Calcite Reinforced Silicasilica Joints in the Biocomposite Skeleton of Deepsea Glass Sponges

Home , Hexactinellid

www.afm-journal.de www.MaterialsViews.com FULL PAPER Calcite Reinforced Silica–Silica Joints in the Biocomposite Skeleton of Deep-Sea Glass Sponges

Hermann Ehrlich ,* Eike Brunner , Paul Simon , Vasily V. Bazhenov , Joseph P. Botting , Kontantin R. Tabachnick , Armin Springer , Kurt Kummer , Denis V. Vyalikh , Serguei L. Molodtsov , Denis Kurek , Martin Kammer , René Born , Alexander Kovalev , Stanislav N. Gorb , Petros G. Koutsoukos , and Adam Summers

palaeontologists, geologists, and biologists. The hierarchically structured glass sponge Caulophacus species uses the As the most basal metazoans, they are the fi rst known example of a silica and calcite biocomposite to join the spicules key to understanding the evolution of both of its skeleton together. In the stalk and body skeleton of this poorly known calcium- and silicon-based biomineraliza- tion. The manifestation of this minerali- deep-sea glass sponge siliceous spicules are modifi ed by the addition of zation, a skeleton of spicules embedded conical calcite seeds, which then form the basis for further silica secretion to in the body of the sponge, is typically a form a spinose region. Spinose regions on adjacent spicules are then joined complex arrangement of calcite or silica. by siliceous crosslinks, leading to unusually strong cross-spicule linkages. In For example, the skeletal spicules of glass addition to the biomaterials implications it is now clear, from this fi rst record sponges (Hexactinellida, Porifera) are valu- able model systems for the investigation of a biomineral other than silica, that the hexactinellid sponges are capable of structure-function relationships in bio- of synthesizing calcite, the ancestral skeletal material. We propose that, while materials, with the ultimate goal of iden- the low concentrations of calcium in deep sea waters drove the evolution of tifying design strategies for new synthetic silica skeletons, the brittleness of silica has led to retention of the more resil- materials.[ 1 ] Natural structural composites ient calcite in very low concentrations at the skeletal joints. often show a variety of desirable properties, including transient properties in the presence of water.[ 2 , 3 ] The hexactinellid sponges have silica skeletal structures that are both 1. Introduction fl exible and tough,[ 1 , 4 , 5 ] because of their hierarchically layered structure and the hydrated state of the silica.[ 6 ] Emblematic of By virtue of their embedded, mineralized skeletons, the complexity of sponge skeletons is Euplectella aspergillum , sponges are of great interest to materials scientists, chemists, in which the skeleton is an elaborate cylindrical latticelike

Dr. H. Ehrlich , Prof. Dr. E. Brunner , M. Kammer Dr. K. Kummer , Dr. D. V. Vyalikh Institute of Bioanalytical Chemistry Institute of Solid State Physics Dresden University of Technology Dresden University of Technology Bergstr. 66, D-01069 Dresden, Germany Zellescher Weg 16, D-01069, Dresden, Germany E-mail: [email protected] Prof. Dr. S. L. Molodtsov Dr. P. Simon European XFEL GmbH Max Planck Institute of Chemical Physics of Solids Notkestr. 85, D-22607 Hamburg, Germany Noetnitzer Str. 40, D-01187 Dresden, Germany Dr. D. Kurek V. V. Bazhenov Centre “Bioengineering” RAS, 7/1 Institute of Chemistry and Applied Ecology Prospekt 60-ya Oktiabrya, 117312 Moscow, Russia Far Eastern National University Dr. A. Kovalev , Prof. Dr. S. Gorb Sukhanova 8, 690650 Vladivostok, Russia Functional Morphology and Biomechanics Dr. J. Botting Zoological Institute Leeds Museum Discovery Centre Christian-Albrecht-Universitaet zu Kiel Carlisle Road,1, Leeds LS10 1LB, UK Am Botanischen Garten 9, D-24098 Kiel, Germany Dr. K. Tabachnick Prof. P. Koutsoukos P.P. Shirshov Institute of Oceanology RAS Department of Chemical Engineering Nahimovski Prospect 36, 117312 Moscow, Russia University of Patras Dr. A. Springer , R. Born 1, Stadiou Str., GR-265 04 Patras, Greece MBZ and Institute of Materials Science Prof. A. Summers Dresden University of Technology Friday Harbor Laboratories Budapester Str. 27, D-01069 Dresden, Germany University of Washington 620 University Road, Friday Harbor, WA. 98250, USA DOI: 10.1002/adfm.201100749

Adv. Funct. Mater. 2011, 21, 3473–3481 © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com 3473 3474 FULL PAPER due totheirsize,durability, fl The skeletalstructuresofglassspongesarealsoremarkable the subjectofdetaileddiscussionsinmodernchemicalbiology. skeletons. Thesemoleculesandtheirfunctionsarecurrently icifi ( stalk usingpincers.We observed clublikeconstructions about 15speciesareknown. seas with“normal” salinityatadepthof130–6770m.Currently is cosmopolitan,beingdistributedinalloceansandsome cules) fusedtoeachotheratpointsofcontact.Thesubgenus dictional plate,whichcontainsmostlyhexactines(six-rayedspi- silica deposition.Thestalkisfi (Figure 1 C–E) orbybridges(Figure 1 that areconnectedtotheirneighborsbynumerousarticulations F–G) throughsecondary is primarilyconstructedfromlongdiactine(tworayed)spicules by theuppersurfaceofcap(Figure 1 A,B). Thetubularstalk by alongstalk.Theatrialcavityisturnedout,andrepresented mushroom-shaped spongesthatareattachedtohardsubstrates Caulophacus 2.1. ClublikeStructuresWithintheSponge’sSkeletalFramework Results 2. remarkable fl of deep-seahexactinellids,innaturalconditions,demonstrate on spicularstalksinordertobetterexploitthem.Photographs ambient watercurrents;manyarealsoraisedabovetheseafl particles andresolvedorganicmatter, andtheyrelypartlyon organisms thatfi as itencountersvariationsinterrain. is present,inwhichthecurrentspeedsupandslowsdown but atintermediatedepths(1000to5000m)aissteadyfl interface ofthesemostlyuniqueconstructionsremainopen. however, numerousquestionsregardingthechemistry/biology molecules includingsilicateins, various tissuesencompassingavarietyoffunctions.Organic tion toprovidestructuralsupport,andasaninertscaffoldfor Their skeletonsappeartohavebeenoptimizedbynaturalselec- liest partoftheanimalfossilrecord(Ediacaran,630to542My). interlayers. tric domainsofconsolidatedsilicananoparticlesandorganic proteinaceous axialfi The basicelementsarelaminatedspiculesthatconsistofa spanning thelengthscalefromnanometerstocentimetres. structure withatleastsixhierarchicallevelsoforganization collagen sponge tion inthespicularstalkandbodyspiculesofhexactinellid tigate thechemistryandpeculiaritiesofstructuralorganiza- unexpected. Theprimarygoalofthecurrentstudywastoinves- the spiculesaremadefromabrittlematerialthisfl electron microscopy(SEM).These spinose,clublikestructures wileyonlinelibrary.com www.afm-journal.de Figure Individual spiculeswereisolatedfromthe Deep-ocean currentsaregenerallyperceivedassluggish, Spicules assignedtohexactinellidsareknownfromtheear- cation andforthespecifi

Caulophacus 2 [ 9 A,B) attheendsofseparatedspiculesusingscanning ] andchitin , includingitssubgenus exibility andresilienceofthestalks. lter greatvolumesofseawatertocollectfood

lamentsurroundedbyalternatingconcen- species [ 10–12 ] areresponsibleforboththebiosil- . ( . Figure exibility, andopticalproperties, c mechanicalpropertiesoftheir xedtothesubstratumbyabasy- [ 8 ] ormorerecentlydiscovered

1 ). [ 15–17 [ 14 C. ] Spongesare sessile ]

ow sp [ 13 [ [ 7 5 . ] ] ]

3.78 37 skeletons ofglasssponges. tural integrityproblemsintheconstructionofhierarchical unique andevolvedasanaturalengineeringsolutiontostruc- responding silica-basedarticulation( coupled witheachother, preventingslippageagainstthecor- revealed thatthespinysurfacesonthesespiculescouldbe 105 eV. SiO 9.0 ngmg in thisspicularmaterial terns arewelldescribedinthespongeliterature. cules of troscopy (XAS)(Figure 4 E,F), performedonuntreatedspi- photoemission spectroscopy(PES)andX-ray absorptionspec- club-like structureswastheninvestigatedbymeansof observed at100eVbondenergy, whereasSiO grants aninsightintoitschemicalbonding.Pure siliconis the exactpositionofpeakonbindingenergyscale sion demonstratesthepresenceofsiliconinallspicules,and tion relatedtoahighsilicaconcentrationinwater. hypothesized thatthisphenomenonresultsfromhypersilicifi and SEM(Figure 2 E,F). DetailedanalysisoftheSEMimages within articulationsusingbothlightmicroscopy(Figure 2 D) the clublikeconstructionscouldbeobservedasbeinglocated absorption spectroscopy structure (seetheSupporting Information,Figure S1).Atomic that calciumandnotsiliconisthemaincomponentofthiscentral persive X-ray spectroscopy(EDX)(Figure 4 D) unambiguouslyshow silica (Figure 4 B). Elementalmapping(Figure 4 C) andenergydis- that thespinespossesscentralstructures,coveredwithalayerof shown in disrupted severalspiculesshowingtheclublikeconstructions,as In order toproceedwithmoredetailedstudies,wemechanically Identifi 2.2. are deeplycorrodedundertheseconditions(Figure 3 ). to alkalitreatment,incontrastthesurfacesilicalayers,which that thespinesofclublikestructureswerehighlyresistant during alkali-baseddesilicifi microscopy (TEM)andelectron diffractionanalysis(EDA)of tern closelyresemblesthatof calcite.Transmission electron edge. Thefi (Figure 4 F) showa strongsignalattheCa 2pabsorption Also, XASspectraobtainedforeachofthespongesamples etry rulesoutforeignparticlesasasourceofthesilicasignal. these skeletalstructuresinaboundform.Thisstoichiom- Therefore wehypothesizethatsiliconoxideispresent in pound inthespiculeshasasilicium-to-oxygenratioof1:1,2. bond energyofabout103eVsuggeststhatthesiliconcom- according totheirstoichiometry(Figure 4 E). Theobserved employed gentledesilicifi these specifi To obtainmoredetailedinformationaboutlocalizationof were alsoobservedinlightmicroscopyimages(Figure 2 C). Siliceous monaxonspiculeswithvariousspinationpat- The chemicalnatureofthemineralphaseswithin ° C, asdescribedpreviously. ± 0.90masspercentofcalciuminthewholespicule. Caulophacus Figure − cation ofCalcite-Silica Composite 1 ne structureofthenear-edge X-ray absorptionpat- x (mean c structureswithinthespiculararticulations,we

compoundsareobservedbetween100and105eV

4 A. SEMimagesofthese brokenelementsshow ± standarddeviation,s.d.),correspondingto

sp. [ 18 , atanaverageconcentrationof37.8 cation usinga2.5 TheappearanceofSi 2pphotoemis- ] alsoconﬁ

cation experimentsweobserved [ 9 , 10 Adv. Funct. ] rmed the presence of calcium rmed thepresenceofcalcium After14daysofimmersion, Figure www.MaterialsViews.com

Mater.

M 3 ). Thisstructureis NaOHsolutionat

2011 [ 19 2 isfoundat [ ] 20 , Ithasbeen 21 ] However, , 3473–3481

ca- [ 18 ± ]

FULL PAPER 3475 ks are connected links. g) Computer links. g) Computer wileyonlinelibrary.com www.afm-journal.de rm the calcite nature of cation was achieved by infrared . on the rocky surface, 15 cm tall. b) The stalks of this sponge . on the rocky surface, sp

after alkali-based desiliciﬁ S3) and Raman Information, Figure (IR) (see the Supporting The obtained FTIR spectra as well as F) spectroscopy. 5 (Figure Raman spectra unambiguously conﬁ these crystals. Mechanical testing of the spicules suggests an important testing of the spicules suggests an important Mechanical strengthening role for the observed structures. Micro- indentation tests of the sponge architecture demonstrated only slight plastic deformation (one micrometer or less), compared with tens of micrometers of elastic deformation. Moreover, 3. Discussion

] Caulophacus 22 , rm 18 [ 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2011 WILEY-VCH Verlag . spicular stalks. a) The microstructure © ] sp A,B) further conﬁ

5 21

[ ed in other invertebrates Figure Caulophacus , 3473–3481 21 , 2011

Complex Complex organization of (see Supporting Information, Figure S2). Further analysis Further S2). Information, Figure (see Supporting E) isolated from clublike structures 5 of the crystals (Figure Figure 1 . the untreated clublike structures ( these results. TEM diffraction analysis of the spicule shows C) and 5 the presence of two phases, one amorphous (Figure D). EDA for the spines shown as a 5 one crystalline (Figure B correspond to the (0001) zone axis 5 TEM image in Figure of calcite with d spacings: 100 (1010) 4.32 Å; 010 (0110) 4.32 Å and 210 (2110) 2.49 Å. These data correlate well with those reported previously for calcite identiﬁ using the electron diffraction technique. of the central calcite crystals isolated by alkali treatment from D) is also very similar to the 5 the clublike structures (Figure morphology of calcite crystal nuclei described previously possess a complex network of glassy spicules (c). d,e) Scanning electron microscopy (SEM) images show that spicules within stal possess a complex network of glassy spicules (c). d,e) Scanning electron microscopy (SEM) to each other with scaffoldlike articulations. f) SEM image that shows parallel spicules connected to each other by small cross reconstruction of these articulations. reconstruction of these articulations. www.MaterialsViews.com Adv. Funct. Mater. Funct. Adv. 3476 FULL PAPER tively isotropic.TheREMofthe spongeintheaxialdirection 1.45 MPa. Thebulkmaterialofthespongeissofterandrela- (REM) of14.9 connected moredensely. ThisareahasareducedE-modulus outer layer(thickness150 the Supporting Information,Figure S4A).Thespongehasrigid buckling eventswereclearlyseenontheindentationcurves(see wileyonlinelibrary.com structures. c)Clubliketerminationofaspiculeobservedusinglightmicroscopy. d)Afterimmersionofthestalksin2.5 2. Figure partially dissolvedarticulationsconﬁ rm theobservationsmadeusinglightmicroscopy. dissolution ofsilica-basedarticulationsrevealedthatclub-likeconstruction (arrows)arelocatedwithinarticulationjoints. www.afm-journal.de lbie tutrs ihn h the within structures Clublike ± 4.5MPa withtheminimalmeasuredvalueof μ m) wherethespiculesare inter- Caulophacus © 2011WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim

sp . skeleton.a,b)Isolatedandbroken stalkspicules,separatedusingpincers,showingclublike the innermostpartofsponge (REM4.3 parallel tothesurface,whichexplainssmallREMvalues of number ofcross-linkingspiculesandwithorientation tion, Figure S4).Theinnersurfaceofthespongehasareduced trude, itwasjust3.82 was 614 ever, after100 ± 173 kPa, whereasinareaswheresinglespiculespro- μ m ofcompressionthe softlayer, thesponge ± 1.34kPa (seetheSupporting Informa- Adv. Funct. M NaOHfortwoweeks,partial e,f) SEMimagesofthe e,f) www.MaterialsViews.com

Mater. ±

0.5kPa). How- 2011 , 21 , 3473–3481 FULL PAPER 3477 n of this coupling. wileyonlinelibrary.com www.afm-journal.de at covers the spicules and the tenth day of immersion ooded with ionic solu- ooded with ned structures. The inner is favored at higher pH values. 2 . spicule represents a microchannel sp

C and pH between 7.62 and 8.22 yielded C and pH between m in diameter ﬂ ° μ Caulophacus . sponge stalks. a) SEM images show that clublike structures are sp

Seawater may become supersaturated with respect to calcium carbonate especially in spatially conﬁ space of the of approximately 1.0 tion, while one extremity of the spicule is sealed by the silica. At these conditions, contact with silicic surfaces is expected to favor heterogeneous nucleation of calcite, as in this case lower supersaturation is needed in comparison with the value needed for example, calculation of the saturation of cal- homogeneous. For cite at 2,000 meters, 5 supersaturation with respect to calcite or to any other calcium carbonate polymorph. This in turn depends on the levels of calcium and carbonate ion activities. The latter depend strongly on local pH as the dissolution of CO - Caulophacus The ] C). In 20 ° [ 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2011 WILEY-VCH Verlag ned to an extracel- © 200 kPa (see the Supporting Infor- (see the Supporting 200 kPa ± , 3473–3481 21 , cation in sponges is conﬁ 2011

Clublike spicules determining the structural integrity of Clublike spicules determining the structural The challenge now is to understand the mechanisms by cult to differentiate between organically mediated and inorganic precipitation, and a combination of different mechanisms in the same species cannot be ruled out. A decisive factor for the control of nucleation of calcium carbonate is the development of local sclerocytes secrete a macromolecular matrix to the extracellular space, which comprises a genetically programmed, three- dimensional framework that guides spicule formation. It is diffi most cases, calcifi lular space delineated by tightly adhering sclerocyte cells. material has REM of 618 S4B), equaling that of the bulk sponge material. mation, Figure which this unique biocomposite is initially formed, at high pres- and at low temperature (between 2 and 4 sure (420 ATM) www.MaterialsViews.com c,d) Alkali treatment led to dissolution of silica, allowing observation of the spines of clublike constructions using SEM, on c,d) Alkali treatment led to dissolution of silica, allowing observation of the spines of clublike constructions in alkali. e,f) The spines seem to be resistant to alkali treatment in contrast to the weakly corroded silica-based material th forms the articulations. responsible for mechanical coupling between two thin spicules within one large spicule. b) Corresponding computer reconstructio responsible for mechanical coupling between two thin spicules within one large spicule. b) Corresponding Figure 3 . Adv. Funct. Mater. Funct. Adv. 3478 FULL PAPER assembly oftheminiaturized subunits. bonate crystalswithsilicateanions andbytheself-organized zation ofthegrowthsubunitsbycoveringsurfacecar- crystalline architecturecanbeinducedthroughtheminiaturi- inorganic solids.Ithasalsobeenshowninvitrothatacomplex cite crystalsinthepresenceofpolymericsilica. can beunderstoodastheresultofcrystallizationcal- sponds fortheionactivityofseawatertopH8.00. precipitate atasaturationexceeding100,valuewhichcorre- values between49and184,respectively. cite, layers ofsilicateshavebeendepositedonthesurfacecal- silicate anionsinteractstronglywithcarbonates.Furthermore, metal carbonatesinhigh-pHsilicagel,whichindicatedthat et al. wileyonlinelibrary.com spicules. X-rayabsorptionspectrashowingthatcalciumcarbonateintheformofcalciteissecondmineralcompone silicon oxides.f) presence ofcalciumasthemaincomponentthisnucleus.e)Photoemissionspectraclub-formedspiculeshowing that thespineseachpossessanucleusiscoveredbysilicalayer. b)Magnifi cation of(a).Elementalmapping(c)andEDX 4. Figure www.afm-journal.de The observedmorphologyofthissilica–calcitebiocomposite [ 26 [ 25 , 27 ] obtainednumerouspeculiarformsofalkaline-earth ] Identifi cation ofmineralcomponents withinclublikeconstructions.a)TheSEMimageofamechanicallydisrupteds afactsupportingcrystallographicaffi [ 23 [ ] 28 Calcite isexpectedto ] © Thesilica–calcite- 2011WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim nityforthetwo [ 24 ] Garcia-Ruiz early animalphylogeny. It hasbeenarguedthatthedifferent structed, andthisisofgreatsignifi spicules hasyetbeenfoundtosecretesilicaalso. these constitutebiocomposites,andnospongewithcalcareous based clublikestructuresmadeby ules in additiontosiliceousspicules,orassmallcalcareousspher- known examplesareintheformofsolidcalcareousskeletons very rarelyco-occurinthesamesponge.Theonlypreviously variety ofminerals,calciumcarbonateandsiliceousstructures nano- andmicrolevelsofstructuralhierarchy. solutions fortheirownfunctionalrequirements, eralization isaprocessinwhichorganismsproducemineral natural exampleofthisprinciple,whichconfi It islessclearhow theskeletonsofearlyspongeswerecon- Although poriferanskeletonsmay becomposedofalarge [ 30 ] orgranules [ 31 ] insiliceousspiculatesponges.Noneof Adv. Funct. Caulophacus cance inpalaeontologyand www.MaterialsViews.com

Mater. analysis (d)showthe nt presentwithinthe rms thatbiomin-

2011

sp [ oftwokinds 29 . arethefi ] , startingon picule shows 21 , 3473–3481 rst FULL PAPER 3479 of ] 10 [ wileyonlinelibrary.com www.afm-journal.de ence of two phases: ines shown as a TEM e standard (f, below). e standard (f, below). i-based desilicifi cation i-based desilicifi The presence of a silica–calcite The presence

]

33 [ exible in the precise structures formed, . spicule with a fragment of club-like structure (arrow) . spicule with a fragment of club-like sp

Caulophacus composite in extant hexactinellids suggests that the mecha- nism of secretion is ﬂ and that extant and early sponges share a common biochemical basis for secreting both materials. Although the structures of the silica–carbonate biocomposite.

Demospongiae ( 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2011 WILEY-VCH Verlag © However, the heterac- However, ] 32 [ Silicispongia preclude homologous biomin- shows features characteristic of both shows features Calcarea , 3473–3481 21 ) and , 2011

Eiffelia globosa Eiffelia Calcite crystals within club-like structures. a) TEM image of the native crystals within club-like structures. a) TEM image of the native Calcite Hexactinellida club-like structures are well visible using SEM (e). Their Raman spectra (f, above) are absolutely similar with those of calcit club-like structures are well visible using SEM (e). Their Raman spectra (f, above) are absolutely amorphous with a diffuse amorphous halo at 2.05 Å (c) and crystalline (d). Electron diffraction pattern corresponding to the sp amorphous with a diffuse amorphous halo at 2.05 Å (c) and crystalline (d). Electron diffraction isolated after alkal Crystals image in (b) corresponds to the calcite lattice with d spacings of 4.32 Å (101) and 2.49 Å (210). (B). Spicule chosen for selected area electron diffraction (SAED). c,d) TEM diffraction analysis of this spicule shows the pres (B). Spicule chosen for selected area electron diffraction (SAED). c,d) TEM diffraction analysis Figure 5 . tinid sponge eralization between the sponge classes. secretion mechanisms of spicules in and groups, and spicules have a bilayered structure suggestive of a www.MaterialsViews.com Adv. Funct. Mater. Funct. Adv. 3480 FULL PAPER before thefullimplicationsofsuchfi phenomenon recentlydiscussedbyus. resent anadditionalexampleofmultiphasebiomineralization- chical systems,representsascientifi different biosilica-basedandnanostructurallyorganizedhierar- Caulophacus tial forbiologicalsurvivalofthe fi complex designedskeletalnetwork,whichisdefi Nature decidestousebothmineralphaseswithinonevery the caseofextremedeep-seaenvironmentalconditions,Mother than calciumcarbonateasastructuralmaterial.However, in known whysomeorganismsutilize,forexample,silicarather on theuniversity-diversity-principle(UDP), ingly, theconceptofturningweaknessintostrengthbased mechanically challengingenvironmentalconditions.Intrigu- mechanically stableandsimultaneouslyveryfl construction ofthespongestalk,makeentire locking systembetweenspicules,andthefi spicule (seetheSupporting Information,Figure S5),theinter- A combinationoftwodifferentmaterialsatthelevelsingle for amechanicalinterlockingofsinglespiculewitheachother. of spicule,whicharemadecrystallinecalcite,responsible sipates stress.Additionally, clavate-shaped, spine-coveredends based constructionisanadditionaldegreeoffreedomthatdis- localization ofmicrodamage.Twisting orbendingofthefi like elementsappeartogovernthefracturetoughnessand In foamlikespongeconstruction,thelargeststructuralfi utes tothevolume,butnotstiffnessofconstruction. construction, astiffandbrittlematerialofsinglecellscontrib- microstructure suchastheglassspongestalk.Inafoamlike tration nearholesandnotchesisdissipatedbymaterialswith stituents. structural geometrywithouttheneedtointroducenewcon- cell wallscanbegreatlyalteredbysimplealterationoftheir cies, ofteninadverseenvironments. extreme evolutionarypressurestoensurethesurvivalofaspe- Structural designofbiologicalmaterialshasevolvedunder Conclusions 4. alogy for eans, wouldtendtosupportanancestralcarbonatespiculeminer- ica–calcite compositesinthesilicispongelineagebutnotcalcar- phacus likely tobehomologous.Ifsimilarstructuresthosein cytological architecture,themineralogicalsecretionprocessesare skeletal elementsthemselvesmayhavechanged,aswellthe computational models of smalldimensionarestifferthanexpected. ient structureoftheglassspongestalk.Typically, slenderrods factors thatareimportantforthehighlydeformableandresil- at thenanoscale. material, solelythroughalterationsofitsstructuralarrangement transform abrittlebiosilicaintoductile,strong,andtough In contrasttothissiliceoussponge,diatoms,forexample,can because ofthepresencetwodifferentinorganicmaterials. wileyonlinelibrary.com www.afm-journal.de The silica–calcitecompositestructuresdescribedhererep- areconfi Porifera [ 35 ] Clearly, thefuturedevelopmentofcomparative

sp rmed inothertaxa,thewidespreadpresenceofsil- . sponge,canbediscussedasvery speciﬁ . Moredataareneededfromotherspongegroups [ 35 , 36 ] Thus,mechanicalresponseofdiatoms [ 37 ] ofglassspongesanddiatoms,astwo ts organism. ttest ndings will be known. ndings willbeknown. c task of greatinterest. [ 34 ] We cannowoutlinethe [ 38 ] Uptoknow, isnot © [ 34 2011WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim [ bos composite brous 17 ] inthecaseof ] Stressconcen- nitively essen- nitively eil under exible

Caulo- bre- ber- c in theN-EPacific, offBeringIsland,stn.2316,55 captured duringthe22 5. ExperimentalSection Foundation EUROCORESProgrammeFANAS,bytheGerman Science 2009. Partofthisstudywassupported,asparttheEuropeanScience Nr. 8066),andbyErasmusMundusCo-operation Window Programme Lomonosov -II”ofDAAD(Ref-325;A/08/72558)andRMES(AVCP Grant and bytheBMBF(GrantNo.03WKBH2G),ajointprogram“Mikhail 394/1-1; MO1049/5-1,ME1256/7-11256/13-1,andWO494/17-1) assistance. ThisworkwaspartiallysupportedbytheDFG(GrantNos.EH R. Schulze,H.Meissner, G.Richter, O.Trommer forexcellenttechnical G. Bavestrelloforhelpfuldiscussions.Theauthorsaredeeplygrateful to TU Dresden,Germany. TheauthorsthankR.Lakes,G.Wörheide, Microscopy Laboratoryforhigh-resolutionandholographyatTriebenberg, We thankProf.H.Lichteforuseofthefacilities attheSpecialElectron Acknowledgements from theauthor. Supporting Information isavailablefromtheWileyOnlineLibraryor Information Supporting 45 days infreshsea-water. Thespongewasdriedafterwardsfor4daysat to thefollowingprocedure.Spongematerialwasstoredforseveral 24,46’ E,4200-4294m.Stalksof placed in10mLplasticvesselsforstorageandanalysis. for 48hat25 digest anypossibleexogenouschitincontaminations.Afterincubation vessel containingchitinasesolution(asdescribedbyuspreviously) again washedthreetimesindistilledwater, driedandplacedina15mL incubation for24hat15 to digestanypossiblecollagencontaminationofexogenousnature.After solution containingpurifi ed three timesindistilledwater, cutinto3cmlongpiecesandplacedina and driedagainat45 37 The vesselwascoveredandplacedunderthermostaticconditionsat fragments ofthe (Fluka) solutionwasaddedtothe10mLplasticvessel,whichcontains Caulophacus. determine theconcentrationofcalciumwithinpurifi ed samplesof spectrometer withVarian GTA-96 graphitefurnaceatomizerwasusedto within thecross-sectionalarea. and SEMatdifferentlocationsalongthelengthofspicularmaterial desilicifi cation procedureswasalsomonitoredusingopticalmicroscopy 0.01% solutionofHF(puriss.p.a.,Fluka).Theeffectivenessthealkali SCIENTA SES200electronenergyanalyzer. of theSi2pcore-levelwasobtainedat200eVphotonenergyusinga in totalelectronyield(TEY)modewithaMCPdetector. Photoemission spectra inthevicinityofCa 2pabsorptionthresholdwererecorded dipole beamlinelocatedattheMAXIIstoragering.X-rayabsorption Sweden) usingradiationfromthebeamlineD1011bendingmagnet (XAS) used tomeasureabsorptionbycalcium. purifi ed waterandagainhomogenised.Awavelengthof239.9nmwas homogenised for30sbyshaking.Eachsamplewasdilutedwith900 microvials, dissolvedbyadding100 SpongeSamples ChemicalEtchingofGlassSpongeSkeleton AtomAbsorptionSpectroscopy(AAS) PhotoemissionSpectroscopy(PES)andX-rayAbsorption ° ° C. Finallythespongeskeletalmaterialwascleanedin10%H C withoutshaking.Thesameprocedurewascarryingoutusing : MeasurementswereperformedatMAX-lab(LundUniversity,

sp ° . spicules.ThreesampleswereweighedintoEppendorf C, fragmentsofskeletonwereagainwashed,dried,and Caulophacus : Theinvestigated ° nd C. Tissue-freedriedspongematerialwaswashed cruiseoftheR.V. “Academic MstislavKeldysh” ° C, thepiecesofglassspongeskeletonwere Clostridium histolyticum

sp Caulophacus . stalks,puriﬁ ed asdescribedabove. Adv. Funct. Caulophacus μ L HFtoeachsampleand : AVarian SpectrAA-10

sp : 8mLof2.5 www.MaterialsViews.com

o . weretreatedaccording Mater. 36,08-35’N167

collagenase(Sigma) sp

. specimenswere 2011 , 21 , 3473–3481 M NaOH o 23,04- [ 10 2 ] O to μ L 2

FULL PAPER 3481

, , , , , , 102 105 302 Lang- 2002 2011 1999 , ,

, (Eds.

Colloids Colloids , 1462 . 2005 2003 , 57 . 1992

Nature 54 , , 303 . , . 48 , 51 . , 820 . , 2010 4 Science

2003 2002 wileyonlinelibrary.com , 32 www.afm-journal.de , 699 . , 2 , 15 . 2010 , J. Inorg. Biochem.

1988 , 209 .

2003 202

, 23 . 39 , 2010 Taxonomy of Porifera Taxonomy 3

, , Metall. Mater. Trans. A Trans. Metall. Mater. 2002 Chem. Mater.

2008

, 379 . 1975

5 , 322 . , , 353 . Proc. Natl. Acad. Sci. USA Chem. Lett. 2 84 Oceanol. Acta , , . Micron Comp. Mater. Sci. Mater. Comp. Proc. Biol. Soc. Washington , 511 . 2010

Rev. Mineral. Geochem. Rev. 2002 2006 1987 361

Int. J. Appl. Mech. , Appl. Surf. Sci. Marine Chem. , 8 . Systema Porifera A Guide to Classiﬁ cation of Sponges A Guide to Classiﬁ Systema Porifera 1993

Nano Today 294 , 100 . , Astrobiology 21 Can. J. Zool. Can. , Nature 2007

2005

, 785 . , 410 . 1194 . Surf., A M. Ilan , A. V. , Ereskovsky A. G. , Muricy , Springer H. K. , Worch A. , Kummer M. Mensch , D. , Kammer R. S. V. Born , Paasch , , Vyalikh E. , S. Brunner L. , Molodtsov D. Kurek , 88 muir S. T. , Hyde DOI: 10.1007/s11661-010-0477-y. DOI: 10.1007/s11661-010-0477-y. Kluwer Academic/Plenum Publishers , New York New York , Publishers Kluwer Academic/Plenum online. 401 J. , Vacelet , N. Berlin and Heidelberg Boury-Esnault ), NATO ASI Series, Springer , Verlag 1554 . 148 . [ 26 ] D. S. Kim , [ 27 ] C. K. H. Lee , Bala , Y. , Zhang H. , Ynag C. , Wang [ 28 ] M. Li , A. Kotachi , X. , T. Lv Miura , Z. H. . Wang Imai , [ 38 ] H. Ehrlich , P. Simon , W. , Carrillo-Cabrera V. V. , Bazhenov J. P. Botting , [ 22 ] M. Donnet , P. Bowen , N. Jongen , [ 23 ] J. J. Lemaître , V. H. Levendekkers , [ 24 ] Hofmann , J. , M. Garcia-Ruiz A. [ 25 ] , Carnerup J. A. M. Garcia-Ruiz , G. E. Melero-García , , Christy S. T. N. Hyde , J. , Welham [ 35 ] A. P. Garcia , [ 36 ] M. J. A. , Buehler P. Garcia , D. Sen , M. J. , Buehler [ 37 ] D. Sen , M. J. . Buehler [ 17 ] R. , Lakes [ 18 ] Materials and methods are [ 19 ] available M. as J. Uriz , Supporting [ 20 ] Information M. Maldonado , M. C. , Carmona M. J. Uriz , A. [ 21 ] , Cruzado S. R. Hall , P. D. , Taylor S. A. Davis , S. Mann , [ 31 ] K. , Rützler K. P. Smith , [ 34 ] M. J. , Buehler [ 16 ] K. R. , Tabachnick [ 29 ] J. J. De , [ 30 ] Yoreo P. , J. Vekilov , Vacelet C. , Donadey C. , Froget in [ 32 ] I. Sethmann , G. [ 33 ] , Wörheide J. P. Botting , N. J. eld , Butterﬁ [ 15 ] N. , Boury-Esnault L. De , Vos , Proc. 2007 , 339 . J. Exp. , 611 . , 1 . 333 18 211 , 1084 . , , , 2 ,

2008 2008 1996

, 303 . 2010

25 , 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2011 WILEY-VCH Verlag Received: April 4, 2011 Received: April , 1420 . © Nature , 395 . 16 1997

, 42 , 275 . , Cell. Tiss. Res. Cell. Nat. Chem. Published online: July 29, 2011 Published online: , 93 . 309 2001 ,

2002

Geology Adv. Funct. Mater. Funct. Adv. 158 , , 3358 . 2005

, 1144 . 2007 101

, , 473 . 310 In Porifera Research: Biodiversity, Innovation Research: Biodiversity, Porifera In Exp. Mech. , Science J. Mater. Res. J. Mater. 2004

308B , 3473–3481 2005 , , DOI: 10.1155/2008/670235. , DOI: 10.1155/2008/670235.

21 (Eds: M. R. Custodio , G. Lobo-Hajdu , E. Hajdu , , J. Struct. Biol. 2007 2008

2011

Science & Sustainability G. ), Muricy Série Livros 28, Museu Nacional, Rio de Janeiro , C. , Solazzo Y. C. , Yang Baessmann , T. D. Langrock , P. R. Ashford , Simon , Hoffmann , J. M. G. , , Tsurkan Wörheide S. , Thomas-Oates A. J. Hunoldt , , V. Reitner M. M. , Ereskovsky Mertig , D. Lubeck , H. D. , Kurek , Worch V. V. V. Smetacek , , Vyalikh V. M. S. , Bazhenov J. L. , Collins , Molodtsov K. , Kummer R. Born , C. Zool., Part B Goebel , P. Simon , S. Heinemann , H. , Worch pp. 439–448 . N. P. Shapkin , M. J. Nanomat. Mertig , C. , Erler R. Born , H. , Worch T. Hanke , Natl. Acad. Sci. USA P. Allen , K. Fields , D. M. E. J. Morse , , Porter F. W. Zok , P. K. Hansma , P. , Fratzl D. E. Morse , P. , Fratzl A. , Mescher E. Gaino , D. Brandt , H. C. , Schröder M. Wiens , P. , Fratzl D. E. Morse , F. W. , Zok [ 9 ] H. Ehrlich , R. Deutzmann , E. , Brunner E. , Cappellini H. Koon , [ 10 ] H. Ehrlich , M. Maldonado , K. D. , Spindler [ 11 ] C. H. Eckert , Ehrlich , T. H. Hanke , , Worch [ 12 ] H. Ehrlich , D. Janussen , P. Simon , S. Heinemann , V. V. [ 13 ] , Bazhenov J. Aizenberg , V. C. , Sundar A. D. , Yablon J. C. , Weaver G. Chen , [ 14 ] B. C. , Heezen E. D. , Schneider O. H. , Pilkey [ 2 ] G. , Mayer M. Sarikaya , [ 3 ] G. , Mayer [ 5 ] J. C. , Weaver J. Aizenberg , G. E. , Fantner D. Kisailus , A. , Woesz [ 4 ] J. Aizenberg , J. C. , Weaver M. S. Thanawala , V. C. , Sundar [ 6 ] M. Sarikaya , H. , Fong N. Sunderland , B. D. Flinn , G. , Mayer [ 7 ] M. , Brasier O. Green , [ 8 ] G. W. Shields , E. G. , Müller X. , Wang K. Kropf , A. Boreiko , U. , Schloßmacher [ 1 ] A. Miserez , J. C. , Weaver P. J. , Thurner J. Aizenberg , Y. Dauphin , www.MaterialsViews.com Foundation DFG (contract No GO 995/4-1) and the EC Sixt Framework the EC Sixt Framework No GO 995/4-1) and DFG (contract Foundation to SNG. (contract No ERAS-CT-2003-980409) Programme Adv. Funct. Mater. Funct. Adv.