Kleptoplast distribution, photosynthetic efficiency and sequestration mechanisms in intertidal benthic foraminifera

Foraminifera are ubiquitously distributed in marine habitats, playing a major role in marine sediment carbon sequestration and the nitrogen cycle. They exhibit a wide diversity of feeding and behavioural strategies (heterotrophy, autotrophy and mixotrophy), including species with the ability of sequestering intact functional chloroplasts from their microalgal food source (kleptoplastidy), resulting in a mixotrophic lifestyle. The mechanisms by which kleptoplasts are integrated and kept functional inside foraminiferal cytosol are poorly known. In our study, we investigated relationships between feeding strategies, kleptoplast spatial distribution and photosynthetic functionality in two shallow-water benthic foraminifera (Haynesina germanica and Elphidium williamsoni), both species feeding on benthic diatoms. We used a combination of observations of foraminiferal feeding behaviour, test morphology, cytological TEM-based observations and HPLC pigment analysis, with non-destructive, single-cell level imaging of kleptoplast spatial distribution and PSII quantum efficiency. The two species showed different feeding strategies, with H. germanica removing diatom content at the foraminifer's apertural region and E. williamsoni on the dorsal site. All E. williamsoni parameters showed that this species has higher autotrophic capacity albeit both feeding on benthic diatoms. This might represent two different stages in the evolutionary process of establishing a permanent symbiotic relationship, or may reflect different trophic strategies.

Charlotte Madeleine Nicole Lekieffre

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.

EPFL2017

Dormant or not dormant? Benthic foraminiferal metabolism under anoxia

Emmanuelle Geslin, Charlotte Madeleine Nicole Lekieffre, Anders Meibom

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.

2016

Charlotte Madeleine Nicole Lekieffre

EPFL2017

Dormant or not dormant? Benthic foraminiferal metabolism under anoxia

Emmanuelle Geslin, Charlotte Madeleine Nicole Lekieffre, Anders Meibom

2016

Kleptoplast distribution, photosynthetic efficiency and sequestration mechanisms in intertidal benthic foraminifera

Correlated TEM-NanoSIMS investigation of foraminiferal metabolism

Dormant or not dormant? Benthic foraminiferal metabolism under anoxia

Correlated TEM-NanoSIMS investigation of foraminiferal metabolism

Dormant or not dormant? Benthic foraminiferal metabolism under anoxia