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

Ultrastructure and distribution of kleptoplasts in benthic foraminifera from shallow-water (photic) habitats

© The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Marine Micropaleontology 138 (2018): 46-62, doi:10.1016/j.marmicro.2017.10.003.Assimilation, sequestration and maintenance of foreign chloroplasts inside an organism is termed “chloroplast sequestration” or “kleptoplasty”. This phenomenon is known in certain benthic foraminifera, in which such kleptoplasts can be found both intact and functional, but with different retention times depending on foraminiferal species. In the present study, seven species of benthic foraminifera (Haynesina germanica, Elphidium williamsoni, E. selseyense, E. oceanense, E. aff. E. crispum, Planoglabratella opercularis and Ammonia sp.) were collected from shallow-water benthic habitats and examined with transmission electron microscope (TEM) for cellular ultrastructure to ascertain attributes of kleptoplasts. Results indicate that all these foraminiferal taxa actively obtain kleptoplasts but organized them differently within their endoplasm. In some species, the kleptoplasts were evenly distributed throughout the endoplasm (e.g., H. germanica, E. oceanense, Ammonia sp.), whereas other species consistently had plastids distributed close to the external cell membrane (e.g., Elphidium williamsoni, E. selseyense, P. opercularis). Chloroplast degradation also seemed to differ between species, as many degraded plastids were found in Ammonia sp. and E. oceanense compared to other investigated species. Digestion ability, along with different feeding and sequestration strategies may explain the differences in retention time between taxa. Additionally, the organization of the sequestered plastids within the endoplasm may also suggest behavioral strategies to expose and/or protect the sequestered plastids to/from light and/or to favor gas and/or nutrient exchange with their surrounding habitats.TJ was funded by the “FRESCO” project, a project supported by the Region Pays de Loire and the University of Angers. This work was also supported by a grant no. 200021_149333 from the Swiss National Science Foundation and the French national program EC2CO-LEFE (project ForChlo).JMB acknowledges the Robert W. Morse Chair for Excellence in Oceanography and the Investment in Science Fund at WHOI. Also, KK acknowledges the Academy of Finland (Project numbers: 278827, 283453)

Inorganic carbon and nitrogen assimilation in cellular compartments of a benthic kleptoplastic foraminifer

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 8 (2018): 10140, doi:10.1038/s41598-018-28455-1.Haynesina germanica, an ubiquitous benthic foraminifer in intertidal mudflats, has the remarkable ability to isolate, sequester, and use chloroplasts from microalgae. The photosynthetic functionality of these kleptoplasts has been demonstrated by measuring photosystem II quantum efficiency and O2 production rates, but the precise role of the kleptoplasts in foraminiferal metabolism is poorly understood. Thus, the mechanism and dynamics of C and N assimilation and translocation from the kleptoplasts to the foraminiferal host requires study. The objective of this study was to investigate, using correlated TEM and NanoSIMS imaging, the assimilation of inorganic C and N (here ammonium, NH4+) in individuals of a kleptoplastic benthic foraminiferal species. H. germanica specimens were incubated for 20 h in artificial seawater enriched with H13CO3− and 15NH4+ during a light/dark cycle. All specimens (n = 12) incorporated 13C into their endoplasm stored primarily in the form of lipid droplets. A control incubation in darkness resulted in no 13C-uptake, strongly suggesting that photosynthesis is the process dominating inorganic C assimilation. Ammonium assimilation was observed both with and without light, with diffuse 15N-enrichment throughout the cytoplasm and distinct 15N-hotspots in fibrillar vesicles, electron-opaque bodies, tubulin paracrystals, bacterial associates, and, rarely and at moderate levels, in kleptoplasts. The latter observation might indicate that the kleptoplasts are involved in N assimilation. However, the higher N assimilation observed in the foraminiferal endoplasm incubated without light suggests that another cytoplasmic pathway is dominant, at least in darkness. This study clearly shows the advantage provided by the kleptoplasts as an additional source of carbon and provides observations of ammonium uptake by the foraminiferal cell.This work was supported by the Swiss National Science Foundation (grant no. 200021_149333) and was part of the CNRS EC2CO-Lefe project ForChlo. It was also supported by the Region Pays de la Loire (Post-doc position of TJ, on FRESCO project) as well as the WHOI Robert W. Morse Chair for Excellence in Oceanography and The Investment in Science Fund at WHOI

Innovative TEM-coupled approaches to study foraminiferal cells

© The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Marine Micropaleontology 138 (2018): 90-104, doi:10.1016/j.marmicro.2017.10.002.Transmission electron microscope (TEM) observation has revealed much about the basic cell biology of foraminifera. Yet, there remains much we do not know about foraminiferal cytology and physiology, especially for smaller benthic foraminifera, which inhabit a wide range of habitats. Recently, some TEM-coupled approaches have been developed to study correlative foraminiferal ecology and physiology in detail: Fluorescently Labeled Embedded Core (FLEC)-TEM for observing foraminiferal life-position together with their cytoplasmic ultrastructure, micro-X-ray computed tomography (CT)-TEM for observing and reconstructing foraminiferal cytoplasm in three dimensions (3D), and TEM-Nanometer-scale secondary ion mass spectrometry (NanoSIMS) for mapping of elemental and isotopic compositions at sub-micrometer resolutions with known ultrastructure. In this contribution, we review and illustrate these recent advances of TEM-coupled methods.This work was financially supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Scientific Research (C) grant number 17K05697 to HN) and the Swiss National Science Foundation (grant no. 200021_149333). JMB’s contributions were funded by US NSF grants OCE-0551001 and OCE-1634469, the WHOI Robert W. Morse Chair for Excellence in Oceanography, and The Investment in Science Fund at WHOI. The micro-X-ray CT imaging was performed under the cooperative research program of Center for Advanced Marine Core Research (CMCR), Kochi University (accept No. 17A021)

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

Cytoklepty in the plankton: A host strategy to optimize the bioenergetic machinery of endosymbiotic algae

Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts (Phaeocystis) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition

Inorganic carbon and nitrogen assimilation in cellular compartments of a benthic kleptoplastic foraminifer

Haynesina germanica, an ubiquitous benthic foraminifer in intertidal mudflats, has the remarkable ability to isolate, sequester, and use chloroplasts from microalgae. The photosynthetic functionality of these kleptoplasts has been demonstrated by measuring photosystem II quantum efficiency and O2 production rates, but the precise role of the kleptoplasts in foraminiferal metabolism is poorly understood. Thus, the mechanism and dynamics of C and N assimilation and translocation from the kleptoplasts to the foraminiferal host requires study. The objective of this study was to investigate, using correlated TEM and NanoSIMS imaging, the assimilation of inorganic C and N (here ammonium, NH4+) in individuals of a kleptoplastic benthic foraminiferal species. H. germanica specimens were incubated for 20 h in artificial seawater enriched with H13CO3- and 15NH4+ during a light/dark cycle

Feeding behaviour of a benthic species (Ammonia tepida) under oxic and anoxic conditions: TEM-NanoSIMS correlation

More and more marine areas are subjected to depleted-O2 concentration, mainly due to eutrophication induced by human activities. These phenomena affect benthic ecosystems, in particular continental shelves and coastal areas where the renewal of the bottom waters is low and/or the organic matter flux is high. Some species of benthic foraminifera are resistant to this hypoxic/anoxic events, surviving weeks to months without oxygen. One of the species known to survive to such extreme condition is the benthic one Ammonia tepida. However the metabolic process by which it succeed in it remains unknown. One hypothesis is that this species would be able to lower its metabolism under oxygen depleted conditions, until the return of better conditions. The aim of the present study is to understand the feeding behaviors of A. tepida under anoxia. For this purpose a laboratory experiment involving incubation with living A. tepida was carried out under controlled oxygen concentrations. The individuals were fed with a pulse of 13C-labeled diatoms (Navicula sp.) and then incubate in oxic or anoxic conditions during 28 days. Along the incubation time, observation of the cell ultrastructure with Transmission Electronic Microscope (TEM) and detection in the cell of the isotopic 13C signal using nanoSIMS, were made. The nanoSIMS (nanoscale Secondary-Ion Mass Spectrometry) is an analytical technique that allows to visualize the incorporation and transfer of isotopically labeled compounds in organisms; thus in our case to follow the ingestion of labeled-diatoms and further transfer of carbon in the cytoplasm of the foraminifera. According to the results acquired in this study, in both conditions the foraminifera directly integrate the diatoms in their cytoplasm during the first hours of the incubation. Then, in oxic conditions, these diatoms are quickly fully degraded, within 3 days, and the 13C-labeled compounds are transferred in the organelles of the cytoplasm. Whereas in anoxia, only a fraction of the diatoms are degraded and the transfer to the foraminifera cytoplasm is weak. These results are confirmed by the δ13C of the foraminifera, measured in bulk by mass spectrometry (GC-MS). In anoxia the δ13C is slightly increasing the first 24h, meaning that the 13C content of the cell increased, and then it remains stable until the end of the experiment. This leads us to assume that the foraminifera stop, or at least strongly lower their metabolic rate under anoxic condition