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.

Subcellular architecture and metabolic connection in the planktonic photosymbiosis between Collodaria (radiolarians) and their microalgae.

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium). We showed that this symbiosis is very dynamic whereby symbionts interact with different host cells via extracellular vesicles within the colony. 3D electron microscopy revealed that the photosynthetic apparatus of the microalgae was more voluminous in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were stored in the symbiotic microalga in starch granules and purine crystals respectively. Nitrogen was also allocated to the algal nucleolus. In the host, low 13 C transfer was detected in the Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (c. 40 ppm) and twofold higher in the host, whereas copper concentration increased in symbionts and was detected in the host cell and extracellular vesicles. Sulfur concentration was around two times higher in symbionts (chromatin and pyrenoid) than their host. This study improves our understanding on the functioning of this oceanic photosymbiosis and paves the way for more studies to further assess its biogeochemical significance.

Photosynthesis from stolen chloroplasts can support sea slug reproductive fitness.

Some sea slugs are able to steal functional chloroplasts (kleptoplasts) from their algal food sources, but the role and relevance of photosynthesis to the animal host remain controversial. While some researchers claim that kleptoplasts are slowly digestible 'snacks', others advocate that they enhance the overall fitness of sea slugs much more profoundly. Our analysis shows light-dependent incorporation of 13C and 15N in the albumen gland and gonadal follicles of the sea slug Elysia timida, representing translocation of photosynthates to kleptoplast-free reproductive organs. Long-chain polyunsaturated fatty acids with reported roles in reproduction were produced in the sea slug cells using labelled precursors translocated from the kleptoplasts. Finally, we report reduced fecundity of E. timida by limiting kleptoplast photosynthesis. The present study indicates that photosynthesis enhances the reproductive fitness of kleptoplast-bearing sea slugs, confirming the biological relevance of this remarkable association between a metazoan and an algal-derived organelle.

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.

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.

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.

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.

Functional kleptoplasts intermediate incorporation of carbon and nitrogen in cells of the Sacoglossa sea slug Elysia viridis.

Some sacoglossan sea slugs incorporate intracellular functional algal chloroplasts, a process termed kleptoplasty. "Stolen" chloroplasts (kleptoplasts) can remain photosynthetically active up to several months, contributing to animal nutrition. Whether this contribution occurs by means of translocation of photosynthesis-derived metabolites from functional kleptoplasts to the animal host or by simple digestion of such organelles remains controversial. Imaging of 13C and 15N assimilation over a 12-h incubation period of Elysia viridis sea slugs showed a light-dependent incorporation of carbon and nitrogen, observed first in digestive tubules and followed by a rapid accumulation into chloroplast-free organs. Furthermore, this work revealed the presence of 13C-labeled long-chain fatty acids (FA) typical of marine invertebrates, such as arachidonic (20:4n-6) and adrenic (22:4n-6) acids. The time frame and level of 13C- and 15N-labeling in chloroplast-free organs indicate that photosynthesis-derived primary metabolites were made available to the host through functional kleptoplasts. The presence of specific 13C-labeled long-chain FA, absent from E. viridis algal food, indicates animal based-elongation using kleptoplast-derived FA precursors. Finally, carbon and nitrogen were incorporated in organs and tissues involved in reproductive functions (albumin gland and gonadal follicles), implying a putative role of kleptoplast photosynthesis in the reproductive fitness of the animal host.

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.

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.