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SummaryNutrients from the host diet and microbial cross-feeding allow diverse bacteria to colonize the animal gut. Less is known about the role of host-derived nutrients in enabling gut bacterial colonization. We examined metabolic interactions within the evolutionary ancient symbiosis between the honey bee (Apis mellifera) and the core gut microbiota memberSnodgrassella alvi. This Betaproteobacteria is incapable of metabolizing saccharides, yet colonizes the honey bee gut in the presence of only a sugar diet. Using comparative metabolomics,13C tracers, and Nanoscale secondary ion mass spectrometry (NanoSIMS), we showin vivothatS. alvigrows on host-derived organic acids, including citrate, glycerate and 3-hydroxy-3-methylglutarate which are actively secreted by the host into the gut lumen.S. alviadditionally modulates tryptophan metabolism in the gut, primarily by converting kynurenine to anthranilate. These results suggest thatSnodgrassellais adapted to a specific metabolic niche in the gut that depends on host-derived nutritional resources.

Efficient nutrient recycling underpins the ecological success of the cnidarian-algal symbiosis in oligotrophic waters that forms the basis of coral reef ecosystems. In a stable symbiosis, nitrogen limitation restricts the growth of algal endosymbionts and stimulates their release of photosynthates to the cnidarian animal host. However, the detailed mechanisms controlling nitrogen availabilityin hospiteand their role in symbiosis regulation remain poorly understood. Here, we studied the metabolic regulation of symbiotic nitrogen cycling in the sea anemone Aiptasia by experimentally altering labile carbon availability in a series of experiments. Using isotope labeling experiments and NanoSIMS imaging, we show that the competition for environmental ammonium between the host and its algal symbionts is regulated by carbon availability. Light regimes that were optimal for algal photosynthesis increased carbon availability in the holobiont and enhanced symbiotic competition for nitrogen. Consequently, algal symbiont densities were lowest under optimal environmental conditions and increased toward the tolerance limits of the symbiosis. This metabolic regulation promotes efficient carbon recycling in a stable symbiosis across a wide range of environmental conditions. At the same time, the dependence on resource competition can favor parasitic interactions that may explain the instability of the symbiosis as the environmental conditions in the Anthropocene shift towards its tolerance limits.

Ocean paleotemperatures have been reconstructed for almost the entirety of the Phanerozoic using the oxygen isotope compositions of calcium carbonates formed by marine organisms and preserved in ocean sediments. However, the isotopic composition of these calcitic tests and shells can be substantially altered through diagenetic processes. Here, we used 18O as an isotopic tracer in controlled experiments designed to simulate early diagenesis of modern benthic foraminifera tests to investigate how fluids penetrate into and exchange oxygen isotopes with these biogenic calcites. Initially pristine tests of Ammonia sp., Haynesina germanica, and Amphistegina lessonii were immersed in an 18O-enriched artificial seawater at 90 °C for hours to days. High-resolution SEM images of the tests before and after the experiments were indistinguishable yet the bulk oxygen isotope compositions of reacted tests revealed rapid and species-dependent isotopic exchange with the water. Correlated SEM, TEM and NanoSIMS imaging of 18O intra-test distributions showed that fluid penetration and exchange is ubiquitous yet heterogenous, and is intimately tied to test ultrastructure and associated organic matter. Species level differences in ultrastructure, quantified through image analysis, explained the observed species-dependent rates of isotopic exchange. Consequently, even calcitic skeletons considered texturally pristine for paleo-climatic reconstruction purposes may have experienced substantial isotopic exchange and hence a critical re-examination of the paleo-temperature record is warranted.

Significance Empirical laws predicting metabolic rates of a species by its average body mass neglect intraspecies variability arising from the range of physiologically feasible rates and phenotypic heterogeneity. We describe an exploratory experimental test of a theory that explicitly accounts for such variations. We show, by single-cell joint measurements of mass and uptake rates, how marginal distributions of mass and rates collapse onto a common master distribution for species spanning 3 orders of magnitude in cell volume. These results demonstrate the potential of a generalized scaling theory that goes beyond population averages to incorporate within-species variation., Kleiber’s law describes the scaling of metabolic rate with body size across several orders of magnitude in size and across taxa and is widely regarded as a fundamental law in biology. The physiological origins of Kleiber’s law are still debated and generalizations of the law accounting for deviations from the scaling behavior have been proposed. Most theoretical and experimental studies of Kleiber’s law, however, have focused on the relationship between the average body size of a species and its mean metabolic rate, neglecting intraspecific variation of these 2 traits. Here, we propose a theoretical characterization of such variation and report on proof-of-concept experiments with freshwater phytoplankton supporting such framework. We performed joint measurements at the single-cell level of cell volume and nitrogen/carbon uptake rates, as proxies of metabolic rates, of 3 phytoplankton species using nanoscale secondary ion mass spectrometry (NanoSIMS) and stable isotope labeling. Common scaling features of the distribution of nutrient uptake rates and cell volume are found to hold across 3 orders of magnitude in cell size. Once individual measurements of cell volume and nutrient uptake rate within a species are appropriately rescaled by a function of the average cell volume within each species, we find that intraspecific distributions of cell volume and metabolic rates collapse onto a universal curve. Based on the experimental results, this work provides the building blocks for a generalized form of Kleiber’s law incorporating intraspecific, correlated variations of nutrient-uptake rates and body sizes.

Some sea slugs are able to steal functional chloroplasts (kleptoplasts) from their algal food sources, but the role and relevance of photosynthesis to the animal host remain controversial. While some researchers claim that kleptoplasts are slowly digestible ‘snacks’, others advocate that they enhance the overall fitness of sea slugs much more profoundly. Our analysis show light-dependent incorporation of 13C and 15N in the albumen gland and gonadal follicles of the sea slug Elysia timida, representing translocation of photosynthates to kleptoplast-free reproductive organs. Long-chain polyunsaturated fatty acids with reported roles in reproduction were produced in the sea slug cells using labelled precursors translocated from the kleptoplasts. Finally, we report reduced fecundity of E. timida by suppressing kleptoplast photosynthesis. The present study provides the first thorough experimental evidence that photosynthesis enhances the reproductive fitness of kleptoplast-bearing sea slugs, confirming the biological relevance of this remarkable association between a metazoan and an algal-derived organelle. Teaser Sea slugs incorporate functional chloroplasts from algae and use products of photosynthesis to maximize reproductive output.

For our understanding of Alzheimer’s disease (AD) pathophysiology, it is of critical importance to determine how key pathological factors, specifically amyloid β (Aβ) plaque formation, are interconnected and implicated in neurodegeneration, disease progression and the development of clinical symptoms. Exactly how Aβ plaque formation is initiated and how the ongoing plaque deposition proceeds is not well understood. This is partly because we can only examine details of the molecular pathology after death in humans, and in mice, we can only examine a particular point in time without any longitudinal information on the fate of individually formed deposits. Herein, we used metabolic labelling of proteins with stable isotopes, together with multimodal imaging mass spectrometry (IMS) for imaging stable isotope labelling kinetics (iSILK) in the APPNL-G-F knock-in mouse model of AD. The aim was to monitor the earliest seeds of Aβ deposition through ongoing plaque development and track the deposition of Aβ that is produced later in relation to already deposited plaques. This allowed us to visualize Aβ peptide aggregation dynamics within individual deposits across different brain regions. We identified the cortex as a primary site of deposition in precipitating plaque pathology. Further, our data show that structural plaque heterogeneity is associated with differential peptide deposition. Specifically, Aβ1-42 is forming an initial core seed followed by radial outgrowth and late secretion and deposition of Aβ1-38.Together these data prove the potential of iSILK for probing amyloid protein secretion, processing and aggregation dynamics in AD pathology.

Correlative light and electron microscopy allows localization of specific molecules at the ultrastructural level in biological tissue but does not provide information about metabolic turnover or the distribution of labile molecules, such as micronutrients. We present a method to directly correlate (immuno)fluorescent microscopy, (immuno)TEM imaging and NanoSIMS isotopic mapping of the same tissue section, with nanometer-scale spatial precision. The process involves chemical fixation of the tissue, cryo sectioning, thawing, and air-drying under a thin film of polyvinyl alcohol. It permits to effectively retain labile compounds and strongly increases NanoSIMS sensitivity for 13C-enrichment. The method is illustrated here with correlated distribution maps of a carbonic anhydrase enzyme isotype, β-tubulin proteins, and 13C- and 15N-labeled labile micronutrients (and their anabolic derivates) within the tissue of a reef-building symbiotic coral. This broadly applicable workflow expands the wealth of information that can be obtained from multi-modal, sub-cellular observation of biological tissue., Loussert-Fonta et al. have developed a method to directly correlate fluorescent microscopy, (immuno)TEM imaging and NanoSIMS isotopic mapping of the same tissue section, with nanometer-scale spatial precision. They illustrate the technique by imaging tissue from a symbiotic coral, Stylophora pistillata.

Cellular uptake and intracellular trafficking of polymer conjugates or polymer nanoparticles is typically monitored using fluorescence-based techniques such as confocal microscopy. While these methods have provided a wealth of insight into the internalization and trafficking of polymers and polymer nanoparticles, they require fluorescent labeling of the polymer or polymer nanoparticle. Because in biological media fluorescent dyes may degrade, be cleaved from the polymer or particle, or even change uptake and trafficking pathways, there is an interest in fluorescent label-free methods to study the interactions between cells and polymer nanomedicines. This article presents a first proof-of-concept that demonstrates the feasibility of NanoSIMS to monitor the intracellular localization of polymer conjugates. For the experiments reported here, poly( N-(2-hydroxypropyl) methacrylamide)) (PHPMA) was selected as a prototypical polymer-drug conjugate. This PHPMA polymer contained a 19F-label at the α-terminus, which was introduced in order to allow NanoSIMS analysis. Prior to the NanoSIMS experiments, the uptake and intracellular trafficking of the polymer was established using confocal microscopy and flow cytometry. These experiments not only provided detailed insight into the kinetics of these processes but were also important to select time points for the NanoSIMS analysis. For the NanoSIMS experiments, HeLa cells were investigated that had been exposed to the PHPMA polymer for a period of 4 or 15 h, which was known to lead to predominant lysosomal accumulation of the polymer. NanoSIMS analysis of resin-embedded and microtomed samples of the cells revealed a punctuated fluorine signal, which was found to colocalize with the sulfur signal that was attributed to the lysosomal compartments. The localization of the polymer in the endolysosomal compartments was confirmed by TEM analysis on the same cell samples. The results of this study illustrate the potential of NanoSIMS to study the uptake and intracellular trafficking of polymer nanomedicines.

Populations of genetically identical cells can display marked variation in phenotypic traits; such variation is termed phenotypic heterogeneity. Here, we investigate the effect of substrate and electron donor limitation on phenotypic heterogeneity in N2 and CO2 fixation in the green sulphur bacterium Chlorobium phaeobacteroides. We grew populations in chemostats and batch cultures and used stable isotope labelling combined with nanometer-scale secondary ion mass spectrometry (NanoSIMS) to quantify phenotypic heterogeneity. Experiments in H2 S (i.e. electron donor) limited chemostats show that varying levels of NH4+ limitation induce heterogeneity in N2 fixation. Comparison of phenotypic heterogeneity between chemostats and batch (unlimited for H2 S) populations indicates that electron donor limitation drives heterogeneity in N2 and CO2 fixation. Our results demonstrate that phenotypic heterogeneity in a certain metabolic activity can be driven by different modes of limitation and that heterogeneity can emerge in different metabolic processes upon the same mode of limitation. In conclusion, our data suggest that limitation is a general driver of phenotypic heterogeneity in microbial populations.

The composition of the convecting asthenospheric mantle that feeds the mantle wedge can be investigated via rear-arc lavas that have minimal slab influence. This “ambient mantle wedge” composition (the composition of the wedge prior to the addition of a slab component) varies substantially both worldwide and within individual arcs. 143Nd / 144 Nd measurements of rear-arc samples that have minimal slab influence are similar to 143Nd / 144 Nd in the stratovolcanoes of the adjacent volcanic fronts, suggesting that 143Nd / 144 Nd of arc-front volcanics are largely inherited from the ambient mantle composition. 143Nd / 144 Nd correlates with ratios such as Th/U, Zr/Nb, and La/Sm, indicating that these ratios also are strongly influenced by ambient wedge heterogeneity. The same phenomenon is observed among individual volcanoes from the Chilean Southern Volcanic Zone (SVZ), where along-strike variability of the volcanic front tracks that of rear-arc monogenetic volcanics. Depleted mantle wedges are more strongly influenced by slab-derived components than are enriched wedges. This leads to surprising trace element correlations in the global dataset, such as between Pb/Nb and Zr/Nb, which are not explicable by variable compositions or fluxes of slab components. Depleted ambient mantle is present beneath arcs with back-arc spreading; relatively enriched mantle is present adjacent to continents. Ambient mantle wedge heterogeneity both globally and regionally forms isotope mixing trajectories for Sr, Nd and Hf between depleted mantle and EM1-type enriched compositions as represented by Gough Island basalts. Making use of this relationship permits a quantitative match with the SVZ data. It has been suggested that EM1-type mantle reservoirs are the result of recycled lower continental crust, though such models do not account for certain trace element ratios such as Ce/Pb and Nb/U or the surprisingly homogeneous trace element compositions of EM1 volcanics. A model in which the EM1 end-member found in continental arcs is produced by low-degree melt-metasomatism of the sub-continental lithospheric mantle may be more plausible. The 143Nd / 144 Nd maximum along the SVZ may be a consequence of either rifting and collision of two ancient lithospheric domains or a slab tear. The correspondence of mantle wedge variations with EM1 suggests a potential role for metasomatized sub-continental lithosphere in creating EM1 sources globally.

Sponges are the oldest known extant animal-microbe symbiosis. These ubiquitous benthic animals play an important role in marine ecosystems in the cycling of dissolved organic matter (DOM), the largest source of organic matter on Earth. The conventional view on DOM cycling through microbial processing has been challenged by the interaction between this efficient filter-feeding host and its diverse and abundant microbiome. Here we quantify, for the first time, the role of host cells and microbial symbionts in sponge heterotrophy. We combined stable isotope probing and nanoscale secondary ion mass spectrometry to compare the processing of different sources of DOM (glucose, amino acids, algal-produced) and particulate organic matter (POM) by a high-microbial abundance (HMA) and low-microbial abundance (LMA) sponge with single-cell resolution. Contrary to common notion, we found that both microbial symbionts and host choanocyte (i.e. filter) cells and were active in DOM uptake. Although all DOM sources were assimilated by both sponges, higher microbial biomass in the HMA sponge corresponded to an increased capacity to process a greater variety of dissolved compounds. Nevertheless, in situ feeding data demonstrated that DOM was the primary carbon source for both the LMA and HMA sponge, accounting for ~90% of their heterotrophic diets. Microbes accounted for the majority (65–87%) of DOM assimilated by the HMA sponge (and ~60% of its total heterotrophic diet) but

Extracellular bacterial symbionts communicate biochemically with their hosts to establish niches that foster the partnership. Using quantitative ion microprobe isotopic imaging (nanoscale secondary ion mass spectrometry [NanoSIMS]), we surveyed localization of 15 N-labelled molecules produced by the bacterium Vibrio fischeri within the cells of the symbiotic organ of its host, the Hawaiian bobtail squid, and compared that with either labelled non-specific species or amino acids. In all cases, two areas of the organ's epithelia were significantly more 15 N enriched: (a) surface ciliated cells, where environmental symbionts are recruited, and (b) the organ's crypts, where the symbiont population resides in the host. Label enrichment in all cases was strongest inside host cell nuclei, preferentially in the euchromatin regions and the nucleoli. This permissiveness demonstrated that uptake of biomolecules is a general mechanism of the epithelia, but the specific responses to V. fischeri cells recruited to the organ's surface are due to some property exclusive to this species. Similarly, in the organ's deeper crypts, the host responds to common bacterial products that only the specific symbiont can present in that location. The application of NanoSIMS allows the discovery of such distinct modes of downstream signalling dependent on location within the host and provides a unique opportunity to study the microbiogeographical patterns of symbiotic dialogue.

Changes in seawater chemistry have affected the evolution of calcifying marine organisms, including their skeletal polymorph (calcite versus aragonite), which is believed to have been strongly influenced by the Mg/Ca ratio at the time these animals first emerged. However, we show that micrabaciids, a scleractinian coral clade that first appeared in the fossil record of the Cretaceous, when the ocean Mg/Ca ratio was near the lowest in the Phanerozoic (thus a priori favoring calcitic mineralogy), formed skeletons composed exclusively of aragonite. Exceptionally preserved aragonitic coralla of Micrabacia from the Late Cretaceous Ripley Formation (southeastern USA) have skeletal microstructures identical to their modern representatives. In addition, skeletons of Micrabacia from Cretaceous chalk deposits of eastern Poland are clearly diagenetically altered in a manner consistent with originally aragonitic mineralogy. These deposits have also preserved fossils of the scleractinian Coelosmilia, the skeleton of which is interpreted as originally calcitic. These findings show that if changes in seawater Mg/Ca ratio influenced the mineralogy of scleractinian corals, the outcome was taxon specific. The aragonitic mineralogy, unique skeletal microstructures and ultrastructures, and low Mg/Ca ratios in both fossil and living micrabaciids indicate that their biomineralization process is strongly controlled and has withstood major fluctuations in seawater chemistry during the past 70 m.y.

Cisplatin is a widely used anti-cancer drug, but its effect is often limited by acquired resistance to the compound during treatment. Here, we use a combination of transmission electron microscopy (TEM) and nanoscale-secondary ion mass spectrometry (NanoSIMS) to reveal differences between cisplatin uptake in human ovarian cancers cells, which are known to be susceptible to acquired resistance to cisplatin. Both cisplatin sensitive and resistant cell lines were studied, revealing markedly less cisplatin in the resistant cell line. In cisplatin sensitive cells, Pt was seen to distribute diffusely in the cells with hotspots in the nucleolus, mitochondria, and autophagosomes. Inductively coupled plasma mass spectrometry (ICP-MS) was used to validate the NanoSIMS results.

The processes that lead to the fourfold variation in arc-averaged compositions of mafic arc iavas remain controversial. Control by the mantle-wedge thermal structure is supported by chemical correlations with the thickness of the underlying arc crust(1-3), which affects the thermal state of the wedge. Control by down-going slab temperature is supported by correlations with the slab thermal parameter(3-7). The Chilean Southern Volcanic Zone provides a test of these hypotheses. Here we use chemical data to demonstrate that the Southern Volcanic Zone and global arc averages define the same chemical trends, both among elements and between elements and crustal thickness. But in contrast to the global arc system, the Southern Volcanic Zone is built on crust of variable thickness with a constant slab thermal parameter. This natural experiment, along with a set of numerical simulations, shows that global arc compositional variability is dominated by different extents of melting that are controlled by the thermal structure of the mantle wedge. Slab temperatures play a subordinate role. Variations in the subducting slab's fluid flux and sediment compositions, as well as mantle-wedge heterogeneities, produce second-order effects that are manifested as distinctive trace element and isotopic signatures; these can be more clearly elucidated once the importance of wedge thermal structure is recognized.

The upside-down jellyfishCassiopeaengages in symbiosis with photosynthetic microalgae that facilitate uptake and recycling of inorganic nutrients. By contrast to most other symbiotic cnidarians, algal endosymbionts inCassiopeaare not restricted to the gastroderm but are found in amoebocyte cells within the mesoglea. While symbiont-bearing amoebocytes are highly abundant, their role in nutrient uptake and cycling inCassiopearemains unknown. By combining isotopic labelling experiments with correlated scanning electron microscopy, and Nano-scale secondary ion mass spectrometry (NanoSIMS) imaging, we quantified the anabolic assimilation of inorganic carbon and nitrogen at the subcellular level in juvenileCassiopeamedusae bell tissue. Amoebocytes were clustered near the sub-umbrella epidermis and facilitated efficient assimilation of inorganic nutrients. Photosynthetically fixed carbon was efficiently translocated between endosymbionts, amoebocytes and host epidermis at rates similar to or exceeding those observed in corals. TheCassiopeaholobionts efficiently assimilated ammonium, while no nitrate assimilation was detected, possibly reflecting adaptation to highly dynamic environmental conditions of their natural habitat. The motile amoebocytes allowCassiopeamedusae to distribute their endosymbiont population to optimize access to light and nutrients, and transport nutrition between tissue areas. Amoebocytes thus play a vital role for the assimilation and translocation of nutrients inCassiopea, providing an interesting new model for studies of metabolic interactions in photosymbiotic marine organisms.

Corals access inorganic seawater nutrients through their autotrophic endosymbiotic dinoflagellates, but also capture planktonic prey through heterotrophic feeding. Correlating NanoSIMS and TEM imaging, we visualized and quantified the subcellular fate of autotrophic and heterotrophic C and N in the coral Stylophora pistillata using stable isotopes. Six scenarios were compared after 6 h: autotrophic pulse (13C-bicarbonate, 15N-nitrate) in either unfed or regularly fed corals, and heterotrophic pulse (13C-, 15N-labelled brine shrimps) in regularly fed corals; each at ambient and elevated temperature. Host assimilation of photosynthates was similar under fed and unfed conditions, but symbionts assimilated 10% more C in fed corals. Photoautotrophic C was primarily channelled into host lipid bodies, whereas heterotrophic C and N were generally co-allocated to the tissue. Food-derived label was detected in some subcellular structures associated with the remobilisation of host lipid stores. While heterotrophic input generally exceeded autotrophic input, it was more negatively affected by elevated temperature. The reduced input from both modes of nutrition at elevated temperature was accompanied by a shift in the partitioning of C and N, benefiting epidermis and symbionts. This study provides a unique view into the nutrient partitioning in corals and highlights the tight connection of nutrient fluxes in symbiotic partners.

Textural and chemical differences in coeval rhyolitic effusive and explosive eruptions are commonly observed, and numerical models predict different timescales for the eruption of crystal-rich mushes versus crystal-poor magmas. We compare quartz zonation and diffusion timescales of crystal-rich rhyolitic ignimbrites and crystal-poor rhyolitic lava flows from the Jurassic Chon Aike Province exposed in Patagonia (Argentina). The timescales are assessed by using diffusion modeling based on nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of titanium (Ti) concentration profiles in quartz crystals oriented by image analysis using micro-tomography. Quantitative Ti-data were acquired by SIMS to estimate crystallization temperatures. The textural and geochemical analysis revealed clear differences between crystal-poor rhyolitic lava flows and crystal-rich rhyolitic ignimbrites. Quartz crystals from rhyolitic lava flows display simple oscillatory cathodoluminescence (CL) zoning interpreted to be magmatic and diffusion chronometry suggest a short timescale for quartz crystallization from 5.6 ± 2.2 to 41.6 ± 9.8 years. Resorption textures are rare, and hence crystals in rhyolitic lava flows recorded a simple, rapid extraction, transport and eruption history for these crystal-poor melts. Rhyolitic ignimbrites, in contrast, reveal complex zoning patterns, reflecting several episodes of partial resorption and growth throughout their crystallization history. The complex quartz zoning textures together with longer diffusion times (

A key parameter in the study of magma evolution is the time scale on which magmatic processes occur. Using nanoscale secondary ion mass spectrometry (NanoSIMS), SIMS, and cathodoluminescence (CL) analyses, we have measured titanium (Ti) diffusion profiles in quartz phenocrysts from a Jurassic rhyolite of the El Quemado Complex (Patagonia, Argentina), providing new insights into the time scales of the associated volcanic processes. CL imaging of quartz phenocrysts reveals oscillatory magmatic zoning. We determined Ti concentrations with SIMS and acquired multiple NanoSIMS profiles across growth zones from core to rim. All transects show sharp changes in the Ti-48/Si-29 ratio, which correlate reasonably well with changes in CL intensity. Diffusion modeling of Ti in quartz yields a surprisingly short time scale for quartz crystallization of 5.6 +/- 2.2 yr and a rapid crystal growth rate of 2.3 x 10(-12) m/s. Based on the observed quartz textures, we suggest that the rhyolite erupted shortly after initial onset of crystallization, followed by decompression-driven quartz dissolution during fast magma ascent. We further argue that the observed oscillatory zoning and the variation of the Ti concentration of the quartz phenocryst does not reflect temperature, pressure, or titanium activity (a(Ti)) changes of the magmatic system, but rather is the result of growth kinetics, which has important implications for the Ti-in-quartz thermometry.

Growth dynamics of the primary spine of the cidaroid sea urchin Phyllacanthus imperialis was assessed for the first time using pulsed (26) Mg-labeling and NanoSIMS isotopic imaging. The sea urchin was incubated twice (for 48 h) in artificial seawater with elevated level of (26) Mg. After each labeling event, the sea urchin was returned for 72 h to seawater with natural isotopic abundance of (26) Mg. NanoSIMS ion microprobe was subsequently used to visualize the labeled regions of the spine with submicrometer lateral resolution. The growth of the new skeleton was restricted to the distalmost and peripheral portions of the spine. Skeletogenesis involved mostly the deposition of continuous thickening layers and lateral growth involving bridges between previously formed trabeculae. The timescale of formation of individual thickening layers (ca. 1 µm in width) on the stereom trabeculae was on the order of 1 day. Longitudinal growth occurred mainly at the periphery in the form of small portions of the thickening deposits or more massive microspines that appeared to branch and fuse with those above and below. These microspines were found to grow at about 10 µm/day. These results reveal that the skeletal growth of a juvenile cidaroid spine is complex and highly heterogeneous, with different extension rates depending on the stage of the stereom development and/or direction of the growth fronts. The growth pattern observed here at the submicrometer scale provides direct evidence supporting the earlier suggestions that the lamellar structure of echinoderm stereom is formed by periodic deposition of continuous mineral layers.

Most microorganisms live in environments where nutrients are limited and fluctuate over time. Cells respond to nutrient fluctuations by sensing and adapting their physiological state. Recent studies suggest phenotypic heterogeneity1 in isogenic populations as an alternative strategy in fluctuating environments, where a subpopulation of cells express a function that allows growth under conditions that might arise in the future2–9. It is unknown how environmental factors such as nutrient limitation shape phenotypic heterogeneity in metabolism and whether this allows cells to respond to nutrient fluctuations. Here, we show that substrate limitation increases phenotypic heterogeneity in metabolism, and this heterogeneity allows cells to cope with substrate fluctuations. We subjected the N2-fixing bacterium Klebsiella oxytoca to different levels of substrate limitation and substrate shifts, and obtained time-resolved single-cell measurements of metabolic activities using nanometre-scale secondary ion mass spectrometry (NanoSIMS). We found that the level of NH4+ limitation shapes phenotypic heterogeneity in N2 fixation. In turn, the N2 fixation rate of single cells during NH4+ limitation correlates positively with their growth rate after a shift to NH4+ depletion, experimentally demonstrating the benefit of heterogeneity. The results indicate that phenotypic heterogeneity is a general solution to two important ecological challenges—nutrient limitation and fluctuations—that many microorganisms face. Single-cell measurements of metabolic activities using NanoSIMS reveals that substrate limitation increases phenotypic heterogeneity in Klebsiella oxytoca metabolism, which allows cells to cope with nutrient fluctuations.

The in vitro inter- and intra-cellular distribution of an isotopically labelled ruthenium(II)-arene (RAPTA) anti-metastatic compound in human ovarian cancer cells was imaged using nano-scale secondary ion mass spectrometry (NanoSIMS). Ultra-high resolution isotopic images of (13)C, (15)N, and Ru indicate that the phosphine ligand remains coordinated to the ruthenium(II) ion whereas the arene detaches. The complex localizes mainly on the membrane or at the interface between cells which correlates with its anti-metastatic effects.

Os and Nd isotopic ratios as well as major and trace element compositions have been measured in 17 mafic lavas from Fogo Island, Cape Verde. These new data complement a previous archipelago-scale study (Southern and Northern Islands) by Doucelance et al. [Doucelance, R., Escrig, S., Moreira, M., Gariepy, C., Kurz, M., 2003. Pb-Sr-He isotope and trace element geochemistry of the Cape Verde Archipelago. Geochim. Cosmochim. Acta, 67 (19), 3717-3733], in which major, trace element contents and Sr–Pb–He isotopic compositions of lavas were determined. We also report Os–Sr–Nd–Pb isotopic ratios of 2 carbonatites from Fogo. Fogo mafic lavas have Os–Sr–Nd–Pb isotopic ratios that define correlations similar to those previously observed for the Cape Verde Southern Islands and interpreted to reflect a mixture between a moderate HIMU end-member (206Pb / 204Pb ∼ 20), common to the Northern and Southern Islands of the archipelago, and an EM1-like end-member. Similar isotopic correlations are observed within the different lava flows which requires that the two end-members are mixed during the lava differentiation at shallow depth. The increasing contribution of the enriched end-member through time in Fogo lavas confirms the shallow origin for the EM1 signature. As with Pb, Sr and He, the Os and Nd isotopic compositions of the moderate HIMU end-member are explained by the mixing of 1.6 Ga recycled oceanic crust (ROC) and lower mantle material (LM) and interpreted to represent the Cape Verde mantle plume expression. For the EM1-like end-member, the high 187Os / 188Os ratios that are measured in mafic lavas preclude the sub-continental lithospheric mantle from being its origin, as previously proposed based on trace element and Pb, Sr and Nd isotopic compositions. Our new isotopic and trace element data still indicate that the enriched component is related to continental lithospheric material incorporated in the oceanic lithosphere during the opening of the Atlantic Ocean but requires the involvement of lower continental crust fragments.

The isotopic compositions of mid-ocean-ridge basalts (MORB) from the Indian Ocean have led to the identification of a large-scale isotopic anomaly relative to Pacific and Atlantic ocean MORB1. Constraining the origin of this so-called DUPAL anomaly2 may lead to a better understanding of the genesis of upper-mantle heterogeneity. Previous isotopic studies3,4,5,6,7,8,9,10 have proposed recycling of ancient subcontinental lithospheric mantle or sediments with oceanic crust to be responsible for the DUPAL signature. Here we report Os, Pb, Sr and Nd isotopic compositions of Indian MORB from the Central Indian ridge, the Rodriguez triple junction and the South West Indian ridge. All measured samples have higher 187Os/188Os ratios than the depleted upper-mantle value11,12 and Pb, Sr and Nd isotopic compositions that imply the involvement of at least two distinct enriched components in the Indian upper-mantle. Using isotopic and geodynamical arguments, we reject both subcontinental lithospheric mantle and recycled sediments with oceanic crust as the cause of the DUPAL anomaly. Instead, we argue that delamination of lower continental crust may explain the DUPAL isotopic signature of Indian MORB.

Nanoscale secondary ion mass spectrometry (NanoSIMS) combined with transmission electron microscopy (TEM) can be a powerful approach to visualize the exact distribution of drugs at the sub-cellular level. In this work, we exploit this approach to identify the distribution and localisation of the organometallic ruthenium(II)-arene drug Ru((6)-C6H5Me)(pta)Cl-2, termed RAPTA-T, in MDA-MB-231 and MCF-7 human breast cancer cells. These cell lines have been chosen because the former cell lines are highly invasive and resistant to most chemotherapeutic agents and the latter ones are very sensitive to hormonal-based therapies. In the MDA-MB-231 cells, RAPTA-T was found to predominantly localise on the cell membrane and to a lesser extent in the nucleolus. These findings are consistent with the previously reported anti-metastatic properties of RAPTA-T and the observation that once internalized RAPTA-T is associated with chromatin. RAPTA-T shows a lack of membrane accumulation on the non-invasive MCF-7 cells, which correlates well with its selective anti-metastatic properties on invasive cell lines.

International audience; High input of organic carbon and/or slowly renewing bottom waters frequently create periods with low dissolved oxygen concentrations on continental shelves and in coastal areas; such events can have strong impacts on benthic ecosystems. Among the meiofauna living in these environments, benthic foraminifera are often the most tolerant to low oxygen levels. Indeed, some species are able to survive complete anoxia for weeks to months. One known mechanism for this, observed in several species, is denitrification. For other species, a state of highly reduced metabolism, essentially a state of dormancy, has been proposed but never demonstrated. Here, we combined a 4 weeks feeding experiment, using 13C-enriched diatom biofilm, with correlated TEM and NanoSIMS imaging, plus bulk analysis of concentration and stable carbon isotopic composition of total organic matter and individual fatty acids, to study metabolic differences in the intertidal species Ammonia tepida exposed to oxic and anoxic conditions. Strongly contrasting cellular-level dynamics of ingestion and transfer of the ingested biofilm components were observed between the two conditions. Under oxic conditions, within a few days, intact diatoms were ingested, degraded, and their components assimilated, in part for biosynthesis of different cellular components: 13C-labeled lipid droplets formed after a few days and were subsequently lost (partially) through respiration. In contrast, in anoxia, fewer diatoms were initially ingested and these were not assimilated or metabolized further, but remained visible within the foraminiferal cytoplasm even after 4 weeks. Under oxic conditions, compound specific 13C analyses showed substantial de novo synthesis by the foraminifera of specific polyunsaturated fatty acids (PUFAs), such as 20:4(n-6). Very limited PUFA synthesis was observed under anoxia. Together, our results show that anoxia induced a greatly reduced rate of heterotrophic metabolism in Ammonia tepida on a time scale of less than 24 hours, these observations are consistent with a state of dormancy.

Growth dynamics of the primary spine of the cidaroid sea urchin Phyllacanthus imperialis was assessed for the first time using pulsed (26) Mg-labeling and NanoSIMS isotopic imaging. The sea urchin was incubated twice (for 48 h) in artificial seawater with elevated level of (26) Mg. After each labeling event, the sea urchin was returned for 72 h to seawater with natural isotopic abundance of (26) Mg. NanoSIMS ion microprobe was subsequently used to visualize the labeled regions of the spine with submicrometer lateral resolution. The growth of the new skeleton was restricted to the distalmost and peripheral portions of the spine. Skeletogenesis involved mostly the deposition of continuous thickening layers and lateral growth involving bridges between previously formed trabeculae. The timescale of formation of individual thickening layers (ca. 1 µm in width) on the stereom trabeculae was on the order of 1 day. Longitudinal growth occurred mainly at the periphery in the form of small portions of the thickening deposits or more massive microspines that appeared to branch and fuse with those above and below. These microspines were found to grow at about 10 µm/day. These results reveal that the skeletal growth of a juvenile cidaroid spine is complex and highly heterogeneous, with different extension rates depending on the stage of the stereom development and/or direction of the growth fronts. The growth pattern observed here at the submicrometer scale provides direct evidence supporting the earlier suggestions that the lamellar structure of echinoderm stereom is formed by periodic deposition of continuous mineral layers.

[1] New major element, trace element and Pb-Sr-Nd isotope data for glasses from the Fonualei Spreading Center (FSC) constrain the genesis of back-arc basin basalts and the origins of boninites. The FSC is an end-member for global back-arc lavas in terms of low Ti8.0 and Na8.0, and contains lavas with a boninitic signature. Latitudinal variations reveal a correspondence in location between back-arc and adjacent arc volcanism. The locations of spikes in subduction input and positive bathymetric anomalies along the FSC correspond to the projected location of the arc volcanoes, likely reflecting 3-D convective structure of the mantle wedge. Non-mobile trace elements in arc and back-arc lavas reveal an increasing proportion northward of a re-enriched refractory mantle source, which is supported by isotope data. Quantitative modeling constrains the extents of melting, fraction of enriched mantle and subduction input. For the FSC, extents of melting are exceptionally large. We show a general relationship between extent of melting, subduction input and distance from the arc that applies to both the Eastern Lau Spreading Center and the FSC segment closer to the arc. In the center of the FSC where the arc volcanism is captured by the back-arc, exceptionally high subduction input and even greater extents of melting are observed, producing melts with boninitic signature. Boninitic samples require the juxtaposition of high subduction input and refractory mantle, leading to large integrated extents of melting, an occurrence that can be produced by multiple causes.

Basalts from the Mid-Atlantic Ridge change progressively in composition with increasing distance from the Azores platform. Study of the Lucky Strike and Menez Gwen segments reveals much complexity in the gradient. Both segments contain only basalts enriched relative to normal mid-oceanic ridge basalt, but in two distinct groups. Moderately enriched basalts occur throughout the segments, with proximal Menez Gwen enriched relative to Lucky Strike. Highly enriched basalts occur at segment centers. Incompatible element ratios of the highly enriched basalts exceed those of the Azores platform, while isotopic compositions are less enriched. These observations can be explained by a low-degree melt of garnet-bearing Azores mantle added to mantle depleted by previous melt extraction. Melting this “metasomatized” mantle produces lavas that match the enriched samples. The Azores gradient cannot be explained by simple two-component mixing; rather, it reflects recent melt extraction and addition processes related to southward flow of the Azores plume. The Azores gradient also permits tests of segmentation models. Central supply models predict step functions in chemical compositions between segments. Within-segment gradients require vertical supply. Central supply is supported by robust central volcanoes, thicker crust at segment centers, and a step function in isotopes between the segments. The lava diversity at segment centers, however, requires batches of distinct magma that are preserved through melting and melt delivery. Within-segment gradients in moderately incompatible element ratios support a component of multiple supply. The data suggest partial homogenization of magma within a segment and preferential melt focusing to segment centers with some vertical transport.

[1] New high-density sampling of the Eastern Lau Spreading Center provides constraints on the processes that affect the mantle wedge beneath a back-arc environment, including the effect of the subduction input on basalt petrogenesis and the change in subduction input with distance from the Tonga arc. We obtained trace element and Pb-Sr-Nd isotopic compositions of 64 samples distributed between 20.2°S and 22.3°S with an average spacing of ∼3.6 km. The trace element and isotope variations do not vary simply with distance from the arc and reflect variations in the mantle wedge composition and the presence of multiple components in the subduction input. The mantle wedge composition varies form north to south, owing to the southward migration of Indian-like mantle, progressively replacing the initially Pacific-like mantle wedge. The mantle wedge compositions also require an enriched mid-ocean ridge basalt–like trace element enrichment that has little effect on isotope ratios, suggesting recent low-degree melt enrichment events. The composition of the subduction input added to the mantle wedge is geographically variable and mirrors the changes observed in the Tonga arc island lavas. The combination of the back-arc and arc data allows identification of several components contributing to the subduction input. These are a fluid derived from the altered oceanic crust with a possible sedimentary contribution, a pelagic sediment partial melt, and, in the southern Lau basin, a volcaniclastic sediment partial melt. While on a regional scale, there is a rough decrease in subduction influence with the distance from the arc, on smaller scales, the distribution of the subduction input reflects different mechanisms of the addition of the subduction input to a variable mantle wedge.

This paper explores the chemical systematics of back-arc basins and the physical processes that give rise to them, making use of published data from the Scotia, Mariana, Lau, and Manus Basins. A new low-pressure fractionation model is used to back-correct data with greater than 5.5 wt.% MgO. Even after hydrous correction, back-arc basin basalts (BABB) have low TiO 2 and FeO contents relative to basalts from other ridges. The low TiO 2 both absolutely and relative to Na 2 O requires a source depletion followed by a Na enrichment. This signature is critical to evaluate the range of mantle temperature at back-arc basins, which is about 100°C. In addition to a subduction component and wedge depletion, BABB reflect a prevalent enriched component akin to enriched ocean ridge basalts worldwide, despite the absence of mantle plumes. Data from the Mariana Basin suggest this component arises from very recent addition of low-degree (low-F) melts, which may be an important general agent of mantle heterogeneity. Important aspects of the back-arc data are the linear relationships among all major element parameters with each other and with water. Previous models involving isothermal, isobaric melting with increasing water contents do not account for these relationships. A constraint on permissible physical models is that both trace element and major element data show no inherited effects from garnet in back-arc basins, which constrains generation and transport of melts from great depth to the base of the melting regime. Quantitative modeling of the effects of water on mantle melting shows that previous conclusions based on the MELTS thermodynamic approach are not consistent with experimental data for the mantle. Our new models can account for the back-arc systematics by mixing between dry, pooled fractional melts, formed similarly to open ocean ridges, with hydrous melts generated from sources enriched in H 2 O, Na 2 O, and K 2 O that have equilibrated at low pressure. Thus, successful physical models must be able to produce melts by these two different mechanisms. The effects of H 2 O in back-arc basin ridges and open ocean ridges contrast markedly. In the open ocean, increased water is associated with lower mean extents of melting, increased TiO 2 contents, and an increased garnet effect. In back-arc basins, increased water is associated with increased extents of melting, lower TiO 2 , and no garnet influence. These differences can be accounted for by contrasts in the melting regimes and tectonic setting of the two environments. In the open ocean, deep, low-degree, hydrous melts are produced in the "wings" of the melting regime and combine with higher degree drier melts produced at a range of shallower pressures. At back-arcs, geometrically and thermally, there is no room for "wings" on the arc side of back-arc spreading centers. On the arc side of the spreading center, where water is added, shallow hydrous melting is important, and melt must get to the surface in the context of descending mantle flow. On the back side, dry melting under relatively anhydrous conditions occurs, similar to open ocean ridges. Mixing between melts from the dry side and the wet side should then lead to the characteristic spectra of parental BABB compositions. Both the geometry of melting and the fact of continual rifting of young lithosphere may contribute to the very different water signatures in the open ocean and back-arc settings.

Correction for ‘NanoSIMS analysis of an isotopically labelled organometallic ruthenium(ii) drug to probe its distribution and state in vitro’ by Ronald F. S. Lee et al., Chem. Commun., 2015, DOI: 10.1039/c5cc06983a.

The density of dinoflagellate microalgae in the tissue of symbiotic corals is an important determinant for health and productivity of the coral animal. Yet, the specific mechanism for their regulation and the consequence for coral nutrition are insufficiently understood due to past methodological limitations to resolve the fine-scale metabolic consequences of fluctuating densities. Here, we characterized the physiological and nutritional consequences of symbiont density variations on the colony and tissue level inStylophora pistillatafrom the Red Sea. Alterations in symbiont photophysiology maintained coral productivity and host nutrition across a broad range of symbiont densities. However, we demonstrate that density-dependent nutrient competition between individual symbiont cells, manifested as reduced nitrogen assimilation and cell biomass, probably creates the negative feedback mechanism for symbiont population growth that ultimately defines the steady-state density. Despite fundamental changes in symbiont nitrogen assimilation, we found no density-related metabolic optimum beyond which host nutrient assimilation or tissue biomass declined, indicating that host nutrient demand is sufficiently met across the typically observed range of symbiont densities under ambient conditions.

The human gut hosts trillions of microorganisms that exert a profound influence on human biology. Gut bacteria communicate with their host by secreting small molecules that can signal to distant organs in the body. Bile acids are one class of these signaling molecules, synthesized by the host and chemically transformed by the gut microbiota. Among bile acid metabolizers, bile acid 7-dehydroxylating bacteria are commensals of particular importance as they carry out the 7-dehydroxylation of liver-derived primary bile acids to 7-dehydroxylated bile acids. The latter represents a major fraction of the secondary bile acid pool. The microbiology of this group of gut microorganisms is understudied and warrants more attention. Here, we detail the bile acid transformations carried out by the 7-dehydroxylating bacterium Clostridium scindens in vitro and in vivo. In vitro, C. scindens exhibits not only 7α-dehydroxylating capabilities but also, the ability to oxidize other hydroxyl groups and reduce ketone groups in primary and secondary bile acids. This study revealed 12-oxolithocholic acid as a major transient product in the 7α-dehydroxylation of cholic acid. Furthermore, the in vivo study included complementing a gnotobiotic mouse line (devoid of the ability to 7-dehydroxylate bile acids) with C. scindens and investigating its colonization dynamics and bile acid transformations. Using NanoSIMS (Nanoscale Secondary Ion Mass Spectrometry), we demonstrate that the large intestine constitutes a niche for C. scindens, where it efficiently 7-dehydroxylates cholic acid to deoxycholic acid. Overall, this work reveals a novel transient species during 7-dehydroxylation as well as provides direct evidence for the colonization and growth of 7-dehydroxylating bacteria in the large intestine.

Two fundamental requirements of transparent conductive oxides are high conductivity and low optical absorptance, properties strongly dependent on the free-carrier concentration of the film. The free carrier concentration is usually-tuned by the addition of dopant atoms; which are commonly assumed to be uniformly distributed in the films or partially segregated at grain boundaries. Here, the combination of secondary ion mass spectroscopy at the nanometric scale (NanoSIMS) and Kelvin probe force microscopy (KPFM) allows direct imaging of boron-dopant distribution in polycrystalline zinc oxide (ZnO) films. This work demonstrates that the boron atoms have a bimodal spatial distribution within each grain of the ZnO films. NanoSIMS analysis shows that boron atoms are preferentially incorporated into one of the two sides of each ZnO grain. KPFM measurements confirm that boron atoms are electrically active, locally increasing the free-carrier concentration" in the film. The proposed cause of this nonuniform dopant EF conduction distribution is the different sticking coefficient of Zn adatoms on the two distinct surface terminations of the ZnO grains. The higher sticking coefficient of Zn on the c+ surface restricts the boron incorporation on this side of the grains, resulting in preferential boron incorporation on the c- side and causing the bimodal distribution.

Glucose is the primary source of energy for the brain but also an important source of building blocks for proteins, lipids, and nucleic acids. Little is known about the use of glucose for biosynthesis in tissues at the cellular level. We demonstrate that local cerebral metabolic activity can be mapped in mouse brain tissue by quantitatively imaging the biosynthetic products deriving from [U-(13)C]glucose metabolism using a combination of in situ electron microscopy and secondary ion mass-spectroscopy (NanoSIMS). Images of the (13)C-label incorporated into cerebral ultrastructure with ca. 100 nm resolution allowed us to determine the timescale on which the metabolic products of glucose are incorporated into different cells, their sub-compartments and organelles. These were mapped in astrocytes and neurons in the different layers of the motor cortex. We see evidence for high metabolic activity in neurons via the nucleus (13)C enrichment. We observe that in all the major cell compartments, such as e.g. nucleus and Golgi apparatus, neurons incorporate substantially higher concentrations of (13)C-label than astrocytes.

In mammals, glycogen synthesis and degradation are dynamic processes regulating blood and cerebral glucose-levels within a well-defined physiological range. Despite the essential role of glycogen in hepatic and cerebral metabolism, its spatiotemporal distribution at the molecular and cellular level is unclear. By correlating electron microscopy and ultra-high resolution ion microprobe (NanoSIMS) imaging of tissue from fasted mice injected with (13)C-labeled glucose, we demonstrate that liver glycogenesis initiates in the hepatocyte perinuclear region before spreading toward the cell membrane. In the mouse brain, we observe that (13)C is inhomogeneously incorporated into astrocytic glycogen at a rate ~25 times slower than in the liver, in agreement with prior bulk studies. This experiment, using temporally resolved, nanometer-scale imaging of glycogen synthesis and degradation, provides greater insight into glucose metabolism in mammalian organs and shows how this technique can be used to explore biochemical pathways in healthy and diseased states.