MULTISCALE EXPERIMENTS AND MODELING IN BIOMATERIALS AND BIOLOGICAL MATERIALS

Jaws of Platynereis dumerilii: Miniature Biogenic Structureswith Hardness Properties Similar to Those of Crystalline Metals

LUIS ZELAYA-LAINEZ,1,4 GIUSEPPE BALDUZZI,1,5 OLAF LAHAYNE,1,6

KYOJIRO N. IKEDA ,2,7 FLORIAN RAIBLE ,2,8

CHRISTOPHER HERZIG,3,9 WINFRIED NISCHKAUER,3,10

ANDREAS LIMBECK ,3,11 and CHRISTIAN HELLMICH 1,12

1.—Institute for Mechanics of Materials and Structures, TU Wien-Vienna University ofTechnology, Vienna, Austria. 2.—Max Perutz Labs, University of Vienna, ViennaBioCenter, Vienna, Austria. 3.—Institute of Chemical Technologies and Analytics, TU Wien-Vienna University of Technology, Vienna, Austria. 4.—e-mail: luis.zelaya@tuwien.ac.at.5.—e-mail: giuseppe.balduzzi@tuwien.ac.at. 6.—e-mail: olaf.lahayne@tuwien.ac.at.7.—e-mail: kyojiro.ikeda@univie.ac.at. 8.—e-mail: florian.raible@mfpl.ac.at.9.—e-mail: christopher.herzig@tuwien.ac.at. 10.—e-mail: winfried.nischkauer@tuwien.ac.at.11.—e-mail: andreas.limbeck@tuwien.ac.at. 12.—e-mail: christian.hellmich@tuwien.ac.at

Nanoindentation, laser ablation inductively coupled plasma mass spec-troscopy and weighing ion-spiked organic matrix standards revealed struc-ture-property relations in the microscopic jaw structures of a cosmopolitanbristle worm, Platynereis dumerilii. Hardness and elasticity values in thejaws’ tip region, exceeding those in the center region, can be traced back tomore metal and halogen ions built into the structural protein matrix. Still,structure size appears as an even more relevant factor governing the hardnessvalues measured on bristle worm jaws across the genera Platynereis, Glyceraand Nereis. The square of the hardness scales with the inverse of the inden-tation depth, indicating a Nix-Gao size effect as known for crystalline metals.The limit hardness for the indentation depth going to infinity, amounting to0.53 GPa, appears to be an invariant material property of the ion-spikedstructural proteins likely used by all types of bristle worms. Such a metal-likebiogenic material is a major source of bio-inspiration.

INTRODUCTION

Bristle worms (Polychaeta) are a highly successfulgroup of invertebrate worms with a broad geo-graphic distribution, in particular across the entiremarine benthos. While they are generally soft-bodied, they also synthesize specific classes of hardstructures such as segmented bristles (chaetae) andjaw apparatuses.1,2 These typically lack the miner-alized calcium composition found in vertebratebones, instead employing alternative biogenic mate-rials, such as polysaccharide chitin (in the bristles)or secreted proteins enriched with glycine andhistidine (in the jaws).3–8 These structures fulfillimportant mechanical functionalities thanks totheir specific chemical composition. Their chemical

resistance allowed them to be preserved in the fossilrecord,9 with jaws forming their own characteristictype of microfossils, scolecodonts.10 The abundanceof scolecodonts in the Ordovician (490–440 millionyears ago) and their additional presence in lateCambrian rocks11 are consistent with the notionthat the evolution of jaws marked an early step inPolychaeta phylogeny. In turn, the evolutionaryinnovation that enabled the production of jaws andbristles likely contributed to the subsequent successand diversification of Polychaeta. This has spurredinterest in the material scientific investigation ofjaws, primarily in larger species of the Nereis andGlycera genus.4–7 Whereas these species offer theadvantage of large jaw apparatuses, measuringseveral millimeters in length, they are currentlynot available as standard laboratory cultures.

(Received January 14, 2021; accepted April 19, 2021;published online June 1, 2021)

JOM, Vol. 73, No. 8, 2021

https://doi.org/10.1007/s11837-021-04702-1� 2021 The Author(s)

2390

Over the past decades, the marine bristle wormPlatynereis dumerilii has emerged as a highlyaccessible model system for Polychaeta research.Namely, Platynereis cultures can be bred in thelaboratory under controlled conditions, providing aconstant supply of larvae that exhibit stereotypicaldevelopment.12 Thanks to a variety of moleculartools,13–15 Platynereis dumerilii has become animportant invertebrate reference model system fordevelopmental, evolutionary, reproductive, neuro-and chronobiology.16 As part of the larger Nereididfamily of Polychaeta, Platynereis possesses fang-likejaws reminiscent of the jaws of its larger Nereisrelatives. Jaw development has been described byFischer et al.12: A pair of jaws starts forming duringthe mid-nectochaetae development stage, i.e., 3 to 4days post-fertilization at 18 �C water temperature.At this point in time, the primary tooth of each jawbecomes visible, which will later define the anteriorend of the jaw. The jaws continue to grow in thesubsequent development stages, with additionalteeth being continuously added. The final numberof one primary and nine secondary teeth, see Fig. 1aand b, is reached in the tubiculous juvenile stage,with the jaws eventually measuring 800 lm inlength. They are then used during a remaining lifespan of typically 6–18 months, before the wormnormally dies after the relatively short (only a fewhours long) nuptial dance.

Despite the insight into the timing and morphol-ogy of jaw biogenesis, other features of Platynereisdumerilii jaws have remained unexplored. Wetherefore set out to characterize the chemical andmechanical properties of Platynereis dumerilii jaws.As outlined above, mineral phases are virtuallyabsent in the chewing instruments of Polychaeta.Instead, as in other invertebrate groups, such hardmouth parts typically rely on a protein matrix. Thisprotein matrix may be reinforced by chitin fibers, aswas shown for beaks of the jumbo squid Dosidicusgigas,17 and it may incorporate metal or halogenions, such as zinc, chloride or magnesium, as wasshown for the mandibles of six species from fourfamilies of Isoptera.18 Intensive studies have beendevoted to the deciphering of the relation betweenhardness properties and the presence of metal andhalogen ions in the jaws of several bristle wormspecies. Lichtenegger and co-workers, in their stud-ies of jaws from the genera Glycera5 and Nereis,6

showed that nanoindentation-derived elastic andhardness properties are positively correlated withconcentrations of metal ions. In this context, zincconcentrations were quantified through x-rayabsorption and fluorescence imaging of Nereis jaws,and electron micro-probe experiments on Glycerajaws provided access to copper concentrations. Lateron, partial peptide mapping and molecular cloningof a partial cDNA from a jaw pulp library4 allowedfor identification of Nvjp� 1 as the key proteinmaking up the organic matrix of Nereis jaws. Thelatter matrix hosted not only zinc ions, but also

halogen ions, such as chloride, bromide and iodine.Corresponding ionic concentrations were quantifiedthrough energy dispersive x-ray spectroscopy(EDS).19–21 In the case of Glycera jaws,7,22 EDSevidenced copper and chloride as the key ions wereincorporated into the structural protein matrix.

The present article extends these studies towardthe significantly smaller species Platynereis dumer-ilii, introducing unprecedented miniaturizationsteps to the nano-indentation protocol, including arefined polishing procedure; in this context, we donot only invest into the identification of metal andhalogen ions as drivers of the mechanical proper-ties, but we complement the current state of the artin bristle worm mechanics by the key topic of sizeeffects—elucidating astonishing similaritiesbetween the nanoindenter-probed hardness of crys-talline metals23 and those of ion-enriched proteincomplexes making up Polychaeta jaws.

Fig. 1. Imaging of Platynereis dumerilii jaw: (a) axonometric sketchwith indication of anatomical axes, (b) light micrograph of upper side(dorsal view), (c) volume-rendered l CT image of upper side (dorsalview), (d) volume-rendered lCT image of lower side (ventral view).

Jaws of Platynereis dumerilii: Miniature Biogenic Structures 2391

MATERIALS AND METHODS

Sample Selection and Preparation

Jaws were harvested from adult Platynereisdumerilii worms by mild dissociation. Briefly, wormtrunks were lysed in a buffer containing 0.2% (w/v)sodium dodecyl sulfate (SDS), 50 mM tris-(hydrox-ymethyl)-aminomethan-hydrochloride (Tris, pH8)and 0.5 M sodium chloride (NaCl) for 5–6 days withfrequent agitation. Jaws and other sedimentedmaterial were washed twice in 50 mL lysis buffer.Jaws were isolated from cellular debris by manualelutriation. Isolated jaws were washed twice morein 50 mL lysis buffer and subsequently in 0.5 MNaCl to remove residual detergent. Finally, jawswere stored in 0.5 M NaCl supplemented with0.05% sodium azide until testing. To check theintegrity and intactness of the jaws, they were thenobserved by light microscopy, being mounted into aZeiss Axio Imager Z1m (Carl Zeiss, Germany), withimages taken by a Zeiss AxioCam MRc5 camera, asseen in Fig. 1b. Afterwards, 90 jaws were embeddedin a two-component EpoFix resin (Struers, Den-mark), ten each in 5-ml Eppendorf microtubes(Eppendorf, Germany). Air pockets within the resinwere removed using a desiccator and a pump. Next,a micro-tomography device (lCT 100, Scanco,Switzerland) was employed at a resolution of1.2 lm to identify the orientation of the jaw planesorthogonal to the dorsal-ventral axis with respect to

the tube axis. A single intact jaw, the dorsal ventralaxis of which was lying perfectly parallel to the tubeaxis (within the precision provided by the micro-tomography device), was further analyzed: There-fore, the jaw, together with a surrounding 3-mm-thick slice with faces orthogonal to the tube axis,was cut out of the tube using an Isomet low-speedsaw (Buehler, USA); see Fig. 2a. This slice wasattached with resin to a glass microscope slide. Thelatter was mounted onto a Logitech PM5 circularpolishing system (Logitech, Scotland); see Fig. 2b.There, it was polished with a Microtex 300-mmpolishing disc in a suspension of agglomeratedpolycrystals made of alpha aluminum oxide(Struers, Denmark). The individual polycrystallineagglomerates initially exhibited a particle size of1 lm, diminishing during the polishing process. Thelatter process was terminated once the slice thick-ness had been reduced to 2 mm; see Fig. 2c. At thisstage, a surface roughness of 15 nm, as measured ina Triboindenter TI 900 device in scanning probemicroscopy (SPM) mode, was reached. In moredetail, raster scanning of the polished surface withthe Berkovich diamond tip yields topographicalimages like the one seen in Fig. 2e; it is made upof pixels of 78 nm size. Such images also allowcomputing the root-mean-squared average (RMS) ofthe topography of the surface, Rq, according toMiller et al.:24

Fig. 2. Sample preparation steps and outcome: (a) cutting resin-embedded jaws out of the microtube, (b) circular polishing of the indentationsurface, (c) sample preparation completed with (d) indentation areas ‘‘middle’’ and ‘‘tip’’ indicated in a light micrograph taken by a nanoindenter-inbuilt microscope and with (e) scanning probe-microscopic topography quantification of the polished surface portion measuring 400 lm2andlying within the middle region; the pixel-wise heights are given in nanometers.

Zelaya-Lainez, Balduzzi, Lahayne, Ikeda, Raible, Herzig, Nischkauer, Limbeck, and Hellmich2392

Rq ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

1

P2

X

P

m¼1

X

P

n¼1

z2mn

v

u

u

t ð1Þ

where P is the number of scanned pixels from theimage obtained by scanning probe microscopy, andzmn is the height difference between the pixels andthe mean scanned plane.24,25

Nanoindentation of Platynereis dumerilii Jaw

The polished surface, with its outward normalpointing in the ventral direction, was indented by aBerkovich tip attached to a Hysitron TriboindenterTI900, according the following protocol for loadcontrol: At a rate of 0.01 mN/s, the load wasincreased up to 100 lN maximum load; the latterwas held for 5 s before unloading took place, again

at a rate of 0.01 mN/s. This indentation process wasrepeated 156 times to realize two grids of 6 � 6 andone grid of 6 � 5 indents in the middle region of thejaw, depicted as a red area in Fig. 2d, and one gridof 6 � 9 indents in the tip region of the jaw, depictedas the green area in Fig. 2d. Thereby, the spacingbetween the indents always amounted to 5 lm. Thecorresponding maximum indentation depths hmax

ranged from 80 nm to 120 nm; hence, they were atleast 2.5 to 5 times larger than the roughness of15 nm, as is required for the experimental realiza-tion of an elasto-plastic half-space.24,26 Oliver andPharr27 computed the maximum indentation depthhmax as follows:

hmax ¼ hc þ hs ð2Þ

where hc is the contact indentation depth and hs isthe displacement of the surface at the perimeter of

Fig. 3. Intensity-to-weight fraction conversion for the ions in the matrix-matched standards: (a) bromide, (b) copper, (c) iron, (d) iodine and (e)zinc.

Jaws of Platynereis dumerilii: Miniature Biogenic Structures 2393

the contact. For such a half-space, the recordedload-displacement curves were evaluated accordingto the method of Oliver and Pharr:27 The reducedmodulus E and the hardness H were computed fromthe unloading stiffness S and the contact area Ac

according to:

E ¼ffiffiffi

pp

S

2ffiffiffiffiffiffi

Ac

p ð3Þ

H ¼ Fmax

Acð4Þ

Chemical Analysis of Platynereis dumeriliiJaw

To quantify the spatial concentration distribu-tions of halogen and metal ions in the Platynereisdumerilii jaw, the polished jaw surface was furtheranalyzed after the nanoindentation campaign. Moreprecisely, it was scanned, in line mode, by a laserablation inductively coupled plasma mass spectrom-eter (LA-ICP-MS). This device analyzed the upper-most 12 lm below the surface, while keeping therest of the jaw sample virtually unaltered. The LA-ICP-MS set-up consisted of a New Wave 213 laserablation system (Elemental Scientific Inc., USA)with a frequency quintupled neodymium-dopedyttrium aluminum garnet laser operating at awavelength of 213 nm, coupled, through polyte-trafluoroethylene tubing, to a quadropole iCAPQ-induced coupled plasma mass spectrometer (ICP-

MS; ThermoFisher Scientific, USA). The laser spotsize was set to 10 lm, and the scan speed waschosen as 10 lm/s. Helium was used as the ablationgas, and argon was admixed as make-up gas beforeentering the ICP-MS. The measurements of the LA-ICP-MS provided intensities I of the isotopes ofbromide, copper, iodine, iron and zinc in counts persecond. These counts needed to be related to theionic ‘‘concentrations,’’ which were approached herevia weight fractions in matrix-matched standards.28

In more detail, a Mettler Toledo precision balancePGH403-S (Mettler-Toledo International Inc.,Switzerland) was employed to define the amountsof selected salts in terms of well defined weight,namely: ammonium bromide, copper(II) chloride,iron(II) chloride, potassium iodide and zinc chloride.Then, a certain weight-defined amount of the fluidorganic compound N-methyl-2-pyrrolidon (MerckKGaA, Germany) was put into 15-ml polycarbonatetest tubes together with the aforementioned saltportions. These components were then well mixed,in the sense that crystal formations could no longerbe discerned by the naked eye. Thereafter, weadded, again at a well-defined weight, the sametwo-compound resin acid which we had alreadyused for the embedding of the worm jaws. Theresulting fluid was then stirred by a vibrating mixerfor 10 min and by a centrifuge for another 10 min.The weight fractions of the metal ions with respectto the weight of the overall mixture are given in theupper part of Table I. Thereafter, our mixing prod-ucts were cured, for 2 days, in the Duran Borosil-icate Glass 3.3 Complete Vacuum Desiccator.Subsequently, all standards, the four spiked ones

Table I. Blank (ion-free) and ion-spiked calibration standards: weight fractions WF per weight of entirecompound, LA-ICP-MS protocol-generated ionic intensities Is, and weight-fraction-to-intensity conversionfactors b; for the ions of interest: bromide, copper, iron, iodine and zinc

Standard WFBr WFCu WFFe WFI WFZn

[10�6] [10�6] [10�6] [10�6] [10�6]

Blank 0 0 0 0 01 142 64 51 165 592 290 129 105 336 1203 1322 590 477 1531 5454 2721 1213 982 3151 1122

Standard ISBr ISCu ISFe ISI ISZn[cps] [cps] [cps] [cps] [cps]

Blank 0 0 0 0 01 123 225 950 1890 992 208 385 1628 3193 1563 1148 1752 7539 16564 7224 2292 3088 13550 31581 1231

Slope bBr bCu bFe bI bZn[10�6 cps�1] [10�6 cps�1] [10�6 cps�1] [10�6 cps�1] [10�6 cps�1]

Equation 5 1.1774 0.3887 0.0718 0.0989 0.8981

Zelaya-Lainez, Balduzzi, Lahayne, Ikeda, Raible, Herzig, Nischkauer, Limbeck, and Hellmich2394

as well as the blank one, were cut by an Isomet low-speed saw (Buehler, USA) into 3-mm-thick circularslices and then attached, using resin, to a glassslide. The free surfaces of these slices underwent apolishing protocol as described in:29 They weremachined using a Leica SM2500 heavy-duty sec-tioning system and the Leica SP2600 ultramiller(Leica Biosystems GmbH, Germany), equipped witha diamond cutting edge rotating at 1000 rpm, at afeeding speed (i.e., a speed orthogonal to the sur-face) of 2 nm/polishing cycle and an advancing speed(i.e., a speed within the plane of the surface) of1.5 mm/s, until a roughness measure Rq accordingto Eq. 1, of around 15 nm was reached. Precautionswere taken to avoid cross-contamination during thepolishing process. Therefore, whenever a suffi-ciently fine surface was realized, the diamond bladewas cleaned twice, 15 min in an ultrasonic bathcontaining 99% ethanol followed by a rinse withdistilled water.

The polished surfaces of the four spiked and oneblank standard then underwent the same LA-ICP-MS protocol as the Platynereis dumerilii jaw, yield-ing the intensities Is given in the middle part ofTable I. The ionic intensities and ionic weightfractions turned out to be almost perfectly propor-tional to each other, so the weight fractions could be

expressed as a linear function, through the origin, ofthe intensities. Mathematically, this reads as

WFi ¼ biIsi for i ¼ Br;Cu;Fe; I;Zn ð5Þ

with ion-specific slope values bi assembled in thelower portion of Table I, representing the slopesseen in Fig. 3. These slope factors b were then usedto convert the ionic intensity distributions mea-sured on the jaws into corresponding weight frac-tion distributions, employed here as ‘‘concentrationquantities.’’

Size Effect-Related Re-evaluationof Nanoindentation Tests on Jaws of Nereisand Glycera

Nanoindentation-tested hardness may not onlydepend on the chemical composition and themicrostructure of the investigated material, butalso on the indentation depths and the correspond-ingly tested three-dimensional domain below theindenting tip. Considering that the strength of thetested material is governed, according to Taylor,30

by the amount of dislocations in the aforementionedthree-dimensional domain, Nix and Gao23 derivedthe following relation between tested hardness andthe contact indentation depths hc (see, e.g., Figure 2of Nix and Gao23)

Fig. 4. Compilation of 156 nanoindentation-derived data sets: (a) hardness versus maximum indentation depth hmax , (b) reduced elastic modulusversus maximum indentation depth hmax and (c) reduced elastic modulus versus hardness values.

Jaws of Platynereis dumerilii: Miniature Biogenic Structures 2395

H2 ¼ H20 1 þ h�

hc

� �

ð6Þ

with a size-independent hardness value H0 associ-ated with infinitely large indentation depths and acharacteristic length h� depending on indentershape and material properties. Relation (6) wasexperimentally validated through tests on polycrys-talline copper31 and silver.32

Herein, we check whether relation (6) may alsohold for the material making up Polychaeta jaws. Inthis context, hardness values have been reported forGlycera and Nereis genera by Broomell et al.,21

Pontin et al.,22 Moses et al.7 and Lichtenegger et al.6

These authors do not directly provide the indenta-tion depths hc; however, they still give sufficientinformation from which the latter can be assessed.

Namely, in case of a Berkovich indenter consideredby Oliver and Pharr,27 the indentation depth hc isrelated to the maximum indentation force Fmax via

hc ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffi

Fmax

24:5H

r

ð7Þ

and the maximum indentation forces applied tojaws of Glycera and Nereis have been reportedin;7,21,22 see Fig. 7a. Lichtenegger et al.6 do notprovide explicit information on the maximum inden-tation force; however, the indenter contact area Ac

can be assessed from Fig. 6a given in,6 and also thisarea gives access to the indentation depth hc, via,27

hc ¼ffiffiffiffiffiffiffiffiffiffi

Ac

24:5

r

ð8Þ

Fig. 5. Statistical evaluation of 156 nanoindentation experiments, 54 in the tip region and 102 in the middle region: (a) mean values and standarddeviations of reduced elastic modulus data and hardness data, (b) histogram over 156 reduced modulus values, (c) histogram over 156 hardnessdata values, (d) tip- and middle-related histograms of reduced modulus values and (e) tip- and middle-related histograms of hardness values.

Zelaya-Lainez, Balduzzi, Lahayne, Ikeda, Raible, Herzig, Nischkauer, Limbeck, and Hellmich2396

RESULTS

Mechanical and Chemical PropertyDistributions in Platynereis dumerilii Jaw

The nanoindentation-probed hardness valuesaccording to Eq. 4 are only weakly correlated withthe corresponding maximum indentation depthshmax; see Fig. 4a. This holds true for the total setof all measurements, as it does for the subsets‘‘middle’’ and ‘‘tip’’ according to Fig. 2d. This is evenmore the case for the reduced elastic modulusvalues according to Eq. 3, where hardly any

correlation can be observed in Fig. 4b. Moreover,no significant correlation between reduced modulusand hardness values can be found; see Fig. 4c. It isinstructive to evaluate these nanoindentation-derived values in a statistical way, first by meansof computing mean values and standard deviationsand of plotting histograms according to Scott’srule:33 It turns out that the the mean value overall 156 reduced elastic modulus values, 11.7 GPa,see Fig. 5a, is slightly higher than the characteristicvalue of the most frequently inhabited histogrambin, amounting to 10.5 GPa; see Fig. 5b. The

Fig. 6. Ion concentration distribution in Platynereis dumerilii jaw, in terms of ion weight fractions in %, resolved down to pixel size of 10 microns:(a) bromide, (b) copper, (c) iron, (d) iodine and (e) zinc.

Jaws of Platynereis dumerilii: Miniature Biogenic Structures 2397

Fig. 7. Nix-Gao size effect law across different species of Polychaeta: (a) tabular representation of experimental sources used for thedetermination of hardness and indentation depth hc ; (b) graphical representation. The bars represent the 95% confidence interval for the meanvalues of hardness and contact indentation depth data; (c) sketch of histidine enriched proteins with metal coordinated bonds.

Zelaya-Lainez, Balduzzi, Lahayne, Ikeda, Raible, Herzig, Nischkauer, Limbeck, and Hellmich2398

situation for the hardness values is different, wherethe mean value of 0.9 GPa lies within the interval ofthe most inhabited histogram bin; see Fig. 5c.

Once we distinguish between the middle and thetip indentation regions, a slightly different pictureemerges: As for the tip region, the mean value of thereduced modulus data, 12.7 GPa, see Fig. 5a, lieswithin the most populated histogram bin, seeFig. 5d, while the mean value of the hardness data,1.08 GPa, is slightly larger than the characteristicvalue of the most frequently inhabited histogrambin, amounting to 0.9 GPa; see Fig. 5e. As for themiddle region, the mean values of both hardnessand modulus data, 0.81 GPa and 11.2 GPa, seeFig. 5a, are slightly larger than the characteristicvalues of the most frequently populated histogrambins, which are 0.7 GPa and 10.5 GPa; see Fig. 5dand e.

Deeper statistical analysis realized through soft-ware package IBM SPSS Statistics34 concerns thequestion whether the data collected from the tipregion may be significantly different from thosecollected in the middle region: Parametric tests, i.e.,analysis of variance (ANOVA), cannot be appliedbecause of the non-normal distribution nature of thedata. Graphical interpretations of the data such ashistograms are extremely helpful when judgingnormality. However, Mishra et al.35 recommendthe combination of graphical interpretations with amore rigorous numerical statistical test to concludeon the normal distribution of data. Thus, thenormality of E and H were further tested by meansof the Kolmogorov-Smirnov and Shapiro-Wilktests,36,37 with the latter one being regarded asthe most powerful normality test.38 However, bothtests provided similar outcomes when applied to theindentation data recollected from both the tip andthe middle regions of the investigated jaw: The datasets turned out to be not normally distributed.Hence, non-parametric approaches were needed todetermine whether or not the data arising from thetip and middle indentation regions are similar.Accordingly, we resorted to the Mann-Whitney Uand the Kruskal-Wallis H tests, which are non-parametric equivalents of parametric tests such asStudent’s t-test and ANOVA.39–43 Our correspond-ingly tested null hypothesis was that there is nostatistically significant difference between theindentation results obtained at the tip region andthe middle region. However, examining the E and Hresults from both regions with the Mann-Whitney Uand Kruskal-Wallis H tests showed that they bothrejected our null hypothesis.

With these local differences in mind, it is instruc-tive to study the ion distributions across the pol-ished surface layer determined according toSect. 2.3: Among all tested ions, zinc and iodineare much more concentrated in the jaw tip regionthan they are in the middle region, with maximumlocal weight fractions of up to almost 5% in the caseof zinc and up to more than 10% in the case of

iodine; see Fig. 6d and e. Hence, the higher modulusand hardness values in the tip region compared tothe middle region may well stem from the corre-spondingly higher concentrations of iodine and zinc.The situation is different when it comes to bromide,iron and copper ions: Bromide ions are fairlyuniformly distributed across the jaw and locallyreach very high weight fractions of > 20%, seeFig. 6a; copper ions are hardly present at all in theinvestigated Platynereis dumerilii jaw, see Fig. 6b.Iron ions, at weight fractions reaching almost 9%,are more frequently present in the middle regionthan they are in the tip region; see Fig. 6c.

Trans-species Size Effect Law for Hardnessof Jaws Across Different Polychaeta

The species-specifically averaged hardness valuesreported by Lichtenegger et al.6 for Nereis limbata,by Broomell et al.21 for Nereis virens, by Pontinet al.22 and by Moses et al.7 for Glycera dibranchi-ata, as well as by us, in Fig. 5a, for Platynereisdumerilii virtually perfectly follow Nix-Gao’s sizeeffect law (Eq. 6); see Fig. 7b. This is underlined byan impressively large coefficient of determinationamounting to R2 ¼ 99:7%. The size-independenthardness and indentation quantities amount toH0 ¼ 0:53 GPa and h� ¼ 0:13lm. The limiting hard-ness H0 is very close to that of crystalline copper(0.58 GPa).23

The potential validity of Nix-Gao’s size effectcannot be checked from a series of nanoindentationtests characterized by only one maximum indenta-tion force. This is shown by Eq. 7: Within such a testseries, the hardness square would scale with (1/h4

c ),and not with (1/hc). Hence, for checking the rele-vance of the Nix-Gao relation, results associatedwith different maximum indentation forces need tocompared. In this sense, given the available data,our analysis was naturally restricted to the meanvalues associated with one and same indentationforce each. To further elucidate this issue, and toshow the high quality of the mean values used forconfirming the relevance of Eq. 6, we complementedour analysis by the 95% confidence intervals for themean values of the hardness and indentation depthdata; see Fig. 7b. These intervals were computed asthe mean values �1:96 times the standard error(SE),44 the latter being the standard deviationdivided by the square root of the number of tests;see the table labeled in Fig. 7a. Correspondinghardness data are assembled in Appendix A, Sup-plementary Material.

DISCUSSION

To the best knowledge of the authors, this is thefirst systematic experimental study on the chemicaland mechanical properties, namely nanoindenta-tion-probed hardness and reduced modulus as wellas LA-ICP-MS- and calibration standard-derived

Jaws of Platynereis dumerilii: Miniature Biogenic Structures 2399

ionic concentration distributions, across a jaw har-vested from a bristle worm of species Platynereisdumerilii, a key model organism in marine biology.

The novel experimental results may be evaluatedand discussed in terms of three major traditionsfound in the nanoindentation and applied materialmechanics literature:

1. histogram representations2. chemo-mechanical couplings3. size effect laws for metals

With respect to histogram representations,45 distri-butions with only one peak were observed, seeFig. 5, indicating that only one material phase waspresent at the observation scale tested by thenanoindentation device, which is about one half toone third of the indentation depth,25 i.e., around 33to 50 nm.

With respect to chemo-mechanical couplings,larger zinc and iodine concentrations found in thetip region of the jaw are correlated with slightly butstill statistically significantly larger hardness andelasticity properties in the tip region; an effectwhich has been extensively discussed in the litera-ture on other bristle worm jaws.5–7,19–22

However, the probably most remarkable originalinsight comes from setting our novel data in contextwith existing data that were obtained from largerbristle worm species tested at larger indentationdepths hc. The differences between the hardnesstested herein, and the hardness values reportedearlier, exceed the variations reported as results oflocal ionic concentration variations. As shown inFig. 7b, these observed differences likely do notresult from significant species-to-species differencesin the jaw material itself, but can be well explainedas consequences of the different indentation depthshc used in the different studies. More precisely, thesquare of the hardness being proportional to theinverse of the indentation depth indicates variationsin the dislocation density of the investigated mate-rial domain, as explained by Nix and Gao23 in thecontext of crystalline metals. An important biolog-ical implication of this finding is that the matterthat forms the jaws of diverse species within thelarge Polychaeta class may be less variable thancommonly assumed. Rather, our data suggest thation-spiked structural proteins likely represent anearly innovation in Polychaeta phylogeny andallowed the formation of a basic architecturalbuilding block with ‘‘universal,’’ i.e., species-invari-ant, hardness properties. The latter exhibit unex-pected parallels with crystalline metals, withrespect to both magnitude and Nix-Gao-type sizeeffect features which are characteristic for ductilematerials. However, metals are produced at hightemperatures with high energy consumption, whilethe ion-spiked high performance structural proteinsare products of regular biological synthesis. Of note,the biosynthesis of Polychaeta jaws appears to be

markedly different from the situation in verte-brates. There, two main types of building blocks,structural proteins (such as collagen) and metal ionforming minerals (such as calcium being part ofhydroxyapatite), are combined into nano-compositesgoverning the mechanical properties of tissues suchas bone.46 Thereby, the minerals with size effectproperties47 far off the Nix-Gao law are markedlymore brittle than ion-spiked proteins present inPolychaeta jaws. The latter therefore provide novel,unprecedented opportunities in the wide field ofbioengineering.

This motivates a re-discussion of our results froma mechano-chemical perspective: The interesting‘‘universal’’ feature integrating largely differingspecies- and testing protocol-specific hardness val-ues is their obedience of the Nix-Gao size effect law.This means that the Platynereis jaw tested herein isnot necessarily harder than the previously testedNereis and Glycera jaws; it rather means that themechanical properties of all these different jawmaterials are actually fairly similar. At the sametime, the validity of the Nix-Gao size effect lawindicates an important qualitative feature sharedby all of these materials: the occurrence of disloca-tion-type dissipative processes within their nanos-tructures—such dislocations are the veryfundament based on which Nix and Gao havemathematically derived their size effect law. Clas-sically, dislocations are considered the atomic scaleorigin of metal plasticity,48 i.e., of ductile materialbehavior. Interestingly, the idea of dislocations canbe reconciled with the knowledge on metal coordi-nation bonds occurring in all of the tested anddiscussed jaws.49,50 After mechanical rupture ofsomewhat aligned metal coordination bonds, seeFig. 7c, these bonds can quickly re-form under anew geometrical configuration associated with acorresponding ‘‘dislocation’’ emerging along the re-bonded line of metal ions. Such reformation pro-cesses along ‘‘dislocation-like lines’’ are beautifullyconsistent with the overall plastic or ductile behav-ior, as reflected by the Nix-Gao size effect law. Inthis context, the essential feature is the existence ofthe rupturing and re-forming metal coordinationbonds rather than the actually used metal ions ororganic ligands. The latter vary largely betweenspecies. In Platynereis, the dominant ions arebromide, iron, iodine and zinc; copper is the dom-inant ion in Glycera, and in Nereis, bromide, iodineand zinc dominate. Again, while the actually usedions and ligands are only of secondary importance,their local chemical concentrations have a remark-able impact on the locally prevailing stiffness andhardness values. In this sense, Nereis jaw materialhardens with increasing zinc concentration;6,20

Glycera jaw material hardens with copper concen-tration,7,22 an effect which is further enhanced bycrystalline atacamite fibers. Platynereis jaw mate-rial hardens with increasing zinc and iodine con-centrations, as was shown in the present study.

Zelaya-Lainez, Balduzzi, Lahayne, Ikeda, Raible, Herzig, Nischkauer, Limbeck, and Hellmich2400

ACKNOWLEDGEMENTS

This project was financially supported by theAustrian Academy of Sciences (OEAW), through theproject Bio3DPrint.

FUNDING

Open access funding provided by TU Wien(TUW).

CONFLICTS OF INTEREST

The authors declare that they have no conflict ofinterest.

OPEN ACCESS

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SUPPLEMENTARY INFORMATION

The online version contains supplementarymaterial available at https://doi.org/10.1007/s11837-021-04702-1.

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