The pteropod Creseis acicula forms its shell through a disordered

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The pteropod Creseis acicula forms its shell through a disordered nascent aragonite phase Odelia Sibony-Nevo, Iddo Pinkas, Viviana Farstey, Hen Baron, Lia Addadi, and Steve Weiner Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b01400 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 8, 2019

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Crystal Growth & Design

The pteropod Creseis acicula forms its shell through a disordered nascent aragonite phase Odelia Sibony-Nevoa, Iddo Pinkasb, Viviana Farsteyc, Hen Barona, Lia Addadia and Steve Weinera* a

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel

E-mail: [email protected]; [email protected]; [email protected]; [email protected] b

Department of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel

E-mail: [email protected] c

The Interuniversity Institute for Marine Sciences, Eilat 88103, Israel

E-mail: [email protected]

Corresponding Author: Steve Weiner Weizmann Institute of Science, Department of Structural Biology Herzl Street, Rehovot 7610001 Israel . +972 8 9342552 E-mail: [email protected] This manuscript is submitted as part of the Crystal Growth and Design Israel Goldberg Memorial virtual issue

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Keywords: Amorphous calcium carbonate, Raman spectroscopy, Cryo-SEM, Mollusk shell

Abstract Shelled pteropods are holoplanktonic mollusks that build lightweight shells containing aragonite crystals. The complex shell architecture is composed of well aligned curved aragonitic fibers. Each curved fiber is continuously crystalline. We used in-vivo microRaman spectroscopy to study the mineral composition of shells of living Creseis acicula pteropods at the larval (veliger) and adult stages. The spectra obtained from the growing edge have weak and broad peaks indicative of a highly disordered nascent aragonite phase. The disordered precursor phase is detected in the newly formed regions both at the shell edge, and during thickening in the internal part of the shell. As the shell grows and thickens throughout the life of the animal, the mineral matures from a disordered transient precursor phase to crystalline aragonite. We conclude that the shell of C. acicula is formed via a disordered nascent form of aragonite, which, being isotropic, facilitates the formation of the convoluted morphology in the continuously crystalline fibers of aragonite.

Introduction Shelled pteropods (suborder Euthecosomata) are holoplanktonic gastropods belonging to the phylum Mollusca 1. The shelled pteropods are one of the major marine calcifiers and are widely distributed in all the oceans2-4. Extant species of the two main superfamilies, the Limacinoidea and Cavolinioidea, are adapted to a pelagic lifestyle, namely they swim freely in the water column during their whole life cycle5. Presumably for this reason, 2 ACS Paragon Plus Environment

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pteropods evolved very thin, lightweight and mechanically tough shells 6, 7. The shells of both superfamilies are composed of aragonite, but their shell shapes and microstructures are different. The Limacinoidea have a crossed lamellar shell microstructure 8, 9 that is the most common shell microstructure in gastropods

10,

whereas the Cavolinioidea shell

microstructure is composed of a unique arc-shaped or helical microstructure. This microstructure is not present in other molluscan groups

9, 11.

The microstructure is

composed of elongated curved fibers of continuously crystalline aragonite fibers

6, 12.

In

the shells of the Cavolinioid Clio pyramidata, adjacent fibers are crystallographically well aligned with each other 7. Checa et al.

13

showed that even though fibers are interlocked

and can even cross each other, the fibers maintain their integrity. Checa et al. 13 also noted that the tips of the fibers emerging on the growing surface are protein rich and have granulated textures. How can the fibers in such a complex superstructure achieve such a high degree of alignment and how can each curved fiber be continuously crystalline? In order to better understand this complex process, we focus on the nature of the newly deposited mineral phase. In mollusks the mantle is responsible for shell formation. The mantle is a soft tissue that is located on the inner surface of the shell 14. The mantle cells secrete a matrix of proteins 15, chitin 16, polysaccharides 14 17, 18 and lipids 19 , and shell secretion starts with the formation of an organic layer (the periostracum) 20. This organic layer covers and protects the outer surface of the mollusk shell

21,

and provides the substrate for initial shell formation

20.

Mineral is induced to form within the preformed matrix. The first mineral formed in bivalve and gastropod larvae is a highly disordered form of calcium carbonate, called amorphous calcium carbonate (ACC)

22, 23.

This ACC then transforms into the stable crystalline 3

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aragonite polymorph. An ACC precursor phase has also been observed in adult mollusks2426.

Hasse et al. 22 noted using EXAFS that the ACC first formed in gastropod larvae already

has a nascent short range order of at least 4 Å around the calcium ions in aragonite. This phase does not diffract X-rays 22. Nascent phases have also been observed in other mollusks 25, 27.

Ramesh et al.

28

and Jardillier et al.

29

made similar observations in larval shells of

gastropods, but concluded that ACC as such is not the first deposited mineral phase. Based on the reduction in volume observed between the tip of the fibers and the fiber main body and on their granulated surface morphology, Checa et al. 13 considered the possibility that in the Cavolinioidea pteropod shells the fibers initially consist of ACC, which subsequently transforms into aragonite. They found, however, no additional evidence in support of this hypothesis. Here we study the shell of Creseis acicula (previously known as Creseis clava)

30,

a

member of the superfamily Cavolinioidea. The C. acicula shell has a needle-like cone shape and the wings and mouth protrude out of the shell, but can contract back into the shell 9. The larval shell does not differ in morphology from the adult shell 31. The C. acicula shell elongates throughout the entire lifecycle of the animal by addition of new material at the shell edge 32. In addition, the shell thickens by adding new material on the inner shell surface 32. Thus in C. acicula each shell contains material deposited first during the larval stage, and then during the juvenile and adult life stages. We use in-vivo micro Raman spectroscopy on living individuals in order to determine whether or not C. acicula forms its shell via an ACC precursor phase, and to characterize the mineral in the mature parts of the shell.


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Experimental section Animals Living larvae (veliger) and adult individuals of Creseis acicula of the superfamily Cavolinioidea were collected in Eilat, the Red Sea, during the period of November to March. The pteropods were collected from the upper 30m of the water column by towing plankton nets of 200 μm mesh. After collection, live individuals were separated from the plankton sample under a stereoscopic microscope. Veliger specimens were identified by their four lobed ciliated velum. After metamorphosis two wings are visible while the velum is lost 31. Collected animals ranged in size (longitudinal axis of the animal) from 0.6 mm for veligers up to 6 mm for adults. Each animal was placed in a 50 ml tube filled with offshore 0.2 μm filtered seawater. The tubes were kept in an incubator with a day/light cycle at 23° C. The animals were kept alive for up to 5 days. SEM of the shell Living animals were preserved in 70% ethanol and air dried prior to imaging. The shells were broken into fragments using a scalpel and mounted on an aluminum stub with double sided carbon tape. High resolution scanning electron microscopy observations were made using a Leo-Supra 55 VP FEG SEM or Ultra 55 SEM (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Cryo-SEM Living Creseis acicula specimens were sandwiched between two metal discs (with 0.05, 0.1 or 0.15 mm cavities, depending on sample thickness), immersed in filtered sea water



containing 10% dextran (Fluka) used as a cryo-protection agent. The sample was cryoimmobilized in a high-pressure freezing device (HPM10; Bal-Tec, Liechtenstein) and then freeze fractured (BAF60, Leica Microsystems, Vienna, Austria) at −120 °C, in a vacuum of 5 × 10−7 mbar. The sample was transferred under vacuum using a VCT 100 shuttle (Leica Microsystems, Vienna, Austria) to an Ultra 55 SEM microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). The samples were “etched” (mild water sublimation), if necessary (10 min, −110 °C), to better expose the tissues and observed with an Inlens secondary electron detector and an Inlens Energy Selected Backscatter detector (EsB). Adobe Photoshop was used to adjust the image brightness and contrast levels. X-ray Diffraction Dry shells of C. acicula were washed with 6% NaOCl, cut in half and mounted using Epoxy, onto a glass fiber holder. Diffraction patterns were collected with a Rigaku RUH3R rotating anode diffractometer equipped with an Osmic Blue mirror X-ray optics. The shell was exposed to the X-ray beam perpendicular to the external shell surface. CuKα radiation was used and the shell was rocked by ± 1 degree during the measurement. Irradiation was performed at room temperature at χ=0° for 3 minutes. A 2D diffraction pattern was collected on the Rigaku RaxisIV++ image plate detector at θ =0°, kept at a distance of 70 mm from the sample. Raman Spectroscopy Living C. acicula veligers and adults were anesthetized with 1.5% MgCl2 in seawater in a glass bottom Petri dish, and were then examined in vivo. Raman measurements were conducted using a LabRAM HR Evolution instrument (Horiba, France) configured with 4


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lasers (325, 532, 632, and 785 nm). Raman spectra from 50 cm−1 onward were obtained. The system incorporates an open confocal microscope (Olympus BXFM) with a spatial resolution of about 0.6 μm using the 60X NA 1 objective and 785nm laser, and a spatial resolution of 0.3 μm using the 60X NA 1 objective and 532 nm laser. The raw data from the Raman measurements were subjected to polynomial baseline subtraction. The Raman measurements on the living animals were performed with the 785 nm laser. At the end of the experiments that lasted between 5 to 10 hours, the animals were still alive and appeared in good condition, judging from their heartbeat and their ability to swim after recovering from the anesthesia. The animals were then transferred to 100% ethanol to preserve the samples, and were then air-dried and kept in a desiccator. The dried shells were measured with Raman spectroscopy after they were rehydrated with filtered sea water. The Raman measurements were performed using the same settings used for the living animals or using the 532 nm laser to obtain higher spatial resolution for measuring the thickening region of the shell.



Results Shell morphology and structure The Creseis acicula shell has an elongated cone-shaped morphology (Figure 1A). The shell can be divided into 3 regions; the protoconch is formed at the larval stage, and corresponds to the veliger shell. This is the oldest part of the shell. The teleoconch is the shell that forms after the transition of the animal from the larva to the adult (Figure 1A). The shell edge is one location where new shell is added, as was demonstrated by in vivo calcein labeling33, 34. The shell length varies between 0.6 mm to 6 mm 4 and the shell thickness varies between 300 nm to ~12 μm (Figure 2B) for veligers and adults respectively. We confirmed that the shell of C. acicula is composed mainly of densely packed curved nanofibers of aragonite, approximately 100-200 nm thick, as was shown in another species of the superfamily Cavolinioidea, Clio pyramidata 7. The nanofibers bend clockwise when observed from the outer shell surface (Figure 1B). X-ray diffraction patterns were obtained from a fragment of the teleoconch (Figure 1A). The X-ray beam was oriented parallel to the shell surface and illuminated an area of around 100 µm diameter (Figure 1C). The diffraction pattern consists of sharp reflections with well delimited arcs having an angular spread ranging between 10 degrees for the (002) diffraction and 50 degrees for the (012) diffraction. There is thus a relatively high degree of alignment among the fibers. The (002) diffraction shows that the c axes of the aragonite crystals are aligned perpendicular to the surface, as was observed by Checa et al. 13. The fact that the diffraction pattern contains peaks from (0kl) planes together with peaks from the (hkl) planes suggests that there are at least two crystal orientations, rotated around the c axis. 8 ACS Paragon Plus Environment

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Figure 1. (A) Creseis acicula adult observed under a stereoscopic microscope. The shell has a needle-like cone shape (about 4 mm long). The protoconch, teleoconch and growing edge regions of the shell are designated. Scale bar 500 μm. (B) Longitudinal fracture of the shell in the teleoconch region, observed by SEM. The shell microstructure is composed of curved aragonite nanofibers. A thin periostracal organic layer covers the outer shell. Scale bar 1 μm. (C) X-ray diffraction pattern obtained from the mature part of a C. acicula shell. The beam was perpendicular to the shell surface. Image from Baron

35.

Used with

permission of the author. The light micrograph image in Figure 2A shows the locations of 3 areas close to the shell growing edge examined in cryo-SEM. Cryo SEM is used to obtain high resolution images of freeze-fractured high pressure frozen samples. High pressure freezing preserves the samples in a state, which is as close as possible to the physiological conditions. Figure 2B shows a longitudinal fracture of the growing edge region. The whole region is mineralized, as evidenced by the backscattered electron (BSE) image (inset Figure 2B). Surprisingly there is no curved nanofiber morphology characteristic of pteropod shells. A polygonal regularly arranged blocky structure is located just below the outer shell surface (Figure 2B). The polygonal layer is roughly 100 nm thick (Figure 2B). A similar incipient



polygonal structure was observed in the Arca imbricata (bivalve) growth surface 36 and in gastropod larvae 37. The image of the shell microstructure about 14 μm from the growing edge shows the presence of short somewhat irregular aragonite fibers (Figure 2C). The polygonal layer seen in the growing edge persists even when the fibers are being formed (Figure 2C). In a region a few tens of microns from the shell edge, the aragonite fibers begin to develop their characteristic curved structure underneath the thin polygonal outer surface layer (Figure 2D). Thickening of the shell occurs by extension of the curved nanofibers on the inner shell surface.

Figure 2. (A) Light microscope image of C. acicula veliger showing the 3 regions examined with cryo-SEM. Scale bar 100 μm. (B) Cryo-SEM image of the longitudinal fracture of the growing edge located in area 1 in Figure 2A. P: thin polygonal structured layer below the outer surface of the shell. OS: the outer shell surface. The curved aragonite


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fibers are absent in this part of the shell. Inset: Backscattered electron image of the same region showing that the electron dense mineral is the major component of the growing edge. Scale bar 200 nm. (C) Short aragonite fibers are present about 14 μm from the growing edge. The aragonite fibers form below the polygonal layer (indicated by arrow 2 in Figure 2A). Scale bar 200 nm. (D) Curved aragonite fibers in a region a few tens of microns from the shell edge (indicated by arrow 3 in Figure 2A). Scale bar 200 nm. It was shown previously that the internal growth surfaces of the aragonite fibers of Cuvierina columnella have a nanogranular texture 13. High magnification of the growing edge fracture surface in C. acicula (Figure S1) shows that it contains both smooth areas (probably organic material, but possibly in part due to beam damage), as well as nanospheres with a size range of a few tens of nanometers. The BSE image of the same area shows that most of the visible surface, and in particular the nano-spheres, is composed of mineral rather than organic material. Raman micro-spectroscopy Raman micro-spectroscopy was performed in order to characterize the mineral phase(s) of the Creseis acicula shell, and especially the newly formed mineral. As mollusks and many other organisms form their crystalline calcium carbonate shells by first depositing an unstable and disordered precursor phase24,

38-43

we took the precaution of studying C.

acicula specimens that were alive during the Raman analysis. The animals were caught usually a day before the analysis in the Red Sea off the coast of Eilat, were maintained alive under anesthesia during the analysis and were still alive after the analysis.



The aragonite Raman spectrum has been calculated by ab-initio techniques and measured in single crystal and powder form 44. Group theory predicts 30 Raman lines which can be divided into internal (modes mostly characterized by carbonate motions) and external (modes mostly characterized by lattice motions). Some of the 30 modes are more pronounced in the measured spectrum and we use these to identify aragonite in the Raman spectra that were measured from the C. acicula shell. Geological aragonite is readily identifiable by the six lattice modes at 153 (B1g), 180 (B2g), 190 (B2g), 206 (B2g) cm−1, 248 (B2g) cm−1 and 260 (B2g) cm−1 and the two internal modes at 702 and 706 (doublet Ag and B3g) and 1085 (Ag) cm−1 (Figure 3A) 44. The lattice modes in the spectra are very sensitive to the degree of crystal order. The peaks in this range become more distinct as the atomic order increases 25, 45. The living C. acicula specimens were measured using the 785 nm laser in order to reduce the likeliness of generating fluorescence that might hide the Raman signal, and is less likely to heat the sample, thus minimizing possible crystallization artifacts. The spatial resolution of the Raman confocal microscope is limited in x and y by the diffraction limit and the depth of acquisition of the scattered light by the numerical aperture of the objective collecting the light scattered from within the shell and the confocal pinhole. Under optimal optical settings, spatial resolution in the xy plane is ~0.5µm and the z resolution is ~1.0 μm. The Raman laser beam was oriented perpendicular to the shell surface. Assuming these optimal conditions, we sampled the entire volume of the growing edge, that is about 300 nm thick (Figure 2), and the upper ~1.0 μm of the more mature regions of the shell.


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Figure 3B is a spectrum of mature aragonite from C. acicula adult obtained from the tip of the most mature region of the shell, namely the protoconch tip. All the peaks are similar to those observed in the geological aragonite (Figure 4A), but the peaks in the lattice mode region of the biogenic aragonite are relatively broad compared to the geological aragonite (Figure 3B). The broader peaks show that even the mature shell mineral is significantly less ordered than geological aragonite 25, 45. Figures 3C-H show the Raman shift signals obtained from three regions along the shells of C. acicula. The Raman spectra were obtained from the growing edge, teleoconch and the protoconch-tip regions of C. acicula adult and from the growing edge, protoconch middle region (protoconch- middle) and protoconch- tip regions of C. acicula veliger. These positions represent different maturation phases of the shell. All spectra are normalized to the 1085 cm-1 (Ag) vibrational mode. We focus on the mineral vibrational modes. Some Raman signals are presumably from the organic components and especially the periostracum (peak at 1100 cm-1) 23. The carbonate vibrational modes, i.e. 1085 (Ag), 706 (Ag) and 702 (B3g) cm-1 peaks, are similar in all spectra (Figurers 3D and G). Peaks in the lattice mode range, 150-300 cm-1 (Figures 3E and H) are different in the 3 regions of the shell. The individual lattice mode peaks from the growing edge of the shell are barely discernible and the entire lattice mode range appears as a hump due to the merging of broad peaks (Figures 3D, G1 spectrum and 3G, G1-2 spectra) or as broad peaks (Figure 3D, G2-4 spectra and 3G, G2-5 spectra). The peaks in the lattice mode range become more distinct and narrower in the teleoconch and protoconch-middle region, and even narrower in the protoconch- tip region. The absence of well separated peaks in the lattice mode range



indicates that the mineral is highly disordered, but still has short range order characteristic of aragonite. In order to make sure that the loss of peak distinction at the growing edge is not due to the small amount of material in this region, we measured the growing edge using very long exposures to verify that this is not just an issue of signal to noise ratio (SNR). SNR is a function of signal, and becomes higher when more signal is collected at a rate following the square root of the number of counts (or the acquisition time). By measuring for much longer times (50 sec scans averaged 10 times for the growing edge and 20 sec scans averaged 10 times in the mature regions), we showed that the broadness of the signal was inherent to the condition of the sample and not due to low SNR. We therefore conclude that the shell in the growing edge region is composed of a highly disordered aragonite phase. As the peaks become sharper further away from the growing edge, we refer to this initially deposited mineral as a nascent aragonite phase. Long range order increases with shell age, indicating a transformation from a nascent disordered aragonite phase into a more ordered phase of mature aragonite.


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Figure 3. Raman spectrum of geological aragonite (from Cuenca, Castile-La Mancha, Spain ) (A) and from the protoconch-tip region of a living C. acicula adult (B) showing the 4 lattice mode peaks at 153, 180, 190 and 206 cm−1 (marked by grey rectangle), and the two internal mode peaks at 702(B3g), 706(Ag) (doublet) and 1085(Ag) cm−1. Low-resolution (10×) images of the C. acicula veliger (C) and adult (F) showing the locations of the areas analyzed in the growing edge (G-blue), teleoconch (T-purple), protoconch-middle (PmRed) and the protoconch- tip (Pt-green). The images are a composite of several 15 ACS Paragon Plus Environment


micrographs. Scale bars 100 μm. Raman spectra from the shell of a living C. acicula veliger (D) and adult (G). All spectra are color-coded according to the shell region and normalized to the 1085 cm-1 carbonate vibrational mode. Pr; periostracum. (E) and (H) enlargement of the lattice mode range in D and G respectively. It is interesting to note that in the lattice region of the C. acicula adult the peaks in the protoconch-tip, the oldest shell region, resemble more the geological aragonite peaks (Figure 3G, P2-3 spectra compared to Figure 3A) as opposed to less distinct peaks in the protoconch-tip of the C. acicula veliger (Figure 3D). This implies that there is a continuous process of maturation of the aragonite shell, namely the older the shell of the animal, the more ordered is the aragonite. It is also of interest to note that even in the growing edge where the aragonite is least ordered, the 1085 cm-1 vibrational mode peak is narrow. In stable ACC this peak is broad, whereas in transient ACC this peak is narrow 23, 46. Overall, we measured 3 living veligers and 3 living adults of C. acicula. In order to compare the data from all the animals we determined the peak height ratios of the 206 cm-1 and 180 cm-1 peaks (I206/I180 ratio) and defined the ratio for geological aragonite as 1.0. We used this ratio as an indicator of how distinct the lattice mode vibrational peaks are in each spectrum assuming that no other factors influence this ratio. A smaller ratio indicates that the peaks in the lattice mode region are less distinguishable and the mineral phase is more disordered. We observed that the peak height ratios increase in spectra obtained from more mature regions of the shell, namely the teleoconch and the protoconch, relative to the ratio at the growing edge in both developmental stages of C. acicula (Figure 4). The ratio increases from the growing edge to the protoconch-tip, but in only a few analyses of the protoconch-tip is the shell atomic order comparable to geological aragonite (Figures 4A 16 ACS Paragon Plus Environment

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and B). Note that the spectra acquired from the teleoconch region were closer to the growing edge in the C. acicula adult (Figure 4B), than the spectra acquired from the protoconch-middle in the C. acicula veliger (Figure 4A).



Figure 4. Normalized peak height ratios from 3 C. acicula veligers (A) and 3 adults (B) in the growing edge, teleoconch, protoconch- middle and the protoconch- tip regions of the shells (indicated by orange dots). The y-axis shows the peak height ratios of 206 cm-1/180 cm-1. Values were normalized to the peak height ratio obtained for the geological aragonite. n is the number of spectra obtained in each region. Blue dots are the average of the normalized peaks obtained in each region of the shell. After examination of the living pteropods, the animals were placed in 100 % ethanol overnight, air dried and then kept in a desiccator awaiting further examination. Raman spectra obtained from the growing edge region, 1.5 months after the samples were dried still demonstrate the presence of the nascent aragonite phase similar to the living animals (Figure 5). Figure 6 also shows that the Raman shift spectra of air-dried C. acicula adult demonstrate the same trends observed in the living animals (Figure 3E). Thus this nascent phase of aragonite appears to be stable over time when the shell is dry.


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Figure 5. Raman spectra from the shell of a C. acicula air dried adult. (A) Low-resolution (10×) image of the C. acicula adult (the same specimen as in Figure 3F) showing the locations of the analyses of the growing edge (G), the teleoconch (T) and the protoconch (P) regions. The image is a composite of several micrographs. Scale bar 100 μm. (B) Raman spectra color-coded according to the shell region: Inset, enlargement of the lattice mode range (marked by grey rectangle). All spectra are normalized to the 1085 cm-1 carbonate vibrational mode.

We also measured the mineral disorder through the shell thickness of an air dried C. acicula veliger (Figure 6). This experiment was performed by progressively lowering the 19 ACS Paragon Plus Environment


objective from outside the shell towards the C. acicula shell until a spectrum was obtained. This located the outer shell surface. We subsequently systematically moved the objective focal spot towards the inner surface of the shell. Spectrum 1 is from the first location that produced a spectrum, and spectrum 5 was taken from the inner surface of the shell. Figure 6 shows Raman z-stack spectra from 2 locations at different distances from the growing edge and therefore differing also in the shell thickness. The shell thickness is about 1.5 μm at distance of 20 μm from the growing edge, and 3.5 μm at a distance of 60 μm from the growing edge (estimated from another C. acicula veliger fractured shell observed with cryo SEM). Both the degree of order as manifested by the distinction of peaks in the lattice mode range, and the intensities of the peaks, decrease from the shell outer surface to the shell inner surface. The trend shows that the thickening of the shell is also achieved by deposition of a disordered precursor phase. Note that the spectra are not normalized. Some z-stacks did not show this trend, either because of technical difficulties or because the inner shell mineral in these regions was already more crystallized.


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Figure 6. (A) Low-resolution (10×) image of the air dried C. acicula veliger analyzed. The image is a composite of several micrographs. Scale bar: 100 μm. Raman spectra were obtained 20 μm (B) and 60 μm (C) away from the growing edge region. White arrows indicate these locations. (D, E) Inset: magnification of the 150-300 lattice mode range spectra (marked with grey rectangle in B and C). Spectrum 1 is the spectrum obtained from the outer shell surface and spectrum 5 is the spectrum obtained from the inner shell surface.



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Discussion Here we show that the mineral phase at the growing edges, as well as the inner surfaces of C. acicula shells, is a disordered phase of aragonite. As this phase becomes more ordered as the distance from the growing edge increases, we refer to this phase as a nascent form of disordered aragonite, following the terminology of Politi et al. 47 and Wehrmeister et al. 25.

The first identification of such a nascent mineral form was by Hasse et al. 22 who showed that even though the first formed mineral in the larval shell of a freshwater gastropod was amorphous in X-ray measurements, the EXAFS spectrum showed the presence of short range atomic order characteristic of aragonite. The mature larval shell is aragonite. A similar phenomenon was demonstrated for the first mineral deposits of sea urchin larvae, but here the short range order resembled calcite 48. Calcite is the crystalline phase of the mature larval skeleton. A challenging question that arises from the observations of Hasse et al. 22 and Politi et al. 48,

as well as the observations reported here for the shell edge mineral deposits of the C.

acicula shell, is whether or not the nascent disordered structure formed from an even more disordered phase of ACC. The involvement of an ACC precursor phase can be inferred, when the crystalline mineral phase preserves a nano-sphere texture. A nano-sphere texture is indicative of crystalline biominerals that formed via an amorphous mineral phase

49.

Such a nano-sphere texture was clearly seen by Weiss et al. 23. The presence of ACC was also inferred for the pteropod species Cuvierina columnella based on the observation of a nano-sphere texture of the mineral on the growing surface (Figure S1), and from an


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observed volume reduction as the tips are incorporated into the shell 13. We did note the presence of sparse nano-spheres in the C. acicula growing edge region, and BSE imaging confirmed that they are composed of a mineral phase (Figure S1). Spectroscopic evidence that provides information on the short range order of initially deposited mineral phases is more compelling (reviewed in Wehrmeister et al. 25). Weiss et al. 23 show a Raman spectrum of a 9 day old mollusk larva that has no lattice mode peaks (their Figure 3B). They concluded that this is indicative of ACC. Wehrmeister et al.

25

identified pure ACC in two biogenic mollusk samples: a cultured pearl and the shell of a bivalve. Jacob et al. 24 also identified ACC in the initial prisms of adult bivalves based on the absence of other peaks besides the 1080 cm-1 peak. This was based on the lack of definitive lattice mode absorptions, and a broad peak in this area. The other biogenic samples analyzed contained a mixture of ACC and a more crystalline phase. Jardillier et al. 29 state that the very early mineral deposited by the larval gastropod Haliotis tuberculata is aragonite based on Raman spectra showing the presence of the 1086 cm-1 peak, but we note the absence of the doublet around 700 cm-1 which is consistent with the presence of ACC. The infrared spectrum shows the presence of a broad 875 cm-1 peak, but the absence of the 712 cm-1 peak. These are all characteristics of ACC rather than a more ordered mineral phase. Ramesh et al. 28 studied the larvae of the bivalve Mytilus edulis, and reported results very similar to those reported here for C. acicula, namely the presence of a nascent partly ordered phase and no evidence for ACC. The above studies do show that in certain cases ACC, as opposed to a nascent partly ordered first mineral deposit, can be detected spectroscopically in mollusks. In all of the Raman measurements we could not spatially separate the spectra of the nano-spherical structures from those of the larger deposits, 23 ACS Paragon Plus Environment


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which is inherent to micro-Raman instrumentation. This may in part be the reason for our inability to clearly see the spectral signature of ACC. Even though we could not detect ACC spectroscopically in C. acicula, we cannot exclude the possibility that the very first deposit is indeed ACC. We also note that ACC can be regarded as a group of different mineral phases 50, and that some may have more short range order than others 22, 51, 52. All spectra obtained from the C. acicula veliger and adult shell show sharp peaks in the carbonate vibrational mode 1085 cm-1 peak. The presence of this sharp peak in the disordered phase is consistent with a transient form of non-hydrated ACC rather than stable ACC phase 46, 53. This is consistent with the notion that C. acicula aragonite shell is formed via a transient precursor phase. We were surprised to discover that the fresh shells that were stored dry for 6 weeks did not transform into a more crystalline phase even at the growing edge. As the samples were first placed overnight in 100% ethanol and then stored in a desiccator, it is possible that the absence of water is responsible for preserving the disordered aragonite phase in its original state. Formation of the crystals from a disordered initial phase may well facilitate the crystallization, alignment and interlocking of the curved crystal fibers. As the amorphous phase is isotropic, it can be shaped more easily by the space in which it forms

46.

This

mineralization process may well be important for the formation of the complex ordered crystal super-structure. The C. acicula Raman spectra clearly show that the mineral phase becomes more ordered over time. This is expressed by the sharpening and increased intensity of the lattice mode


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aragonite peaks from the outer shell surface as a function of increasing distance from the shell edge (Figures. 3-4). The high crystallinity of the mature part of the shell is confirmed by the sharp X-ray diffraction arcs, which do not show any hint of the presence of amorphous material. We did however note that the Raman spectra of the shell material from the protoconch-tip that was formed very early during development, and is hence the oldest mineral in an individual, is for the most part still less ordered than geological aragonite (Figures 3A and B). As more disorder implies that the mineral phase is more soluble, we infer that C. acicula shells, and possibly the shells of many other species of the Cavolinioidea, will dissolve more readily in sea water than geological aragonite. We do note however that a measurement of the aragonite stoichiometric solubility product of a mixed assemblage of fossil pteropod shells from a deep-sea core produced the same solubility product as synthetic aragonite54. This may indicate that these fossil pteropod shells diagenetically continued to crystallize after burial.



Conclusions Here we show using in vivo micro Raman spectroscopy that the aragonitic shell of Creseis acicula forms via a disordered transient nascent aragonite phase. This nascent phase forms during shell elongation at the growing edge and at the inner surface during shell thickening. This phase transforms to a more ordered phase of aragonite in the more mature parts of the shell. The authors declare no competing financial interest.


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Acknowledgements We thank Dr. Eyal Shimoni for his help with freezing the living specimens and Dr. Linda Shimon for her help with X-ray diffraction. Super resolution microscopy was performed at the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science. Micro Raman spectroscopy was performed at the Time Domain Spectroscopy and Microscopy Unit, Department of Chemical Research Support. L.A. is the recipient of the Dorothy and Patrick Gorman Professorial Chair of Biological Ultrastructure.



Supporting Information Supporting information 1: Cryo-SEM images of a longitudinal cross section of C. acicula veliger


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For Table of Contents Use Only The pteropod Creseis acicula forms its shell through a disordered nascent aragonite phase Odelia Sibony-Nevoa, Iddo Pinkasb, Viviana Farsteyc, Hen Barona, Lia Addadia and Steve Weinera* In vivo Raman micro-spectroscopy provides insights into the mineral phases of the Creseis acicula pteropod shell, and especially the newly formed mineral. C. acicula forms its shell via a disordered transient nascent aragonite phase that transforms to a more ordered phase of aragonite in the more mature parts of the shell.



Figure 1. (A) Creseis acicula adult observed under a stereoscopic microscope. The shell has a needle-like cone shape (about 4 mm long). The protoconch, teleoconch and growing edge regions of the shell are designated. Scale bar 500 μm. (B) Longitudinal fracture of the shell in the teleoconch region, observed by SEM. The shell microstructure is composed of curved aragonite nanofibers. A thin periostracal organic layer covers the outer shell. Scale bar 1 μm. (C) X-ray diffraction pattern obtained from the mature part of a C. acicula shell. The beam was perpendicular to the shell surface. Image from Baron 35. Used with permission of the author. 177x61mm (300 x 300 DPI)


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Figure 2. (A) Light microscope image of C. acicula veliger showing the 3 regions examined with cryo-SEM. Scale bar 100 μm. (B) Cryo-SEM image of the longitudinal fracture of the growing edge located in area 1 in Figure 2A. P: thin polygonal structured layer below the outer surface of the shell. OS: the outer shell surface. The curved aragonite fibers are absent in this part of the shell. Inset: Backscattered electron image of the same region showing that the electron dense mineral is the major component of the growing edge. Scale bar 200 nm. (C) Short aragonite fibers are present about 14 μm from the growing edge. The aragonite fibers form below the polygonal layer (indicated by arrow 2 in Figure 2A). Scale bar 200 nm. (D) Curved aragonite fibers in a region a few tens of microns from the shell edge (indicated by arrow 3 in Figure 2A). Scale bar 200 nm. 176x108mm (300 x 300 DPI)



Figure 3. Raman spectrum of geological aragonite (from Cuenca, Castile-La Mancha, Spain ) (A) and from the protoconch-tip region of a living C. acicula adult (B) showing the 4 lattice mode peaks at 153, 180, 190 and 206 cm−1 (marked by grey rectangle), and the two internal mode peaks at 702(B3g), 706(Ag) (doublet) and 1085(Ag) cm−1. Low-resolution (10×) images of the C. acicula veliger (C) and adult (F) showing the locations of the areas analyzed in the growing edge (G-blue), teleoconch (T-purple), protoconch-middle (Pm-Red) and the protoconch- tip (Pt-green). The images are a composite of several micrographs. Scale bars 100 μm. Raman spectra from the shell of a living C. acicula veliger (D) and adult (G). All spectra are color-coded according to the shell region and normalized to the 1085 cm-1 carbonate vibrational mode. Pr; periostracum. (E) and (H) enlargement of the lattice mode range in D and G respectively. 172x183mm (299 x 299 DPI)


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Figure 4. Normalized peak height ratios from 3 C. acicula veligers (A) and 3 adults (B) in the growing edge, teleoconch, protoconch- middle and the protoconch- tip regions of the shells (indicated by orange dots). The y-axis shows the peak height ratios of 206 cm-1/180 cm-1. Values were normalized to the peak height ratio obtained for the geological aragonite. n is the number of spectra obtained in each region. Blue dots are the average of the normalized peaks obtained in each region of the shell. 172x195mm (300 x 300 DPI)



Figure 5. Raman spectra from the shell of a C. acicula air dried adult. (A) Low-resolution (10×) image of the C. acicula adult (the same specimen as in Figure 3F) showing the locations of the analyses of the growing edge (G), the teleoconch (T) and the protoconch (P) regions. The image is a composite of several micrographs. Scale bar 100 μm. (B) Raman spectra color-coded according to the shell region: Inset, enlargement of the lattice mode range (marked by grey rectangle). All spectra are normalized to the 1085 cm-1 carbonate vibrational mode. 172x146mm (299 x 299 DPI)


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Figure 6. (A) Low-resolution (10×) image of the air dried C. acicula veliger analyzed. The image is a composite of several micrographs. Scale bar: 100 μm. Raman spectra were obtained 20 μm (B) and 60 μm (C) away from the growing edge region. White arrows indicate these locations. (D, E) Inset: magnification of the 150-300 lattice mode range spectra (marked with grey rectangle in B and C). Spectrum 1 is the spectrum obtained from the outer shell surface and spectrum 5 is the spectrum obtained from the inner shell surface. 167x180mm (299 x 299 DPI)



88x30mm (300 x 300 DPI)


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The pteropod Creseis acicula forms its shell through a disordered

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