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Trace elemental alterations of bivalve shells following transgenerational exposure to ocean acidification: Implications for geographical traceability and environmental reconstruction
Journal Pre-proof Trace elemental alterations of bivalve shells following transgenerational exposure to ocean acidification: Implications for geographical traceability and environmental reconstruction
Liqiang Zhao, Stefania Milano, Kentaro Tanaka, Jian Liang, Yuewen Deng, Feng Yang, Eric O. Walliser, Bernd R. Schöne PII:
S0048-9697(19)35495-6
DOI:
https://doi.org/10.1016/j.scitotenv.2019.135501
Reference:
STOTEN 135501
To appear in:
Science of the Total Environment
Received date:
8 July 2019
Revised date:
9 November 2019
Accepted date:
11 November 2019
Please cite this article as: L. Zhao, S. Milano, K. Tanaka, et al., Trace elemental alterations of bivalve shells following transgenerational exposure to ocean acidification: Implications for geographical traceability and environmental reconstruction, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.135501
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© 2019 Published by Elsevier.
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Trace elemental alterations of bivalve shells following transgenerational exposure to ocean acidification: implications for geographical traceability and environmental reconstruction
Liqiang Zhao 1, 2, 3, *, Stefania Milano 2, 4, Kentaro Tanaka 3, Jian Liang 5, Yuewen Deng 1, Feng Yang 6, Eric O. Walliser 2, Bernd R. Schöne 2 College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China
2
Institute of Geosciences, University of Mainz, Mainz 55128, Germany
3
Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba 277 -8564, Japan
4
Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Leipzig 04103, Germany
5
Department of Fisheries, Tianjin Agricultural University, Tianjin 300384, China
6
College of Life Science and Fisheries, Dalian Ocean University, Dalian 116023, China
* Corresponding author.
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E-mail address: [email protected]; [email protected] (L. Zhao)
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Journal Pre-proof Abstract Trace elements of bivalve shells can potentially record the physical and chemical properties of the ambient seawater during shell formation, thereby providing valuable information on environmental conditions and provenance of the bivalves. In an acidifying ocean, whether and how seawater acidification affects the trace elemental composition of bivalve shells is largely unknown. Here, we investigated the transgenerational effects of OA projected for the
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end of the 21st century on the incorporation of trace elements into shells of the Manila clam,
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Ruditapes philippinarum. Neither seawater pH nor transgenerational exposure affected the
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Mg and Sr composition of the shells. Compared with clams grown under ambient conditions,
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specimens exposed to elevated CO2 levels incorporated significantly higher amounts of Cu,
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Zn, Ba and Pb into their shells, in line with the fact that at lower pH, these elements in seawater occur at higher fractions in free forms which are biologically available.
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Transgenerational effects manifested themselves significantly during the incorporation of Cu
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and Zn into the shells, most likely because Cu and Zn are biologically essential trace elements
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for metabolic processes. In addition, the plasticity of metabolism toward energetic efficiency following transgenerational exposure confers the clams enhanced ability to discriminate against Cu and Zn during the uptake from the ambient environment to the site of calcification. In the context of near-future OA scenarios, these findings may provide unique insights into the two primary applications of trace elements of bivalve shells as geographical tracers and proxies of environmental conditions.
Keywords: Bivalve shells, trace elements, ocean acidification, transgenerational acclimation, Ruditapes philippinarum
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1. Introduction Trace elemental signatures of bivalve shells can potentially provide a wealth of environmental information. Physical and chemical properties of the surrounding environment (temperature, salinity, food availability, oxygen, pH, etc.) that prevail during shell growth are preserved in the shells in the form of variations of trace elements (Schöne, 2013; Prendergast et al., 2017). Using periodic shell growth patterns as a calendar and time
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gauge, these environmental proxy records can be placed in a precise temporal context (Butler and Schöne, 2017). As such, trace elemental signatures of bivalve shells , over the last
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decades, have attracted much attention as natural fingerprints to trace the geographical
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origin (e.g., Iguchi et al., 2013; Ricardo et al., 2015; Zhao et al., 2019a) and identify the
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population connectivity (e.g., Becker et al., 2007; Fodrie et al., 2011; Gomes et al., 2016; Norrie et al., 2016), and as ultra-high-resolution proxies to reconstruct past environmental
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changes (e.g., Yan et al., 2013; Marali et al., 2017; Zhao et al., 2017a).
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However, gap of knowledge regarding how rapidly changing marine environments affect the incorporation of trace elements into the shells exists, likely hindering the promise of trace elemental signatures of bivalve shells as both geographical tracers and environmental proxies. Ocean acidification (OA), for example, decreases the availability of carbonate at an unprecedented rate (IPCC, 2014). These changes in seawater carbonate chemistry not only have severely affected many calcifying organisms (Gazeau et al., 2013; Kroeker et al., 2013), but also have profoundly modified the supply, cycling and availability of biologically relevant trace elements in the ocean (Millero et al., 2009; Hoffmann et al., 2012). Hence, OA may affect the trace elemental signatures of bivalve shells by directly altering the bioavailability of trace elements in seawater or indirectly by affecting the
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Journal Pre-proof physiological uptake and incorporation into the shells. In particular, many coastal and estuarine ecosystems already experience large natural pH fluctuations on diel, seasonal and interannual time-scales (Baumann et al., 2015; Law et al., 2017; Zhao et al., 2018a), which can complicate the interpretation of proxy records without a holistic understanding of how pH affects the trace elemental signatures of bivalve shells.
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In a rapidly acidifying ocean, environmental reconstructions and geographical
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traceability based on bivalve shell geochemistry may be biased by effects arising from the
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plasticity of physiological metabolism. There is accumulating evidence that demonstrates the
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rapid transgenerational acclimation and corresponding metabolic plasticity to near-future
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OA scenarios projected for the end of this century observed in short-lived marine bivalve species, such as the Sydney rock oyster, Saccostrea glomerata (Parker et al., 2015), the
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Manila clam, Ruditapes philippinarum (Zhao et al., 2018b), the blue mussel, Mytilus edulis
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(Thomsen et al., 2017) and the Asian date mussel, Musculista senhousia (Zhao et al., 2019b,
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2019c). If this is the case, and given the well-documented metabolic controls over the trace elemental composition of bivalve shells (Gillikin et al., 2005; Planchon et al., 2013; Poulain et al., 2015; Zhao et al., 2017b) as well as the metabolic plasticity, then one would expect that the degree to which metabolic activities affect the trace elemental uptake and incorporation into bivalve shells changes following transgenerational exposure to OA. In order to underpin the application of trace elements of bivalve shells as appropriate tracers and proxies in the face of OA, such gap of knowledge (i.e., the influence of metabolic plasticity) should thus be closed.
The present study aims to tackle the question of whether, and to what extent, long-
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Journal Pre-proof term, transgenerational exposure to near-future OA scenarios projected by the end of this century affects the incorporation of trace elements into bivalve shells. More recently, our studies have demonstrated the great capacity of the juvenile Manila clam, R. philippinarum to cope with OA following transgenerational acclimation by maintaining the acid-base homeostasis (Zhao et al., 2017c) and generating the plasticity of metabolism (Zhao et al., 2018b). In this study, we test whether and how the trace elemental composition of R.
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philippinarum shells varies following transgenerational exposure to OA. These findings will
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provide valuable information for both environmental reconstructions and geographical
2. Materials and methods
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2.1. Clam collection and maintenance
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traceability based on bivalve shell geochemistry in an acidifying ocean.
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Adult R. philippinarum specimens (32 ± 2.3 mm shell length) were collected, on 2 April 2014
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from the intertidal zone of Liangshui Bay, Yellow Sea (39°04′14.41″ N, 122°01′47.70″ E). Upon arrival in the laboratory within 2 h, the clams were acclimated in 600-L circulating seawater systems for two weeks, during which environmental conditions were maintained at levels comparable to those at the site of sampling and day of collection (i.e., temperature of 9 °C, salinity of 32, pH 8.1). They were fed with the marine algae Chlorella vulgaris every two days. During the acclimation period no mortality occurred.
2.2. Experimental setup The transgenerational experiment has been described in detail by Zhao et al. (2017c). In brief, following laboratory acclimation, the clams were exposed to two scenarios of OA (i.e.,
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Journal Pre-proof pH 8.1 and 7.7), the latter corresponding to the seawater pH level projected for the year 2100. Two experimental systems, as schematically illustrated by Xu et al. (2016), were built (Fig. 1). Each system contained about 600-L circulating seawater and 450 clams. Elevated seawater pCO2 and corresponding lowered pH were adjusted by continuously bubbling CO 2 gas into the system. To facilitate gonadal maturation, a well-established protocol (i.e., water temperature progressively increasing from 10 to 20 °C within 30 days and subsequently kept
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constant for 40 days; Xu et al., 2016) was employed. To meet the nutritional requirement
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during gonadal ripening, the clams were fed with an equal mixture of microalgal species , C.
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vulgaris, Isochrysis galbana, Chaetoceros muelleri and Nitzschia closterium at a daily ration
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of approximate 40,000 cells mL-1. To maintain seawater quality, half of the system volume
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was renewed every 4-5 days.
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Following 70 days of artificial induction of gonadal maturation, 100 clams were
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randomly collected from each system and induced to spawn by temperature shock (+ 4 °C).
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About 95 % and 96 % of individuals exposed to pH 8.1 and 7.7 spawned within 8 h, respectively (Xu et al., 2016). Fertilized eggs were filtered through a nylon mesh (60 µm), rinsed three times in sand-filtered seawater to get rid of excessive sperms and other gonadal tissues and then incubated in pre-prepared 100-L aquaria (about 50 eggs mL-1). After 26 h of fertilization, newly hatched D-veliger larvae were collected and randomized into 100-L aquaria at a density of 10 larvae mL-1, according to the transgenerationally experimental design (as shown in Fig. 1). Three replicates were carried out for each experimental treatment. Room temperature was kept stable at 20 °C. From day 1 to day 3, larvae were fed with I. galbana at a daily ration of 20,000 cells mL-1 and, from day 4 onward with a mixture of I. galbana and C. vulgaris at a daily ration of 40,000 cells mL-1. 100% of the aquarium was
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Journal Pre-proof renewed every two days using pre-adjusted sand-filtered seawater (with consistent environmental conditions) to maintain water quality. While larvae developed into the pediveliger stage (12-15 days after fertilization, 180-210 µm shell length, characterized with eyespot and protruding foot), all aquaria were renewed twice daily so as to facilitate larval settlement. Following larval metamorphosis which took about 5 days, newly settled juveniles were quantified and approximate 1000 individuals for each aquarium were randomly
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sampled for the following 30-days nursery (Fig. 1), during which the clams were fed with C.
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vulgaris at a daily ration of 80,000 cells mL-1. Experimental conditions were maintained
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comparable to those during larval stage, and daily cares were also handled in the same
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manner.
2.3. Trace elemental analysis via LA-ICP-MS
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In the present study, six elements (Mg, Cu, Zn, Sr, Ba and Pb) which are widely used as
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proxies for past environmental reconstruction and tracers for geographical traceability
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(Schöne and Krause, 2016; Prendergast et al., 2017) were analyzed in R. philippinarum shells. Specifically, following 30 days of nursery, a total of 20 juveniles from each experimental treatment were randomly collected to analyze the trace elemental composition of the shells. The right valve of each juvenile was carefully taken, soaked in Teflon vials with 15 ml 15 % H2O2 buffered with 0.05 M NaOH for 14 h to remove organic matter, and then rinsed with deionized water, air-dried and mounted on a glass slide. Trace elements were analyzed using an ESI NWR 193 ArF excimer laser ablation system coupled to an Agilent 7500ce qua drupole ICP-MS in ‘spot’ mode (Zhao et al., 2017c). Ablation was achieved using a beam diameter of 100 µm, an energy density of ~2.8 J cm-2 and a pulse repetition rate of 5 Hz. Backgrounds were analyzed for 15 s, followed by 25 s ablation time and then 20 s wash out. Monitored
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Journal Pre-proof isotopes were 26Mg, 65Cu, 66Zn, 88Sr, 137Ba and 208Pb. NIST SRM 610 (synthetic glass) was used as calibration material, and the preferred values reported in GeoReM database (http://georem.mpch-mainz.gwdg.de/, application version 18) (Jochum et al., 2005, 2011) were used as the ‘true’ concentrations to calculate the elemental concentrations in juvenile shells. MACS-3 (synthetic calcium carbonate) and USGS BCR-2G (basalt glass) were simultaneously measured as quality control materials (QCM) so as to monitor accuracy and
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reproducibility of the measurements. All reference materials were analyzed at the beginning
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and at the end of a sequence and after around 60 measurements of the shells. For all
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materials, 43Ca was used as internal standard. For the reference materials, we applied the Ca
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concentrations reported in the GeoReM database, and for the samples 56.03 wt %., the
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stoichiometric CaO content of aragonite. The time-resolved signal was processed using the program GLITTER 4.4.1 (www.glitter-gemoc.com, Macquarie University, Sydney, Australia).
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Reproducibility expressed as the relative standard deviation based on repeated
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measurements of the QCM was always better than 7 % for MACS-3 and 3 % for USGS BCR-2G.
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The measured Mg, Cu, Zn, Sr, Ba and Pb concentrations of MACS-3 agreed within 3.2 %, 2.5 %, 9.5 %, 4.1 %, 10.1 % and 14.0 % with the preliminary reference values (personal communication S. Wilson, USGS, in Jochum et al. 2012) and within 12.1 %, 12 %, 32 %, 0.7 %, 1.5 % 3.0 % for USGS BCR-2G, respectively. Minimum detection limits were 0.70 µg g-1, 0.26 µg g -1, 0.19 µg g -1, 0.012 µg g-1, 0.063 µg g-1, and 0.016 µg g -1 for Mg, Cu, Zn, Sr, Ba and Pb, respectively.
2.4. Statistical analysis All experimental data were statistically analyzed using SPSS Statistics 19.0. The ShapiroWilk’s test and Levene’s F-test were employed to examine the normality and homogeneity
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Journal Pre-proof of the data, respectively. Subsequently, the combined effects of adult treatment and seawater pH on the trace elemental values (expressed as molar ratios relative to calcium) of juvenile shells were examined by means of two-way analysis of variance (two-way ANOVA) followed by the LSD post-hoc test. In the present study, statistically significant difference
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was set at p < 0.05.
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3. Results
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In the present study, all experimental data are normally distributed and treatment variances
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are homogeneous. According to two-way ANOVA analysis, transgenerational exposure,
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seawater pH, as well as interaction between these two factors, did not significantly affect the incorporation of Mg (Fig. 2A) and Sr (Fig. 2B) into juvenile R. philippinarum shells (p >
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0.05). On average, the amount of Cu (Fig. 2C) incorporated into the shells was significantly
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affected by both transgenerational exposure and seawater pH (p < 0.05), yet no significant
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interaction between these two factors was observed (p > 0.05). With decreasing seawater pH from 8.1 to 7.7, the amount of Zn (Fig. 2D) incorporated into the shells increased significantly (p < 0.05), however transgenerational exposure did not significantly affect the incorporation of Zn into the shells (Fig. 2D; p > 0.05). Interestingly, the interaction between transgenerational exposure and seawater pH on the Zn composition of the shells was significant (Fig. 2D; p < 0.05). Specifically, when exposed to pH 7.7, juvenile clams with a prior history of transgenerational exposure to pH 7.7 incorporated significantly lower amounts of Zn into the shells than those originating from parents reared at pH 8.1 during the gonadal conditioning (Fig. 2D; p < 0.05). Irrespective of transgenerational exposure which exhibited no significant effects on the incorporation of Ba (Fig. 2E) and Pb (Fig. 2F) into
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Journal Pre-proof juvenile shells (p > 0.05), the shells forming at pH 7.7 contained significantly larger amounts of Ba and Pb than those grown at pH 8.1 (p < 0.05).
4. Discussion The present study represents the first investigation of transgenerational effects of OA on the
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trace elemental composition of marine bivalves. Our results demonstrate different responses of elemental incorporation processes into R. philippinarum shells. To better
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elucidate effects of transgenerational exposure, seawater pH as well as their interaction on
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the Mg, Sr, Cu, Zn, Ba and Pb composition of the shells, a schematic model illustrated in Fig.
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3, which is based on the findings of the OA effects on the speciation of metals in seawater (Millero et al., 2009) and the pathways of trace elements from the environment into bivalve
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shells (Zhao et al., 2017d), is built. Given that the juveniles were grown under strictly
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controlled experimental conditions, influences of factors such as seawater temperature,
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salinity, food availability, trace elemental concentrations were minimized. Therefore, seawater pH and corresponding pH-dependent speciation of trace elements (i.e., the percent of free ions for a given element) are considered environmental factors affecting trace elemental records of R. philippinarum shells in the model. The ability of bivalves to discriminate divalent ions with similar ionic radii and electrochemical properties as Ca ion during shell formation is mainly metabolically dependent (Zhao et al., 2017d). It seems, therefore, reasonable to incorporate the metabolic performance (shown in Zhao et al., 2018b) into the model as an important physiological factor.
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Journal Pre-proof According to this proposed schematic model, it is not surprising that neither seawater pH nor transgenerational exposure affected the amount of Mg and Sr incorporated into the shells. In seawater, Mg and Sr behave conservatively and exist exclusively as free ions (Bruland, 1983), the latter known as major bioavailable forms which are taken up by bivalves (Campbell, 1995). In particular, it has been shown that the solubility and bioavailability of metals in the free form are not significantly affected by changes of seawater pH and
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carbonate system (Millero et al., 2009). As such, it seems reasonable to conclude that
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seawater pH does not affect the uptake of Mg and Sr into bivalve shells. A recent study
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examining the effects of elevated pCO2 on the trace elemental composition of shells of the
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green-lipped mussel, Perna canaliculus (Norrie et al., 2018) has also come to a similar conclusion. On the other hand, during the uptake from the surrounding seawater to the site
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of calcification, Mg 2+ and Sr2+ ions are not regarded simply as metabolic analogues of Ca2+
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and consequently mainly transported by intercellular Ca 2+ pathways (Zhao et al., 2017d), the
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latter demonstrating less metabolic interferences with the transport mechanisms of Mg and
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Sr. Hence, the possibility that metabolic plasticity following transgenerational exposure to OA affects the uptake of Mg and Sr during shell building can be reasonably ruled out.
Unlike Mg and Sr which occur predominantly in the free form in seawater, Cu, Zn, Ba and Pb tend to form strong complexes with both hydroxide and carbonate (Bruland, 1983), and thereby likely experience significant changes of speciation as seawater pH decreases (Millero et al., 2009). In particular, under acidified conditions, the solubility and bioavailable fraction (i.e., the percent of free ions) of trace elements mentioned above increase rapidly as schematically illustrated in Figure 3. The pH-dependent nature of the bioavailability of Cu, Zn, Ba and Pb probably offers the most likely explanation for observations of increased amounts
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Journal Pre-proof of these elements incorporated into shells of CO2-exposed R. philippinarum. In addition, these findings compare well with observations in larval shells of mussels , Mytilus californianus and M. galloprovincialis (Frieder et al., 2014) and juvenile shells of P. canaliculus (Norrie et al., 2018).
Another possible explanation for increasing incorporation of Cu, Zn and Pb into the
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shells with decreasing seawater pH is related to metabolic effects. Divalent ions including
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Cu2+, Zn2+ and Pb2+ are largely taken up by bivalves as analogues of Ca2+ (Markich and Jeffree,
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1994). In particular, intracellular Ca2+ transport pathways such as Ca2+-ATPase and Ca 2+-
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channel are mainly involved in the uptake of Cu, Zn and Pb from the environment to the site
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of calcification (Zhao et al., 2017d). Under acidified conditions, the clams may shift the energy budget toward compensating for the acid-base imbalance in the calcifying fluid by
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active removal of excessive protons (Zhao et al., 2017c), consequently reducing the amount
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of energy allocated to discriminate against Cu2+, Zn2+ and Pb2+ during intracellular transport
shells.
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of Ca2+ and eventually resulting in higher amounts of these elements incorporated into the
Transgenerational effects are most pronounced during the uptake and incorporation of Cu and Zn into R. philippinarum shells, which may be related to the plasticity of metabolism as shown in transgenerationally acclimated clams under acidified conditions (Zhao et al., 2018b). As biologically essential trace elements for metabolic processes in marine bivalves, Cu and Zn can be highly metabolically regulated even when taken in low doses (Pan and Wang, 2012). Rapid improved tolerance of Cu and Zn following transgenerational exposure to metal stress has been found in the oyster, Crassostrea sikamea (Weng and Wang, 2014),
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Journal Pre-proof which can be related to the increased induction of metallothionein-like proteins. If this is the case, then one would expect an increase of energy allocated to the protein synthesis and secretion. This assumption is in line with recent observations
that, following
transgenerational acclimation, CO 2-exposed juvenile clams are capable of implementing more ATP-efficient and less costly ionic regulatory mechanisms to compensate for the acidbase imbalance at the site of calcification (Zhao et al., 2017c) and preferentially extracting
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metabolically generated CO2 rather than active transport seawater dissolved inorganic
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carbon which is energetically expensive to calcify (Zhao et al., 2018b), thereby generating
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more energy available for the discrimination and elimination of Cu and Zn in the intracellular
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Ca2+ transport pathways. In this regard, omics technologies (genomics, transcriptomics,
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metabolomics and proteomics) may open an avenue in elucidating the underlying mechanisms at lower levels of biological organization, especially regarding the role of
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specific proteins related to metabolic pathways and ionic transporters in the face of long -
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term and transgenerational exposure to seawater acidification.
Transgenerational effects did not manifest themselves when the clams took up Pb from the environment to the site of calcification. Lead could act as a metabolic poison and produce intracellular changes (Pan and Wang, 2012). Yet, marine bivalves are usually well equipped to detoxify the impact of Pb via energetically efficient processes (such as cellular redistribution) rather than active efflux systems (Viarengo and Nott, 1993). If the detoxification of Pb is not extremely energetically expensive and in particular not closely correlated to the synthesis and secretion of metallothionein-like proteins, then one would not be surprised to see the limited role of the plasticity of energy metabolism generated
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Journal Pre-proof following transgenerational acclimation in the uptake and incorporation of Pb into R. philippinarum shells.
Overall, findings of the present study have important implications for the application of trace elemental signatures of bivalve shells as geographical tracers and environmental proxies in an acidifying ocean. The incorporation of Mg and Sr remained virtually unaffected
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following long-term transgenerational exposure to OA, suggesting that environmental
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information such as seawater temperature deduced from shell Mg/Ca and Sr/Ca values
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should be unbiased in the face of rapidly changing environment. However, the influence of
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pH on the incorporation of Ba and Pb into bivalve shells should be taken into consideration
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when shell Ba/Ca and Pb/Ca ratios are explored as proxies for primary productivity and pollution (Thébault et al., 2009; Holland et al., 2014), especially in coastal waters where diel,
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seasonal and annual variations in seawater pH are remarkable. While a number of studies
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have reported the potential of shell Cu/Ca and Zn/Ca ratios as indicators of water Cu and Zn
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pollution (Schöne and Krause, 2016), given transgenerational effects of OA on their incorporation processes into the shells, caution should be taken in terms of both geographical traceability and environmental reconstruction.
5. Conclusions The present study provides valuable information on the transgenerational effects of elevated pCO2 on the trace elemental signatures of marine bivalve shells, which may further foster the two primary applications of trace element data of bivalve shells in the field of geographical traceability and environmental reconstruction. Specifically, transgenerational
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Journal Pre-proof acclimation and elevated pCO2 do not affect the incorporation of Mg and Sr into the shells, thereby ensuring their robustness as both geographical tracers and environmental proxies. Due to the pH-dependence of Ba and Pb in free forms in seawater, the combination of Ba and Pb records of bivalve shells (which are also less metabolically affected) holds the potential to reconstruct the history of seawater pH and carbonate chemistry. In many estuarine and coastal habitats which naturally experience large pH fluctuations on different-
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time scales, it remains possible to discriminate the geographical origin of bivalves using shell
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Ba and Pb signatures. It is evident that the incorporation of biologically essential trace
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elements (such as Cu and Zn) is affected by both seawater pH and the history of
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transgenerational exposure, consequently hindering their potential as geographical tracers
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and environmental proxies. Yet, studies on the Cu and Zn composition of bivalve shells can likely open an existing new avenue in the research of biomineralization which is highly
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biologically controlled.
Acknowledgments
The authors would like to thank Regina Mertz-Kraus at the University of Mainz for assistance with LA-ICP-MS analyses. The current study has been made possible by a research grant from the framework (FP7) of the Marie Curie International Training Network ARAMACC (604802) to BRS and LZ and by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants to LZ (17P17333) and KT (17K17669).
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Law, C.S., Bell, J.J., Bostock, H.C., Cornwall, C.E., Cummings, V.J., Currie, K., Davy, S.K.,
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Journal Pre-proof Norrie, C.R., Dunphy, B.J., Baker, J.A., Lundquist, C.J., 2016. Local-scale variation in trace elemental fingerprints of the estuarine bivalve Austrovenus stutchburyi within and between estuaries. Mar. Ecol. Prog. Ser. 559, 89–102. Pan, K., Wang, W.X., 2012. Trace metal contamination in estuarine and coastal environments in China. Sci. Total Environ. 421–422, 3–16. Parker, L.M., O'Connor, W.A., Raftos, D.A., Pörtner, H.O., Ross, P.M., 2015. Persistence of
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positive carryover effects in oysters following transgenerational exposure to ocean
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acidification. PLoS One. 10, e0132276.
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Planchon, F., Poulain, C., Langlet, D., Paulet, Y.M., André, L., 2013. Mg-isotopic fractionation
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pathway and calcification process of bivalves. Geochim. Cosmochim. Acta 121, 374–397. Prendergast, A.L., Versteegh, E.A.A., Schöne, B.R., 2017. New research on the development
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biogenic carbonates. Palaeogeogr. Palaeoclimatol. Palaeoecol. 484, 1–6.
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Ricardo, F., Genio, L., Leal, M.C., Albuquerque, R., Queiroga, H., Rosa, R., Calado, R., 2015. Trace element fingerprinting of cockle (Cerastoderma edule) shells can reveal harvesting location in adjacent areas. Sci. Rep. 5, 11932. Schöne, B.R., 2013. Arctica islandica (bivalvia): a unique paleoenvironmental archive of the northern north Atlantic Ocean. Glob. Planet. Chang. 111, 199–225. Schöne, B.R., Krause, R.A., 2016. Retrospective environmental biomonitoring – Mussel Watch expanded. Global and Planetary Change 144, 228-251. Thomsen, J., Stapp, L.S., Haynert, K., Schade, H., Danelli, M., Lannig, G., Wegner, K.M., Melzner, F., 2017. Naturally acidified habitat selects for ocean acidification-tolerant mussels. Sci. Adv. 3, e1602411.
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Journal Pre-proof Thébault, J., Chauvaud, L., L'Helguen, S., Clavier, J., Barats, A., Jacquet, S., Pécheyran, C., Amouroux, D., 2009. Barium and molybdenum records in bivalve shells: geochemical proxies for phytoplankton dynamics in coastal environments? Limnol. Oceanogr. 54, 1002–1014. Viarengo, A., Nott, J.A., 1993. Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comp. Biochem. Physiol. 104C, 355–372.
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Wen, N., Wang, W.X., 2014. Improved tolerance of metals in contaminated oyster larvae.
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Aquat. Toxicol. 146, 61–69.
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Xu, X., Yang, F., Zhao, L., Yan, X., 2016. Seawater acidification affects the physiological
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energetics and spawning capacity of the Manila clam Ruditapes philippinarum during
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gonadal maturation. Comp. Biochem. Phys. 196A, 20-29. Yan, H., Shao, D., Wang, Y., Sun, L., 2013. Sr/Ca profile of long-lived Tridacna gigas bivalves
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from South China Sea: a new high-resolution SST proxy. Geochim. Cosmochim. Acta 112,
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Zhao, L., Liu, B., An, W., Deng, Y., Lu, Y., Liu, B., Wang, L., Cong, Y., Sun, X., 2019b. Assessing the impact of elevated pCO2 within and across generations in a highly invasive fouling mussel (Musculista senhousia). Sci. Total Environ. 689, 322–331. Zhao, L., Liu, L., Liu, B., Liang, J., Lu, Y., Yang, F., 2019c. Antioxidant responses to seawater acidification in an invasive fouling mussel are alleviated by transgenerational acclimation. Aquat. Toxicol. 217, 105331. Zhao, L., Schöne, B.R., Mertz-Kraus, R., 2017b. Controls on strontium and barium incorporation into freshwater bivalve shells (Corbicula fluminea). Palaeogeogr. Palaeoclimatol. Palaeoecol. 465, 386–394.
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Journal Pre-proof Zhao, L.., Schöne, B.R., Mertz-Kraus, R., 2017d. Delineating the role of calcium in shell formation and elemental composition of Corbicula fluminea (Bivalvia). Hydrobiologia 790, 259–272. Zhao, L., Schöne, B.R., Mertz-Kraus, R., Yang, F., 2017c. Sodium provides unique insights into transgenerational effects of ocean acidification on bivalve shell formation. Sci. Total. Environ. 577, 360–366.
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Nd/144Nd ratios. Mar. Environ. Res. 148, 12–18.
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Determination of the geographical origin of marine mussels (Mytilus spp.) using
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Zhao, L., Walliser, E.O., Mertz-Kraus, R., Schöne, B.R., 2017a. Unionid shells (Hyriopsis
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cumingii) record manganese cycling at the sediment-water interface in a shallow eutrophic lake in China (Lake Taihu). Palaeogeogr. Palaeoclimatol. Palaeoecol. 484, 97–
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Zhao, L., Milano, S., Walliser, E.O., Schöne, B.R., 2018a. Bivalve shell formation in a naturally
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CO2-enriched habitat: unraveling the resilience mechanisms from elemental signatures. Chemosphere 203, 132–138. Zhao, L., Yang, F., Milano, S., Han, T., Walliser, E.O., Schöne, B.R., 2018b. Transgenerational acclimation to seawater acidification in the Manila clam Ruditapes philippinarum: Preferential uptake of metabolic carbon. Sci. Total. Environ. 627, 95–103.
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Figure captions Figure 1 Flow chart of the transgenerational experimental design (for more details refer to the text).
Figure 2 The amount of Mg (A), Sr (B), Cu (C), Zn (D), Ba (E) and Pb (F) incorporated into the shells of the Manila clam, Ruditapes philippinarum, following transgenerational exposure to
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pH 8.1 (i.e., non-acclimated line) and pH 7.7 (i.e., transgenerational-acclimated line). Trans. = transgenerational. Results of two-way ANOVA analysis were given in corresponding panels.
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expressed as mean ± standard deviation (n = 20)
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Trace elemental concentrations of shells are reported as molar ratios relative to calcium and
Figure 3 A schematic model developed to demonstrate how transgenerational exposure to
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seawater acidification affects the incorporation of trace elements into the shells of the
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Manila clam, Ruditapes philippinarum. This model is primary built on the pathways of trace
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elements from the environment into bivalve shells (as schematically illustrated by Zhao et al. 2017d), according to which Ca 2+ from the ambient seawater is largely transported via intracellular pathways (Ca2+-ATPase and Ca2+-channel) and in part via intercellular pathway (passive diffusion) to the calcifying fluid. Divalent ions Cu2+, Zn2+ and Pb2+ are taken up by the bivalves via intracellular Ca2+ pathways, thus highly metabolically regulated, whereas intercellular passive diffusion is considered the predominant transport pathway of Mg 2+, Sr2+ and Ba2+ (Zhao et al., 2017d). In addition, the bioavailable forms (i.e., free forms) of Cu, Zn, Ba and Pb in seawater are highly pH-dependent, i.e., with decreasing seawater pH concentrations of their free ions increase (Millero et al., 2009). For more details regarding mechanisms of shell calcification please refer to Zhao et al. (2018b) and Lu et al. (2018).
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Journal Pre-proof Graphical abstract
Research highlights: 1.
Neither seawater pH and transgenerational exposure affects the Mg and Sr
composition of bivalve shells. 2.
With decreasing seawater pH, the amounts of Cu, Zn, Ba and Pb taken up in clam
Transgenerational exposure to seawater acidification affects the Cu and Zn
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3.
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shells increase.
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composition of bivalve shells.
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Figure 1
Figure 2
Figure 3
Liqiang Zhao, Stefania Milano, Kentaro Tanaka, Jian Liang, Yuewen Deng, Feng Yang, Eric O. Walliser, Bernd R. Schöne PII:
S0048-9697(19)35495-6
DOI:
https://doi.org/10.1016/j.scitotenv.2019.135501
Reference:
STOTEN 135501
To appear in:
Science of the Total Environment
Received date:
8 July 2019
Revised date:
9 November 2019
Accepted date:
11 November 2019
Please cite this article as: L. Zhao, S. Milano, K. Tanaka, et al., Trace elemental alterations of bivalve shells following transgenerational exposure to ocean acidification: Implications for geographical traceability and environmental reconstruction, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.135501
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© 2019 Published by Elsevier.
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Trace elemental alterations of bivalve shells following transgenerational exposure to ocean acidification: implications for geographical traceability and environmental reconstruction
Liqiang Zhao 1, 2, 3, *, Stefania Milano 2, 4, Kentaro Tanaka 3, Jian Liang 5, Yuewen Deng 1, Feng Yang 6, Eric O. Walliser 2, Bernd R. Schöne 2 College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China
2
Institute of Geosciences, University of Mainz, Mainz 55128, Germany
3
Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba 277 -8564, Japan
4
Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Leipzig 04103, Germany
5
Department of Fisheries, Tianjin Agricultural University, Tianjin 300384, China
6
College of Life Science and Fisheries, Dalian Ocean University, Dalian 116023, China
* Corresponding author.
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E-mail address: [email protected]; [email protected] (L. Zhao)
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Journal Pre-proof Abstract Trace elements of bivalve shells can potentially record the physical and chemical properties of the ambient seawater during shell formation, thereby providing valuable information on environmental conditions and provenance of the bivalves. In an acidifying ocean, whether and how seawater acidification affects the trace elemental composition of bivalve shells is largely unknown. Here, we investigated the transgenerational effects of OA projected for the
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end of the 21st century on the incorporation of trace elements into shells of the Manila clam,
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Ruditapes philippinarum. Neither seawater pH nor transgenerational exposure affected the
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Mg and Sr composition of the shells. Compared with clams grown under ambient conditions,
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specimens exposed to elevated CO2 levels incorporated significantly higher amounts of Cu,
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Zn, Ba and Pb into their shells, in line with the fact that at lower pH, these elements in seawater occur at higher fractions in free forms which are biologically available.
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Transgenerational effects manifested themselves significantly during the incorporation of Cu
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and Zn into the shells, most likely because Cu and Zn are biologically essential trace elements
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for metabolic processes. In addition, the plasticity of metabolism toward energetic efficiency following transgenerational exposure confers the clams enhanced ability to discriminate against Cu and Zn during the uptake from the ambient environment to the site of calcification. In the context of near-future OA scenarios, these findings may provide unique insights into the two primary applications of trace elements of bivalve shells as geographical tracers and proxies of environmental conditions.
Keywords: Bivalve shells, trace elements, ocean acidification, transgenerational acclimation, Ruditapes philippinarum
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1. Introduction Trace elemental signatures of bivalve shells can potentially provide a wealth of environmental information. Physical and chemical properties of the surrounding environment (temperature, salinity, food availability, oxygen, pH, etc.) that prevail during shell growth are preserved in the shells in the form of variations of trace elements (Schöne, 2013; Prendergast et al., 2017). Using periodic shell growth patterns as a calendar and time
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gauge, these environmental proxy records can be placed in a precise temporal context (Butler and Schöne, 2017). As such, trace elemental signatures of bivalve shells , over the last
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decades, have attracted much attention as natural fingerprints to trace the geographical
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origin (e.g., Iguchi et al., 2013; Ricardo et al., 2015; Zhao et al., 2019a) and identify the
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population connectivity (e.g., Becker et al., 2007; Fodrie et al., 2011; Gomes et al., 2016; Norrie et al., 2016), and as ultra-high-resolution proxies to reconstruct past environmental
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changes (e.g., Yan et al., 2013; Marali et al., 2017; Zhao et al., 2017a).
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However, gap of knowledge regarding how rapidly changing marine environments affect the incorporation of trace elements into the shells exists, likely hindering the promise of trace elemental signatures of bivalve shells as both geographical tracers and environmental proxies. Ocean acidification (OA), for example, decreases the availability of carbonate at an unprecedented rate (IPCC, 2014). These changes in seawater carbonate chemistry not only have severely affected many calcifying organisms (Gazeau et al., 2013; Kroeker et al., 2013), but also have profoundly modified the supply, cycling and availability of biologically relevant trace elements in the ocean (Millero et al., 2009; Hoffmann et al., 2012). Hence, OA may affect the trace elemental signatures of bivalve shells by directly altering the bioavailability of trace elements in seawater or indirectly by affecting the
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Journal Pre-proof physiological uptake and incorporation into the shells. In particular, many coastal and estuarine ecosystems already experience large natural pH fluctuations on diel, seasonal and interannual time-scales (Baumann et al., 2015; Law et al., 2017; Zhao et al., 2018a), which can complicate the interpretation of proxy records without a holistic understanding of how pH affects the trace elemental signatures of bivalve shells.
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In a rapidly acidifying ocean, environmental reconstructions and geographical
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traceability based on bivalve shell geochemistry may be biased by effects arising from the
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plasticity of physiological metabolism. There is accumulating evidence that demonstrates the
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rapid transgenerational acclimation and corresponding metabolic plasticity to near-future
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OA scenarios projected for the end of this century observed in short-lived marine bivalve species, such as the Sydney rock oyster, Saccostrea glomerata (Parker et al., 2015), the
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Manila clam, Ruditapes philippinarum (Zhao et al., 2018b), the blue mussel, Mytilus edulis
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(Thomsen et al., 2017) and the Asian date mussel, Musculista senhousia (Zhao et al., 2019b,
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2019c). If this is the case, and given the well-documented metabolic controls over the trace elemental composition of bivalve shells (Gillikin et al., 2005; Planchon et al., 2013; Poulain et al., 2015; Zhao et al., 2017b) as well as the metabolic plasticity, then one would expect that the degree to which metabolic activities affect the trace elemental uptake and incorporation into bivalve shells changes following transgenerational exposure to OA. In order to underpin the application of trace elements of bivalve shells as appropriate tracers and proxies in the face of OA, such gap of knowledge (i.e., the influence of metabolic plasticity) should thus be closed.
The present study aims to tackle the question of whether, and to what extent, long-
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Journal Pre-proof term, transgenerational exposure to near-future OA scenarios projected by the end of this century affects the incorporation of trace elements into bivalve shells. More recently, our studies have demonstrated the great capacity of the juvenile Manila clam, R. philippinarum to cope with OA following transgenerational acclimation by maintaining the acid-base homeostasis (Zhao et al., 2017c) and generating the plasticity of metabolism (Zhao et al., 2018b). In this study, we test whether and how the trace elemental composition of R.
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philippinarum shells varies following transgenerational exposure to OA. These findings will
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provide valuable information for both environmental reconstructions and geographical
2. Materials and methods
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2.1. Clam collection and maintenance
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traceability based on bivalve shell geochemistry in an acidifying ocean.
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Adult R. philippinarum specimens (32 ± 2.3 mm shell length) were collected, on 2 April 2014
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from the intertidal zone of Liangshui Bay, Yellow Sea (39°04′14.41″ N, 122°01′47.70″ E). Upon arrival in the laboratory within 2 h, the clams were acclimated in 600-L circulating seawater systems for two weeks, during which environmental conditions were maintained at levels comparable to those at the site of sampling and day of collection (i.e., temperature of 9 °C, salinity of 32, pH 8.1). They were fed with the marine algae Chlorella vulgaris every two days. During the acclimation period no mortality occurred.
2.2. Experimental setup The transgenerational experiment has been described in detail by Zhao et al. (2017c). In brief, following laboratory acclimation, the clams were exposed to two scenarios of OA (i.e.,
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Journal Pre-proof pH 8.1 and 7.7), the latter corresponding to the seawater pH level projected for the year 2100. Two experimental systems, as schematically illustrated by Xu et al. (2016), were built (Fig. 1). Each system contained about 600-L circulating seawater and 450 clams. Elevated seawater pCO2 and corresponding lowered pH were adjusted by continuously bubbling CO 2 gas into the system. To facilitate gonadal maturation, a well-established protocol (i.e., water temperature progressively increasing from 10 to 20 °C within 30 days and subsequently kept
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constant for 40 days; Xu et al., 2016) was employed. To meet the nutritional requirement
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during gonadal ripening, the clams were fed with an equal mixture of microalgal species , C.
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vulgaris, Isochrysis galbana, Chaetoceros muelleri and Nitzschia closterium at a daily ration
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of approximate 40,000 cells mL-1. To maintain seawater quality, half of the system volume
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was renewed every 4-5 days.
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Following 70 days of artificial induction of gonadal maturation, 100 clams were
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randomly collected from each system and induced to spawn by temperature shock (+ 4 °C).
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About 95 % and 96 % of individuals exposed to pH 8.1 and 7.7 spawned within 8 h, respectively (Xu et al., 2016). Fertilized eggs were filtered through a nylon mesh (60 µm), rinsed three times in sand-filtered seawater to get rid of excessive sperms and other gonadal tissues and then incubated in pre-prepared 100-L aquaria (about 50 eggs mL-1). After 26 h of fertilization, newly hatched D-veliger larvae were collected and randomized into 100-L aquaria at a density of 10 larvae mL-1, according to the transgenerationally experimental design (as shown in Fig. 1). Three replicates were carried out for each experimental treatment. Room temperature was kept stable at 20 °C. From day 1 to day 3, larvae were fed with I. galbana at a daily ration of 20,000 cells mL-1 and, from day 4 onward with a mixture of I. galbana and C. vulgaris at a daily ration of 40,000 cells mL-1. 100% of the aquarium was
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Journal Pre-proof renewed every two days using pre-adjusted sand-filtered seawater (with consistent environmental conditions) to maintain water quality. While larvae developed into the pediveliger stage (12-15 days after fertilization, 180-210 µm shell length, characterized with eyespot and protruding foot), all aquaria were renewed twice daily so as to facilitate larval settlement. Following larval metamorphosis which took about 5 days, newly settled juveniles were quantified and approximate 1000 individuals for each aquarium were randomly
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sampled for the following 30-days nursery (Fig. 1), during which the clams were fed with C.
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vulgaris at a daily ration of 80,000 cells mL-1. Experimental conditions were maintained
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comparable to those during larval stage, and daily cares were also handled in the same
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manner.
2.3. Trace elemental analysis via LA-ICP-MS
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In the present study, six elements (Mg, Cu, Zn, Sr, Ba and Pb) which are widely used as
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proxies for past environmental reconstruction and tracers for geographical traceability
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(Schöne and Krause, 2016; Prendergast et al., 2017) were analyzed in R. philippinarum shells. Specifically, following 30 days of nursery, a total of 20 juveniles from each experimental treatment were randomly collected to analyze the trace elemental composition of the shells. The right valve of each juvenile was carefully taken, soaked in Teflon vials with 15 ml 15 % H2O2 buffered with 0.05 M NaOH for 14 h to remove organic matter, and then rinsed with deionized water, air-dried and mounted on a glass slide. Trace elements were analyzed using an ESI NWR 193 ArF excimer laser ablation system coupled to an Agilent 7500ce qua drupole ICP-MS in ‘spot’ mode (Zhao et al., 2017c). Ablation was achieved using a beam diameter of 100 µm, an energy density of ~2.8 J cm-2 and a pulse repetition rate of 5 Hz. Backgrounds were analyzed for 15 s, followed by 25 s ablation time and then 20 s wash out. Monitored
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Journal Pre-proof isotopes were 26Mg, 65Cu, 66Zn, 88Sr, 137Ba and 208Pb. NIST SRM 610 (synthetic glass) was used as calibration material, and the preferred values reported in GeoReM database (http://georem.mpch-mainz.gwdg.de/, application version 18) (Jochum et al., 2005, 2011) were used as the ‘true’ concentrations to calculate the elemental concentrations in juvenile shells. MACS-3 (synthetic calcium carbonate) and USGS BCR-2G (basalt glass) were simultaneously measured as quality control materials (QCM) so as to monitor accuracy and
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reproducibility of the measurements. All reference materials were analyzed at the beginning
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and at the end of a sequence and after around 60 measurements of the shells. For all
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materials, 43Ca was used as internal standard. For the reference materials, we applied the Ca
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concentrations reported in the GeoReM database, and for the samples 56.03 wt %., the
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stoichiometric CaO content of aragonite. The time-resolved signal was processed using the program GLITTER 4.4.1 (www.glitter-gemoc.com, Macquarie University, Sydney, Australia).
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Reproducibility expressed as the relative standard deviation based on repeated
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measurements of the QCM was always better than 7 % for MACS-3 and 3 % for USGS BCR-2G.
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The measured Mg, Cu, Zn, Sr, Ba and Pb concentrations of MACS-3 agreed within 3.2 %, 2.5 %, 9.5 %, 4.1 %, 10.1 % and 14.0 % with the preliminary reference values (personal communication S. Wilson, USGS, in Jochum et al. 2012) and within 12.1 %, 12 %, 32 %, 0.7 %, 1.5 % 3.0 % for USGS BCR-2G, respectively. Minimum detection limits were 0.70 µg g-1, 0.26 µg g -1, 0.19 µg g -1, 0.012 µg g-1, 0.063 µg g-1, and 0.016 µg g -1 for Mg, Cu, Zn, Sr, Ba and Pb, respectively.
2.4. Statistical analysis All experimental data were statistically analyzed using SPSS Statistics 19.0. The ShapiroWilk’s test and Levene’s F-test were employed to examine the normality and homogeneity
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Journal Pre-proof of the data, respectively. Subsequently, the combined effects of adult treatment and seawater pH on the trace elemental values (expressed as molar ratios relative to calcium) of juvenile shells were examined by means of two-way analysis of variance (two-way ANOVA) followed by the LSD post-hoc test. In the present study, statistically significant difference
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was set at p < 0.05.
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3. Results
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In the present study, all experimental data are normally distributed and treatment variances
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are homogeneous. According to two-way ANOVA analysis, transgenerational exposure,
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seawater pH, as well as interaction between these two factors, did not significantly affect the incorporation of Mg (Fig. 2A) and Sr (Fig. 2B) into juvenile R. philippinarum shells (p >
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0.05). On average, the amount of Cu (Fig. 2C) incorporated into the shells was significantly
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affected by both transgenerational exposure and seawater pH (p < 0.05), yet no significant
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interaction between these two factors was observed (p > 0.05). With decreasing seawater pH from 8.1 to 7.7, the amount of Zn (Fig. 2D) incorporated into the shells increased significantly (p < 0.05), however transgenerational exposure did not significantly affect the incorporation of Zn into the shells (Fig. 2D; p > 0.05). Interestingly, the interaction between transgenerational exposure and seawater pH on the Zn composition of the shells was significant (Fig. 2D; p < 0.05). Specifically, when exposed to pH 7.7, juvenile clams with a prior history of transgenerational exposure to pH 7.7 incorporated significantly lower amounts of Zn into the shells than those originating from parents reared at pH 8.1 during the gonadal conditioning (Fig. 2D; p < 0.05). Irrespective of transgenerational exposure which exhibited no significant effects on the incorporation of Ba (Fig. 2E) and Pb (Fig. 2F) into
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Journal Pre-proof juvenile shells (p > 0.05), the shells forming at pH 7.7 contained significantly larger amounts of Ba and Pb than those grown at pH 8.1 (p < 0.05).
4. Discussion The present study represents the first investigation of transgenerational effects of OA on the
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trace elemental composition of marine bivalves. Our results demonstrate different responses of elemental incorporation processes into R. philippinarum shells. To better
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elucidate effects of transgenerational exposure, seawater pH as well as their interaction on
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the Mg, Sr, Cu, Zn, Ba and Pb composition of the shells, a schematic model illustrated in Fig.
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3, which is based on the findings of the OA effects on the speciation of metals in seawater (Millero et al., 2009) and the pathways of trace elements from the environment into bivalve
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shells (Zhao et al., 2017d), is built. Given that the juveniles were grown under strictly
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controlled experimental conditions, influences of factors such as seawater temperature,
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salinity, food availability, trace elemental concentrations were minimized. Therefore, seawater pH and corresponding pH-dependent speciation of trace elements (i.e., the percent of free ions for a given element) are considered environmental factors affecting trace elemental records of R. philippinarum shells in the model. The ability of bivalves to discriminate divalent ions with similar ionic radii and electrochemical properties as Ca ion during shell formation is mainly metabolically dependent (Zhao et al., 2017d). It seems, therefore, reasonable to incorporate the metabolic performance (shown in Zhao et al., 2018b) into the model as an important physiological factor.
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Journal Pre-proof According to this proposed schematic model, it is not surprising that neither seawater pH nor transgenerational exposure affected the amount of Mg and Sr incorporated into the shells. In seawater, Mg and Sr behave conservatively and exist exclusively as free ions (Bruland, 1983), the latter known as major bioavailable forms which are taken up by bivalves (Campbell, 1995). In particular, it has been shown that the solubility and bioavailability of metals in the free form are not significantly affected by changes of seawater pH and
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carbonate system (Millero et al., 2009). As such, it seems reasonable to conclude that
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seawater pH does not affect the uptake of Mg and Sr into bivalve shells. A recent study
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examining the effects of elevated pCO2 on the trace elemental composition of shells of the
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green-lipped mussel, Perna canaliculus (Norrie et al., 2018) has also come to a similar conclusion. On the other hand, during the uptake from the surrounding seawater to the site
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of calcification, Mg 2+ and Sr2+ ions are not regarded simply as metabolic analogues of Ca2+
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and consequently mainly transported by intercellular Ca 2+ pathways (Zhao et al., 2017d), the
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latter demonstrating less metabolic interferences with the transport mechanisms of Mg and
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Sr. Hence, the possibility that metabolic plasticity following transgenerational exposure to OA affects the uptake of Mg and Sr during shell building can be reasonably ruled out.
Unlike Mg and Sr which occur predominantly in the free form in seawater, Cu, Zn, Ba and Pb tend to form strong complexes with both hydroxide and carbonate (Bruland, 1983), and thereby likely experience significant changes of speciation as seawater pH decreases (Millero et al., 2009). In particular, under acidified conditions, the solubility and bioavailable fraction (i.e., the percent of free ions) of trace elements mentioned above increase rapidly as schematically illustrated in Figure 3. The pH-dependent nature of the bioavailability of Cu, Zn, Ba and Pb probably offers the most likely explanation for observations of increased amounts
11
Journal Pre-proof of these elements incorporated into shells of CO2-exposed R. philippinarum. In addition, these findings compare well with observations in larval shells of mussels , Mytilus californianus and M. galloprovincialis (Frieder et al., 2014) and juvenile shells of P. canaliculus (Norrie et al., 2018).
Another possible explanation for increasing incorporation of Cu, Zn and Pb into the
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shells with decreasing seawater pH is related to metabolic effects. Divalent ions including
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Cu2+, Zn2+ and Pb2+ are largely taken up by bivalves as analogues of Ca2+ (Markich and Jeffree,
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1994). In particular, intracellular Ca2+ transport pathways such as Ca2+-ATPase and Ca 2+-
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channel are mainly involved in the uptake of Cu, Zn and Pb from the environment to the site
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of calcification (Zhao et al., 2017d). Under acidified conditions, the clams may shift the energy budget toward compensating for the acid-base imbalance in the calcifying fluid by
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active removal of excessive protons (Zhao et al., 2017c), consequently reducing the amount
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of energy allocated to discriminate against Cu2+, Zn2+ and Pb2+ during intracellular transport
shells.
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of Ca2+ and eventually resulting in higher amounts of these elements incorporated into the
Transgenerational effects are most pronounced during the uptake and incorporation of Cu and Zn into R. philippinarum shells, which may be related to the plasticity of metabolism as shown in transgenerationally acclimated clams under acidified conditions (Zhao et al., 2018b). As biologically essential trace elements for metabolic processes in marine bivalves, Cu and Zn can be highly metabolically regulated even when taken in low doses (Pan and Wang, 2012). Rapid improved tolerance of Cu and Zn following transgenerational exposure to metal stress has been found in the oyster, Crassostrea sikamea (Weng and Wang, 2014),
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Journal Pre-proof which can be related to the increased induction of metallothionein-like proteins. If this is the case, then one would expect an increase of energy allocated to the protein synthesis and secretion. This assumption is in line with recent observations
that, following
transgenerational acclimation, CO 2-exposed juvenile clams are capable of implementing more ATP-efficient and less costly ionic regulatory mechanisms to compensate for the acidbase imbalance at the site of calcification (Zhao et al., 2017c) and preferentially extracting
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metabolically generated CO2 rather than active transport seawater dissolved inorganic
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carbon which is energetically expensive to calcify (Zhao et al., 2018b), thereby generating
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more energy available for the discrimination and elimination of Cu and Zn in the intracellular
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Ca2+ transport pathways. In this regard, omics technologies (genomics, transcriptomics,
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metabolomics and proteomics) may open an avenue in elucidating the underlying mechanisms at lower levels of biological organization, especially regarding the role of
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specific proteins related to metabolic pathways and ionic transporters in the face of long -
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term and transgenerational exposure to seawater acidification.
Transgenerational effects did not manifest themselves when the clams took up Pb from the environment to the site of calcification. Lead could act as a metabolic poison and produce intracellular changes (Pan and Wang, 2012). Yet, marine bivalves are usually well equipped to detoxify the impact of Pb via energetically efficient processes (such as cellular redistribution) rather than active efflux systems (Viarengo and Nott, 1993). If the detoxification of Pb is not extremely energetically expensive and in particular not closely correlated to the synthesis and secretion of metallothionein-like proteins, then one would not be surprised to see the limited role of the plasticity of energy metabolism generated
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Journal Pre-proof following transgenerational acclimation in the uptake and incorporation of Pb into R. philippinarum shells.
Overall, findings of the present study have important implications for the application of trace elemental signatures of bivalve shells as geographical tracers and environmental proxies in an acidifying ocean. The incorporation of Mg and Sr remained virtually unaffected
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following long-term transgenerational exposure to OA, suggesting that environmental
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information such as seawater temperature deduced from shell Mg/Ca and Sr/Ca values
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should be unbiased in the face of rapidly changing environment. However, the influence of
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pH on the incorporation of Ba and Pb into bivalve shells should be taken into consideration
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when shell Ba/Ca and Pb/Ca ratios are explored as proxies for primary productivity and pollution (Thébault et al., 2009; Holland et al., 2014), especially in coastal waters where diel,
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seasonal and annual variations in seawater pH are remarkable. While a number of studies
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have reported the potential of shell Cu/Ca and Zn/Ca ratios as indicators of water Cu and Zn
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pollution (Schöne and Krause, 2016), given transgenerational effects of OA on their incorporation processes into the shells, caution should be taken in terms of both geographical traceability and environmental reconstruction.
5. Conclusions The present study provides valuable information on the transgenerational effects of elevated pCO2 on the trace elemental signatures of marine bivalve shells, which may further foster the two primary applications of trace element data of bivalve shells in the field of geographical traceability and environmental reconstruction. Specifically, transgenerational
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Journal Pre-proof acclimation and elevated pCO2 do not affect the incorporation of Mg and Sr into the shells, thereby ensuring their robustness as both geographical tracers and environmental proxies. Due to the pH-dependence of Ba and Pb in free forms in seawater, the combination of Ba and Pb records of bivalve shells (which are also less metabolically affected) holds the potential to reconstruct the history of seawater pH and carbonate chemistry. In many estuarine and coastal habitats which naturally experience large pH fluctuations on different-
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time scales, it remains possible to discriminate the geographical origin of bivalves using shell
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Ba and Pb signatures. It is evident that the incorporation of biologically essential trace
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elements (such as Cu and Zn) is affected by both seawater pH and the history of
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transgenerational exposure, consequently hindering their potential as geographical tracers
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and environmental proxies. Yet, studies on the Cu and Zn composition of bivalve shells can likely open an existing new avenue in the research of biomineralization which is highly
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biologically controlled.
Acknowledgments
The authors would like to thank Regina Mertz-Kraus at the University of Mainz for assistance with LA-ICP-MS analyses. The current study has been made possible by a research grant from the framework (FP7) of the Marie Curie International Training Network ARAMACC (604802) to BRS and LZ and by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants to LZ (17P17333) and KT (17K17669).
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Figure captions Figure 1 Flow chart of the transgenerational experimental design (for more details refer to the text).
Figure 2 The amount of Mg (A), Sr (B), Cu (C), Zn (D), Ba (E) and Pb (F) incorporated into the shells of the Manila clam, Ruditapes philippinarum, following transgenerational exposure to
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pH 8.1 (i.e., non-acclimated line) and pH 7.7 (i.e., transgenerational-acclimated line). Trans. = transgenerational. Results of two-way ANOVA analysis were given in corresponding panels.
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expressed as mean ± standard deviation (n = 20)
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Trace elemental concentrations of shells are reported as molar ratios relative to calcium and
Figure 3 A schematic model developed to demonstrate how transgenerational exposure to
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seawater acidification affects the incorporation of trace elements into the shells of the
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Manila clam, Ruditapes philippinarum. This model is primary built on the pathways of trace
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elements from the environment into bivalve shells (as schematically illustrated by Zhao et al. 2017d), according to which Ca 2+ from the ambient seawater is largely transported via intracellular pathways (Ca2+-ATPase and Ca2+-channel) and in part via intercellular pathway (passive diffusion) to the calcifying fluid. Divalent ions Cu2+, Zn2+ and Pb2+ are taken up by the bivalves via intracellular Ca2+ pathways, thus highly metabolically regulated, whereas intercellular passive diffusion is considered the predominant transport pathway of Mg 2+, Sr2+ and Ba2+ (Zhao et al., 2017d). In addition, the bioavailable forms (i.e., free forms) of Cu, Zn, Ba and Pb in seawater are highly pH-dependent, i.e., with decreasing seawater pH concentrations of their free ions increase (Millero et al., 2009). For more details regarding mechanisms of shell calcification please refer to Zhao et al. (2018b) and Lu et al. (2018).
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Journal Pre-proof Graphical abstract
Research highlights: 1.
Neither seawater pH and transgenerational exposure affects the Mg and Sr
composition of bivalve shells. 2.
With decreasing seawater pH, the amounts of Cu, Zn, Ba and Pb taken up in clam
Transgenerational exposure to seawater acidification affects the Cu and Zn
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3.
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shells increase.
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composition of bivalve shells.
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Figure 1
Figure 2
Figure 3