Pre-treatment methods for stable oxygen and carbon isotope analyses of structural carbonates of bones in marine teleost fishes.

RATIONALE: Although the proportion of structural carbonates in vertebrate bones is low, the values of isotopes, namely stable oxygen (δ18 O) and carbon (δ13 C), in structural carbonates provide environmental and physiological information, which can be beneficial for estimating the palaeontological and ecological parameters of vertebrates. However, a few studies have analysed the isotopes of structural carbonates in modern teleost fishes, and a well-developed protocol for sample preparation is lacking. METHODS: We examined different pre-treatment methods of preparing bone samples of three marine teleost fishes (Japanese flounder, Pacific bluefin tuna and yellowtail) and investigated the effects of the cleaning methods on the stable isotope values of structural carbonates among vertebrae in the same individual. Isotope values were analysed using isotope ratio mass spectrometry. RESULTS: Physical cleaning was the most promising pre-treatment method and resulted in δ18 O values that were comparable to those of otoliths. Chemical treatments with NaOH and H2 O2 changed the percentage of structural carbonates in the bone and affected δ18 O and δ13 C values. High-temperature treatments, such as boiling and roasting, altered δ18 O values due to the exchange of oxygen with environmental water or vapour. CONCLUSIONS: Our results suggest that chemical cleaning methods used to prepare bone phosphate or collagen samples for isotope analyses are not suitable for structural carbonates. Physical cleaning is the appropriate pre-treatment method for analysing the isotopes of structural carbonates. Also, we emphasise that standardising the vertebral number is necessary to make δ13 C values comparable between specimens in the same species.

Ocean acidification stunts molluscan growth at CO2 seeps.

Ocean acidification can severely affect bivalve molluscs, especially their shell calcification. Assessing the fate of this vulnerable group in a rapidly acidifying ocean is therefore a pressing challenge. Volcanic CO2 seeps are natural analogues of future ocean conditions that offer unique insights into the scope of marine bivalves to cope with acidification. Here, we used a 2-month reciprocal transplantation of the coastal mussel Septifer bilocularis collected from reference and elevated pCO2 habitats to explore how they calcify and grow at CO2 seeps on the Pacific coast of Japan. We found significant decreases in condition index (an indication of tissue energy reserves) and shell growth of mussels living under elevated pCO2 conditions. These negative responses in their physiological performance under acidified conditions were closely associated with changes in their food sources (shown by changes to the soft tissue δ13C and δ15N ratios) and changes in their calcifying fluid carbonate chemistry (based on shell carbonate isotopic and elemental signatures). The reduced shell growth rate during the transplantation experiment was further supported by shell δ13C records along their incremental growth layers, as well as their smaller shell size despite being of comparable ontogenetic ages (5-7 years old, based on shell δ18O records). Taken together, these findings demonstrate how ocean acidification at CO2 seeps affects mussel growth and reveal that lowered shell growth helps them survive stressful conditions.

Periostracum of bivalve mollusk shells for sampling engineered metal nanoparticles: A case study of silver-based nanoparticles in Canada's experimental lake.

Given the ability of engineered metal nanoparticles to be transformed in natural waters in unpredictable manners, various sampling methods must be developed. Here, we took a novel approach to collection silver nanoparticles (AgNPs) that involved the use of the intact periostracum, the outer proteinaceous organic layer, of freshwater unionid mussels Pyganodon sp. Eight adult mussels were collected in August 2019 from a small boreal lake (L222) at the International Institute for Sustainable Development - Experimental Lakes Area (northwestern Ontario), which had been dosed with 15 kg of poly(vinylpyrrolidone)-coated silver nanoparticles (PVP-AgNPs) in 2014-2015. Additionally, three adult mussels were collected from a control lake (L375). Numerous silica (SiO2) diatom frustules were adhered to periostracum of all mussels. Intact periostracum promotes the formation of layer composed of diatoms and sand grains. The Ag content in soft tissues and shells of the mussels from L375 was as low as ≤ 0.1 µg/g. In mussels from L222, Ag concentrations in the periostracum of five shells were in detectable amounts (1-4 µg/g); in three shells concentrations were as high as 86, 122, and 494 µg/g. The underlying mineral shell is depleted in Ag (<0.1 µg/g). The Ag content in soft tissue organs (whole body) ranged from 44 to 191 µg/g. AgNPs occur on the surface of both periostracum and diatoms. Single AgNPs (d = 20-60 nm) were partly sulfidized to Ag2S. The observed AgNPs often form aggregates with an average and a maximal size of circa 100 nm and 1.5 µm, respectively. Scraping small fragments of intact periostracum of unionid shell is non-lethal to mussels, and is easy to do under field conditions. This simple sampling protocol could be used to detect metal-based nanoparticles (engineered or accidental) with the use of unionid and dreissenid bivalves.

Using neodymium isotope ratio in Ruditapes philippinarum shells for tracking the geographical origin.

Geographical traceability of marine bivalves is becoming increasingly important to assure their quality and to defend the interest of consumers and producers. This study verifies the neodymium isotopic ratio (143Nd/144Nd) in Ruditapes philippinarum shells as a tracer of the geographic origin, based on the geochemical aspect that 143Nd/144Nd of their habitats strongly depends on the geology of its catchment areas. The 143Nd/144Nd ratios of clam shells from the Japanese and Chinese coastal areas displayed a heterogeneous pattern from local to international scales, reflecting the geological age of the catchment area. The blind test suggested that a part of Manila clam was sold with mislabeling in the Japanese market, demonstrating the high potential of 143Nd/144Nd to unmask the fraud labeling in a food market. Our findings emphasize the potential of 143Nd/144Nd as a tracer for the geographical origin of marine bivalves, and also as a strong deterrent against mislabeling.

Influence of normal tide and the Great Tsunami as recorded through hourly-resolution micro-analysis of a mussel shell.

We report here hourly variations of Mg/Ca, Sr/Ca, and Ba/Ca ratios in a Mediterranean mussel shell (Mytilus galloprovincialis) collected at the Otsuchi bay, on the Pacific coast of northeastern Japan. This bivalve was living in the intertidal zone, where such organisms are known to form a daily or bidaily growth line comprised of abundant organic matter. Mg/Ca ratios of the inner surface of the outer shell layer, corresponding to the most recent date, show cyclic changes at 25-90 µm intervals, while no interpretable variations are observed in Sr/Ca and Ba/Ca ratios. High Mg/Ca ratios were probably established by (1) cessation of the external supply of Ca and organic layer forming when the shell is closed at low tide, and (2) the strong binding of Mg to the organic layer, but not of Sr and Ba. Immediately following the great tsunami induced by the 2011 Tohoku earthquake, Mg/Ca enrichment occurred, up to 10 times that of normal low tide, while apparent Ba/Ca enrichment was observed for only a few days following the event, therefore serving a proxy of the past tsunami. Following the tsunami, periodic peaks and troughs in Mg/Ca continued, perhaps due to a biological memory effect as an endogenous clock.

Spatiotemporal change of cesium-137 in the Pacific coast of Tohoku, Japan: The mussel watch approach.

We measured radiocesium in mussel tissue collected from the Pacific coast of Tohoku from 2011 to 2015 to investigate the temporal and spatial dynamics of radiocesium in the coastal area. Radioactive 137Cs was detected in all the samples collected in 2011, but it was not found in samples from localities north of Sendai after 2012. In contrast, 137Cs was detected in many sites in the Fukushima area even from 2012 to 2015. The fluctuation of 137Cs concentration in mussel tissue seems to reflect the 137Cs concentration in suspended particles in the seawater, suggesting that there was an influx of soil deposition and resuspension of seabed sediment. These results suggest that the 137Cs concentration in mussel tissue sensitively indicates the 137Cs concentration in the environment, and that the "mussel watch" approach is an effective way to understand the dynamics of radiocesium concentrations in coastal areas.

Divalent metal transporter-related protein restricts animals to marine habitats.

Utilization and regulation of metals from seawater by marine organisms are important physiological processes. To better understand metal regulation, we searched the crown-of-thorns starfish genome for the divalent metal transporter (DMT) gene, a membrane protein responsible for uptake of divalent cations. We found two DMT-like sequences. One is an ortholog of vertebrate DMT, but the other is an unknown protein, which we named DMT-related protein (DMTRP). Functional analysis using a yeast expression system demonstrated that DMT transports various metals, like known DMTs, but DMTRP does not. In contrast, DMTRP reduced the intracellular concentration of some metals, especially zinc, suggesting its involvement in negative regulation of metal uptake. Phylogenetic distribution of the DMTRP gene in various metazoans, including sponges, protostomes, and deuterostomes, indicates that it originated early in metazoan evolution. However, the DMTRP gene is only retained in marine species, and its loss seems to have occurred independently in ecdysozoan and vertebrate lineages from which major freshwater and land animals appeared. DMTRP may be an evolutionary and ecological limitation, restricting organisms that possess it to marine habitats, whereas its loss may have allowed other organisms to invade freshwater and terrestrial habitats.

Identification of timing of scallop morphological deformity and mortality from shell oxygen isotope records.

The Yesso scallop, Patinopecten yessoensis (Jay), is one of the most important bivalve species in the Japanese and Chinese mariculture industry. In recent years, however, high incidences of scallop shell deformity and mortality have occurred with increasing frequency, but timing of onset and underlying causes are often unclear. Here, we proposed a promising δ18Oshell-based method for constraining the onset of shell deformity and mortality of P. yessoensis. Following six months of intermediate suspension culture in Funka Bay, Northern Japan, shells from healthy, deformed and dead scallops were randomly sampled. High-resolution seawater temperature time-series computed from healthy scallop shell δ18O profiles were precisely and temporally aligned to the instrumental temperature curve, thus allowing δ18Oshell-derived temperature time-series from deformed and dead scallops to be contextualized and allowing timing of scallop deformity and death to be retrieved. Irrespective of scallop shell length, onsets of deformity were anchored in February, and since then deformed scallops grew slowly in comparison to healthy individuals. Without exception, however, dead scallops had already ceased their shell building and died before February, indicating different underlying causes of scallop deformity and mortality. Perhaps most promisingly, considering that shells do not have any isotopic turn-over and once formed, temperature information is locked in. Thus, this approach holds great promise for identifying time anchor points (onsets of deformity and death) in archived scallops collected over different time scales, especially during massive mortality events.

Determination of the geographical origin of marine mussels (Mytilus spp.) using 143Nd/144Nd ratios.

Geographical traceability of marine bivalves is critical to guarantee their quality and safeguard the interest of both consumers and producers. The neodymium isotopic ratio (143Nd/144Nd) of the coastal water mainly reflects the geology of its neighboring watershed, displaying the distinct and systematic variability at high level of geographical detail and thereby shedding light on its potential as a geochemical tracer. For the first time, the present study investigated the utility and robustness of 143Nd/144Nd archived in mytilid mussel shells for geographical traceability purposes. The reproducibility of 143Nd/144Nd ratios maintained in mussels shells from the same cohort demonstrates that the Nd isotopic ratio meets the major requirement for an ideal geochemical tracer, i.e., the biologically induced variation should be rather minimal. The distribution and variability of mussel shell 143Nd/144Nd patterns were subsequently mapped along the Japanese and Chinese coastal waters. Neodymium isotopes of mussel shells record 143Nd/144Nd variations among local regions and between the two countries, which are rather compatible with the ages and lithology of the continental bedrocks. These findings highlight the great potential of 143Nd/144Nd for tracing the geographical origin of marine bivalves.

Retrospective monitoring of salinity in coastal waters with mussel shells.

Sea surface salinity (SSS) is a key parameter to understand and predict many physical, chemical and biological processes in dynamic coastal environments. Yet, in many regions, instrumental measurements are spatially sparse and insufficiently long, hindering our ability to document changes, causes, and consequences of SSS across different time scales. Therefore, there is an need to develop a robust proxy to extend SSS records back in time. Here, we test whether SSS can be reconstructed reliably and quantitatively from shell oxygen isotopic ratios (δ18Oshell) of the mussel Mytilus galloprovincialis (Lamarck, 1819) in Otsuchi Bay, Northern Japan. δ18Oshell ratios vary spatially and temporally and exhibit strong linear correlations with both sea surface temperature (SST) and SSS measurements, indicating that the composite signal recorded by δ18Oshell measurably responds to variations in both parameters. By combining contemporaneous variations of SST and δ18Oshell, SSS records encoded into mussel shells are deconvolved that significantly correlate with in situ SSS values. To further validate the robustness of δ18Oshell as a quantitative SSS proxy, high-resolution and temporally aligned time-series of δ18Oshell-derived SSS are reconstructed that are highly synchronous with the instrumental records. In particular, two lowered SSS scenarios occur concomitantly with periods of the summer monsoon and typhoon events. δ18Oshell-derived SSS time-series are also comparable to those from numerical modeling. In conclusion, our findings demonstrate that mussel δ18Oshell signatures can be used as a useful tool to construct high-resolution records of SSS in the coastal regions.

Stimpson's hard clam Mercenaria stimpsoni; A multi-decadal climate recorder for the northwest Pacific coast.

A sclerochronological and radiocarbon-based study of life history traits of Stimpson's hard clam (Mercenaria stimpsoni), collected alive from Funakoshi Bay, northeast Japan, showed the lifespan of the species to be at least 92 years (determined from annual growth line counts). Three M. stimpsoni specimens exhibited the following synchronous growth pattern, suggestive of environmental control; annual increment width increasing after 1955 to a maximum value between 1970 and 1980, subsequently decreasing gradually until 2000, and thereafter remaining constant or increasing slightly. Variations on annual growth patterns, as well as standardized growth indices chronology, were relatively closely linked to the Atlantic Multidecadal Oscillation (AMO), but less so to Pacific Decadal Oscillation (PDO). Carbonate samples collected from ontogenetically younger shell portions, estimated from growth line counts to have been deposited before 1950, contained no nuclear bomb-test radiocarbon, thereby supporting the accuracy of annual growth line counts (versus overcounting from ventral margin). Together with the synchronous annual increment width patterns, this indicated that age and annual growth rate estimations for M. stimpsoni based on growth line counts were reliable and applicable to high-resolution sclerochronological analyses, which should contribute to a deeper understanding of multi-decadal northwest Pacific climate variability.

Using tsunami deposits to determine the maximum depth of benthic burrowing.

The maximum depth of sediment biomixing is directly related to the vertical extent of post-depositional environmental alteration in the sediment; consequently, it is important to determine the maximum burrowing depth. This study examined the maximum depth of bioturbation in a natural marine environment in Funakoshi Bay, northeastern Japan, using observations of bioturbation structures developed in an event layer (tsunami deposits of the 2011 Tohoku-Oki earthquake) and measurements of the radioactive cesium concentrations in this layer. The observations revealed that the depth of bioturbation (i.e., the thickness of the biomixing layer) ranged between 11 and 22 cm, and varied among the sampling sites. In contrast, the radioactive cesium concentrations showed that the processing of radioactive cesium in coastal environments may include other pathways in addition to bioturbation. The data also revealed the nature of the bioturbation by the heart urchin Echinocardium cordatum (Echinoidea: Loveniidae), which is one of the important ecosystem engineers in seafloor environments. The maximum burrowing depth of E. cordatum in Funakoshi Bay was 22 cm from the seafloor surface.

Spatial Distribution and Temporal Trend of Anthropogenic Organic Compounds Derived from the 2011 East Japan Earthquake.

The tsunami caused by the Great East Japan Earthquake on March 11, 2011 disturbed coastal environments in the eastern Tohoku region in Japan. Numerous terrestrial materials, including anthropogenic organic compounds, were deposited in the coastal zone. To evaluate the impacts of the disaster, we analyzed PCBs, LABs, PAHs, and hopanes in mussels collected from 12 locations in the east of Tohoku during 2011-2015 (series A) by GC-ECD or GC-MS and compared them with results from mussels collected from 22 locations around Japan during 2001-2004 (series B). Early LAB concentrations in series A at some locations were higher than the maximum concentrations in series B but decreased during the 5 years. Because LABs are molecular markers for sewage, these decreases are consistent with the recovery of sewage treatment plants in these areas. Early PAH concentrations at several locations were higher than the maximum concentrations in series B but also decreased. These high concentrations would have been derived from oil spills. The decreases of both LABs and PAHs indicate that these locations were affected by the tsunami but recovered. In contrast, later high concentrations of target compounds were detected sporadically at several locations. This pattern suggests that environmental pollution was caused by human activities, such as reconstruction. To understand the long-term trend of environmental pollution induced by the disaster, continuous monitoring along the Tohoku coast is required.