Growth of nacre in abalone: Seasonal and feeding effectsIntroductionExperimental techniquesResults and discussionCharacterization of growth surfacesEpithelium observationsDemineralized shell and organic layerConclusionsAcknowledgementsReferencesGrowth of nacre in abalone: Seasonal and feeding effectsM.I. Lopez⁎, P.Y. Chen, J. McKittrick, M.A. MeyersUniversity of California, San Diego, La Jolla, California, USAabstractarticle infoArticle history:Received 20 March 2010Received in revised form 16 July 2010Accepted 3 September 2010Available online 9 September 2010Keywords:AbaloneNacreGrowthHaliotisChitinBiomineralizationThe processes of aggregation of mineral and organic materials to the growing surfaces in red abalone (Haliotisrufescens) are analyzed. The flat pearl implantation method is used to observe the transient stages of calciumcarbonate deposition, the structure of the organic interlayer, and the steady-state growth of aragonite tiles.The morphology of the organic interlayer is characterized by scanning electron microscopy. These resultsenable a realistic depiction of the formation of the terraced cones that comprise the principal biomin-eralization mechanism in this gastropod. In all cases, the growth initiated through spherulites, followed by tileformation. The transient stage with spherulitic formation was shorter at higher temperature; this is indicativeof a greater activity of the animal at 21 °C. The growth rate in a normally fed gastropod was found to be highercompared with one provided with limited food. The effect of water temperature (seasonal) was alsoestablished, with growth proceeding faster in the summer (T ~21 °C) than in winter (15 °C). The structures ofthe organic interlayer and of the epithelium are revealed by scanning electron microscopy.Published by Elsevier B.V.1. IntroductionUnderstanding the process in which living organisms control thegrowth of structured inorganic materials can inspire new and bettersynthetic materials [1–5]. Indeed, there have been recent successes insynthesizing a ceramic/polymer composite with outstanding tough-ness inspired by the structure of nacre in the abalone shell [6–9].The growth of nacre is a well studied subject characterized bymany researchers [10–35]. In particular, the growth and structurerelationship has been studied in detail [16,20–24,31]. Results showthat aragonite crystals first radiate from nucleation sites forming aspherulitic pattern, and then, columnar aragonite crystals formpreferentially in the c direction (perpendicular to the growth surface).This morphology is then replaced by the aragonite tile pattern. Linet al. [31] examined the structure during a period of 1 to 6 weeks. Inthe third week, the columnar growth still dominated and by the sixthweek growth cones of the aragonite nacre became present. Further-more, the role of the organic layer in the growth of the abalonenacre has been studied by Belcher et al. [22,23,26], Zaremba et al. [27],Sarikaya et al. [28–30], Lin et al. [15,31], Meyers et al. [16,17], andBezares et al. [34,35], which has led to proposed mechanisms ofgrowth.However, little attention has been paid to factors that affect thedevelopment of these transient phases. Changes in the feedingpatterns may limit the source of ions for mineral formation in theabalone shell. Moreover, changes in its environment, such astemperature of the sea water, might affect the nucleation rate andgrowth rate of the transitory phases of calcium carbonate. Thus, theenvironment may play an important role in the mineral formation.Additionally, past studies suggest a large involvement of the mantleand epithelial cell layer to form the intricate structure of the growingfront of the shell. Calcium radioisotope movement studies on theoyster Crassostrea virginica show that movement of the45Ca out ofthe mantle correlated with the amount of45Ca deposited on the shellgrowth front. Additional mollusk ion transport studies on the isolatedmantle indicate ion movements from the mantle to the shell, whileother studies suggest that Ca2+transport occurs by diffusion throughthis mantle [38]. However, this process is not fully understood andstudies of this soft tissue can give insights into this biomineralizationprocess.This study intends to investigate the process of mineralizationfollowing periods of growth interruption, taking into considerationimportant environmental factors (access to food and temperature)and to employ high-magnification characterization techniques tobetter understand how the soft tissue (e.g. epithelium and organicmembrane) influences the mechanism of growth.2. Experimental techniquesTwo labeled red abalone (Haliotis rufescens) were held in a 45 literfish tank in an open water facility at the Scripps Institution ofOceanography. The tank had direct access to continuously circulatingsea water, providing a natural environment with steady pH. Animalswere fed giant kelp (Macrocystis pyrifera) at different schedules andthe mean temperature was controlled. Three experiments werecarried out, varying average temperature and feeding rate of theMaterials Science and Engineering C 31 (2011) 238–245⁎ Corresponding author.E-mail address: [email protected] (M.I. Lopez).0928-4931/$ – see front matter. Published by Elsevier B.V.doi:10.1016/j.msec.2010.09.003Contents lists available at ScienceDirectMaterials Science and Engineering Cjournal homepage: www.elsevier.com/locate/msecanimal (Table 1). The ‘flat pearl’ technique, first used in the US by theU.C. Santa Barbara group [20,21,25,27] and latter applied by Lin et al.[31], was utilized to extract specimens for growth observations.Circular glass slides (15 mm in diameter) were implanted in liveabalone for periods of 1–3 weeks and then extracted weekly forexamination. The mantle was pressed back (retracted) with a flatscalpel and the slides were glued to the growing edge of the animal(Fig. 1). The largest quantity of slides allowed by the size and surfaceof the animal was implanted on each abalone. In Fig. 1, six slides wereimplanted and are shown by arrows. Once securely adhered, themantle relocated itself over slides over the period of approximately24 h. At least one slide from each of the abalone was removed weeklyand prepared for scanning electron microscopy (SEM) and atomicforce microscopy (AFM) characterization.For SEM preparation, the slides where air dried and sputter coatedwith gold-platinum. The specimens were observed both from