The effects of environment on Arctica islandica shell formation and architecture

Mollusks record valuable information in their hard parts that reflect ambient environmental conditions. For this reason, shells can serve as excellent archives to reconstruct past climate and environmental variability. However, animal physiology and biomineralization, which are often poorly understood, can make the decoding of environmental signals a challenging task. Many of the routinely used shell-based proxies are sensitive to multiple different environmental and physiological variables. Therefore, the identification and interpretation of individual environmental signals (e.g., water temperature) often is particularly difficult. Additional proxies not influenced by multiple environmental variables or animal physiology would be a great asset in the field of paleoclimatology. The aim of this study is to investigate the potential use of structural properties of Arctica islandica shells as an environmental proxy. A total of 11 specimens were analyzed to study if changes of the microstructural organization of this marine bivalve are related to environmental conditions. In order to limit the interference of multiple parameters, the samples were cultured under controlled conditions. Three specimens presented here were grown at two different water temperatures (10 and 15 C) for multiple weeks and exposed only to ambient food conditions. An additional eight specimens were reared under three different dietary regimes. Shell material was analyzed with two techniques; (1) confocal Raman microscopy (CRM) was used to quantify changes of the orientation of microstructural units and pigment distribution, and (2) scanning electron microscopy (SEM) was used to detect changes in microstructural organization. Our results indicate that A. islandica microstructure is not sensitive to changes in the food source and, likely, shell pigment are not altered by diet. However, seawater temperature had a statistically significant effect on the orientation of the biomineral. Although additional work is required, the results presented here suggest that the crystallographic orientation of biomineral units of A. islandica may serve as an alternative and independent proxy for seawater temperature.

parts that reflect ambient environmental conditions.For this reason, shells can serve as excellent archives to reconstruct past climate and environmental variability.However, animal physiology and biomineralization, which are often poorly understood, can make the decoding of environmental signals a challenging task.Many of the routinely used shell-based proxies are sensitive to multiple different environmental and physiological variables.Therefore, the identification and interpretation of individual environmental signals (e.g., water temperature) often is particularly difficult.Additional proxies not influenced by multiple environmental variables or animal physiology would be a great asset in the field of paleoclimatology.The aim of this study is to investigate the potential use of structural properties of Arctica islandica shells as an environmental proxy.A total of 11 specimens were analyzed to study if changes of the microstructural organization of this marine bivalve are related to environmental conditions.In order to limit the interference of multiple parameters, the samples were cultured under controlled conditions.Three specimens presented here were grown at two different water temperatures (10 and 15 • C) for multiple weeks and exposed only to ambient food conditions.An additional eight specimens were reared under three different dietary regimes.Shell material was analyzed with two techniques; (1) confocal Raman microscopy (CRM) was used to quantify changes of the orientation of microstructural units and pigment dis-tribution, and (2) scanning electron microscopy (SEM) was used to detect changes in microstructural organization.Our results indicate that A. islandica microstructure is not sensitive to changes in the food source and, likely, shell pigment are not altered by diet.However, seawater temperature had a statistically significant effect on the orientation of the biomineral.Although additional work is required, the results presented here suggest that the crystallographic orientation of biomineral units of A. islandica may serve as an alternative and independent proxy for seawater temperature.

Introduction
Biomineralization is a process through which living organisms produce a protective, mineralized hard tissue.The considerable diversity of biomineralizing species contributes to high variability in terms of shape, organization and mineralogy of the structures produced (Lowenstam and Weiner, 1989;Carter et al., 2012).Different architectures at the micrometer and nanometer scale and different biochemical compositions determine material properties that serve specific functions (Weiner and Addadi, 1997;Currey, 1999;Merkel et al., 2007).Besides these differences, all mineralized tissues are hybrid materials consisting in hierarchical arrangements of biomineral units surrounded by organic matrix, also known as "microstructures" (Bøggild, 1930;Carter and Clark, 1985;Rodriguez-Navarro et al., 2006), "ultrastructures" (Blackwell et al., 1977;Olson et al., 2012) or overall "fabrics" (Schöne, 2013;Schöne et al., 2013).The carbonate and organic phases represent the fundamental level of the organization of biomaterials (Aizenberg et al., 2005;Meyers et al., 2006).The mechanisms of microstructure formation and shaping, especially in mollusks, has attracted increasing attention during recent decades.At present, it is commonly accepted that the organic compounds play an important role in the formation of the inorganic phases of biominerals (Weiner and Addadi, 1991;Berman et al., 1993;Dauphin et al., 2003;Nudelman et al., 2006).However, the identification of the exact mechanisms driving biomineralization is still an open research question.Previous studies conducted on mollusks show that environmental parameters can influence microstructure formation (Lutz, 1984;Tan Tiu and Prezant, 1987;Tan Tiu, 1988;Nishida et al., 2012).These results set the stage for research that focuses on the use of shell microstructures as proxies for reconstructing environmental conditions (Tan Tiu, 1988;Tan Tiu and Prezant, 1989;Olson et al., 2012;Milano et al., 2017).
Mollusks are routinely used as climate and environmental proxy archives because they can record a large amount of environmental information in their shells (Richardson, 2001;Wanamaker et al., 2011a;Schöne and Gillikin, 2013).Whereas structures at nanometric levels are still underexplored as potential environmental recorders, shell patterns at lower magnification, such as individual growth increments, are commonly used for this purpose (Jones, 1983;Schöne et al., 2005;Marali and Schöne, 2015;Mette et al., 2016).Mollusks deposit skeletal material on a periodic basis and at different rates (Thompson et al., 1980;Deith, 1985).During periods of fast growth, growth increments are formed, whereas during periods of slower growth, growth lines are formed (Schöne, 2008;Schöne and Gillikin, 2013).The periodicity of such structures ranges from tidal to annual (Gordon and Carriker, 1978;Schöne and Surge, 2012).By crossdating time series with similar growth patterns it is possible to construct century-and millennia-long master chronologies (Marchitto et al., 2000;Black et al., 2008Black et al., , 2016;;Butler et al., 2013).This basic approach, in combination with geochemical methods, has great potential in reconstructing past climatic conditions (Wanamaker et al., 2011b).At present, the most frequently used and well-accepted geochemical proxy is oxygen isotopic composition of shell material (δ 18 O shell ) (Epstein, 1953;Grossman and Ku, 1986;Schöne et al., 2004;Wanamaker et al., 2007), which may serve as a paleothermometer and/or paleosalinometer (Mook, 1971;Andrus, 2011); however, δ 18 O shell value is influenced by both seawater temperature and the isotopic composition of seawater (δ 18 O water ; related to salinity).Thus, δ 18 O shell -based temperature reconstructions are particularly challenging in habitats with fluctuating δ 18 O water conditions such as estuaries or restricted basins (Gillikin et al., 2005).Because of the multiple impacts on δ 18 O shell values, there have been substantial efforts to develop alternative techniques to reconstruct environmental variables from mollusk shells (Schöne et al., 2010;Milano et al., 2017).
This study investigates the possibility using shell microstructure properties to serve as a new environmental proxy.For this purpose, the effects of seawater temperature (grown at 10 and 15 • C) and dietary regime on the microstructural units of Arctica islandica cultured under controlled conditions were analyzed and quantified. A. islandica was chosen as model species because of its great potential in paleoclimatology and paleoceanography (see Schöne, 2013;Wanamaker et al., 2016).Its extreme longevity of up to more than 500 years makes this species a highly suitable archive for long-term paleoclimate and environmental reconstructions (Schöne et al., 2005;Wanamaker et al., 2008Wanamaker et al., , 2012;;Butler et al., 2013).

Materials and methods
The analyses were conducted on 11 A. islandica shells.Three juvenile A. islandica shells, sampled for the seawater temperature experiment, were collected alive on 21 November 2009 aboard the F.V. Three of a Kind off Jonesport, Maine, USA (44 • 26 9.829 N, 67 • 26 18.045 W), in 82 m water depth.From 2009 to 2011, all animals were kept in a flowing seawater laboratory at the Darling Marine Center, Walpole, Maine, USA (see Beirne et al., 2012, for additional details).In 2011, clams were grown at two different temperature regimes for 16 weeks (Table 1).At the completion of the experiment, shells were estimated to be between 4 to 5 years old.Eight 1-year old juveniles were collected in July 2014 from Kiel Bay, Baltic Sea (54 • 32 N, 10 • 42 E; Fig. 1) and kept alive in tanks at 7 • C for 6 months at the Alfred Wegener Institute (AWI) for Polar and Marine Research, Bremerhaven, Germany.During this time interval, the animals were fed with an algal mix of Nannochloropsis sp., Isochrysis galbana and Pavlova lutheri.Then, they were transferred to the Royal Netherlands Institute for Sea Research (NIOZ), Texel, the Netherlands, and cultured in tanks at three different dietary conditions for 11 weeks (Table 1).

Seawater temperature experiment
The seawater temperature experiment started on 27 March and ended on 21 July 2011.Prior to the start of the experiment the animals were marked with calcein.The staining leaves a clear fluorescent marker in the shells that can be used to identify which shell material has formed prior to and during the experiment.Initially, the animals were kept at 10.3 ± 0.2 • C for 48 days.Then, they were briefly removed from the tanks and marked again.Subsequently, the clams were cultured for 69 more days at 15.0 ± 0.3 • C. Ambient seawater was pumped from the adjacent Damariscotta River estuary and adjusted to the desired temperature.The salin- ity was measured with a Hydrolab ® MiniSonde.It ranged between 30.2 ± 0.7 and 30.7 ± 0.7, in the two experimental phases, respectively.During the entire culture period, all clams were exposed to ambient food conditions.At the end of the experiment the soft tissues were removed.

Food experiment
The food experiment was carried out from 9 February to 29 April 2015.The animals were placed in aquaria inside a climate room at 9 • C. Water temperature in the tanks ranged between 8 and 10 • C. Water salinity was measured by using an ENDECO 102 refractometer and ranged between 29.6 and 29.9 ± 0.1 in each aquarium.The 15 L tanks were constantly supplied with aerated water from the Wadden Sea.The clams were acclimated for 3 weeks before the start of the experiment.Three dietary regimes were chosen.One treatment consisted of feeding the animals with Microalgae Mix (food type 1), a ready-made solution of four marine microalgae (25 % Isochrysis, 25 % Tetraselmis, 25 % Thalassiosira and 25 % Nannochloropsis) with a particle size range of 2-30 µm.A second treatment was based on PhytoMaxx (food type 2), a solution of living Nannochloropsis algae with a 2-5 µm size range.A third treatment served as control; i.e., the animals were not fed with any additional food.In treatments with food type 1 and 2, microalgae were provided at the con- stant optimum concentration of 20 × 10 6 cells L −1 (Winter, 1969).A dispenser equipped with a timer was used to distribute the food 5 times per day.At the end of the experiment the soft tissues were removed.A distinct dark line in the shells indicated the transposition to the NIOZ aquaria and the associated stress.This line marks the beginning of the tank experiment.

Sample preparation
The right valve of each specimen was cut into two 1.5 mm thick sections along the axis of maximum growth.For this purpose, a low-speed precision saw (Buehler Isomet 1000) was used.Given the small size and fragility of the juvenile shells used in the food experiment, the valves were fully embedded in a block of Struers EpoFix (epoxy) and air-dried overnight prior the sectioning.Sections of the clams used in the temperature experiment were embedded in epoxy after the cutting.All samples were ground using a Buehler Metaserv 2000 machine equipped with Buehler silicon carbide papers of different grit sizes (P320, P600, P1200, P2500).In addition, the samples were manually ground with Buehler P4000 grit paper and polished with a Buehler diamond polycrystalline suspension (3 µm).Sample surfaces were rinsed in demineralized water and air-dried.In the samples of the temperature experiment, the calcein marks were located under a fluorescence light microscope (Zeiss Axio Imager.A1m microscope equipped with a Zeiss HBO100 mercury lamp and filter set 38: excitation wavelength, ca.450-500 nm; emission wavelength, ca.500-550 nm).

A. islandica shell organization
The shell of A. islandica consists of pure aragonite and it is divided in two major layers, an outer shell layer (OSL) and the inner shell layer (ISL).The OSL is further subdivided in outer portion (oOSL) and inner portion (iOSL) (Schöne, 2013).These layers are characterized by specific microstructures (Ropes et al., 1984;Fig. 2).The oOSL largely con- sists of homogenous microstructures with a granular aspect (Schöne et al., 2013).This type of architecture is characterized by approximately equidimensional units of about 5 µm in width.The unit shape tends to be irregular with a bulky aspect.The organization lacks of specific structural arrangement typical of other types of microstructures such as the crossed-lamellar and crossed-acicular microstructures.The latter are the main component characterizing the iOSL and ISL (Dunca et al., 2009).Here, elongated units are arranged with two main dip directions, resulting in a relative oblique alignment.As shown in Fig. 2, the elongation of the structures becomes more evident in the ISL.The present study focuses on ventral margin of the shells.Analyses were carried out exclusively in the OSL.
Similar to other mollusks, the shell of A. islandica contains pigment polyenes, which are obviously visible when using CRM (Hedegaard et al., 2006).Polyenes are organic compounds containing single (C−C) and double (C=C) carboncarbon bonds forming a polyenic chain.Their distribution across the shell is not homogenous.The pigments are abundant in the oOSL, whereas they become scarce in the iOSL.Furthermore, an enrichment in polyenes has been observed in the growth lines, potentially indicating their involvement in the biomineralization process (Stemmer and Nehrke, 2014).However, the specific functions of these organic compounds have not been disclosed yet (Hedegaard et al., 2006;Karampelas et al., 2009).Given the high phenotypic variation in pigmentation among and within mollusk species, it has been proposed that coloration does not have a primary function as an adaptive tool (i.e., camouflage, warning signaling) as in other animals (Seilacher, 1972;Evans et al., 2009).This, in turn, can indicate a certain degree of influence of the environment on the pigments, in particular by diet (Hedegaard et al., 2006;Soldatov et al., 2013).In the current study, the effect of different dietary regimes was tested in order to explore the potential of polyenes as environmental proxy.

Confocal Raman microscopy and image processing
Shells were mapped with a WITec alpha 300 R (WITec GmbH, Germany) confocal Raman microscope.Scans of 50 × 50 µm, 100 × 50 µm and 150 × 50 µm were performed using a piezoelectric scanner table.All Raman measurements were carried out using a 488 nm diode laser.A spectrometer (UHTS 300, WITec, Germany) was used with a 600 mm −1 grating, a 500 nm blaze and an integration time of 0.03 s.On each sample two to six scans were made, depending of the thickness of the shell.For instance, in juvenile shells (food experiment), two scans of each sample were made.On larger shells used in the temperature experiment, six maps were completed, i.e., two maps in the oOSL, two in the middle of the iOSL and two in the inner portion of the iOSL.Each scan contained between 40 000 and 120 000 data points, depending on the map size.The spatial resolution equaled 250 nm.Half of the maps were performed on the shell portion formed before the experiments.The other half were made on the shell portion formed under experimental conditions.In order to avoid areas affected by transplantation or marking stress, the scans were located far off the calcein and stress lines.Raman maps on food experiment shells were performed 300 µm away from the stress line.In the shells from the temperature experiment, the scans were made 1 mm away from the calcein mark.
Polarized Raman microscopy is known to provide comprehensive information about the crystallographic properties of the materials (Hopkins and Farrow, 1985).The aragonite spectrum is characterized by two lattice modes (translation mode T a , 152 cm −1 and librational mode L a , 206 cm −1 ) and the two internal modes (in-plane band ν 4 , 705 cm −1 and symmetric stretch ν 1 , 1085 cm −1 ).The ratio (R ν1/T a ) between peak intensities belonging to ν 1 and T a is caused by different crystallographic orientations of the aragonitic units (Hopkins and Farrow, 1985;Nehrke and Nouet, 2011).Within each scan, R ν1/T a was calculated for each data point.New spectral images were generated using WITecProject software (version 4.1, WITec GmbH, Germany).These images were then binarized by replacing all values above 2.5 with 1 and the others with 0. The orientation was quantified by calculating the area formed by pixels of value 1 over the total scan area.The imaging software Gwyddion (http://gwyddion.net/;last access: June 2016) was used for this purpose.The results were expressed in percentage.
The Raman scans of the food experiment shells were analyzed to investigate the pigment composition.Polyene peaks have definite positions in the spectrum according to the number of the C−C and C=C bonds of the chain, which are specific for certain types of pigments.The two major polyene peaks at ∼ 1130 (R 1 ) and 1520 cm −1 (R 4 ) were identified by using the "multipeak fitting 2" routine of IGOR Pro (version 7.00, WaveMetrics, USA).Their exact position was determined adopting a Gaussian fitting function (Fig. 3).The number of single (N 1 ) and double carbon bonds (N 4 ) was calculated by applying the equations by Schaffer et al. (1991): (1) (2) Spectral images of the R 4 band were used to locate the polyenes in the shell and measure the thickness of the pigmented layer.The images were analyzed using the software Panopea (© Schöne and Peinl).The thickness of the pigmented layer was calculated as distance between the outer shell margin and the point where the concentration of polyenes suddenly declined.The measurements were taken perpendicular to the shell outer margin.This analysis was conducted only on the shells of the food experiment.Given the larger size of the shells used in the temperature experiment, the spectral maps were not sufficient for a correct localization of the pigmented layer boundaries and estimation of its thickness.
To quantify changes of the orientation of individual biomineral units of the juvenile shells (food experiment), the spectral maps were subdivided into two portions.The outermost shell portion (oOSL) was enriched in pigments whereas the iOSL showed a decrease in polyene content.

Scanning electron microscopy
After performing Raman measurements, the samples were prepared for SEM analysis.Each shell slab was ground with a Buehler Metaserv 2000 machine and Buehler silicon F2500 grit carbide paper.To reduce the impact of grinding on the sample surface of juvenile shells, extra grinding was done by hand.Then, the slabs were polished with a Buehler diamond polycrystalline suspension (3 µm).Afterward, shell surfaces were etched in 0.12 N HCl solution for 10 (food experiment samples) to 90 s (temperature experiment samples) and subsequently placed in 6 vol % NaClO solution for 30 min.After being rinsed in demineralized water, air-dried samples were sputter coated with a 2 nm thick platinum film by using a Low Vacuum Coater Leica EM ACE200.
A scanning electron microscope (LOT Quantum Design 2nd generation Phenom Pro desktop SEM) with backscattered electron detector and 10 kV accelerating voltage was used to analyze the shells.Images were taken at the same distances from the calcein and stress lines as in the case of the Raman measurements to assure comparability of the data.
In addition, stitched SEM images of the ventral margins were used to accurately determine the shell growth advance during the culturing experiments.Growth increment widths were measured with the software Panopea.Given the difference in duration of the two phases of the temperature experiment, the measurements were expressed as total growth and instantaneous growth rate (Fig. 4a, b).The latter was calculated using the following equation (Brey et al., 1990;Witbaard et al., 1997): Instantaneous growth rate = (ln(y t /y 0 )/a), where y 0 represents the initial shell height, y t is the final shell height and a is the duration of the experiment.In the case of the food experiment, only the total growth was calculated (Fig. 4c).  3 Results

Effect of seawater temperature and diet on A. islandica shell growth
When exposed to a water temperature of 10 • C, the shells grew between 11.67 and 14.17 mm during a period of 48 days.During a period of 69 days at 15 • C, the growth ranged between 2.32 and 5.77 mm (Fig. 4a).The instantaneous growth rate showed a decrease between the two experimental phases.At 10 and 15 • C, the average instantaneous growth per day was 0.0091 and 0.0013, respectively (Fig. 4b).The decrease in total growth and growth rate between the two temperatures was statistically significant (t test, p < 0.01).
During the food experiment, shells grew between 0.37 and 3.71 mm with large differences due to the different food types.Growth of specimens exposed to food type 1 ranged between 1.87 to 3.71 mm, whereas those cultured with food type 2 grew between 0.55 to 0.96 mm.Both control specimens added 0.37 mm of shell during the experimental phase (Fig. 4c).ANOVA and Tukey's HSD post hoc tests showed significant differences between specimens cultured with food type 1 and 2 (p < 0.05) and between food type 1 and control shells (p < 0.05).

Effect of seawater temperature on A. islandica microstructure
At a water temperature of 10 • C, the area occupied by microstructural units oriented with R ν1/T a higher than 2.5 a.u.(= arbitrary units) ranged between 31.3 and 50.6 % in the oOSL and between 21.3 and 33.5 % in the iOSL.When exposed to 15 • C, values ranged between 25.6 and 48.7 and between 45.7 and 55.9 % in the oOSL and iOSL, respectively (Fig. 5).Whereas the slight difference of area with R ν1/T a > 2.5 in the oOSL was not significant between the two water temperatures (t test, p = 0.62), the area with R ν1/T a >2.5 in the iOSL significantly increased at 15 • C (t test, p = 0.02).
Under the SEM, no difference was visible between units formed at 10 and 15 • C (Fig. 6).

Effect of food on A. islandica microstructure and pigments
In the shells cultured with food type 1, the area occupied by biomineral units oriented with R ν1/T a higher than 2.5 a.u.during the experiment ranged between 24.8 % (oOSL) and 43.0 % (iOSL).In the shell portion deposited during the acclimation phase, the ratio varied between 19.4 % (oOSL) and 36.2 % (iOSL).Although a trend was recognized, these variations were not statistically different (t tests OSL: p = 0.43; ISL: p = 0.57; Fig. 7a).On the contrary, in the clams exposed to food type 2, the area occupied by units oriented with R ν1/T a > 2.5 ranged between 11.7 % (oOSL) and 20.4 % (iOSL).Before the experiment, the proportions were higher, i.e., 18.1 % (oOSL) and 26.3 % (iOSL) (Fig. 7b).As for the other treatment, the difference was not significant (t tests oOSL: p = 0.34; iOSL: p = 0.28).In the control shells grown with no extra food supply, the area with R ν1/T a > 2.5 ranged between 24.6 % (oOSL) and 44.8 % (iOSL) during the experiment and 21.2 % (oOSL) and 44.5 % (iOSL) before the experiment (Fig. 7c).Hence, no trend was visible and the two portions did not show significant differences (t tests oOSL: p = 0.59; iOSL: p = 0.99).As for the temperature experiment, under the SEM, the microstructure of the shells from the food experiment did not show any change (Fig. 8).
All treatments showed a slightly thicker pigmented layer formed during the experiment than during the acclimation phase (Fig. 9a).During the experiment, clams cultured with Table 2. Details of the pigment composition of the Arctica islandica shells used in the food experiment.The position of the major polyene peaks R 1 and R 4 in the Raman spectrum is indicated together with the number of single and double carbon bonds of the pigment molecular chain (N 1 and N 4 ).Each shell was analyzed in the portions formed before and during the experimental phase.food type 1 showed, on average, a thickening by 6.4 %.In the food type 2 specimens, the layer thickness increased by 9.9 %.Control shells showed an increase of 10.4 % (Fig. 9b).However, none of these differences was statistically significant (t test.Food type 1: p = 0.43; food type 2: p = 0.39; control: p = 0.10).According to the position of the polyene peaks, the number of single carbon bonds in the pigment chain did not change between the acclimation and experimental phase (N 1 = 10.1 ± 1.3 and N 1 = 10.0 ± 0.9, respectively).Likely, no significant variation was observed in the number of double carbon bonds (N 4 = 10.5 ± 0.2 and N 4 = 10.4 ± 0.3, respectively; Table 2).

Discussion
According to the results, variations of both food type and water temperature can influence the shell production rate of A. islandica.However, the shell microstructure and pigmentation react differently to these two environmental variables.Whereas changes of the dietary conditions do not affect the shell architecture and pigment composition, the crystallographic orientation of the biomineral units responds to seawater temperature fluctuations.

Environmental influence on shell microstructure
The environmental conditions experienced by mollusks during the process of biomineralization appear to influence shell organization (Carter, 1980).Among the different environmental variables, water temperature is the most studied driving force of structural changes of the shell.For instance, shell mineralogy can vary depending on water temperature (Carter, 1980).According to the thermal potentiation hypothesis, nucleation and growth of calcitic structural units is favored at low temperatures by kinetic factors (Carter et al., 1998).As a consequence, bivalve species living in cold water environments exhibit additional or thicker calcitic layers compared to the corresponding species from warm waters (Lowenstam, 1954;Taylor and Kennedy, 1969).Changes in the calcium carbonate polymorph also affect the type of microstructures (Milano et al., 2016a).However, architectural variations often occur without mineralogical impact (Carter, 1980).The present results indicate that temperature induces a change in the crystallographic orientation of the biomineral units of A. islandica.Although water temperature was previously shown to have an impact on microstructure formation, the attention has been mainly focused on the effects on the morphometric characteristics (e.g., size and shape) or on the type of microstructure.Milano et al. (2017) demonstrated that size and elongation of prismatic structural units of Cerastoderma edule were positively correlated to seawater temperature variation throughout the growing season.Likely, low temperatures induced the formation of small nacre tablets in Geukensia demissa (Lutz, 1984).Seasonal changes of the microstructural type were reported in the freshwater bivalve Corbicula fluminea (Prezant and Tan Tiu, 1986;Tan Tiu and Prezant, 1989).During the warm months, crossed-acicular structure was produced, whereas simple crossed-lamellar were formed during the winter period.So far, variations of the crystallographic properties of bivalve biominerals have been exclusively investigated as a response to hypercapnic (acidified) conditions.Mytilus galloprovincialis and Mytilus edulis showed a significant change in the orientation of the prisms forming shell calcitic layer when subjected to hypercapnia (Hahn et al., 2012;Fitzer et al., 2014a).Altered crystallographic organization may derive from the animal exposure to suboptimal conditions.These findings together with the present results suggest that thermal-and hypercapnic-induced stress are likely to affect the ability of the bivalves to preserve the orientation of their microstructural units (Fitzer et al., 2014b).
Different food sources do not significantly influence the orientation of the biomineral units or the composition and distribution of pigments in shells of A. islandica.In previous studies, the relationship between microstructure and diet was virtually overlooked resulting in a lack of data in the literature.As suggested by Hedegaard et al. (2006), however, the type of polyenes is influenced by food.The ingestion of pigment-enriched microalgae potentially leads to an accumulation of pigments in mollusk tissues and the shell (Soldatov et al., 2013).On the other hand, it has been argued that polyenes do not generate from food sources like other pigments (i.e., carotenoids), but they are locally synthesized (Karampelas et al., 2009).In accordance to Stemmer and Nehrke (2014), the results presented here support the view that the specific diets on which the animals rely on do not influence shell pigment composition.The chemical characteristics of the polyenes are likely to be species-specific and independent from the habitats.

Confocal Raman microscopy as tool for microstructural analysis
From a methodological perspective, the present study represents an innovative approach in the investigation of shell microstructural organization.Electron backscatter diffraction (EBSD) has been previously used to determine the crystallographic orientation of gastropod (Fryda et al., 2009;Pérez-Huerta et al., 2011) and bivalve microstructural units (Checa et al., 2006;Frenzel et al., 2012;Karney et al., 2012).Whereas, CRM on mollusk shells is generally applied within studies on taphonomic mineralogical alteration and pigment identification (Stemmer and Nehrke, 2014;Beierlein et al., 2015).Both techniques provide considerably high spatially resolved analysis up to 250 nm, allowing for the identification of individual structural units at micrometer and nanometer scale (Cusack et al., 2008;Karney et al., 2012).CRM offers important advantages supporting a broader application of this methodology in the biomineralization research field.For instance, samples do not require any pre-treatment.Unlike EBSD, there is no need for preparing thin sections (∼ 150 µm thick) or etching the shell surface (Griesshaber et al., 2010;Hahn et al., 2012).Therefore, further structural and geochemical analyses can be easily performed on the same sections (Nehrke et al., 2012).In addition, the size of CRM scans can be remarkably large (∼ 7-8 mm 2 ) without compromising the achievable resolution.By overlapping adjacent scans, it is possible to produce stitched scans allowing one to further increase the region of interest on the shell surface.On the other side, EBSD provides a relevant advantage to take into consideration.It allows for absolute measures of the crystallographic orientation of the carbonate structures.The CRM, instead, determines the relative change in the orientation between the single units without providing absolute values.SEM has previously been demonstrated to provide a convenient approach for the identification of individual structural units and the quantification of potential changes occurring within them (Milano et al., 2017(Milano et al., , 2016b)).However, SEM exclusively provides information about the morphometric characteristics of the microstructural units.As highlighted by the present study, to achieve an exhaustive examination, it is suggested to combine SEM with techniques assessing crystallographic properties of the biomaterials.For instance, our results show that the effect of water temperature is detectable in crystallographic orientation but not in morphometric features of the biomineral units.

Environmental influence on shell growth
Numerous previous studies demonstrated that the growth rate of A. islandica is linked to environmental variables (e.g., Witbaard et al., 1997Witbaard et al., , 1999;;Schöne et al., 2004;Butler et al., 2010;Mette et al., 2016).However, the relative importance of the main factors, temperature and food supply/quality driving shell formation are still not well understood.Positive correlations between shell growth and water temperature have been identified (i.e., Schöne et al., 2005;Wanamaker et al., 2009;Marali et al., 2015), but the relationship between shell growth and environment is more complex (Marchitto et al., 2010;Stott et al., 2010;Schöne et al., 2013) and likely dependent on the synergic effect of food availability and water temperature (Butler et al., 2013;Lohmann and Schöne, 2013;Mette et al., 2016).Tank experiments were run in order to precisely identify the role of these two parameters on the shell growth of A. islandica (Witbaard et al., 1997;Hiebenthal et al., 2012).A 10-fold increase in instantaneous growth rate was observed between 1 and 12 • C, with the greatest variation occurring below 6 • C (Witbaard et al., 1997).On the contrary, a temperature increase between 4 and 16 • C was shown to produce a slowdown of shell production (Hiebenthal et al., 2012).Our results are in agreement with the latter study and show a decrease in the instantaneous growth rate between 10 and 15 • C. High temperatures are often associated with an increase of free radical production (Abele et al., 2002).A large amount of energy then has to be allocated to limit oxidative cellular damage (Abele and Puntarulo, 2004).This translates into a higher accumulation of lipofuscin and slower shell production rate (Hiebenthal et al., 2013).The contrasting results of previous studies may be explained by individual differences in the tolerance toward temperature change (Marchitto et al., 2000).
Along with water temperature, food availability was also shown to influence A. islandica shell growth (Witbaard et al., 1997).At high algal cell densities, the siphon activity increased.This, in turn, was positively correlated to shell growth.Previous experiments used different combinations of algae, such as Isochrysis galbana and Dunaliella marina (Witbaard et al., 1997), or Nannochloropsis oculata, Phaeodactylum tricornutum and Chlorella sp.(Hiebenthal et al., 2012) to grow the clams.However, there are still uncertainties about the composition of the primary food source for this species (Butler et al., 2010).Even though it is challenging to determine the preferred algal species, our results show that the use of a mixture of different algal species results in significantly faster shell growth than the used of just one algal species.In the natural environment, suspension feeders such as A. islandica preferentially ingest certain particle sizes (Rubenstein and Koehl, 1977;Jorgensen, 1996;Baker et al., 1998).The exposure to a limited algal size range, as in the case of food type 2, may affect shell growth.Furthermore, multispecific solutions contain a higher variability of biochemical components that better meet the nutritional requirements of the animal (Widdows, 1991).Our results are in good agreement with previous findings.For instance, it has been shown by Strömgren and Cary (1984) that Mytilus edulis shell growth increased as a result of a diet based on three different algal species.Furthermore, Epifanio (1979) tested the differences on the growth of Crasssostrea virginica and Mercenaria mercenaria of a mixed diet composed by Isochrysis galbana and Thalassiosira pseudonana and diets consisting of the single species.Faster growth was measured in the mixed diet treatment, indicating a synergic effect of the relative food composition (Epifanio, 1979).Likely, Mytilus edulis grew faster when reared with different types of mixed diets as opposed to monospecific diets (Galley et al., 2010).

Conclusions
Arctica islandica shell growth and biomineral orientation vary with changes in seawater temperature.However, exposure to different food sources affect shell deposition rate but do not influence the organization of the biomineral units.Given the exclusive sensitivity to one environmental variable, the orientation of biomineral units may represent a promising new temperature proxy for paleoenvironmental reconstructions.However, additional studies are needed to further explore the subject.In particular, intra-individual variability influence on the results needs to be assessed.In the present study, a variation in the orientation between individuals was well visible and the risks associated have to be taken in acwww.biogeosciences.net/14/1577/2017/Biogeosciences, 14, 1577-1591, 2017 count when considering further application of the possible proxy.Furthermore, the effect of other environmental variables such as salinity needs to be tested.The innovative application of CRM for microstructural orientation and proxy development proved that the technique has large potential in this research direction.More studies are needed to validate its suitability in paleoclimatology experimental works.
Data availability.The data are available from the article itself.CRM output data are in form of binary images and the original files can only be opened when operating with the CRM software associate to the machine specifically used in this study.Similarly, the SEM images represent the direct outputs of the microscope.
Competing interests.The authors declare that they have no conflict of interest.

Figure 1 .
Figure 1.Shell of adult Arctica islandica used in the temperature experiment (top) and juvenile from the Baltic Sea used in the food experiment (bottom).The map indicates the localities where the two sets of shells were collected: Jonesport, Maine (circle), and Kiel Bay (square).

Figure 2 .
Figure 2. Sketch showing the microstructures characterizing the different shell layers of Arctica islandica.The oOSL is formed by homogenous microstructure (HOM), whereas the oOSL and ISL are composed of crossed acicular structure (CA); dog is direction of growth.

Figure 3 .
Figure 3. Raman spectrum of Arctica islandica showing the typical aragonite peaks (gray line).The exact position of the polyene peaks R 1 and R 4 was determined by using a peak fitting routine based on a Gaussian function (black line).

Figure 4 .
Figure 4. Arctica islandica shell growth under controlled conditions.(a) Total growth and (b) instantaneous growth rate during the temperature experiment.(c) Total growth during the food experiment.

Figure 5 .
Figure 5.Effect of temperature increase on biomineral orientation.(a) Position of the Raman maps of the three specimens reared at 10 and 15 • C. Dotted lines indicate the location of the calcein marks; dog is direction of growth.(b) Raman spectral maps of R ν1/T a .Left images of each column represents shell portion formed at 10 • C, right images represent shell portions formed at 15 • C. First row of pairs refers to oOSL, the other two represent the iOSL; scale bars = 10 µm.(c) Proportions of biominerals with R ν1/T a > 2.5 a.u. with respect to the total map area.Asterisks indicate significant difference between the orientation of iOSL microstructures formed at 10 and 15 • C (p < 0.05).

Figure 6 .
Figure 6.SEM images of Arctica islandica shell microstructures formed at 10 • C (left column) and at 15 • C (right column).The sketch indicates the position of the images 1 mm away from the calcein mark (gray line).The first row of images refers to the oOSL, the other two row refers to the iOSL.Scale bars if not otherwise indicated = 5 µm.

Figure 7 .
Figure 7. Effect of different diets based on (a) food type 1, (b) food type 2 and (c) ambient food on biomineral orientation.The optical microscope images indicate the position of the Raman scans.Dotted line marks the start of the experiment.The portion of shell prior the line was formed during the acclimation phase; dog is direction of growth.The Raman spectral maps indicate the ratio R ν1/T a for each data point of the scan.For each shell, maps on the left represent shell portions during the experiment, maps on the right represent shell portions formed during the acclimation phase.In the acclimation portion of the sample reared with ambient food, a significant change in the microstructure orientation is visible.The respective area of the Raman map was not considered in further calculations because it was influenced by the emersion and transportation stress at the start of the experiment.Scale bars = 10 µm.The graphs show the proportions of biominerals of oOSL and iOSL with R ν1/T a > 2.5 a.u. with respect to the total map area.

Figure 8 .
Figure 8. SEM images of Arctica islandica shell microstructures formed during the acclimation phase at AWI (left column) and during the food experiment (right column).Scale bars = 4 µm.

Figure 9 .
Figure 9. Effects of diet on shell pigment distribution.(a) Raman spectral maps of the 1524 cm −1 band representing the distribution of the polyenes in the shell cultured with food type 2. Dotted line marks the start of the experiment; dog is direction of growth.(b) The graph shows the thickness of the pigmented layer over the whole shell thickness before and during the food experiments.

Table 1 .
List of the studied specimens of Arctica islandica and experimental conditions.