Correlated TEM-NanoSIMS investigation of foraminiferal metabolism

Foraminifera are ubiquitous eukaryotic protists inhabiting all types of marine environments. The chemical and isotopic compositions of their carbonate tests are commonly used as proxies for paleo-environmental conditions. However, while foraminifera represent a large fraction of the meiofauna and could therefore play a significant role in biogeochemical cycles, little is known about their biology. For the last 30 years, studies have revealed a wide range of physiological functions and metabolic pathways, in both planktic and benthic foraminifera: symbiosis, denitrification, kleptoplasty, dormancy, etc. However, the detailed metabolic processes involved in this large variety of physiological functions remain poorly understood. NanoSIMS, the main analytical technique used in this work, is a powerful analytical technique to simultaneously visualize, with a high spatial resolution (ca 100 nm), and quantify the incorporation of isotopically labeled compounds in organisms. In this study, NanoSIMS was combined with TEM to investigate the spatio-temporal dynamics of isotopically labeled compound assimilation at a sub-cellular scale. The first chapter presents an inventory of TEM pictures of the main organelles found in benthic foraminifera based on the literature, complemented by new TEM observations of nine benthic species. This work is essential to interpret the data of the chapters that follow. Using NanoSIMS combined with TEM, the second chapter investigates the heterotrophic metabolism, under oxic and anoxic conditions, of the intertidal benthic foraminifera, Ammonia cf. tepida. A sharp decrease of the metabolic activity observed in anoxia strongly suggests dormancy in response to the lack of oxygen. The third chapter is dedicated to kleptoplasty in benthic species. Incubation with labeled 13C-bicarbonate, 15N-ammonium, and 34S-sulfate were made, and the assimilation and fate of these molecules and their metabolites within the foraminiferal cell were traced with correlated TEM-NanoSIMS. A number of key observations were made: (1) assimilation of inorganic C was shown in the kleptoplastic Haynesina germanica under light conditions, but was not observed under dark conditions, indicating a photosynthetic uptake via the kleptoplasts. (2) In a different species, Elphidium williamsoni, photosynthetic assimilation of inorganic C was also observed, but the observed 13C-enrichments were much lower and not found in the same organelles as in H. germanica, indicating differences in the metabolic pathways among kleptoplastic species. (3) Assimilation of NH4+ and SO42- was documented in both kleptoplastic and akleptoplastic species, strongly suggesting the existence of a cytoplasmic pathway for NH4+ and SO42- assimilation. Thus, the role of kleptoplasts in N and S foraminiferal metabolism remains unclear and need further investigations. Finally the last chapter applied a similar protocol to study the C assimilation dynamics in symbiotic dinoflagellates and subsequent transfer the planktonic foraminiferal host cell. Dinoflagellates are transferring large amounts of photosynthates to the foraminifera, mainly in the form of lipid droplets. In conclusion, correlated TEM and NanoSIMS imaging is an efficient tool to study foraminiferal metabolism. Through this study it has led to progress in the knowledge of their ultrastructure and metabolic pathways, and ultimately shed light on their potential role in the biogeochemical cycles of marine ecosystems

Dormant or not dormant? Benthic foraminiferal metabolism under anoxia

Hypoxic events particularly affect benthic ecosystems on continental shelves and in coastal areas where renewal of bottom waters slow. Foraminifera living in such environments are among the most tolerant to hypoxia in the meiofauna. Some foraminifera species are able to survive hypoxia, and even anoxia, for weeks to months. Different species must have developed different mechanisms for survival - hypotheses include reduction of the metabolism, symbiosis with bacteria, or denitrification. NanoSIMS (Secondary Ion Mass Spectrometry) imaging is a powerful analytical technique to visualize and quantify the incorporation and transfer of isotopically labeled compounds in organisms with subcellular resolution. We used NanoSIMS imaging, correlated with TEM ultrastructural observations of individual foraminifera, to study the metabolism of intertidal Ammonia tepida, which has shown strongly reduced metabolism under anoxia. Individuals were fed with a 13C-labeled microalgal biofilm and incubated for 4 weeks in oxic and anoxic conditions, respectively. NanoSIMS imaging reveal strongly contrasting cellular-level dynamics of integration and transfer of the ingested biofilm components under the two conditions. In oxic conditions, ingested biofilm components are internalized, metabolized, and used for biosynthesis of different cellular components on a time scale of 24 hours: Lipid droplets are formed, then consumed through respiration. In contrast, upon the onset of anoxia, individual internalized biofilm components remain visible within the cytoplasm after 4 weeks. Lipids of different compositions are initially formed but then not respired. These observations indicate that foraminifera do initially have an active heterotrophic metabolism in the absence of oxygen, but this it is strongly reduced when oxygen is no longer available. Isotopic labeling experiments, NanoSIMS and TEM imaging, and GC-MS will be key to study metabolic mechanisms under anoxic conditions in marine environments

A benthic foraminifera species respond to anoxia with a strong metabolic shift suggesting a state of dormancy

Low-oxygenation events are more and more frequent and strong on continental shelves and in coastal areas where renewal of bottom waters is slow. Among the meiofauna living in such environments, foraminifera are among the most tolerant to the lack of oxygen. Some benthic foraminiferal species are able to survive hypoxia, and even anoxia, for weeks to months. Different species must have developed different mechanisms for survival - hypotheses include reduction of the metabolism, symbiosis with bacteria, or denitrification. Ammonia tepida, one of the most abundant species in intertidal environments, is able to survive up to 60 days in anoxia. Here we combined a 4 week feeding experiment using 13C-enriched microalgae (diatoms), with correlated transmission electron microscopy (TEM) and NanoSIMS (Secondary Ion Mass Spectrometry) imaging, and concentrations (GC/MS, GC/FFID), as well as bulk and compound specific carbon isotope ratios (13C/12C obtained by EA/IRMS and GC/C/IRMS) of individual fatty acids (FAs) to study the metabolic differences in intertidal Ammonia tepida exposed to oxic and anoxia conditions, respectively. Strongly contrasting cellular-level dynamics of integration and transfer of the ingested biofilm components were observed under the two conditions. Under oxic conditions, within a few days, intact diatoms (i.e. including the frustule) were ingested, assimilated and consumed, in part for biosynthesis of different cellular components: 13C-labeled lipid droplets formed over a timescale of a few days and were then partly lost 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. The compound specific 13C/12C ratios indicated substantial de novo synthesis by the foraminifera of polyunsaturated FAs (PUFAs), such as 20:4(n-6), in oxic conditions; very limited PUFA synthesis was observed under anoxia. Together, our results indicate that anoxia induced a greatly reduced rate of heterotrophic metabolism in Ammonia tepida on a time scale of about 24 hours, which seems consistent with a state of dormancy

Photosynthesis in the kleptoplastidic foraminiferal species "Haynesina germanica"

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Carbon integration and transfer by the photosynthetic symbiotic dinoflagellates of the planktonic foraminifer Orbulina universa observed by TEM-nanoSIMS techniques

The planktonic foraminifera Orbulina universa is living in the photic zone of marine water columns and it possesses photosynthetic microalgae symbionts in its cytoplasm (dinoflagellates). The transfer of carbon compounds from the symbiotic microalgae to the foraminifer host cell is a continuous process. During day microalgae might migrate on the spines to start to transfer carbon compounds from their starch vesicles to the lipid droplets of the foraminifer. During night, nearly the total amount of photoassimilates accumulated during the light phase is transferred from the symbiont starch vesicles to the host lipid droplets

Kleptoplasty in a shallow water benthic foraminifer, Haynesina germanica

Some benthic foraminifera have the ability to incorporate functional chloroplasts from diatoms (kleptoplasty). Our objective was to investigate kleptoplast cellular organisation, functionality (O2 flux, C and N incorporation), pigment composition and photoprotection mechanism in one shallow water benthic foraminiferal species (Haynesina germanica). Different experimental strategies (light regimes, stable isotopes) and analytical methods (TEM observations, O2 microsensor, NanoSIMS, PAM fluorometry, reflectance and HPLC pigment analysis) were used.Haynesina germanica showed net oxygen production up to 1000 pmol.O2.cell-1.day-1. Its Fv/Fm (ratio informing about photosystem II functionality) slowly decreased from 0.65 to 0.55 in 7 days in darkness and quickly decreased to 0.2 under high light (70 µmol photons.m-2.s-1). Kleptoplast functional time was estimated between 11-21 days in darkness and between 7-8 days at HL. The H. germanica cells are able to assimilate 1) labelled bicarbonate( 13C) in some chloroplasts, lipid droplets, fibrillar vacuoles; and 2) ammonium (15N), in some chloroplasts, fibrillar vacuoles, nucleus, dense bodies, crystalline structures and unidentified organelles (NanoSIMS observations coupled to TEM). Pigment composition was similar to diatoms.A “functional” xanthophyll cycle was observed on H. germanica exposed to different short term light regimes. However, on long term experiments total pigment content decreases or d by more than 50% after 5 days of starvation and then slowly decreases over time (15 days). These results showed that Haynesina germanica kleptoplasts are able to be completely functional but only over a relatively short time. This strategy may be an advantage for this species if,continuous chloroplast resupply from food source