The gizzard plates in the Cephalaspidean gastropod Philine quadripartita: Analysis of structure and function

Quaternary International 390 (2015) 4–14

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The gizzard plates in the Cephalaspidean gastropod Philine quadripartita: Analysis of structure and function Margarita Shepelenko a, Vlad Brumfeld b, Sidney R. Cohen b, Eugenia Klein b, Hadas Lubinevsky c, Lia Addadi a, Steve Weiner a,∗ a b c

Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, 76100, Israel Israel Oceanographic & Limnological Research (IOLR) and National Institute of Oceanography, Tel- Shikmona, P.O.B. 8030, Haifa 31080, Israel

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Article history: Available online 26 June 2015 Keywords: Crushing Elasticity index Micro-CT Amorphous calcium carbonate Amorphous calcium phosphate Chitin matrix

a b s t r a c t Cephalaspidean gastropods are common marine mollusks with a unique digestive apparatus containing 3 hardened plates of millimeter size inside the muscular esophageal crop (gizzard). The gizzard plates are reported to either grind or crush shelled prey. The current study aims at better understanding the manner in which the gizzard plates of the cephalaspid Philine quadripartita function in the overall digestion process by relating their structural and mechanical properties. Philine quadripartita possesses 3 gizzard plates which have one of the common configurations of cephalaspidean gizzard plates: two paired plates that are mirror images of each other and one smaller unpaired plate. We used micro-CT to characterize the gizzard musculature, the food which is present at different stages of the digestion process and the working surface of the gizzard plates. We show that the gizzard plates are used to crush the shelled prey, and that the functional mode of the small unpaired plate is different from the larger plates. All 3 plates are composed of a mixture of amorphous calcium carbonate and amorphous calcium phosphate embedded in a chitinous matrix. The proportions of these two mineral phases vary systematically within the plate. The plates have a complex layered structure, whose elastic moduli and hardness also vary in a continuous systematic manner. We observed that the stiffest layer is below the working surface, unlike most teeth where the stiffest layer is at the surface. Rigorous analysis of the elasticity indices of the gizzard plates as compared with sea urchin teeth and synthetic calcite provided insights into the connection between the biological function and the mechanical properties of biological composites. Specifically, we show that materials used for grinding require harder surfaces to avoid excessive wear compared to materials for crushing, whereas both of these functions require high toughness. © 2015 Elsevier Ltd and INQUA. All rights reserved.

1. Introduction In animals, the grinding of food is usually carried out by teeth. In taxa that lack teeth, such as birds, and even in some groups that have teeth, such as seals, sea lions and extinct sauropods, grinding of food may take place in the muscular stomach or gizzard with the help of stones (gizzard stones or gastroliths) that are deliberately swallowed by the animal (Wings, 2007). Certain crustaceans that also lack teeth often possess an elaborate gastric mill inside their cardiac stomach, which contains a median tooth and two lateral teeth capable of grinding ingested food items (Thistle and Watling, 1989). Another interesting adaptation of this approach to digestion has evolved in most of the Cephalaspidean gastropods,

∗

Corresponding author. E-mail address: [email protected] (S. Weiner).

http://dx.doi.org/10.1016/j.quaint.2015.04.060 1040-6182/© 2015 Elsevier Ltd and INQUA. All rights reserved.

namely the formation of hardened plates that line the gizzard, and are used for crushing and/or grinding (Wägele, 2004). The gizzard is located between the crop, where the food is stored, and the stomach of the mollusk (Mikkelsen, 1996). Note that a minority of cephalaspids do not contain plates. The Cephalaspidea is a taxon of marine slugs and snails that inhabit soft sediments. The taxon includes about 840 different species (Wägele, 2004) and comprises up to 2% of all gastropod mollusks (Ponder and Lindberg, 1997). Some are herbivores, but about 500 species are predatory including the Philinidae, which are reported to prey on vagile invertebrates (Wägele, 2004). Platebearing cephalaspids possess 3 mm-sized gizzard plates (Malaquias et al., 2009), which vary in shape between species (Marcus and Marcus, 1969; Rudman, 1972a; Malaquias and Reid, 2008; Price et al., 2011; Ohnheiser and Malaquias, 2013; Eilertsen and Malaquias, 2013a; Too et al., 2014). Some gizzard plates are composed entirely of chitin, whereas others are mineralized with

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calcium-bearing minerals embedded in a matrix of chitin (Rudman, 1971, 1972a, 1972b). Table 3 summarizes the mineralogical and elemental composition of the gizzard plates in 15 mineral bearing species of cephalaspids studied to date (references are cited in the table). The minerals formed are diverse, and can vary from species to species even within a taxonomic family. An invariable component of all the gizzard plates is a non-diffracting mineral, defined here as ‘amorphous’. All these amorphous minerals are reported to contain calcium and phosphate. Some species also produce rather uncommon crystalline minerals, such as fluorite, weddellite and monohydrocalcite (Table 3). Some studies shed light on specific structural features of the gizzard plates (Lowenstam, 1968; Price et al., 2011; Ohnheiser and Malaquias, 2013; Eilertsen and Malaquias, 2013b). These studies, however, focus on colordistinguishable zones either on the dorsal side of the gizzard plates (Eilertsen and Malaquias, 2013b) or in a median transverse cut through a gizzard plate (Lowenstam, 1968). Special attention has been attributed to the microsculpture of the ventral surface of gizzard plates (Price et al., 2011; Ohnheiser and Malaquias, 2013), whereas the ultrastructure of the gizzard plates still needs to be clarified. The biomechanical function of the gizzard plates has been referred to as being analogous to millstones which grind food items (Jörger et al., 2010), or are thought to have a crushing function (Hurst, 1965; Marcus and Marcus, 1969; Lowenstam, 1972; Rudman, 1972b; Lowenstam and Weiner, 1989). Some studies make no clear distinction between grinding and crushing (Malaquias et al., 2004; Wägele, 2004; Wägele and Klussmann-Kolb, 2005). Thus the expression ‘masticatory plates’ is sometimes used, which implies both crushing and grinding functions (Marcus and Marcus, 1969). We report here a detailed study of the ultrastructure and the elastic modulus (E) and hardness (H) variations of the gizzard plates of cephalaspid Philine quadripartita Ascanius, 1772, together with an analysis of the gizzard musculature and of the state of the food which is present at different stages of the digestion process.

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cleaned with 6% sodium hypochlorite and subsequently washed with ethanol and acetone. 2.2. Micro-CT Prior to examination, whole animals where either preserved in 70% ethanol solution or stained with iodine. For iodine staining fresh specimens were fixed in 4% PFA and 2.5% GA as above, washed with double distilled water (DDW) and gradually dehydrated (30 min/step) in a series of 25%, 50%, 70%, and twice in 100% ethanol solutions. The dehydrated specimens were immersed in 2% iodine solution in ethanol for two days at 4 °C, washed in ethanol and kept in ethanol at −20 °C until the micro-CT scan. Both whole animals and isolated gizzard plates were positioned within home-made plastic holders and placed in the standard sample holder of the micro-CT instrument (XRadia MICRO XCT-400, Zeiss X-Ray Microscopy, Pleasanton, CA, USA). The samples were scanned using a voltage source of 40 kV and current of 200 μA. Measurements of whole animals were made with a voxel size of 34 μm3 , whereas higher resolution scans of the gizzard and isolated gizzard plates were made with a voxel size of 12 μm3 and 7 μm3 , respectively. After recording 1500 projections over 180° the volume was reconstructed with the XRadia software that uses a filtered back projection algorithm. 3D surface rendering was carried out with Avizo software (FEI, Hillsboro, OR, USA). 2.3. Electron microscopy and elemental analysis 2.3.1. Cross-sections Gizzard plates fixed in 4% PFA and 2.5% GA were dehydrated in a series of ethanol solutions, and critical-point-dried using a critical-point dryer (Baltec CPD_030). After CPD, the gizzard plates were broken to expose the desired cross-section, mounted on a sample stub using conductive carbon tape and coated by sputtering with a 15 nm layer of Au/Pd. The specimen was imaged using an Ultra-55/Supra-55 scanning electron microscope (Zeiss).

2. Materials and methods 2.1. Animals Live Philine mollusks collected from the southeast Mediterranean off the sandy coast of Israel were collected by the R/V Shikmona using a beam trawl carried out between 36 and 40 m depths. This trawl has a horizontal width of 1.2 m and 40 cm of vertical opening, with a 25 mm stretched mesh. The speed over the seafloor was about 2.5 knots. A total of 6 trawl hauls were made. Each haul lasted 20 min. We consider that the specimens are conspecific with the Atlantic/Mediterranean species P. quadripartita Ascanius, 1772 (Price et al., 2011; Ohnheiser and Malaquias, 2013). For preservation of the fresh specimens different methods were used, as follows: 1) some fresh specimens were washed and kept in 70% ethanol solution; 2) some fresh specimens were flash-frozen in liquid nitrogen and preserved in a freezer at −20 °C; 3) some fresh specimens were fixed in a mixture of 4% paraformaldehyde (PFA) and 2.5% glutaraldehyde (GA). For this last procedure 11.2 g PFA was placed in a beaker, 270 ml of sea water was added and the beaker was covered with aluminum foil. The solution was heated while stirring for >30 min at ∼70 °C until it became transparent. After cooling, 10 ml of 70% GA in water was added. Whole animals, as well as dissected gizzard plates cleaned from the enclosing tissues, were examined within 4 months after collection. Museum specimens were collected from the southeast Mediterranean off the sandy coast of Israel, similarly to the fresh ones, at depths in the range of 7–12 m and were preserved for 10 years in 70% ethanol solution. For observation of isolated gizzard plates, the plates (fresh and preserved) were surgically extracted,

2.3.2. Working surface The samples were fixed in 4% PFA and 2.5% GA, dried in air, and imaged using an environmental scanning electron microscope (ESEM) XL 30 ESEM FEG (Philips/FEI) while keeping the specimen under pressure/temperature conditions relative to the water dew point in order to avoid drying. 2.3.3. Elemental analysis by energy-dispersive X-ray spectroscopy (EDS) For EDS analysis gizzard plates were embedded in EpoFix (Struers) and polished on the plane parallel to the short or the long axis of the gizzard plates using SiC papers of grit 1000/1200 and 4000 and 70% ethanol solution as lubricant. For final polishing 0.05 μm Nanoalumina MasterPolishTM (Buehler, A4010084) suspension for water sensitive materials and HACOSILK VB-EB (A/S Hartfelt & Co., HF18442000M) silk polishing cloth were used. The polished samples were coated with carbon. EDS measurements were made with an Oxford ISIS at 10 kV. Polished titanium was used to calibrate the energy scale. Oxygen was calculated based on stoichiometry by measuring the oxygen-bound cations and normalized. Base on this, the elemental concentrations were automatically inferred by the software. Carbon was not considered because of the impossibility to distinguish between the organic and mineralbound carbon. 2.4. Infrared spectroscopy (FT-IR) The gizzard plates and the acid insoluble fractions of the gizzard plates were ground and homogenized separately using an

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agate mortar and pestle, then mixed with KBr powder and pressed into either 7 mm or 3 mm diameter pellets. FT-IR spectra were obtained with either Nicolet 380 or a Nicolet iS5 instrument using either 32 or 64 scans at a resolution of 4 cm−1 . Standards for amorphous calcium carbonate (ACC) and amorphous calcium phosphate (ACP) were downloaded from the web site of the Kimmel Center for Archaeological Science, Weizmann Institute. Chitin standard from crab shells was purchased from Sigma Aldrich. Acid insoluble fraction preparation: a whole gizzard plate was placed in 1 N HCl in an agate mortar and gently crushed with an agate pestle. After mineral dissolution was complete, the acid insoluble fraction was washed in water, acetone and finally dried under a heat lamp. 2.5. X-ray diffraction (XRD) XRD patterns were obtained in reflection using a TTRAX III (Rigaku) powder diffractometer equipped with a rotating Cu anode operating at 50 kV and 200 mA. Gizzard plates were crushed with a mortar and pestle to a fine powder. The powder was dispersed on a Si zero-background holder and measured under specular reflection conditions in Bragg-Brentano geometry (θ /2θ scans) in step mode (3 s or 12 s per step) with step size 0.025° between 2θ = 20–95°. XRD patterns were collected with a scintillation detector and analyzed using Jade 9.5 software and the PDF-4+ 2013 ICDD database. 2.6. Raman spectroscopy Raman analysis of the polished cross-sections of the gizzard plates (see 2.3.3) was performed on a Renishaw InVia Reflex Raman micro-spectrometer configured with a near infrared excitation source with an excitation wavelength of 785 nm (300 mW). A 50× lens objective (Leica) was used. Spectra were recorded after the accumulation of 15 scans, 1 s exposure time, and 100% laser power in the range of 500–1630 cm−1 . Baseline adjustment, smoothing, peak picking, and mapping of peak integrated intensities were performed with the instrument control software (Renishaw WiRE 3.4). 2.7. Nanoindentation For nanoindentation analysis fresh gizzard plates preserved in 70% ethanol solution were used. The polished samples (see 2.3.3.) were preserved in 70% ethanol solution until the beginning of the measurement and glued to a dedicated sample holder just before the measurement. Nanoindentation measurements were performed with an XPNanoindenter (Agilent) using a Berkovich tip in continuous stiffness measurement (CSM) mode in which a small dither is applied to the displacement while loading. Samples were loaded under displacement control at a constant strain rate of 0.05 s−1 to a depth of 1 μm, so that indentation loading times were about 2 min. Modulus values were then obtained continuously during the loading segment, using the method of Oliver and Pharr (1992). A Poisson ratio of 0.3 was assumed in the calculation. Determining the range of tip penetration depths that reflect the representative mechanical behavior of the gizzard plates for modulus and hardness averaging is a critical part of the data analysis. At low penetration depths, the CMS modulus and hardness values are strongly influenced by various surface effects, notably roughness, which can lead to large variations in the calculated values of modulus and hardness. This is because the Oliver and Pharr technique assumes that the surface is a perfectly flat and uniform plane. At high penetration depth, however, there is an increase in the bulk effect on the modulus and hardness, because of the inhomogeneity of the sample. The range of penetration depths for modulus and

Fig. 1. Representative modulus and hardness vs. displacement profiles of an indentation, showing changes of the modulus and hardness with depth. Initial rise at low depths is due to surface effects. Values reported here were taken in the intermediate region indicated by markers M and N, before the effects of bulk inhomogeneity is detected. The profile was taken from the top of the 3rd layer (see Results section).

hardness averaging is a tradeoff between these two factors. Thus the elastic modulus and hardness vs. displacement plots were used to find the depth at which the modulus and hardness values stabilize after initial surface effects. The depth range between 300 and 500 nm was chosen as shown in Fig. 1. The values of the elastic modulus and the hardness within the 3 relatively thin layers located at and below the working surface represent the average of 6–8 measurements. For the maximum and minimum of this region, denoted as MAX and MIN, respectively (Fig. 11) 12–15 measurements were used for the average. Good statistics in this region were required in order to confirm that the differences between the elastic modulus as well as the hardness values were significant and to obtain maximum spatial resolution for determining the trend in modulus and hardness. A spacing of 20 μm between adjacent indentations was used which was sufficient to identify the global minimum and maximum of this region. Since the 1st layer is only several μm thick and does not uniformly cover the working surface depending on the degree of wear, it was not possible to accurately measure the mechanical properties of this layer. In addition this layer borders on the Epoxy matrix of the embedding template, which also introduces uncertainty due to debonding between the sample and the Epoxy matrix and possible surface effects. In the 4th layer which comprises the bulk of the plate, the values of the elastic modulus and the hardness represent the average of 2–3 individual measurements taken along a single cross-section. A gradual decrease in the mechanical properties towards the non-working surface proved to be highly reproducible. For determination of the elastic modulus and the hardness at least 3 different specimens were used. 2.8. Gizzard plate heating Gizzard plates were heated for 4 h in air in an oven that had been preheated to 500 °C. After cooling, they were gently crushed with a mortar and pestle. 3. Results 3.1. Gizzard plates: anatomical locations, morphologies and function Fig. 2A is a transmitted light image of a fresh specimen of the cephalaspid P. quadripartita. The darker areas in the image correspond to the mineral bearing organs, namely the inner aragonitic shell of the mollusk (in the lower part of the animal) and the

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Fig. 2. A – Transmitted light image of the whole cephalaspidean gastropod mollusk P. quadripartita; B–D – Optical image of the dorsal side of the gizzard plates of P. quadripartita. P: peripheral zone; M: middle zone; C: central zone. In the black and white version of this image, the darker areas in (A) show the locations of the gizzard plates (top) and the internal shell (bottom).

gizzard plates (in the center of the animal). Fig. 2B–D are images of the 3 gizzard plates: two curved paired plates that are nearly triangular in shape and are mirror images of each other, and one smaller gizzard plate that is spindle-shaped. From the 3D reconstructed micro-CT image (Fig. 3) it can be seen that the convex dorsal surfaces of the paired gizzard plates face each other, and the intestinal tract. The white areas in the central zone of the dorsal side (Fig. 2B–D) are therefore the working surfaces that face into the intestinal tract and the brown and ivory colored areas in the middle and peripheral zones respectively are embedded in the tissue. Semi-transparent brown-colored traces of the tissues are clearly seen on the peripheral zone of the paired gizzard plate in Fig. 2D. Fig. 3 also shows the internal shell, almost the entire gastrointestinal tract and the gizzard plates. The food enters the mouth, passes through the esophagus and reaches the gizzard with its surrounding gizzard plates, where it is processed. The processed food items then reach the stomach, pass through the intestines and are finally extruded from the anus. Fig. 4A–D show two P. quadripartita specimens (specimen 1 and 2) that fed on small shelled gastropods and bivalves. The food can be seen at different stages of the digestion process. Fig. 4A (specimen 1) shows the location of the gizzard plates in relation to the food source, with the intact shells present before reaching the gizzard, and the crushed shells exiting from the gizzard. The different stages of digestion can be better observed by digitally removing the gizzard plates (Fig. 4B, specimen 2). Clearly the ingested intact small shelled mollusks are crushed in the gizzard before they reach the stomach. Muscles operating between the gizzard plates lie in blocks between the plates, as described by Förster (1934). The large paired gizzard plates are connected by a muscle (Fig. 4C, specimen 2) which is much narrower than the larger accordion-like muscles connecting the paired and unpaired plates (Fig. 4D, specimen 2). This suggests differential movement/functions of the paired and unpaired gizzard plates in the digestion process. An examination of the working surface of the gizzard plates using SEM shows that the surface is rough and striated (Fig. 5). These results support a crushing function of the gizzard plates in P. quadripartita, suggesting that the shelled prey is pressed by the gizzard plates, resulting in fragmentation of the prey exoskeleton. We saw no evidence of a grinding action such as smeared surfaces that would result from a milling action of the shelled prey into a powder. However, this would depend on whether the surfaces really come in contact with each other. It is conceivable that other species do grind their prey. On the working surface of the gizzard plates we observed socalled mineral protrusions with elongated shapes and smooth surfaces. These protrusions are up to hundreds of microns in size

Fig. 3. Micro-CT-based 3D reconstruction of the shell and the gastrointestinal tract of a specimen preserved in 70% alcohol for 10 years. In this specimen the intestinal tract is almost complete and is filled mainly with sediment. The internal shell is present in the lower part of the image. The intestinal tract is disrupted towards the anus due to poor preservation.

(Fig. 6). The protrusions penetrate to a certain depth into the plate material and in some places are buried beneath the working surface. These protrusions are composed of a high density material, as inferred from the X-ray based micro-CT image and from the SEM image in backscattering electron (BSE) mode compared to other regions of the gizzard plates (Fig. 6). There is a relatively high concentration of the protrusions in the region of the paired gizzard plates facing the unpaired gizzard plate. However they are hardly seen in the region of the contact area of the two paired gizzard

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Fig. 4. A – Micro-CT-based 3D reconstruction of the shell and the gastrointestinal tract above and between the gizzard plates of a freshly caught specimen (specimen 1) preserved for several weeks in 70% ethanol solution. Note the intact bivalve and gastropod shells above the gizzard and fragmented shells below the gizzard; B–D – Segmented micro-CT-based 3D reconstructions of the digestion tract of a different specimen (specimen 2); each image rendering is taken in a different orientation: B – Image of only the food particles, which appear at different stages of the digestion process. The lines on both sides of the food were added manually to mark the estimated borders of the gizzard; C – Iodine-enhanced image of the muscle (digitally colored red) connecting the two large paired gizzard plates; D – Iodine-enhanced image of the muscle connecting one large gizzard plate to the small gizzard plate. Note the crushed food particles after passage between the gizzard plates. In the black and white version of this figure, the muscles are the elongated tissues that connect to the gizzard plates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

plates. It appears that these protrusions are permanent features of the working surface of the plates in P. quadripartita, which implies that they have a function, although yet unknown. 3.2. Composition of the gizzard plates Fig. 7A(a–c) shows FT-IR spectra of the 3 gizzard plates from a freshly dredged specimen preserved in liquid nitrogen. The 3 spectra are almost identical showing that the mineral phases are the same in each of the gizzard plates. Furthermore, the peaks are rel-

atively broad implying the presence of amorphous mineral phases. The weakly split peaks between 1400 and 1500 cm−1 and the peak at 866 cm−1 correspond to the ν 3 and ν 2 vibrations of ACC respectively, while peaks at 1061 cm−1 and 571 cm−1 correspond to the ν 3 and ν 4 vibrations of ACP, respectively. The amorphous nature of the minerals is confirmed by X-ray diffraction (Fig. 7B (a)). Fig. 7A (d) shows a representative FT-IR spectrum of P. quadripartita gizzard plates preserved for 10 years in a museum. The proportions of ACC and ACP are very different with much of the more soluble ACC having been lost. All the spectra were normalized to the height of

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are the major elemental components, which is consistent with the FT-IR analyses. The gizzard plate is thus composed primarily of a mixture of amorphous materials. The amorphous materials have characteristic nanosphere texture, and are intimately mixed with chitin fibrils (Fig. 8). Following the general notion in biomineralization that the organic matrix is first deposited and then the mineral (Lowenstam and Weiner, 1989), it is most likely that the chitinous matrix is responsible for the different textures observed in the 4 layers of each plate (next section). 3.3. Ultrastructure of the gizzard plates Fig. 5. Environmental scanning electron micrograph of the working surface of a gizzard plate imaged with the gas secondary electron detector (GSE) at a pressure of 5.6 Torr. The black arrows mark examples of striations on the working surface.

the ν 3 peak of ACP. After heating the powdered gizzard plates to 500 °C for 4 h, we used both FT-IR and X-ray diffraction to show that the ACC and ACP phases transform into dahllite (carbonated hydroxyapatite), magnesium oxide and calcium oxide (Fig. 7B (b)). An X-ray diffraction pattern taken directly from the protrusions in Fig. 6 by applying reciprocal space mapping showed no evidence for the presence of a crystalline phase (not shown). Fig. 7C shows a representative Raman spectrum taken over the 2nd–4th layers in the polished median transverse cross-section of the paired gizzard plates of a fresh specimen preserved in 70% EtOH. Note the characteristic peaks for ACC at 1086 cm−1 and 708 cm−1 . Peak at 959 cm−1 corresponds to the ACP, while peaks at 1444 cm−1 and 1372 cm−1 correspond to chitin. The FT-IR spectrum of the hydrochloric acid insoluble fraction of fresh gizzard plates includes bands that correspond to the structure of α -chitin (Fig. 7D). The Raman measurements thus confirm that 3 major components, ACC, ACP and α -chitin, are present in layers 2–4 of the gizzard plates (Fig. 9), and are presumably present in the 1st layer. EDS analysis performed on polished cross-sections of gizzard plates from P. quadripartita detected 6 principal elements, namely Mg, Zn, Sr, S, Ca and P, as well as Al and Si. Al and Si are probably contamination from the polishing materials (Table 1). Ca and P

Large and small gizzard plates all have a complex microstructure with 3 relatively thin layers located at and below the working surface, while the 4th thick layer comprises the bulk of the plate (Fig. 9). The 4th layer has a well-developed finely laminated structure. Note that because the gizzard plates are composed almost entirely of amorphous minerals, small particles ranging in size from 20 to 40 nm can be discerned at high resolution. We describe the larger-scale structures starting from the outermost layer facing into the gizzard. This layer is often worn away, and can be found only on the intact flanks of the plates. Fig. 10 A shows the 1st layer of the gizzard plate, which is confined to the working surface of the gizzard plates and varies in thickness from 5 to 10 μm. The layer is densely packed and the fracture surface reveals blocky structures about 0.5–1 μm in diameter, with no preferred orientation. The 2nd layer has a thickness of about 200 μm below the working surface, but increases to about 400 μm in the highly curved flank. The fracture surface shows a dense structure (Fig. 9), but at high magnification an oriented texture can be seen (Fig. 10B). The orientation is oblique to the plate surface. The 3rd layer covers the entire gizzard plate. It is about 100 μm thick and has a prominent arc-like sub-structure (Fig. 10C). The 4th layer comprises the bulk of the plate structure (Fig. 9). At high resolution the texture of the 4th layer resembles that of the 2nd layer (not shown). This layer can reach up to 1 mm in thickness and is composed of multiple sub-layers of about 20 μm in thickness, as shown in Fig. 9. The Ca/P ratios within the layers range from about 1 to 5, implying varying proportions of ACP (with a ratio of 1–1.5

Fig. 6. A – Micro-CT-based 3D reconstruction of an unpaired fresh gizzard plate fixed in 2.5% GA and 4% PFA solution. B – BSE micrograph of the carbon-coated cross-section of the unpaired gizzard plate shown in Fig. 6A embedded in Epoxy resin and polished more or less along the long axis of the plate (black line in Fig. 6A). The white arrows point to the two protrusions found in this region indicated with black arrows in Fig. 6A.

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Fig. 8. Scanning electron micrograph of the 2nd layer (see below) showing the two basic building blocks of the gizzard plates: amorphous materials with their characteristic nanosphere texture and chitin fibrils, as inferred from FT-IR and Raman spectroscopic analysis of the gizzard plates.

Fig. 9. Scanning electron micrograph image showing all 4 layers of the gizzard plate in a section that fractured more or less along the short axis of a large plate, with part of the working surface exposed on the top part of the image. The sample was preserved for 10 years in 70% EtOH solution. 1, 2, 3 and 4 show the locations of the 4 layers.

(Nancollas, 1982; Christoffersen et al., 1989)) and ACC. The Ca/P ratio is minimal at the working surface (the 1st layer) with values around 0.8. This could conceivably be due to the presence of a polyphosphate phase. Ratios ranging from 2 to 5 characterize the 2nd to 4th layers, while at some point in the 4th layer (580 μm in Fig. 11) the Ca/P ratio starts to gradually decrease to about 1.3 as the non-working surface is approached. This implies that the nonworking surface is composed almost entirely of ACP. In the region of the protrusions the Ca/P ratio reaches values in the range of 10– 14. These high values suggest the presence of an almost pure ACC phase with a small amount of occluded phosphate. 3.4. Mechanical properties

Fig. 7. A – FT-IR spectra of the 3 gizzard plates from a freshly dredged specimen treated with liquid nitrogen (a–c); FT-IR spectrum of the museum specimen preserved for 10 years in 70% EtOH solution (d); ACP and ACC standards (ACC, ACP). B – X-ray spectrum of the gizzard plates from a freshly dredged specimen treated with liquid nitrogen (a); X-ray spectrum of gizzard plates after heating to 500 °C: cHA = carbonated hydraxylapatite (dahllite), CaO = lime, MgO = periclase (b). C – Representative Raman spectrum of 2nd to 4th layers of the polished median transverse cross-section of the paired gizzard plate of a fresh specimen preserved in 70% EtOH. D – FT-IR spectra of the (a) acid insoluble fraction of the gizzard plates after dissolution in HCl 1 N; (b); α -chitin standard.

Fig. 11 shows the general trend of the elastic modulus and hardness in median transverse sections through the large gizzard plate. Note that the profiles of the elastic modulus and hardness vs. distance from the working surface show similar behavior. This can be attributed to the Oliver and Pharr method for determination of modulus and hardness (1992) whereby these two quantities are closely related for a given material and a specific indenter, and depend on the material having a well-behaved elastic recovery (Lawn and Howes, 1981; Amitay-Sadovsky and Wagner, 1998). There is an increase in the modulus and hardness from the working surface to the beginning of the 3rd layer until at some point between the top of the 2nd layer and the base of the 3rd layer they reach a maximum. From this point the modulus and hardness gradually decrease until they reach a minimum at the base of the 3rd layer.

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Table 1 Elemental composition of the gizzard plates using electron dispersive spectroscopy (EDS). Element

Al

Si

Mg

Zn

Sr

S

Ca

P

Concentration (at%)

Up to 0.6

Up to 1.2

7–10

Up to 6

Up to 0.7

Up to 1.5

11–27 protrusion: 35–40

5–14 protrusion: 2.5–4

Fig. 10. A–C – Scanning electron micrographs of fracture surfaces of (A) 1st layer, (B) 2nd layer and (C) 3rd layer of large plates from fresh samples that were fixed in 2.5% GA and 4% PFA solution, dehydrated by critical point drying, fractured and coated by sputtering a 15 nm layer of Au/Pd.

The bulk of the gizzard plate is characterized by high fluctuations in the modulus and hardness and by a gradual decrease towards the non-working surface. This decrease in mechanical properties is consistent with the presence of a graded material, presumably produced by variations in the proportions of ACC and ACP described in the previous section. Table 2 summarizes the values of the elastic moduli and hardness of the layers. 4. Discussion Philine quadripartita has two large paired gizzard plates that are mirror images of each other, and one smaller gizzard plate: one of the common configurations of cephalaspidean gizzard plates

Fig. 11. Representative average profile of the elastic modulus and hardness versus distance from the working surface measured on median transverse sections through the large paired gizzard plate of a fresh specimen preserved in 70% ethanol. The measurements were performed by nanoindentation of embedded and polished specimens. The values of the elastic modulus within the 3 relatively thin layers located at and below the working surface represent the average of 6–8 measurements, except for the maximum and minimum of this region, denoted as MAX and MIN, respectively, for which 12 to 15 measurements were used for the average. In the 4th layer the values of the elastic modulus represent the average of 2–3 individual measurements taken along a single cross-section. For calculation of the elastic modulus data were taken from at least 3 different specimens. The positive and negative error bars are 1 standard error of the mean.

(Rudman, 1972a; Price et al., 2011; Ohnheiser and Malaquias, 2013). Direct observation using micro-CT shows that small shelled prey is crushed by the action of the gizzard plates before entering the stomach. We noted the accordion-like structure of the muscles connecting the paired and unpaired gizzard plates, compared to the relatively narrow muscle connecting the two paired plates, in 3 different samples. In addition, we found relatively high concentrations of electron dense and mineralogically distinct protrusions in the region of the paired gizzard plates facing the unpaired plate, but not in the region between the two paired gizzard plates. These observations support a unique role in the crushing mechanism for the small unpaired plate that is different from the larger plates, assuming that the protrusions on the working surface of the gizzard plates are one of the functional adaptations for crushing. Importantly, the protrusions that we found on the working surface of P. quadripartita gizzard plates resemble, in their characteristic elongated shape, the crystal accretions on the working surface of P. indistincta Ohnheiser and Malaquias (2013) gizzard plates (Ohnheiser and Malaquias, 2013). The gizzard plates have a chitinous organic matrix, consistent with previous reports (Rudman, 1971, 1972a, 1972b) and the associated mineral is an intimate mixture of ACC and ACP. ACC has not been reported before in cephalaspidean gizzard plates, while certain amorphous phosphatic constituents of the gizzard plates have been indicated previously (Table 3). This mineral composition is rarely found in organisms. One known example in biomineralization is the exoskeletal carapace of the Pseudosquilla bigelowi (Arthropoda: Malacostraca), which was found to be composed of ACC and amorphous phosphatic hydrogel (Lowenstam, 1972). Another example is the mandible of the crayfish Cherax quadricarinatus which was shown to contain ACC and ACP in its base (Bentov et al., 2012). Interestingly, there is a resemblance in the morphological motif and the biomechanical function between cephalaspidean gizzard with gizzard plates and the teeth-bearing gastric mill of decapod crustaceans. Both cephalaspidean gizzard and crustacean gastric mill often have a configuration of two paired and one unpaired food processing parts (plates and teeth respectively) (Thistle and Watling, 1989). Mineral and elemental compositions of P. quadripartita specimens were previously studied by Lowenstam (1972). Our results differ in some respects to those reported by Lowenstam (1972).

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Table 2 Elastic moduli and hardness of the gizzard plate layers, sea urchin teeth and synthetic calcite. Material Gizzard Gizzard Gizzard Gizzard

plates plates plates plates

(2nd layer) (MAX) (MIN) (4th layer)

Sea urchin teeth (Ma et al., 2008)

Calcite (synthetic) (Ma et al., 2008)

Elastic modulus, [GPa]

Hardness, [GPa]

Fluctuates between 24 and 34 GPa (n = 71) 41.0 ± 0.7 (n = 15) 25 ± 2 (n = 12) Fluctuates between 26 and 34 GPa (n = 81) and gradually decreases to about 10 GPa starting at about 100 μm from the non-working surface 98.5 ± 15.2 (n = 15) for polycrystalline matrix 71.1 ± 8.4 (n = 7) for needles 76 ± 4.6 (n = 7) for plates 73.5 ± 2.9 (n = 11)

Fluctuates between 1 and 1.6 GPa (n = 71) 1.88 ± 0.08 (n = 15) 1.0 ± 0.1 (n = 12) Fluctuates between 1 and 1.6 GPa (n = 80) and gradually decreases to about 0.4 GPa starting at about 100 μm from the non-working surface 5.7 ± 1.1 (n = 15) for polycrystalline matrix 3.5 ± 0.9 (n = 7) for needles 3.8 ± 0.4 (n = 7) for plates 2.7 ± 0.23 (n = 11)

n = number of indentations. The analytical errors are 1 standard error of the mean.

Using EDS analysis we showed that Zn, Sr and S are among the minor constituents of the gizzard plates. These elements were not found by Lowenstam. In addition, we found no evidence for the presence of F reported by Lowenstam. The differences in mineral composition are even more pronounced. Lowenstam reported on the presence of monohydrocalcite crystalline phase, while we found no evidence for the presence of this or any other crystalline constituent. We can think of two reasons for these differences. It could be that we worked with Lessepsian migrants from the Indian Ocean P. aperta rather than P. quadripartita, which are very similar in their morphology and have been often considered synonymous. Another possible reason may be related to the freshness of the analyzed specimens we examined, as opposed to the museum preserved specimens analyzed by Lowenstam. In general the mineralogy of gizzard plates is clearly diverse even within a genus (Table 3). Some of this diversity, especially for the less stable amorphous mineral components, may well be an artifact of preservation. In this study we compared gizzard plates of P. quadripartita that were preserved for 10 years in a museum, with freshly caught specimens. The proportions of ACC and ACP were very different with much of the more soluble ACC having been lost (Fig. 7A). These observations raise questions about the reliability of studying museum specimens of gizzard plates. We report here a detailed study of the ultrastructure and the hardness and elastic modulus variations of the large plates, which presumably reflect the mainly crushing function of the plates. The outermost working surface (1st layer) possesses a dense blocky microstructure that differs from the other 3 layers. In addition, the localized high electron density protrusions on the working surface are also most probably functionally important. We could not however measure the hardness and elastic modulus of the thin 1st layer, but we did observe that the hardness and modulus increase with distance from the surface, and the highest measured hardness and modulus values were not at the surface, but beneath the surface. Below this hard and stiff layer, the values decrease, and vary in a systematic way to the other embedded surface. These variations in hardness and modulus values are consistent with variations in Ca/P ratio within the layers of the gizzard plates. Importantly, in many respects cephalaspidean gizzard plates are quite different from vertebrate teeth or mollusk mineralized teeth that have a crystalline hard and stiff outer layer (Duedall and Buckley, 1971; van der Wal et al., 1989). However, there are definite similarities between the hardness and modulus values of the gizzard plates and the base of the crayfish, mandible. Both are composed of a mixture of ACC and ACP (Bentov et al., 2012). P. quadripartita gizzard plates fulfill a crushing function. The plates are characterized by relatively low values of both elastic modulus and hardness compared to sea urchin teeth used to obtain food by grinding rocky surfaces (Ma et al., 2008). This can be related to the difference in mineral composition of the two: a mixture of amorphous minerals in gizzard plates as opposed

to crystalline magnesium-bearing calcite in sea urchin teeth. This is supported by the modulus and hardness values measured for sea urchin teeth and synthetic calcite (Ma et al., 2008) which are significantly higher than for the mixture of biogenic amorphous minerals in gizzard plates (see Table 2). However, gizzard plates of other cephalaspids have been shown to contain crystalline constituents (Lowenstam, 1968; Lowenstam and McConnell, 1968; Lowenstam, 1972; Eilertsen and Malaquias, 2013b). These facts point to the intricate relation between composition, mechanical characteristics, and biological function. This complex relationship can be better understood by analysis of the elasticity index, which has been considered in wear of materials. Noting that many polymers exhibit excellent wear properties under impact conditions, Leyland and Matthews introduced the elasticity index H/E, which describes the elastic strain to failure and hence predicts wear resistance (Leyland and Matthews, 2000). Materials ranking high on the scale of elasticity index exhibit high toughness. Recently, it has been shown that nanomaterials fashioned to be both hard and compliant have enhanced mechanical properties (GotlibVainshtein et al., 2014). Table 4 summarizes the elasticity indices of P. quadripartita gizzard plates, sea urchin teeth, and synthetic calcite. The elasticity index of the gizzard plates takes on values ranging between 0.040 and 0.047, which is similar to the elasticity indices of plates and needles of sea urchin teeth. The elasticity index of the sea urchin teeth polycrystalline matrix, incorporating the plates and the needles, is higher than that of the gizzard plates, but the difference is significantly smaller than that for both modulus and hardness individually. This finding may reflect the principal difference between the grinding and the crushing functions of biological composite materials. Whereas both of these functions require high toughness, the grinding function, requires harder surfaces to avoid excessive wear. The digestive system of marine mollusks is an excellent example of the interplay between chemical and structural composition with the biological function. Investigation of this unique organism at small scales has revealed how its functionality is optimized for its crushing function. Comparing and contrasting this species to other organisms with varying roles reveal new insights into the specific requirements of biological mechanical functionality. Acknowledgements We dedicate this study to Dr. Henk Mienis from the Tel Aviv University Zoological Museum (TAUZM), who crossed our lives in different ways, but always contributed so positively to our enrichment. We also thank Dr. Henk Mienis for providing the museum specimens of P. quadripartita. We thank Dr. Boaz Mayzel from Tel Aviv University (TAU) for collecting a fresh P. quadripartita specimen in the early stages of the research. We thank Dr. Rivka Elbaum from the Faculty of Agriculture, the Hebrew University of Jerusalem (HUJI), for providing Raman facilities. We thank Dr. Shmuel

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Table 3 The diversity of mineralogical and elemental composition of cephalaspidean gizzard plates. Family/Species

Scaphandridae Scaphander lignarius Linnaeus, 1758

Scaphander punctostriatus Mighels and Adams, 1842

Scaphander watsoni Dall, 1881

Scaphander interruptus Dall, 1889 Scaphander nobilis Verill, 1884

Scaphander bathymophilus Dall, 1881

Scaphander darius Marcus and Marcus, 1967

Scaphander clavus Dall, 1889

Scaphander cylindrellus http://www.marinespecies.org/ aphia.php?p=taxdetails&id= 575361Dall, 1908

Cylichnidae Cylichna cylindracea Pennant, 1777 Cylichna occulta occulta Mighels and Adams, 1842 Cylichna magna Lemche, 1941 Acteocina cf. culcitella Gould, 1853 Philinidae Philine quadripartita Ascanius, 1772 Philine angasi Crosse and Fischer, 1865 a b

Elemental compositiona , b

Crystalline mineral

Amorphous mineral

Reference

CaO 59.5%; F 22.5%; P2 O5 8.1%; MgO 5.9%; FeO 0.03% (E.P. and E.S.) Central zone: F 36%; O 21%; Ca 20%; P, Mg, S, Na, K: 0–2% Middle zone: F 34%; O 25%; Ca 8%; Mg 5%; P, S, Na, Al: 0–2% Peripheral zone: O 32%; Ca 11%; P 5%; Mg 4%; S, Na: 0–1% (XRMA) –

fluorite

indicated

presumably fluorite

inferred

(Lowenstam and McConnell, 1968; Lowenstam, 1972) (Eilertsen and Malaquias, 2013b)

fluorite

inferred

presumably fluorite

inferred

fluorite (central zone)

indicated

(Eilertsen and Malaquias, 2013b)

none

indicated

(Lowenstam, 1972)

fluorite (central, middle and peripheral zones)

indicated

(Eilertsen and Malaquias, 2013b)

presumably fluorite

inferred

(Eilertsen and Malaquias, 2013b)

presumably fluorite

inferred

(Eilertsen and Malaquias, 2013b)

presumably fluorite

inferred

(Eilertsen and Malaquias, 2013b)

weddellite

none

(Lowenstam, 1968)

Inner subunit: P2 O5 24.8%; FeO 18.8%; CaO 11.7%; BaO 4.5%; MnO 2.7%; MgO 2.5%; F 0.3% (E.P.)

none

indicated

(Lowenstam, 1968; Lowenstam, 1972)

Outer subunit:

weddellite

–

(Lowenstam, 1972)

Inner subunit: P2 O5 28.4%; CaO 28.2%; MgO 6.7%; F 0.2%; MnO 0.06% (E.P. and E.S.) CaO 33.7%; P2 O5 31.6%; MgO 4.9%; F 0.2%; MnO 0.05%; FeO 0.01% (E.P. and E.S.) P2 O5 39.7%; CaO 28.5%; MgO 9.4%; F 0.1%; FeO 0.02% (E.P.)

none none

indicated indicated

(Lowenstam, 1972)

none

indicated

(Lowenstam, 1972)

none

indicated

(Lowenstam, 1972)

CaO 32.4%; P2 O5 16.9%; MgO 9.4%; F 1.4% (E.P.)

monohydrocalcite

indicated

(Lowenstam, 1972)

CaO 25.9%; P2 O5 25.0%; MgO 10.7%; F 0.4%; FeO 0.07%; MnO 0.01% (E.P. and E.S.)

monohydrocalcite

indicated

(Lowenstam, 1972)

Central zone: F 28%; Ca 28%; O 22%; P, Mg, S, Na: 0–2% Middle zone: O 44%; Ca 10%; Mg 5%; P, S, Na, Al, K: 0–2% Peripheral zone: O 45%; Ca 11%; P 7%; F 6%; Mg 4%; S, Na, K: 0–1% (XRMA) Central zone: F 38%; O 26%; Ca 16%; P, Mg, S, Na, K: 0–2% Middle zone: O 27%; Ca 12%; Mg 9%; P 5%; Na 4%; S, Al, K: 0–1% Peripheral zone: O 50%; Ca 10%; P 7%; F 5%; Mg 3%; S, K: 0–1% (XRMA) P2 O5 35.8%; CaO 29.0%; MgO 7.7%; MnO 0.2%; F 0.1%; FeO 0.02% (E.P. and E.S.) Central zone: F 46%; O 19%; Ca 16%; P, Mg, S, Na: 0–2% Middle zone: O 55%; Ca 6%; S, Na, K: 0–1% Peripheral zone: O 38%; F 29%; Ca 7%; Mg, P: 4%; Na, Al: 0–1% (XRMA) Central zone: F 45%; O 24%; Ca 14%; P, Mg, S, Na: 0–2% Middle zone: O 23%; F 39%; Ca 12%; P, Mg, S, Na: 0–3% Peripheral zone: O 43%; Ca 11%; P 7%; F 4%; Mg, S, Na, Al: 0–2% (XRMA) Central zone: F 45%; Ca 20%; O 17%; P, Mg, S, Na: 0–2% Middle zone: Ca 31%; O 30%; F 5%; P, Mg: 0–2% Peripheral zone: O 50%; F 17%; Ca 8%; P, Mg: 5%; S, Na: 0–1% (XRMA) Central zone: F 40%; O 25%; Ca 20%; P, Mg, S, Na, Al: 0–2% Middle zone: Ca 45%; O 45%; F 3%; Na 0–1% Peripheral zone: O 36%; Ca 10%; P 6%; Mg, F: 2–3%; S, Al: 0–1% (XRMA) Outer subunit: Ca 32.2%; P, F, Fe, Sr and Mg 0.5% in total (E.P.)

As reported in the corresponding literature sources. E.P. = electron probe, E.S. = emission spectroscopy, XRMA = X-ray microanalysis.

(Lowenstam and McConnell, 1968) (Eilertsen and Malaquias, 2013b)

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Table 4 Elasticity indexes of the gizzard plates, sea urchin teeth and calcite. Material

Elasticity index, H/E

Gizzard plates (2nd layer) Gizzard plates (MAX) Gizzard plates (MIN) Gizzard plates (4th layer) Sea urchin teeth (Ma et al., 2008)

0.042–0.047 0.046 ± 0.002 0.040 ± 0.005 0.038–0.047 0.06 ± 0.01 for polycrystalline matrix 0.05 ± 0.01 for needles 0.050 ± 0.006 for plates 0.037 ± 0.003

Calcite (Ma et al., 2008)

The analytical errors were calculated using error propagation approach.

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