Air-liquid interface culture and modified culture medium promote the differentiation of human induced pluripotent stem cells into intestinal epithelial cells.

An in vitro system that evaluates pharmacokinetics in the small intestine is crucial for the development of oral drugs. We produced human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) with high drug metabolizing enzyme and drug transporter activities. However, the gene expression of our hiSIECs partially differed from that of the human small intestine, with low drug metabolizing enzyme activities. Therefore, we used air-liquid interface (ALI) culture and 5-aza-2'-deoxycytidine (5AZA)-free medium to generate hiSIECs (novel hiSIECs). Novel hiSIECs showed enhanced gene expression of drug metabolizing enzymes, such as cytochrome P450 (CYP)3A4, CYP2C9, CYP2C19, and carboxylesterase 2 that are highly expressed in the small intestine. In addition, the expression of genes involved in nutrient absorption-one of the major functions of the small intestine-also increased. The novel hiSIECs expressed ZO-1 and E-cadherin. Moreover, the novel hiSIECs exhibited a barrier function that allowed low lucifer yellow permeation. The novel hiSIECs showed high activities of CYP3A4, CYP2C9, and CYP2C19, which are abundantly expressed in the small intestine. In conclusion, the novel hiSIECs have great potential as an in vitro system to evaluate pharmacokinetics in the small intestine.

Relationship between stable cesium concentration and body size of Japanese flounder Paralichthys olivaceus and the effect of a size-dependent shift in diet.

To understand the relationship between the radioactive cesium (Cs) concentration in muscle of Japanese flounder Paralichthys olivaceus and the species' biological characteristics (size, sex, and age) under conditions of ecological equilibrium (i.e., distributed among ecosystem components over sufficient time, and with nearly constant ratios of Cs concentration in organisms to the concentration in water) as existed before the accident at the Fukushima Dai-ichi Nuclear Power Station (FDNPS), Japan, in 2011, we examined stable Cs, as it is thought to exist in equilibrium in the environment and behave similarly to radioactive Cs in aquatic animals. The concentration of stable Cs in 241 P. olivaceus (range 216-782 mm total length [TL]) collected in Sendai Bay, approximately 90 km north of the FDNPS, in June-July 2015 was expressed as an exponential function with size as an independent variable; the results show the concentration of stable Cs doubled with an increase in TL of 442 mm. Next, to evaluate the cause of the size-dependent change in stable Cs concentration, we examined 909 individuals (200-770 mm TL) collected in September 2013-July 2015 to determine their feeding habit based on size. Analysis of the frequency of occurrence of prey organisms in stomach contents showed that sand lance Ammodytes japonicus (55-180 mm standard length [SL]) was the most consistently consumed across size classes. Analysis on a wet-mass basis showed that A. japonicus and anchovy Engraulis japonicus (65-130 mm SL) were the main food of P. olivaceus sized 200-599 mm TL, whereas chub mackerel Scomber japonicus (120-230 mm SL) and two species of flatfishes (180-205 mm SL) were abundant in the diet of P. olivaceus sized ≥600 mm TL. All these prey items were presumed to have similar concentrations of stable Cs. Based on the above, the effect of diet on the relationship between stable Cs in muscle and fish size was considered negligible. That the diet of P. olivaceus largely did not change with size was also confirmed by C and N stable isotope ratios in P. olivaceus and their prey species. Therefore, the Cs-size relationship is probably determined by changes in the balance between the rate of Cs intake from food and seawater and the excretion rate during growth, both of which change as functions of body mass. Values of stable Cs concentrations among environmental components and animals appear to be a valid indicator for understanding the radioactive Cs distribution in the marine environment and aquatic animals under the equilibrium state, as existed before the 2011 nuclear accident.

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.

Contrasting life-history responses to climate variability in eastern and western North Pacific sardine populations.

Massive populations of sardines inhabit both the western and eastern boundaries of the world's subtropical ocean basins, supporting both commercial fisheries and populations of marine predators. Sardine populations in western and eastern boundary current systems have responded oppositely to decadal scale anomalies in ocean temperature, but the mechanism for differing variability has remained unclear. Here, based on otolith microstructure and high-resolution stable isotope analyses, we show that habitat temperature, early life growth rates, energy expenditure, metabolically optimal temperature, and, most importantly, the relationship between growth rate and temperature are remarkably different between the two subpopulations in the western and eastern North Pacific. Varying metabolic responses to environmental changes partly explain the contrasting growth responses. Consistent differences in the life-history traits are observed between subpopulations in the western and eastern boundary current systems around South Africa. These growth and survival characteristics can facilitate the contrasting responses of sardine populations to climate change.

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.

Mid-Cretaceous marine Os isotope evidence for heterogeneous cause of oceanic anoxic events.

During the mid-Cretaceous, the Earth experienced several environmental perturbations, including an extremely warm climate and Oceanic Anoxic Events (OAEs). Submarine volcanic episodes associated with formation of large igneous provinces (LIPs) may have triggered these perturbations. The osmium isotopic ratio (187Os/188Os) is a suitable proxy for tracing hydrothermal activity associated with the LIPs formation, but 187Os/188Os data from the mid-Cretaceous are limited to short time intervals. Here we provide a continuous high-resolution marine 187Os/188Os record covering all mid-Cretaceous OAEs. Several OAEs (OAE1a, Wezel and Fallot events, and OAE2) correspond to unradiogenic 187Os/188Os shifts, suggesting that they were triggered by massive submarine volcanic episodes. However, minor OAEs (OAE1c and OAE1d), which do not show pronounced unradiogenic 187Os/188Os shifts, were likely caused by enhanced monsoonal activity. Because the subaerial LIPs volcanic episodes and Circum-Pacific volcanism correspond to the highest temperature and pCO2 during the mid-Cretaceous, they may have caused the hot mid-Cretaceous climate.

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.

Anadromous red-spotted masu salmon (Oncorhynchus masou ishikawae), a southernmost sea-migration form of salmonid, displays low variation in both age at seaward migration and sea age.

We examined variations in age at seaward migration and sea age for the anadromous form of red-spotted masu salmon (Oncorhynchus masou ishikawae) in two Japanese rivers. The anadromous form of red-spotted masu salmon expressed only two sea migration patterns in the two rivers: (a) the majority of the salmon (95%, n = 81) were of age-0, and age-1 migrants were rare (n = 4); and (b) all the salmon examined (n = 22) made a return migration within a year, with 23% of the salmon exhibiting potamodromy in the river. Owing to low variation in their sea migratory patterns, the anadromous form of red-spotted masu salmon is likely vulnerable to environmental fluctuations.

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.

Sulfur assimilation in corals with aposymbiotic and symbiotic zooxanthellae.

Although sulfate ions are the main form of sulfur in the ocean, there is limited knowledge on their use by living organisms. Stable isotope labelling and NanoSIMS analysis were used in this study to clarify how sulfate, in seawater, is assimilated by corals and zooxanthellae at the cellular level. Aposymbiotic and symbiotic coral juveniles from the genus Acropora were incubated for 2 days in filtered seawater with 34 S-labelled sulfate. Further, the labelled corals were incubated for additional 2 days in natural seawater. Mapping of sulfur isotopes (34 S/32 S) showed that the 'hotspots' were enriched in 34 S on a sub-micro level and were heterogeneously distributed in the coral soft tissues. Specifically, 34 S hotspots were found in both the symbiotic zooxanthellae and coral host tissues. In aposymbiotic corals, 34 S was detected in the tissues, indicating that the host corals directly assimilated the sulfate ions without any aid from the zooxanthellae. Even after 2 days in normal seawater, the 34 S label was clearly seen in both symbiotic and aposymbiotic corals, indicating that the assimilated sulfur was retained for at least 2 days.

Tracking long-distance migration of marine fishes using compound-specific stable isotope analysis of amino acids.

The long-distance migrations by marine fishes are difficult to track by field observation. Here, we propose a new method to track such migrations using stable nitrogen isotopic composition at the base of the food web (δ15 NBase ), which can be estimated by using compound-specific isotope analysis. δ15 NBase exclusively reflects the δ15 N of nitrate in the ocean at a regional scale and is not affected by the trophic position of sampled organisms. In other words, δ15 NBase allows for direct comparison of isotope ratios between proxy organisms of the isoscape and the target migratory animal. We initially constructed a δ15 NBase isoscape in the northern North Pacific by bulk and compound-specific isotope analyses of copepods (n = 360 and 24, respectively), and then we determined retrospective δ15 NBase values of spawning chum salmon (Oncorhynchus keta) from their vertebral centra (10 sections from each of two salmon). We then estimated the migration routes of chum salmon during their skeletal growth by using a state-space model. Our isotope tracking method successfully reproduced a known chum salmon migration route between the Okhotsk and Bering seas, and our findings suggest the presence of a new migration route to the Bering Sea Shelf during a later growth stage.

Land Invasion by the Mudskipper, Periophthalmodon septemradiatus, in Fresh and Saline Waters of the Mekong River.

There has been a long-standing controversy about whether vertebrates emerged in the Paleozoic from marine or freshwater environments. Several hypotheses have proposed coastal, estuarine and riparian areas as sites of the transition. Here, we report the ecology of an amphibious fish Periophthalmodon septemradiatus, which we presume is in the process of niche expansion into terrestrial habitats from estuarine to freshwater environments along the Mekong River, Vietnam. Adult fish are highly terrestrial and have not been observed to venture into water during our survey. Courtship behaviour was observed, and fertilised eggs were recovered from burrows in both brackish and freshwater environments. The smallest fish collected at 12, 96, and 148 km from the river mouth were juveniles shortly after starting an amphibious life. These findings suggest reproduction in both brackish and freshwater environments. In contrast, otolith Sr:Ca ratio indicates larval hatching only in brackish water. Analysis of a 940-base pair (bp) segment of the mitochondrial cytochrome c oxidase subunit II and a 934-bp segment of the mitochondrial D-loop demonstrated no genetic segregation between populations. The fish may provide a unique opportunity to study how ambient salinity affects the biology and ecology of a living vertebrate during transition from water to land.

Effects of tourism-derived sewage on coral reefs: Isotopic assessments identify effective bioindicators.

Pulau Redang and Pulau Tioman have experienced huge tourism growth over the last two decades, but minimal sewage treatment may threaten the resilience of their coral reefs. This study uses stable isotope techniques to identify suitable bioindicators of sewage nutrients (δ15N) at these islands by measuring macroalgae (Lobophora spp.), gastropods (Drupella spp.), scleractinian coral (Acropora spp.), and leather coral (Sinularia spp.). At tourist hubs using seepage septic tank systems, enrichment of Acropora δ15N (Redang, +0.7‰) and Sinularia δ15N (Tioman, +0.4‰) compared to pristine background levels indicate enhanced sewage nutrient discharge. Carbon isotopes and survey data suggest that sedimentation did not confound these δ15N trends. Potential damaging effects of sewage discharge on the coral reef communities at both islands are highlighted by strong correlations between Acropora δ15N and regional variation in coral reef community structure, and exclusive occurrence of degraded reefs at regions of high sewage influence.

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.

Distribution of wild and stocked Japanese eels in the lower reaches of the Tone River catchment revealed by otolith stable-isotope ratios.

The natural occurrence of Japanese eels Anguilla japonica in Japan's highly altered catchments is a critical measure of habitat restoration and ecosystem health, yet this metric may be obscured by the incidence of co-occurring cultured eels. Distribution of wild and stocked A. japonica in the lower reaches of the Tone River cachment was investigated using otolith oxygen and carbon stable-isotope ratios (δ18 O and δ13 C). We developed a discrimination model to classify wild and cultured eels for 560 individuals and applied it to eels captured in the lower reaches of the Tone River catchment. Wild eels were found at all study sites of this part of the catchment, indicating natural recruitment of the species occurred from the estuary to third-order streams at least as far as Lake Imbanuma. The proportion of wild eels ranged from 86 to 100% at river and stream sites and was only 23% at upstream Lake Imbanuma sites. This difference probably depends on whether or not eels were released near the corresponding site. More than 50% of stocked eels occurred at sites proximate to release locations, indicating limited dispersal by cultured eels.