Heterotrophic Foraminifera Capable of Inorganic Nitrogen Assimilation.

Nitrogen availability often limits biological productivity in marine systems, where inorganic nitrogen, such as ammonium is assimilated into the food web by bacteria and photoautotrophic eukaryotes. Recently, ammonium assimilation was observed in kleptoplast-containing protists of the phylum foraminifera, possibly via the glutamine synthetase/glutamate synthase (GS/GOGAT) assimilation pathway imported with the kleptoplasts. However, it is not known if the ubiquitous and diverse heterotrophic protists have an innate ability for ammonium assimilation. Using stable isotope incubations (15N-ammonium and 13C-bicarbonate) and combining transmission electron microscopy (TEM) with quantitative nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, we investigated the uptake and assimilation of dissolved inorganic ammonium by two heterotrophic foraminifera; a non-kleptoplastic benthic species, Ammonia sp., and a planktonic species, Globigerina bulloides. These species are heterotrophic and not capable of photosynthesis. Accordingly, they did not assimilate 13C-bicarbonate. However, both species assimilated dissolved 15N-ammonium and incorporated it into organelles of direct importance for ontogenetic growth and development of the cell. These observations demonstrate that at least some heterotrophic protists have an innate cellular mechanism for inorganic ammonium assimilation, highlighting a newly discovered pathway for dissolved inorganic nitrogen (DIN) assimilation within the marine microbial loop.

Ammonium is the preferred source of nitrogen for planktonic foraminifer and their dinoflagellate symbionts.

The symbiotic planktonic foraminifera Orbulina universa inhabits open ocean oligotrophic ecosystems where dissolved nutrients are scarce and often limit biological productivity. It has previously been proposed that O. universa meets its nitrogen (N) requirements by preying on zooplankton, and that its symbiotic dinoflagellates recycle metabolic 'waste ammonium' for their N pool. However, these conclusions were derived from bulk 15N-enrichment experiments and model calculations, and our understanding of N assimilation and exchange between the foraminifer host cell and its symbiotic dinoflagellates remains poorly constrained. Here, we present data from pulse-chase experiments with 13C-enriched inorganic carbon, 15N-nitrate, and 15N-ammonium, as well as a 13C- and 15N- enriched heterotrophic food source, followed by TEM (transmission electron microscopy) coupled to NanoSIMS (nanoscale secondary ion mass spectrometry) isotopic imaging to visualize and quantify C and N assimilation and translocation in the symbiotic system. High levels of 15N-labelling were observed in the dinoflagellates and in foraminiferal organelles and cytoplasm after incubation with 15N-ammonium, indicating efficient ammonium assimilation. Only weak 15N-assimilation was observed after incubation with 15N-nitrate. Feeding foraminifers with 13C- and 15N-labelled food resulted in dinoflagellates that were labelled with 15N, thereby confirming the transfer of 15N-compounds from the digestive vacuoles of the foraminifer to the symbiotic dinoflagellates, likely through recycling of ammonium. These observations are important for N isotope-based palaeoceanographic reconstructions, as they show that δ15N values recorded in the organic matrix in symbiotic species likely reflect ammonium recycling rather than alternative N sources, such as nitrates.

16S rRNA gene metabarcoding and TEM reveals different ecological strategies within the genus Neogloboquadrina (planktonic foraminifer).

Uncovering the complexities of trophic and metabolic interactions among microorganisms is essential for the understanding of marine biogeochemical cycling and modelling climate-driven ecosystem shifts. High-throughput DNA sequencing methods provide valuable tools for examining these complex interactions, although this remains challenging, as many microorganisms are difficult to isolate, identify and culture. We use two species of planktonic foraminifera from the climatically susceptible, palaeoceanographically important genus Neogloboquadrina, as ideal test microorganisms for the application of 16S rRNA gene metabarcoding. Neogloboquadrina dutertrei and Neogloboquadrina incompta were collected from the California Current and subjected to either 16S rRNA gene metabarcoding, fluorescence microscopy, or transmission electron microscopy (TEM) to investigate their species-specific trophic interactions and potential symbiotic associations. 53-99% of 16S rRNA gene sequences recovered from two specimens of N. dutertrei were assigned to a single operational taxonomic unit (OTU) from a chloroplast of the phylum Stramenopile. TEM observations confirmed the presence of numerous intact coccoid algae within the host cell, consistent with algal symbionts. Based on sequence data and observed ultrastructure, we taxonomically assign the putative algal symbionts to Pelagophyceae and not Chrysophyceae, as previously reported in this species. In addition, our data shows that N. dutertrei feeds on protists within particulate organic matter (POM), but not on bacteria as a major food source. In total contrast, of OTUs recovered from three N. incompta specimens, 83-95% were assigned to bacterial classes Alteromonadales and Vibrionales of the order Gammaproteobacteria. TEM demonstrates that these bacteria are a food source, not putative symbionts. Contrary to the current view that non-spinose foraminifera are predominantly herbivorous, neither N. dutertrei nor N. incompta contained significant numbers of phytoplankton OTUs. We present an alternative view of their trophic interactions and discuss these results within the context of modelling global planktonic foraminiferal abundances in response to high-latitude climate change.

Link between light-triggered Mg-banding and chamber formation in the planktic foraminifera Neogloboquadrina dutertrei.

The relationship between seawater temperature and the average Mg/Ca ratios in planktic foraminifera is well established, providing an essential tool for reconstructing past ocean temperatures. However, many species display alternating high and low Mg-bands within their shell walls that cannot be explained by temperature alone. Recent experiments demonstrate that intrashell Mg variability in Orbulina universa, which forms a spherical terminal shell, is paced by the diurnal light/dark cycle. Whether Mg-heterogeneity is also diurnally paced in species with more complex shell morphologies is unknown. Here we show that high Mg/Ca-calcite forms at night in cultured specimens of the multi-chambered species Neogloboquadrina dutertrei. Our results demonstrate that N. dutertrei adds a significant amount of calcite, and nearly all Mg-bands, after the final chamber forms. These results have implications for interpreting patterns of calcification in N. dutertrei and suggest that diurnal Mg-banding is an intrinsic component of biomineralization in planktic foraminifera.

Nanometer-Scale Chemistry of a Calcite Biomineralization Template: Implications for Skeletal Composition and Nucleation.

Plankton, corals, and other organisms produce calcium carbonate skeletons that are integral to their survival, form a key component of the global carbon cycle, and record an archive of past oceanographic conditions in their geochemistry. A key aspect of the formation of these biominerals is the interaction between organic templating structures and mineral precipitation processes. Laboratory-based studies have shown that these atomic-scale processes can profoundly influence the architecture and composition of minerals, but their importance in calcifying organisms is poorly understood because it is difficult to measure the chemistry of in vivo biomineral interfaces at spatially relevant scales. Understanding the role of templates in biomineral nucleation, and their importance in skeletal geochemistry requires an integrated, multiscale approach, which can place atom-scale observations of organic-mineral interfaces within a broader structural and geochemical context. Here we map the chemistry of an embedded organic template structure within a carbonate skeleton of the foraminifera Orbulina universa using both atom probe tomography (APT), a 3D chemical imaging technique with Ångström-level spatial resolution, and time-of-flight secondary ionization mass spectrometry (ToF-SIMS), a 2D chemical imaging technique with submicron resolution. We quantitatively link these observations, revealing that the organic template in O. universa is uniquely enriched in both Na and Mg, and contributes to intraskeletal chemical heterogeneity. Our APT analyses reveal the cation composition of the organic surface, offering evidence to suggest that cations other than Ca2+, previously considered passive spectator ions in biomineral templating, may be important in defining the energetics of carbonate nucleation on organic templates.