Microscale imaging sheds light on species-specific strategies for photo-regulation and photo-acclimation of microphytobenthic diatoms.

Intertidal microphytobenthic (MPB) biofilms are key sites for coastal primary production, predominantly by pennate diatoms exhibiting photo-regulation via non-photochemical quenching (NPQ) and vertical migration. Movement is the main photo-regulation mechanism of motile (epipelic) diatoms and because they can move from light, they show low-light acclimation features such as low NPQ levels, as compared to non-motile (epipsammic) forms. However, most comparisons of MPB species-specific photo-regulation have used low light acclimated monocultures, not mimicking environmental conditions. Here we used variable chlorophyll fluorescence imaging, fluorescent labelling in sediment cores and scanning electron microscopy to compare the movement and NPQ responses to light of four epipelic diatom species from a natural MPB biofilm. The diatoms exhibited different species-specific photo-regulation features and a large NPQ range, exceeding that reported for epipsammic diatoms. This could allow epipelic species to coexist in compacted light niches of MPB communities. We show that diatom cell orientation within MPB can be modulated by light, where diatoms oriented themselves more perpendicular to the sediment surface under high light vs. more parallel under low light, demonstrating behavioural, photo-regulatory response by varying their light absorption cross-section. This highlights the importance of considering species-specific responses and understanding cell orientation and photo-behaviour in MPB research.

Trophic strategies of intertidal foraminifera explored with single-cell microbiome metabarcoding and morphological methods: What is on the menu?

In mudflats, interactions and transfers of nutrients and secondary metabolites may drive ecosystems and biodiversity. Foraminifera have complex trophic strategies as they often rely on bacteria and eukaryotes or on potential symbionts for carbon and nitrogen resources. The capacity of these protists to use a wide range of adaptive mechanisms requires clarifying the relationships between them and their microbial associates. Here, we investigate the interactions of three foraminiferal species with nearby organisms in situ, by coupling molecular (cloning/Sanger and high-throughput sequencing) and direct counting and morphological identification with microscopy. This coupling allows the identification of the organisms found in or around three foraminiferal species through molecular tools combined with a direct counting of foraminifera and diatoms present in situ through microscopy methods. Depending on foraminiferal species, and in addition to diatom biomass, diatom frustule shape, size and species are key factors driving the abundance and diversity of foraminifera in mudflat habitats. Three different trophic strategies were deduced for the foraminifera investigated in this study: Ammonia sp. T6 has an opportunistic strategy and is feeding on bacteria, nematoda, fungi, and diatoms when abundant; Elphidium oceanense is feeding mainly on diatoms, mixed with other preys when they are less abundant; and Haynesina germanica is feeding almost solely on medium-large pennate diatoms. Although there are limitations due to the lack of species coverage in DNA sequence databases and to the difficulty to compare morphological and molecular data, this study highlights the relevance of combining molecular with morphological tools to study trophic interactions and microbiome communities of protists at the single-cell scale.

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.

Relative abundances of benthic foraminifera in response to total organic carbon in sediments: Data from European intertidal areas and transitional waters.

We gathered total organic carbon (%) and relative abundances of benthic foraminifera in intertidal areas and transitional waters from the English Channel/European Atlantic Coast (587 samples) and the Mediterranean Sea (301 samples) regions from published and unpublished datasets. This database allowed to calculate total organic carbon optimum and tolerance range of benthic foraminifera in order to assign them to ecological groups of sensitivity. Optima and tolerance range were obtained by mean of the weighted-averaging method. The data are related to the research article titled "Indicative value of benthic foraminifera for biomonitoring: assignment to ecological groups of sensitivity to total organic carbon of species from European intertidal areas and transitional waters" [1].

Indicative value of benthic foraminifera for biomonitoring: Assignment to ecological groups of sensitivity to total organic carbon of species from European intertidal areas and transitional waters.

This work contributes to the ongoing work aiming at confirming benthic foraminifera as a biological quality element. In this study, benthic foraminifera from intertidal and transitional waters from the English Channel/European Atlantic coast and the Mediterranean Sea were assigned to five ecological groups using the weighted-averaging optimum with respect to TOC of each species. It was however not possible to assign typical salt marsh species due to the presence of labile and refractory organic matter that hampers TOC characterization. Tests of this study species' lists with Foram-AMBI on two independent datasets showed a significant correlation between Foram-AMBI and TOC, confirming the strong relation between foraminifera and TOC. For one of the validation datasets, associated macrofaunal data were available and a significant correlation was found between the foraminiferal Foram-AMBI and the macrofaunal AMBI. The here proposed lists should be further tested with sensitivity-based indices in different European regional settings.

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.

Ammonium is the preferred source of nitrogen for planktonic foraminifer and their dinoflagellate symbionts.

The symbiotic planktonic foraminifera Orbulina universa inhabits open ocean oligotrophic ecosystems where dissolved nutrients are scarce and often limit biological productivity. It has previously been proposed that O. universa meets its nitrogen (N) requirements by preying on zooplankton, and that its symbiotic dinoflagellates recycle metabolic 'waste ammonium' for their N pool. However, these conclusions were derived from bulk 15N-enrichment experiments and model calculations, and our understanding of N assimilation and exchange between the foraminifer host cell and its symbiotic dinoflagellates remains poorly constrained. Here, we present data from pulse-chase experiments with 13C-enriched inorganic carbon, 15N-nitrate, and 15N-ammonium, as well as a 13C- and 15N- enriched heterotrophic food source, followed by TEM (transmission electron microscopy) coupled to NanoSIMS (nanoscale secondary ion mass spectrometry) isotopic imaging to visualize and quantify C and N assimilation and translocation in the symbiotic system. High levels of 15N-labelling were observed in the dinoflagellates and in foraminiferal organelles and cytoplasm after incubation with 15N-ammonium, indicating efficient ammonium assimilation. Only weak 15N-assimilation was observed after incubation with 15N-nitrate. Feeding foraminifers with 13C- and 15N-labelled food resulted in dinoflagellates that were labelled with 15N, thereby confirming the transfer of 15N-compounds from the digestive vacuoles of the foraminifer to the symbiotic dinoflagellates, likely through recycling of ammonium. These observations are important for N isotope-based palaeoceanographic reconstructions, as they show that δ15N values recorded in the organic matrix in symbiotic species likely reflect ammonium recycling rather than alternative N sources, such as nitrates.

Response of a kleptoplastidic foraminifer to heterotrophic starvation: photosynthesis and lipid droplet biogenesis.

The aim of this work is to document the complex nutritional strategy developed by kleptoplastic intertidal foraminifera. We study the mixotrophic ability of a common intertidal foraminifer, Elphidium williamsoni, by (i) investigating the phylogenetic identity of the foraminiferal kleptoplasts, (ii) following their oxygenic photosynthetic capacity and (iii) observing the modification in cellular ultrastructural features in response to photoautotrophic conditions. This was achieved by coupling molecular phylogenetic analyses and TEM observations with non-destructive measurements of kleptoplast O2 production over a 15-day experimental study. Results show that the studied E. williamsoni actively selected kleptoplasts mainly from pennate diatoms and had the ability to produce oxygen, up to 13.4 nmol O2 cell-1 d-1, from low to relatively high irradiance over at least 15 days. Ultrastructural features and photophysiological data showed significant differences over time, the number of lipid droplets, residual bodies and the dark respiration increased; whereas, the number of kleptoplasts decreased accompanied by a minor decrease of the photosynthetic rate. These observations suggest that in E. williamsoni kleptoplasts might provide extra carbon storage through lipid droplets synthesis and highlight the complexity of E. williamsoni feeding strategy and the necessity of further dedicated studies regarding mechanisms developed by kleptoplastidic foraminifera for carbon partitioning and storage.

Kleptoplastidic benthic foraminifera from aphotic habitats: insights into assimilation of inorganic C, N and S studied with sub-cellular resolution.

The assimilation of inorganic compounds in foraminiferal metabolism compared to predation or organic matter assimilation is unknown. Here, we investigate possible inorganic-compound assimilation in Nonionellina labradorica, a common kleptoplastidic benthic foraminifer from Arctic and North Atlantic sublittoral regions. The objectives were to identify the source of the foraminiferal kleptoplasts, assess their photosynthetic functionality in light and darkness and investigate inorganic nitrogen and sulfate assimilation. We used DNA barcoding of a ~ 830 bp fragment from the SSU rDNA to identify the kleptoplasts and correlated transmission electron microscopy and nanometre-scale secondary ion mass spectrometry (TEM-NanoSIMS) isotopic imaging to study 13 C-bicarbonate, 15 N-ammonium and 34 S-sulfate uptake. In addition, respiration rate measurements were determined to assess the response of N. labradorica to light. The DNA sequences established that over 80% of the kleptoplasts belonged to Thalassiosira (with 96%-99% identity), a cosmopolitan planktonic diatom. TEM-NanoSIMS imaging revealed degraded cytoplasm and an absence of 13 C assimilation in foraminifera exposed to light. Oxygen measurements showed higher respiration rates under light than dark conditions, and no O2 production was detected. These results indicate that the photosynthetic pathways in N. labradorica are not functional. Furthermore, N. labradorica assimilated both 15 N-ammonium and 34 S-sulfate into its cytoplasm, which suggests that foraminifera might have several ammonium or sulfate assimilation pathways, involving either the kleptoplasts or bona fide foraminiferal pathway(s) not yet identified.

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. 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.

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.

Functional xanthophyll cycle and pigment content of a kleptoplastic benthic foraminifer: Haynesina germanica.

Some shallow water benthic foraminifera are able to retain functional chloroplasts (kleptoplasts) from their food source, i.e. diatoms. Here we assessed the functionality of the kleptoplast xanthophyll cycle (XC, i.e. the main diatom short-term photo-regulation mechanism) and we surveyed Haynesina germanica kleptoplast pigment composition over time and at different light regimes. Six common diatom lipophilic pigments were detected, two chlorophylls (Chl a, Chl c) and four carotenoids (fucoxanthin and by-products, diadinoxanthin, diatoxanthin and ß-carotene), the same pigment profile as the diatom species frequently isolated at the sampling site. The xanthophyll cycle (XC) was functional with kleptoplast diatoxanthin (DT) content increase with concomitant diadinoxanthin (DD) decrease after short term light exposure. DT/(DT+DD) and DT/DD ratios increased significantly in specimens exposed to low light and high light in comparison to specimens maintained in the dark. Specimens placed in very low light after the light treatments reverted to values close to the initial ones, suggesting that H. germanica XC is functional. A functional XC is an indication of H. germanica kleptoplasts capacity for short-term photo-protection from photo-oxidative damages caused by excess of light. Furthermore, the pigment survey suggests that H. germanica preserved some chloroplasts over a longer time than others and that pigment content is influenced by previous light history. Finally, the current study highlighted seasonal differences, with higher pigment contents in winter specimens (27.35 ± 1.30 ng cell-1) and lower in summer specimens (6.08 ± 1.21 ng cell-1), a quantitative and qualitative composition suggesting light acclimation to low or high light availability, according to the season.

Simultaneous 2D imaging of dissolved iron and reactive phosphorus in sediment porewaters by thin-film and hyperspectral methods.

This study presents a new approach combining diffusive equilibrium in thin-film (DET) and spectrophotometric methods to determine the spatial variability of dissolved iron and dissolved reactive phosphorus (DRP) with a single gel probe. Its originality is (1) to postpone up to three months the colorimetric reaction of DET by freezing and (2) to measure simultaneously dissolved iron and DRP by hyperspectral imaging at a submillimeter resolution. After a few minutes at room temperature, the thawed gel is sandwiched between two monospecific reagent DET gels, leading to magenta and blue coloration for iron and phosphate, respectively. Spatial distribution of the resulting colors is obtained using a hyperspectral camera. Reflectance spectra analysis enables deconvolution of specific colorations by the unmixing method applied to the logarithmic reflectance, leading to an accurate quantification of iron and DRP. This method was applied in the Arcachon lagoon (France) on muddy sediments colonized by eelgrass (Zostera noltei) meadows. The 2D gel probes highlighted microstructures in the spatial distribution of dissolved iron and phosphorus, which are most likely associated with the occurrence of benthic fauna burrows and seagrass roots.

Vertical migration, nitrate uptake and denitrification: survival mechanisms of foraminifers (Globobulimina turgida) under low oxygen conditions.

(15)NO(3)(-) isotope labelling experiments were performed to investigate foraminiferal nitrate uptake strategies and the role of pseudopodial networks in nitrate uptake. Globobulimina turgida were placed below the nitrate penetration depth in homogenized sediment cores incubated in artificial seawater containing (15)NO(3)(-) . A nylon net prevented the vertical migration of foraminifera to strata containing nitrate and oxygen, but allowed potential access to such strata by extension of pseudopods. No (15)NO(3)(-) was found in G. turgida in these cores, suggesting that foraminifera cannot extend their pseudopods for nitrate uptake through several millimetres of sediment, but must physically migrate upwards closer to nitrate-containing strata. However, foraminiferal migration patterns in control cores with no nylon net were erratic, suggesting that individuals move in random orientations until they find favourable conditions (i.e. free nitrate or oxygen). A second experiment showed that foraminifera actively collect nitrate both in the presence and in the absence of oxygen, although uptake was initiated faster if oxygen was absent from the environment. However, no systematic influence of the size of the intracellular nitrate pool on nitrate uptake was observed, as specimens containing a large range of intracellular nitrate (636-19 992 pmol per cell) were measured to take up (15)NO(3)(-) at comparable rates.

Widespread occurrence of nitrate storage and denitrification among Foraminifera and Gromiida.

Benthic foraminifers inhabit a wide range of aquatic environments including open marine, brackish, and freshwater environments. Here we show that several different and diverse foraminiferal groups (miliolids, rotaliids, textulariids) and Gromia, another taxon also belonging to Rhizaria, accumulate and respire nitrates through denitrification. The widespread occurrence among distantly related organisms suggests an ancient origin of the trait. The diverse metabolic capacity of these organisms, which enables them to respire with oxygen and nitrate and to sustain respiratory activity even when electron acceptors are absent from the environment, may be one of the reasons for their successful colonization of diverse marine sediment environments. The contribution of eukaryotes to the removal of fixed nitrogen by respiration may equal the importance of bacterial denitrification in ocean sediments.