninsula, Southwestern Japan. Publ. Seto. Mar. Biol. Lab. 37, 337-343.

[7] Casale, P., D'Addario, M., Freggi, D. Argano, R. 2012 Barnacles (Cirripedia, Thoracica) and associated epibionts from sea turtles in the central Mediterranean. Crustaceana 85, 533- 549. (doi:10.1163/156854012x634393).

[8] Tottrup, A.P., Chan, B.K.K., Koskinen, H. Hoeg, J.T. 2010 'Flying barnacles': implications for the spread of non-indigenous species. Biofouling 26, 577-582. (doi:10.1080/08927014.2010.489203).

[9] Barnes, D.K.A., Warren, N.L., Webb, K., Phalan, B. Reid, K. 2004 Polar pedunculate barnacles piggy-back on pycnogona, penguins, pinniped seals and plastics. Mar. Ecol.-Prog. Ser. 284, 305-310. (doi:10.3354/meps284305).

73 [10] Winston, J.E. 2012 Dispersal in marine organisms without a pelagic larval phase. Integr. Comp. Biol. 52, 447-457. (doi:10.1093/icb/ics040).

[11] Newman, W.A. Abbott, D.P. 1980 Cirripedia: the Barnacles. In Intertidal Invertebrates of California (eds. R.H. Morris, D.P. Abbott & E.C. Haderlie), pp. 504-535. Stanford, CA: Stanford University Press.

[12] Zheden, V., von Byern, J., Kerbl, A., Leisch, N., Staedler, Y., Grunwald, I., Power, A.M. Klepal, W. 2012 Morphology of the cement apparatus and the cement of the buoy barnacle Dosima fascicularis (Crustacea, Cirripedia, Thoracica, Lepadidae). Biol. Bull. 223, 192-204.

[13] Young, P.S. 1990 Lepadomorph cirripeds from the Brazilian coast. I.-Families Lepadidae, Poecilasmatidae and Heteralepadidae. Bull. Mar. Sci. 47, 641-655.

[14] Boëtius, J. 1952-53 Some notes on the relation to the substratum of Lepas anatifera L. and Lepas fascicularis E. et S. Oikos 4, 112-117. (doi:10.2307/3564807).

[15] Churchill, C.K.C., Foighil, D.O., Strong, E.E. Gittenberger, A. 2011 Females floated first in bubble-rafting snails. Curr. Biol. 21, R802-R803. (doi:10.1016/j.cub.2011.08.011).

[16] Churchill, C.K.C., Strong, E.E. Foighil, D.O. 2011 Hitchhiking juveniles in the rare neustonic gastropod Recluzia cf. Jehennei (Janthinidae). J. Mollus. Stud. 77, 441-444. (doi:10.1093/mollus/eyr020).

[17] Iosilevskii, G. Weihs, D. 2009 Hydrodynamics of sailing of the Portuguese man-of-war Physalia physalis. J. R. Soc. Interface 6, 613-626. (doi:10.1098/rsif.2008.0457).

[18] Purcell, J.E., Clarkin, E. Doyle, T.K. 2012 Foods of Velella velella (Cnidaria: Hydrozoa) in algal rafts and its distribution in Irish seas. Hydrobiologia 690, 47-55. (doi:10.1007/s10750-012-1052-x).

[19] Paul, M., Henry, P.-Y.T. Thomas, R.E. 2014 Geometrical and mechanical properties of four species of northern European brown macroalgae. Coast. Eng. 84, 73-80. (doi:10.1016/j.coastaleng.2013.11.007).

[20] Dromgoole, F.I. 1981 Form and function of the pneumatocysts of marine algae. I. Variations in the pressure and composition of internal gases. Bot. Mar. 24, 257-266. (doi:10.1515/botm.1981.24.5.257).

[21] Bravo, M., Astudillo, J.C., Lancellotti, D., Luna-Jorquera, G., Valdivia, N. Thiel, M. 2011 Rafting on abiotic substrata: properties of floating items and their influence on community succession. Mar. Ecol.-Prog. Ser. 439, 1-U26. (doi:10.3354/meps09344).

[22] Zheden, V., Klepal, W., Gorb, S. Kovalev, A. submitted Mechanical properties of the cement of the stalked barnacle Dosima fascicularis (Cirripedia, Crustacea). Interface Focus.

74 [23] Knudsen, S.M., von Muhlen, M.G. Manalis, S.R. 2012 Quantifying particle coatings using high-precision mass measurements. Anal. Chem. 84, 1240-1242. (doi:10.1021/ac300034r).

[24] Fischer, H., Polikarpov, I. Craievich, A.F. 2004 Average protein density is a molecular- weight-dependent function. Protein Sci. 13, 2825-2828. (doi:10.1110/ps.04688204).

[25] Efron, B. 1982 The jackknife, the bootstrap and other resampling plans. Philadelphia, PA: SIAM.

[26] Manly, B.F.J. 2006 Randomization, bootstrap and Monte Carlo methods in biology. London: Chapman & Hall.

[27] Nemeschkal, H.L. 1999 Morphometric correlation patterns of adult birds (Fringillidae: Passeriformes and Columbiformes) mirror the expression of developmental control genes. Evolution 53, 899-918. (doi:10.2307/2640730).

[28] Ryan, P.G. Branch, G.M. 2012 The November 2011 irruption of buoy barnacles Dosima fascicularis in the Western Cape, South Africa. Afr. J. Mar. Sci. 34, 157-162. (doi:10.2989/1814232x.2012.675130).

[29] Zheden, V., Klepal, W., von Byern, J., Bogner, F.R., Thiel, K., Kowalik, T. Grunwald, I. 2014 Biochemical analyses of the cement float of the goose barnacle Dosima fascicularis - a preliminary study. Biofouling 30, 949-963. (doi:10.1080/08927014.2014.954557).

[30] Whitehead, T.O., Biccard, A. Griffiths, C.L. 2011 South African pelagic goose barnacles (Cirripedia, Thoracica): substratum preferences and influence of plastic debris on abundance and distribution. Crustaceana 84, 635-649. (doi:10.1163/001121611x574290).

75 Table caption

Table 1. Summary of the physical values measured in 10 floats to which a varying number of animals were attached.

float animals wet mass of volume of the volume of the buoyant force number attached the float (g) float (ml) gas (ml) (mN) 1 2 1.19 1.1 0.17 1.53

2 2 1.78 2 0.35 3.14

3 2 2.12 2.1 0.13 0.97

4 4 1.19 1.1 0.29 2.69

5 4 1.64 2 0.54 5.06

6 4 3.41 4.2 0.97 9.05

7 5 2.37 3 0.33 2.9

8 7 1.37 1.2 0.27 2.45

9 7 1.69 1.9 0.27 2.33

10 7 2.19 2.9 0.62 5.84

76 Figure captions

Figure 1. (a) Single Dosima fascicularis washed ashore. Scale bar: 1 cm. (b) Several animals share one cement float. Scale bar: 1 cm. (c) Experimental setup for measuring the buoyant force. The cement ball (c) floating in sea water was pushed under the water surface with a thin wire-frame (w) attached to a force transducer (ft). The red arrow indicates the movement of the platform.

Figure 2. (a) Stereo microscopic image of a razor blade section through a cement float (partly torn). The cement formed concentric layers around the stalk (s) and the alga (a). Scale bar: 2 mm. (b) Scanning electron micrograph of a cross section of a cement float. Large bubbles (b) were mainly in the inner region of the float, whereas in the outer region forming a kind of rind small, round bubbles (*) were dominant. Scale bar: 1 mm.

Figure 3. Correlation between the volume of the cement float and its wet mass. The data were fitted by random bootstrap for linear regression: y = -0.57 + 1.43x, R2 = 0.96, P < 0.001; R2 = coefficient of determination.

Figure 4. (a) The histogram shows the wet mass of five floats, which consists of the dry portion (black) and the water content (grey) of the cement. Floats 4 and 8 were air dried, floats 3, 5 and 9 were oven dried. (b) The histogram shows the wet mass (grey), the dry mass

(black) and the mass of the dried floats after three days kept in water (dark grey). After three days the dried floats sometimes took up more than 20% of water but they did not return to their original mass.

Figure 5. (a) Correlation between the volume of the gas inside the cement and the volume of the float. The data were fitted by by random bootstrap for linear regression: y = -0.05 + 0.2x, R2 = 0.65, P =

0.018. (b) Graph of a typical buoyant force measurement. The buoyant force of a cement float was defined as an average force in the area between the two grey lines (in this case 9.05 mN). (c)

Correlation between the volume of the gas inside the float and the buoyant force: y =

-0.19 + 9.6x, R2 = 0.99, P < 0.001.

Figure 6. (a) Differences between the buoyant force of the cement floats at atmospheric pressure (black) and after the exposure to reduced pressure: 700 mbar (grey) and 30 mbar

(grey with red frame). (b) Cement float in a beaker filled with sea water in a vacuum desiccator at 200 mbar. Gas bubbles leaked out of the burst cement (arrows).

80 VI. Discussion

For the first time, this thesis characterises comprehensively the cement of Dosima fascicularis. The topics morphology, biochemistry and mechanical properties are highlighted.

This animal is the most specialized pleustonic goose barnacle (Cheng & Lewin 1976), unique in producing a cement float which gives positive buoyancy to the animal and enables it to drift autonomously at the water surface (Thiel & Gutow 2005).

The first manuscript (Zheden et al. 2012) gives the first ever detailed morphological description of the cement apparatus of D. fascicularis. The morphology of the cement glands is comparable to those of Balanus hameri and Lepas anatifera. In these species the organelles are distributed evenly throughout the cytoplasm of the gland cells (Lacombe & Liguori 1969;

Walker 1970; Jonker et al. 2012) which is considered an original condition. Within the gland cell of more derived barnacles (e.g. B. tintinnabulum, B. psittacus, Elminius modestus) a secretory and a storage region can be distinguished (Lacombe & Liguori 1969; Lacombe

1970; Walker 1970). The shape and the size of the Dosima cement gland cells change with the developmental stage of the barnacle. In Semibalanus balanoides this was interpreted as a primitive condition (Lacombe 1970). The nucleus of the gland cells alters from round to lobed and the number of nucleoli increases during gland development. In more advanced barnacles all stages of gland cell development can be observed at the same time (Lacombe 1970).

D. fascicularis is the only barnacle known so far which produces its own float. The highly specialized capacity to use the cement not only as an adhesive but also as float is unique amongst barnacles and considered a derived characteristic. The gas inside the bubbles of the cement is presumably CO2, a metabolic product. We assume that CO2 diffuses from the hemolymph into the lumen of the cement canal through the cells lining the collector and secondary canals. From there it is transported to the outside together with the cement through the cuticle lined principal canal.

81 The second manuscript (Zheden et al. 2014) deals with the biochemistry of the cement of D. fascicularis. Our data reveal that the Dosima cement consists of 84% proteins. The dominance of proteins is a general characteristic of barnacle cement (Barnes & Blackstock 1974, 1976;

Naldrett & Kaplan 1997; Kamino 2006) and is also known from other marine adhesives

(Stewart et al. 2004; Silverman & Roberto 2007). We identify protein bands with a molecular weight between 47.4 and 250.0 kDa which have their isoelectric points in the acidic range. In agreement with acorn barnacles high amounts of hydrophobic amino acids are in the Dosima cement. These render the cement matrix insoluble (Naldrett & Kaplan 1997; Kamino et al.

2012). So far the sequence analysis reveals that the Dosima cement consists of a de novo set of proteins with low homologies to other barnacle cement proteins. In the cement of Dosima we find sulphur but no disulphide bonds, which are commonly present in the adhesive of acorn barnacles to stabilize the protein complex and contribute to its insolubility (Naldrett

1993; Kamino et al. 2000). Polysaccharides are also detected in the Dosima cement. This could indicate that sulphated polysaccharides might occur in the Dosima cement as in the adhesive of the sandcastle worm Phragmatopoma californica (Wang & Stewart 2013).

The third manuscript (Zheden et al. submitted-a) is the first study of mechanical properties of the cement of a stalked barnacle. It comprises the results of the first mechanical tests of the special cement of D. fascicularis. We show that the values of the elastic modulus, hardness and tenacity of the Dosima cement are considerably lower than in the rigid cement of acorn barnacles (Watermann et al. 1997; Sun et al. 2004; Ramsay et al. 2008; Sullan et al. 2009;

Sangeetha & Kumar 2011). Our results reveal that the Dosima cement is one of the softest biological adhesives. Its elastic modulus (5-20 kPa) lies in the same range as that of the tube foot disc of echinoderms (Santos et al. 2005) or the adhesive pads of ensiferan insects (Perez

Goodwyn et al. 2006). Two regions can be distinguished within the cement float: a softer interior and a compact harder outer zone. The main function of the inner region, which contains large gas-filled bubbles, is to give buoyancy to the animal. The outer cement layers

82 form a kind of rind with small gas-bubbles. This rind acts as protection towards the environment and gives mechanical stability. Similar structural and mechanical characteristics are also found in the adhesives of Phragmatopoma californica and Mytilus edulis, where the densest material is at the interface to the surrounding water (Benedict & Waite 1986; Stewart et al. 2004).

Emphasis of the fourth manuscript (Zheden et al. submitted-b) lies on the buoyancy of the cement float. D. fascicularis is the only barnacle which secretes a high amount of foam-like cement and uses it as a float (Boëtius 1952-53). Apart from the individual use it can form free-floating colonies attached to a central float (Ryan & Branch 2012). The size of the attached animals and not their number determine the volume and the mass of the float. The gas content within the bubbles of the cement is crucial for the positive buoyancy of the whole animal. We detect an average gas volume of 18.5% in the float. The water content of the cement is more than 90%. Our pressure experiments show that the cement float is a porous system closed to the surrounding water. When the float is damaged, water replaces the gas and the float sinks.

In summary the cement apparatus of Dosima fascicularis resembles that of other barnacles.

The cement, one of the softest biological adhesives, is highly specialized and differs from that of other barnacles in its structure, chemical composition and mechanical properties. In comparison with the cement of all other barnacles investigated so far the Dosima cement is an interesting example of how an adhesive material changes its function due to a slight modification of the structure. The inclusion of gas-filled bubbles and the resulting capacity to drift allows extension of the animal’s habitat and the occupation of new ecological niches.

Investigations on the morphology, chemical composition and mechanical properties of biological adhesives will, no doubt, inspire the development of synthetic bio-adhesives with applications in medicine and industry (Smith & Callow 2006; von Byern & Grunwald 2010;

83 Favi et al. 2014). These bio-inspired adhesives are promising because of their non-toxicity, quick binding and their high strength (Favi et al. 2014). Barnacle cement can serve as a powerful dental adhesive (Ang et al. 2011) and can lead to the development of environmentally benign antifouling marine coatings (Callow & Callow 2011). Dosima cement, in particular, can be used to develop scaffolds for tissue growth and wound healing

(Cooper & Kennedy 2010; Kennedy & Cooper 2013). Due to its shock-absorbing properties a possible application in orthopedics seems feasible.

References

Ang QJ, Lee JXC, Sng JHT. 2011. Potential of barnacle cement in dentistry. Innovation. 10:14-19.

Barnes H, Blackstock J. 1974. The biochemical composition of the cement of a pedunculate cirripede. J Exp Mar Biol Ecol. 16:87-91.

Benedict CV, Waite JH. 1986. Composition and ultrastructure of the byssus of Mytilus edulis. J Morphol. 189:261-270.

Boëtius J. 1952-53. Some notes on the relation to the substratum of Lepas anatifera L. and Lepas fascicularis E. et S. Oikos. 4:112-117.

Callow JA, Callow ME. 2011. Trends in the development of environmentally friendly fouling-resistant marine coatings. Nat Commun. 2:244.

Cheng L, Lewin RA. 1976. Goose barnacles (Cirripedia: Thoracica) on flotsam beached at La Jolla, California. Fish Bull. 74:212-217.

Cooper A, Kennedy MW. 2010. Biofoams and natural protein surfactants. Biophys Chem. 151:96-104.

Favi PM, Yi SJ, Lenaghan SC, Xia LJ, Zhang MJ. 2014. Inspiration from the natural world: from bio-adhesives to bio-inspired adhesives. J Adhes Sci Technol. 28:290-319.

Kamino K. 2006. Barnacle underwater attachment. In: Smith AM, Callow JA, editors. Biological Adhesives. Berlin: Springer; p. 145-166.

84 Kamino K, Inoue K, Maruyama T, Takamatsu N, Harayama S, Shizuri Y. 2000. Barnacle cement proteins - Importance of disulfide bonds in their insolubility. J Biol Chem. 275:27360- 27365.

Kamino K, Nakano M, Kanai S. 2012. Significance of the conformation of building blocks in curing of barnacle underwater adhesive. Febs J. 279:1750-1760.

Kennedy MW, Cooper A. 2013. Surfactant proteins and natural biofoams. In: Ruso JM, Pineiro A, editors. Proteins in solution and at interfaces: methods and applications in biotechnology and materials science. Hoboken, NJ: Wiley & Sons; p. 365-378.

Lacombe D. 1970. A comparative study of the cement glands in some balanid barnacles (Cirripedia, Balanidae). Biol Bull. 139:164-179.

Lacombe D, Liguori VR. 1969. Comparative histological studies of the cement apparatus of Lepas anatifera and Balanus tintinnabulum. Biol Bull. 137:170-180.

Naldrett MJ. 1993. The importance of sulphur cross-links and hydrophobic interactions in the polymerization of barnacle cement. J Mar Biol Assoc UK. 73:689-702.

Naldrett MJ, Kaplan DL. 1997. Characterization of barnacle (Balanus eburneus and B. cenatus) adhesive proteins. Mar Biol. 127:629-635.

Perez Goodwyn P, Peressadko A, Schwarz H, Kastner V, Gorb S. 2006. Material structure, stiffness, and adhesion: why attachment pads of the grasshopper (Tettigonia viridissima) adhere more strongly than those of the locust (Locusta migratoria) (Insecta: Orthoptera). J Comp Physiol A. 192:1233-1243.

Ramsay DB, Dickinson GH, Orihuela B, Rittschof D, Wahl KJ. 2008. Base plate mechanics of the barnacle Balanus amphitrite (=Amphibalanus amphitrite). Biofouling. 24:109-118.

Ryan PG, Branch GM. 2012. The November 2011 irruption of buoy barnacles Dosima fascicularis in the Western Cape, South Africa. Afr J Mar Sci. 34:157-162.

Sangeetha R, Kumar R. 2011. Interfacial morphology and nanomechanics of cement of the barnacle, Amphibalanus reticulatus on metallic and non-metallic substrata. Biofouling. 27:569-577.

Santos R, Gorb S, Jamar V, Flammang P. 2005. Adhesion of echinoderm tube feet to rough surfaces. J Exp Biol. 208:2555-2567.

Silverman HG, Roberto FF. 2007. Understanding marine mussel adhesion. Mar Biotechnol. 9:661-681.

Smith AM, Callow JA. 2006. Biological adhesives. Berlin: Springer.

Stewart RJ, Weaver JC, Morse DE, Waite JH. 2004. The tube cement of Phragmatopoma californica: a solid foam. J Exp Biol. 207:4727-4734.

85 Sullan RMA, Gunari N, Tanur AE, Chan Y, Dickinson GH, Orihuela B, Rittschof D, Walker GC. 2009. Nanoscale structures and mechanics of barnacle cement. Biofouling. 25:263-275.

Sun YJ, Guo SL, Walker GC, Kavanagh CJ, Swain GW. 2004. Surface elastic modulus of barnacle adhesive and release characteristics from silicone surfaces. Biofouling. 20:279-289.

Thiel M, Gutow L. 2005. The ecology of rafting in the marine environment. II. The rafting organisms and community. Oceanogr Mar Biol Ann Rev. 43:279-418. von Byern J, Grunwald I. 2010. Biological adhesive systems: from nature to technical and medical application. Vienna: Springer.

Watermann B, Berger H-D, Sönnichsen H, Willemsen P. 1997. Performance and effectiveness of non-stick coatings in seawater. Biofouling. 11:101-118.

Zheden V, Klepal W, Gorb S, Kovalev A. submitted-a. Mechanical properties of the cement of the stalked barnacle Dosima fascicularis (Cirripedia, Crustacea). Interface Focus.

Zheden V, Kovalev A, Gorb S, Klepal W. submitted-b. Characterization of cement float buoyancy in the stalked barnacle Dosima fascicularis (Crustacea, Cirripedia). Interface Focus.

86 VII. Abstract

Cirripedia Thoracica are sessile marine Crustacea. They comprise acorn and stalked barnacles, all of which attach to the substratum with an adhesive (cement) produced by cement glands. The stalked barnacle Dosima fascicularis is unique amongst the Cirripedia, because it uses its adhesive not only for attachment but also as a float. The animal produces an excess amount of proteinaceous, foam-like cement with enclosed gas filled bubbles. With the float the normally sessile animal can drift autonomously at the water surface.

In this thesis the morphology of the cement apparatus and the cement of Dosima fascicularis was analyzed in detail for the first time. The pathway of the cement was followed from its production in the cement gland cells to its secretion through pores at the base of the stalk where it polymerizes in the sea water.

Further the biochemistry of the Dosima cement was investigated. The protein and carbohydrate content of the cement as well as the amino acids and chemical elements were analyzed. In its chemical composition the Dosima cement is similar to that of other barnacles, but it also shows interesting variations.

In this thesis mechanical properties like elastic modulus, hardness and tensile stress of the cement float were tested for the first time. The values of these mechanical properties are considerably lower in the very soft and visco-elastic Dosima cement than in the rigid cement of other barnacles. Of special interest was the buoyancy of the float, which depends on the balance between the volume of the float and the volume of the gas in the cement.

The studies on the morphology, the chemical composition and the mechanical properties of the Dosima cement are of particular interest because of the possible application of this biological adhesive in medicine and industry.

87 VIII. Zusammenfassung

Cirripedia Thoracica sind marine, festsitzende Krebse (Crustacea), welche Seepocken und

Entenmuscheln beinhalten. Diese Tiere setzen sich mit Hilfe eines überwiegend proteinhaltigen Adhäsivs (Zement) am Substrat fest. Die Entenmuschel Dosima fascicularis ist einzigartig unter den Cirripedia, da sie ihren Zement nicht nur zum Festsetzen sondern auch als Floß verwendet. Dieses Tier produziert eine große Menge an schaumigen Zement der mit Gasblasen gefüllt ist. Mit diesem Zementfloß kann das ursprünglich festsitzende Tier autonom an der Wasseroberfläche treiben.

In dieser Doktorarbeit wurde zum ersten Mal detailliert die Morphologie des Zementapparats und des Zements von Dosima fascicularis untersucht. Es wurde der Weg des Zements von seinem Entstehungsort in den Zementdrüsenzellen bis zu seiner Sekretion über Poren an der

Stielbasis des Tieres verfolgt.

Weiter wurde die chemische Zusammensetzung des Zements analysiert, vor allem der

Protein- und Kohlehydratanteil, die involvierten Aminosäuren sowie die chemischen

Elemente. Die chemische Zusammensetzung des Dosima Zements ist ähnlich der anderer

Cirripedia, weist aber einige interessante Unterschiede auf.

In dieser Arbeit wurden das erste Mal mechanische Eigenschaften wie der Elastizitätsmodul, die Härte und die Zugspannung dieses speziellen Zementfloßes getestet. Die Werte dieser mechanischen Eigenschaften sind im sehr weichen und viskoelastischen Dosima Zement deutlich geringer als im harten Zement der anderen Cirripedia. Ein weiteres Hauptaugenmerk dieser Arbeit lag auf dem Auftrieb des Floßes bzw. des Tieres. Der Auftrieb ist abhängig vom

Verhältnis zwischen Volumen des Floßes und dem Volumen des Gases im Zement.

Die Arbeit über die Morphologie, die chemische Zusammensetzung und die mechanischen

Eigenschaften des Dosima Zements sind von besonderem Interesse in Bezug auf mögliche medizinische und industrielle Anwendungen dieses biologischen Materials.

88 IX. Acknowledgments

I am deeply indebted to Prof. Dr. Waltraud Klepal for the opportunity to work on the Dosima project and to the FWF for the financial support. Waltraud Klepal supported me in every way with her expertise from the beginning until the completion of my thesis. I am grateful to Dr. Janek von Byern, who helped to realize this project. I want to thank all members of the Core Facility Cell Imaging and Ultrastructure Research for help in many ways, especially Mag. Daniela Gruber and Mag. Norbert Cyran for their technical support and Dr. Siegfried Reipert, Dr. Marieluise Weidinger, Johannes Suppan MSc and Dr. Jacqueline Montanaro- Punzengruber for helpful discussions. Further I want to thank Gregor Eder for the really beautiful pictures and videos of the barnacles. I am thankful to Mag. Alexandra Kerbl and Mag. Nikolaus Leisch for helping to make many series of semi- and ultra-thin sections and for their support with Adobe Photoshop and Illustrator. I want to thank Dr. Yannick Städler for the µCT scans of my animals.

I am very thankful to Prof. Dr. Hans Leo Nemeschkal for his support in statistics.

My special thanks go to Prof. Dr. Stanislav Gorb of the University of Kiel, who welcomed me in his lab to investigate the mechanical properties of the Dosima cement. Dr. Alexander Kovalev was very helpful with the experimental setup and with the statistical analyses of the results.

I am grateful to Dr. Ingo Grunwald from the Fraunhofer Institute IFAM (Bremen) for giving me the opportunity to work in his lab and who introduced me to the world of biochemistry. I also want to thank Julia Damiano BSc, Anamika Chatterjee MSc and Neeti Sudumbrekar MSc for helping me within this new field and for being my friends in a foreign city.

I want to thank Dr. Annemarie Power of the National University of Ireland, Galway for her collaboration and together with Jaimie-Leigh Jonker PhD and Paul McEvilly MSc for helping collect animals.

I am thankful to all my friends who were always there for me during the last years. I want to say a big “Thank you” to my sister Nadine Zheden, who accompanied me on my field trips to Denmark and helped me find and collect Dosima. Last but not least I would like to thank my parents Christine and Herbert Zheden for their support and motivation during my studies.

89 X. Curriculum vitae

Personal Data Name: Mag. Vanessa Zheden Place of birth: Friesach (Carinthia) Nationality: Austrian

Studies since 2009 PhD thesis supervised by Prof. Dr. Waltraud Klepal: Characterization of the cement of the stalked barnacle Dosima fascicularis: Morphology, biochemistry and mechanical properties 03/07 – 11/08 Diploma thesis supervised by Prof. Dr. Waltraud Klepal: Cuticle structures on different body regions of Semibalanus balanoides (L.) (Thoracica, Cirripedia) with special emphasis of mature and degenerating penis since 2003 Zoology, University of Vienna 10/99 – 05/03 Biology, University of Vienna

Work experience 08/09 –12/13 Employee and collaborator within the Austrian Science Fund (FWF)- project to Prof. Dr. Klepal on the morphology, biochemistry and mechanical properties of the cement apparatus in Dosima fascicularis at the Core Facility Cell Imaging and Ultrastructure Research (CIUS), University of Vienna 10/07 – 07/09 Tutor for “Submicroscopical anatomy and preparatory techniques in electronmicroscopy“ at CIUS 01/04 – 06/07 Preparation and identification of bone fragments from different excavation sites at the archaeozoological collection of the Natural History Museum Vienna by order of the Austrian national heritage agency (Bundesdenkmalamt)

Scholarships 08/14 – 10/14 PhD Completion Grant of the Faculty of Life Sciences, University of Vienna 30.07. – 23.08.13 Short-term scientific mission (COST-action TD 0906) at the Christian- Albrechts-Universität Kiel (Germany), in the group of Prof. Dr. Stanislav Gorb: to test the mechanical properties of the cement of Dosima fascicularis 03/11 – 07/11 DAAD-Scholarship at the Fraunhofer Institute for Manufacturing Technology and Advanced Materials IFAM, Bremen (Germany), in the group of Dr. Ingo Grunwald: to analyse biochemically the cement of Dosima fascicularis

Memberships ASEM – Austrian Society for Electron Microscopy COST-Action TD0906: Biological adhesives: from biology to biomimetics

90 Publications

Zheden V., Klepal W., von Byern J., Bogner F.R., Thiel K., Kowalik T., Grunwald I. (2014): Biochemical analyses of the cement float of the goose barnacle Dosima fascicularis – a preliminary study. Biofouling 30: 949-963.

Zheden V., von Byern J., Kerbl A., Leisch N., Staedler Y., Grunwald I., Power A.M., Klepal W. (2012): Morphology of the cement apparatus and the cement of the buoy barnacle Dosima fascicularis (Crustacea, Cirripedia, Thoracica, Lepadidae). Biological Bulletin 223:192-204.

Klepal W., Rentenberger C., Zheden V., Adam S., Gruber D. (2010): Structural peculiarities of the penis of Semibalanus balanoides (Linnaeus, 1767) and Chthamalus stellatus (Poli, 1791) (Crustacea: Cirripedia: Thoracica), Journal of Experimental Marine Biology and Ecology 392: 228-233.

Zheden V., Klepal W., Gorb S.N., Kovalev A. Mechanical properties of the cement of the stalked barnacle Dosima fascicularis (Cirripedia, Crustacea). Submitted to Interface Focus.

Zheden V., Kovalev A., Gorb S.N., Klepal W. Characterization of cement float buoyancy of the stalked barnacle Dosima fascicularis (Crustacea, Cirripedia). Submitted to Interface Focus.

Book chapters Power A., Klepal W., Zheden V., Jonker J., McEvilly P., von Byern J. (2010): Mechanisms of adhesion in adult barnacles. In: Biological adhesive systems: from nature to technical and medical application; J. von Byern and I. Grunwald, eds. Springer, Vienna; p. 153-168.

Cyran N., Klinger L., Scott R., Griffiths C., Schwaha T., Zheden V., Ploszczanski L., von Byern J. 2010. Characterization of the adhesive systems in cephalopods. In: Biological adhesive systems: from nature to technical and medical application. J. von Byern and I. Grunwald, eds. Springer, Vienna; p. 53-86.

Some popular scientific articles “Superkleber der Meere“ in the online newspaper of the University of Vienna, August 2010

“Die Cuticula im Wandel der Jahreszeiten: Außerordentliche Variabilität von extrazellulärem Material bei Seepocken” in GIT Laborfachzeitschrift 5, May 2011, p. 308-311

91 Conferences

25.08. – 30.08.13 Microscopy Conference MC 2013 in Regensburg (Germany) Talk: “The cement float: morphology, biochemistry and mechanical properties of the barnacle Dosima fascicularis (Crustacea, Cirripedia Thoracica)”

26.05. – 27.05.12 2ndASEM-Workshop Advanced Electron Microscopy in Salzburg (Austria) Talk: “The buoy barnacle Dosima fascicularis with its special cement”

05.09. – 07.09.11 International Conference: Marine Resources and Beyond in Bremerhaven (Germany) Talk: “Characterization of the cement formation in Dosima fascicularis (Crustacea, Cirripedia)”

28.08. – 02.09.11 Microscopy Conference MC 2011 in Kiel (Germany) Talk: “The cement apparatus of Dosima fascicularis (Ellis and Solander, 1786) (Crustacea, Cirripedia Thoracica)”

18.05. – 20.05.11 Biological and Biomimetic Adhesives in Mons (Belgium) Talk: “Dosima fascicularis a barnacle which floats rather than sticks”

21.10. – 24.10.10 3. Graduiertenforum der Fachgruppe Morphologie in Vienna (Austria) Talk: “Morphologie und Ultrastruktur des Zementapparats von Dosima fascicularis (Crustacea, Cirripedia)”

30.08. – 04.09.09 Microscopy Conference MC 2009 in Graz (Austria) Poster: “Temporary adaptation of the cuticle in Semibalanus balanoides (Linnaeus, 1767) (Crustacea, Cirripedia Thoracica)”