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

Surviving anoxia in marine sediments: The metabolic response of ubiquitous benthic foraminifera (Ammonia tepida)

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

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

mRNA Structural Constraints on EBNA1 Synthesis Impact on In Vivo Antigen Presentation and Early Priming of CD8(+) T Cells

Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8(+) T cell epitopes by CD11c(+) dendritic cells in draining lymph nodes and early priming of antigen-specific CD8(+) T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8(+) T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection

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

Changes in ultrastructural features of the foraminifera Ammonia spp. in response to anoxic conditions: Field and laboratory observations

The ultrastructure of the living foraminiferan, Ammonia sp. (phylotype unknown), collected from surficial and deeper, subsurface (anoxic) sediments from the Dutch Wadden Sea, was examined to provide information on the physiology of the foraminiferal cell and its adaptive strategies to low‑oxygen conditions. The observed changes in cell ultrastructure under anoxia were further compared with the cell ultrastructure of Ammonia sp. (phylotype T6), from oxic and anoxic incubation experiments. The ultrastructural evidence indicates that under low‑oxygen conditions Ammonia spp. may accumulate lipid droplets. In addition, the size of the lipid droplets may increase with the duration of anoxic conditions, becoming over 5 μm in size, while the remaining cytosol of the foraminiferan become less electron dense. In some specimens, lipid droplets were also found in the space between the plasma membrane and the organic lining. We expect that the apparent increase in the number and size of the lipid droplets is indicative of a stress response of the foraminifera to the adverse anoxic conditions. Other ultrastructural changes in response to anoxia include the presence of intact bacteria and electron dense opaque bodies within the foraminiferal cytosol, and a possible thickening of the organic lining. The role of the bacteria remains enigmatic but they may be linked to foraminiferal dormancy in anoxia

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

A deep learning model to generate synthetic CT for prostate MR-only radiotherapy dose planning: a multicenter study

IntroductionFor radiotherapy based solely on magnetic resonance imaging (MRI), generating synthetic computed tomography scans (sCT) from MRI is essential for dose calculation. The use of deep learning (DL) methods to generate sCT from MRI has shown encouraging results if the MRI images used for training the deep learning network and the MRI images for sCT generation come from the same MRI device. The objective of this study was to create and evaluate a generic DL model capable of generating sCTs from various MRI devices for prostate radiotherapyMaterials and methodsIn total, 90 patients from three centers (30 CT-MR prostate pairs/center) underwent treatment using volumetric modulated arc therapy for prostate cancer (PCa) (60 Gy in 20 fractions). T2 MRI images were acquired in addition to computed tomography (CT) images for treatment planning. The DL model was a 2D supervised conditional generative adversarial network (Pix2Pix). Patient images underwent preprocessing steps, including nonrigid registration. Seven different supervised models were trained, incorporating patients from one, two, or three centers. Each model was trained on 24 CT-MR prostate pairs. A generic model was trained using patients from all three centers. To compare sCT and CT, the mean absolute error in Hounsfield units was calculated for the entire pelvis, prostate, bladder, rectum, and bones. For dose analysis, mean dose differences of D99% for CTV, V95% for PTV, Dmax for rectum and bladder, and 3D gamma analysis (local, 1%/1 mm) were calculated from CT and sCT. Furthermore, Wilcoxon tests were performed to compare the image and dose results obtained with the generic model to those with the other trained models.ResultsConsidering the image results for the entire pelvis, when the data used for the test comes from the same center as the data used for training, the results were not significantly different from the generic model. Absolute dose differences were less than 1 Gy for the CTV D99% for every trained model and center. The gamma analysis results showed nonsignificant differences between the generic and monocentric models.ConclusionThe accuracy of sCT, in terms of image and dose, is equivalent to whether MRI images are generated using the generic model or the monocentric model. The generic model, using only eight MRI-CT pairs per center, offers robust sCT generation, facilitating PCa MRI-only radiotherapy for routine clinical use