Description: Here we performed pulse-chase experiments with 13C-enriched dissolved inorganic carbon, followed by TEM and quantitative NanoSIMS isotopic imaging to visualize photosynthetic C assimilation by individual symbiotic dinoflagellates and subsequent translocation to their Orbulina universa host. NanoSIMS image processing was carried out as described in LeKieffre et al. (2017) and Nomaki et al. (2018). Briefly, TEM images were aligned with corresponding NanoSIMS 12C14N- images (Online Resource 1) using the software Look@NanoSIMS (Polerecky et al. 2012), which allows a user to hand-draw regions of interest (ROIs) corresponding to different organelles (e.g., dinoflagellate starch grains, foraminiferal lipid droplets, and fibrillar bodies). For each type of organelle and each time point, the average 13C-enrichment and its standard deviation were calculated based on 3 replicate foraminifera (except for the 6 h and 30 h time points, where only 2 replicates were available). The ROIs drawn on TEM images were also used to assess the relative abundance (in %) of lipid droplets in the foraminiferal endoplasm and starch grains in the dinoflagellate cytoplasm, respectively. Lipid droplet abundance was determined as the number of pixels occupied by lipid droplets divided by the total number of pixels of foraminiferal endoplasm. Starch grain abundance was determined as the number of pixels of occupied by starch grains divided by the total number of pixels covering dinoflagellate cytoplasm.

Description: The European Community Marine Strategy Framework Directive (MSFD) was established to provide guidelines for monitoring the quality of marine ecosystems. Monitoring the status of marine environments is traditionally based on macrofauna surveys, for which standardised methods have been established. Benthic foraminifera are also good indicators of environmental status because of their fast turnover rates, high degree of specialisation, and the preservation of dead assemblages in the fossil record. In spite of the growing interest in foraminiferal bio-monitoring during the last decades, no standardised methodology has been proposed until today. The aim of the FOraminiferal BIo-MOnitoring (FOBIMO) expert workshop, held in June 2011 at Fribourg, Switzerland, which assembled 37 scientists from 24 research groups and 13 countries, was to develop a suite of standard methods. This paper presents the main outcome of the workshop, a list of motivated recommendations with respect to sampling devices, sample storage, treatment, faunal analysis and documentation. Our recommendations fulfil the criteria imposed both by scientific rigour and by the practical limitations of routine studies. Hence, our aim is to standardise methodologies used in bio-monitoring only and not to limit the use of different methods in pure scientific studies. Unless otherwise stated, all recommendations concern living (stained) benthic foraminiferal assemblages. We have chosen to propose two types of recommendations. Mandatory recommendations have to be followed if a study wants to qualify as sound and compatible to the norms. The most important of these recommendations are the interval from 0 to 1 cm below the sediment surface has to be sampled, and an interface corer or box corer that keeps the sediment surface intact is to be used for offshore surveys. A grab sampler must not be deployed in soft sediments. Three replicate samples are to be taken and analysed separately. Samples are to be washed on a 63-mu m screen, and the living benthic foraminiferal fauna of the 〉 125 mu m fraction is to be analysed. Splits are to be picked and counted entirely, and all counted foraminifera from at least one replicate per station have to be stored in micropalaeontological slides. Census data, supplementary laboratory data and microslides have to be archived. Advisory recommendations are to sample in autumn, to have a sample size of 50 cm(2) or a tube of 8 cm inner diameter, to use 〉 70% ethanol as a preservative, rose Bengal at a concentration of 2 grams per litre for staining, and a staining time of at least 14 days. The split size should be defined by a target value of 300 specimens, heavy liquid separation should be avoided, and the 63-125 mu m fraction or deeper sediment levels may be considered in some environments. We are convinced that the application of this protocol by a large number of scientists is a necessary first step to a general acceptance of benthic foraminifera as a reliable tool in bio-monitoring studies

Description: © 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.

Description: 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.

Description: 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.

Description: © The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Marine Micropaleontology 138 (2018): 12-32, doi:10.1016/j.marmicro.2017.10.005.

Description: We report systematic transmission electron microscope (TEM) observations of the cellular ultrastructure of selected, small rotalid benthic foraminifera. Nine species from different environments (intertidal mudflat, fjord, and basin) were investigated: Ammonia sp., Elphidium oceanense, Haynesina germanica, Bulimina marginata, Globobulimina sp., Nonionellina labradorica, Nonionella sp., Stainforthia fusiformis and Buliminella tenuata. All the observed specimens were fixed just after collection from their natural habitats allowing description of intact and healthy cells. Foraminiferal organelles can be divided into two broad categories: (1) organelles that are present in all eukaryotes, such as the nuclei, mitochondria, endoplasmic reticulum, Golgi apparatus, and peroxisomes; and (2) organelles observed in all foraminifera but not common in all eukaryotic cells, generally with unknown function, such as fibrillar vesicles or electron-opaque bodies. Although the organelles of the first category were observed in all the observed species, their appearance varies. For example, subcellular compartments linked to feeding and metabolism exhibited different sizes and shapes between species, likely due to differences in their diet and/or trophic mechanisms. The organelles of the second category are common in all foraminiferal species investigated and, according to the literature, are frequently present in the cytoplasm of many different species, both benthic and planktonic. This study, thus, provides a detailed overview of the major ultrastructural components in benthic foraminiferal cells from a variety of marine environments, and also highlights the need for further research to better understand the function and role of the various organelles in these fascinating organisms.

Description: This work was supported by the Swiss National Science Foundation (grant no. 200021_149333), The Investment in Science Fund at WHOI and the French national program EC2CO-LEFE (project ForChlo). TJ was funded by the “FRESCO” project, a project supported by the Region Pays de Loire and the University of Angers.

Description: © 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.

Description: 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.

Description: 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).

Description: © 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): 1-11, doi:10.1016/j.marmicro.2017.11.002.

Description: JMB acknowledges support from the WHOI Robert W. Morse Chair for Excellence in Oceanography and The Investment in Science Fund at WHOI.

Description: © 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.

Description: 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.

Description: 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).

Description: © The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in LeKieffre, C., Jauffrais, T., Bernhard, J., Filipsson, H., Schmidt, C., Roberge, H., Maire, O., Panieri, G., Geslin, E., & Meibom, A. Ammonium and sulfate assimilation is widespread in benthic foraminifera. Frontiers in Marine Science, 9, (2022): 861945, https://doi.org/10.3389/fmars.2022.861945.

Description: Nitrogen and sulfur are key elements in the biogeochemical cycles of marine ecosystems to which benthic foraminifera contribute significantly. Yet, cell-specific assimilation of ammonium, nitrate and sulfate by these protists is poorly characterized and understood across their wide range of species-specific trophic strategies. For example, detailed knowledge about ammonium and sulfate assimilation pathways is lacking and although some benthic foraminifera are known to maintain intracellular pools of nitrate and/or to denitrify, the potential use of nitrate-derived nitrogen for anabolic processes has not been systematically studied. In the present study, NanoSIMS isotopic imaging correlated with transmission electron microscopy was used to trace the incorporation of isotopically labeled inorganic nitrogen (ammonium or nitrate) and sulfate into the biomass of twelve benthic foraminiferal species from different marine environments. On timescales of twenty hours, no detectable 15N-enrichments from nitrate assimilation were observed in species known to perform denitrification, indicating that, while denitrifying foraminifera store intra-cellular nitrate, they do not use nitrate-derived nitrogen to build their biomass. Assimilation of both ammonium and sulfate, with corresponding 15N and 34S-enrichments, were observed in all species investigated (with some individual exceptions for sulfate). Assimilation of ammonium and sulfate thus can be considered widespread among benthic foraminifera. These metabolic capacities may help to underpin the ability of benthic foraminifera to colonize highly diverse marine habitats.

Description: This work was supported by the Swiss National Science Foundation (grant no. 200021_149333), and a postdoctoral fellowship allowed to CL by the University Loire-Bretagne. SBB sampling was funded by US National Science Foundation grant BIO IOS 1557430 to JMB, who also acknowledges NASA grant #80NSSC21K0478 for partial support. HF acknowledges funding from the Swedish Research Council VR (grant number 2017-04190). Svalbard sampling was supported by the Research Council of Norway through CAGE (Center for Excellence in Arctic Gas Hydrate Environment and Climate, project number 223259) and NORCRUST (project number 255150) to GP and the fellowship MOPGA (Make Our Planet Great Again) by CAMPUS France to CS.

Description: © The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Schmidt, C., Geslin, E., Bernhard, J. M., LeKieffre, C., Svenning, M. M., Roberge, H., Schweizer, M., & Panieri, G. Deposit-feeding of Nonionellina labradorica (foraminifera) from an Arctic methane seep site and possible association with a methanotroph. Biogeosciences, 19(16), (2022): 3897–3909, https://doi.org/10.5194/bg-19-3897-2022.

Description: Several foraminifera are deposit feeders that consume organic detritus (dead particulate organic material with entrained bacteria). However, the role of such foraminifera in the benthic food web remains understudied. Foraminifera feeding on methanotrophic bacteria, which are 13C-depleted, may cause negative cytoplasmic and/or calcitic δ13C values. To test whether the foraminiferal diet includes methanotrophs, we performed a short-term (20 h) feeding experiment with Nonionellina labradorica from an active Arctic methane-emission site (Storfjordrenna, Barents Sea) using the marine methanotroph Methyloprofundus sedimenti and analysed N. labradorica cytology via transmission electron microscopy (TEM). We hypothesised that M. sedimenti would be visible post-experiment in degradation vacuoles, as evidenced by their ultrastructure. Sediment grains (mostly clay) occurred inside one or several degradation vacuoles in all foraminifers. In 24 % of the specimens from the feeding experiment degradation vacuoles also contained bacteria, although none could be confirmed to be the offered M. sedimenti. Observations of the apertural area after 20 h incubation revealed three putative methanotrophs, close to clay particles, based on bacterial ultrastructural characteristics. Furthermore, we noted the absence of bacterial endobionts in all examined N. labradorica but confirmed the presence of kleptoplasts, which were often partially degraded. In sum, we suggest that M. sedimenti can be consumed via untargeted grazing in seeps and that N. labradorica can be generally classified as a deposit feeder at this Arctic site.

Description: This research has been supported by the French scientific programme MOPGA (Make our Planet Great Again) managed by the National Research Agency; the Norwegian Research Council through the Centre for Arctic Gas Hydrate, Environment and Climate (project number 223259); NORCRUST (project number 255250); and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 444059848.