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.

www.nature.com/scientificreports www.nature.com/scientificreports/ be crucial for the survival of sacoglossan sea slugs 15 . While the translocation of photosynthesis-derived products from a viable autotrophic unicellular symbiont to its host is well established 16,17 , such translocation from viable kleptoplasts to the sea slug host is still debated 13,15,18,19 . Earlier radiolabeled carbon-based studies suggested translocation of photosynthates into sacoglossan sea slugs cells 20,21 . In Tridachia crispata and Tridachiellia diomedea, 14 C radioactivity was detected within 2 h in what was assumed to be the renopericardium, the cephalic neural tissue and the mucus secreting pedal gland 21 . The detection of radioactivity in kleptoplast-free organs at such sort-time frame was indicative of incorporation via functional kleptoplasts in these two species. In another species, Elysia timida, it was recently proposed that, after some days of starvation, starch accumulated at the kleptoplasts and could be used by the animal after kleptoplast digestion 18,19 . Another potential benefit mediated by kleptoplasty that remains unexplored in photosynthetic sea slugs is nitrogen assimilation. While kleptoplasts can provide carbon substrates to the host, short periods of starvation could rapidly lead to nitrogen deficiency, unless kleptoplasts could somehow mediate nitrogen acquisition 22 . Indeed, previous observations of uptake of 15 N-labeled ammonium, nitrite and urea (but not nitrate) into bulk tissues and specific amino acids enrichment of Elysia viridis hosting functional kleptoplasts, suggests a light-dependent assimilation of nitrogen in this organism 22 .
This work presents new evidence for light-dependent incorporation of inorganic carbon and nitrogen into tissues of the sacoglossan sea slug Elysia viridis, using compound specific isotope analysis (CSIA) of fatty acid methyl esters (FAME) coupled with high-resolution secondary ion mass spectrometry (NanoSIMS), demonstrating spatial and temporal movements of 13 C and 15 N isotopes.

Results
Isotopic dual labeling pulse experiments were conducted with E. viridis individuals incubated for 12 h with 13 C-bicarbonate and 15 N-ammonium. Different animals were sampled at increasing time points up to 12 h of labeled inorganic carbon and nitrogen addition (see Materials and Methods for details).
Microscopy observations. Transversal sections of E. viridis, immediately after the pericardium region, allowed the visualization of digestive tubules, the albumin gland, gland ducts and gonadal follicles (Fig. 1). Histological identification was based on anatomical works of E. viridis and other sacoglossan sea slugs [23][24][25][26][27] . Electron microscopy observations of the digestive tubules revealed intact sequestered kleptoplasts after 1.5 h of incubation (Fig. 2a), with clearly distinguishable ultrastructural features, such as thylakoids, starch grains, pyrenoids and plastoglobuli (Fig. 2b,c). Kleptoplasts were equally abundant in the digestive tubules after 12 h of incubation (Fig. 2d). While some of the kleptoplasts were intact, others had more diffuse thylakoids and membranes (Fig. 2e,f). All kleptoplasts observed in sea slugs incubated for 12 h lacked plastoglobuli but had intact starch grains. After both 1.5 and 12 h of incubation, kleptoplasts were surrounded by flaky electron-lucent cytoplasm (Fig. 2, indicated by asterisks).
imaging of 13 c and 15 n assimilation. All NanoSIMS 13 C/ 12 C and 15 N/ 14 N images from the individuals incubated for 12 h in the light with 13 C-bicarbonate and 15 N-ammonium showed 13 C and 15 N-labeling in all the observed tissues, i.e., digestive tubules ( Fig. 4a), albumin gland ( Fig. 4b) and gland ducts (Fig. 4c), and gonadal follicles (Fig. 4d). All tissues exhibited similar enrichment dynamics; overall they increased from low-enrichment after 1.5 h of incubation to higher enrichment values after 12 h of incubation (Figs. 5 and S1-S3). It was clear from observations of semi-thin sections, combined with NanoSIMS imaging, that 13 C-and 15 N-labeling was not homogenously distributed in the different tissues, instead 13 C and 15 N-hotspots could be identified in all images.
All individuals incubated in the dark for 12 h with 13 C-bicarbonate and 15 N-ammonium displayed no 13 C-enrichments (Fig. S4). In contrast, 15 N-labeling was observed in all sea slug tissues (i.e., digestive tubules, gland ducts, albumin gland and follicles), albeit at a much lower level than in conspecifics incubated under light (compare Figs. 4 and S4).
Electron microscopy observations combined with NanoSIMS imaging of individuals incubated in the light for 1.5 and 12 h allowed the visualization of specific isotopically labeled organelles. In the digestive tubules, after 1.5 h of incubation, only the starch of some kleptoplasts was 13 C-labeled (Figs. 6a and S5). Of the three specimens incubated for 1.5 h, only one exhibited low 15 N-labeling in some unidentified vesicles and secretory vesicles associated with the Golgi apparatus (Fig. S6). After 12 h of incubation, the three specimens analyzed were labeled with 13 C and 15 N. In the digestive tubules, 13 C-labeling was mainly concentrated in the kleptoplast starch and/or pyrenoids, but was also spread in the cytoplasm with a flaky aspect surrounding the photosynthetic organelles (Figs. 6b,c and S5). The lipid droplets observed in one of the specimens in close association with the kleptoplasts membranes were not labeled (Figs. 6c and S5). After 12 h, 15 N-labeling was mainly concentrated in the thylakoids of the kleptoplasts and also spread in the cytoplasm of the digestive tubule, with high variability being recorded among specimens (Figs. 6b,c and S6). In the digestive tubules, a few unidentified vesicles, as well as some unidentified dark circular structures with a diameter of ca. 2 µm, were also 15 N-labeled (Figs. 6d and S5). Finally, some of the dark vesicles on the gland duct were also labeled, both in 13 C and 15 N (Fig. 6e).  Table S2 for FAs δ 13 C-averaged-values.
Chloroplasts are rich in 18:2n-6, which can be transformed in arachidonic acid (20:4n-6) only trough a series of desaturation and elongation steps. It is interesting to note that 13 C incorporation rate (as shown by the slope of the regression curves in Fig. 3) is higher for 18:2n-6 than 20:4n-6, indicating that the carbon donor (Acetyl CoA→ Malonyl-CoA) during the elongation process is not 13 C-enriched and thereby not provided by the chloroplast but rather by the host. However, it is impossible to tell if algal 18:2n-6 was obtained by the host trough chloroplast degradation (likely from Diacylglycerols or phospholipids) or trough lipid transport 35 . We therefore hypothesize the following path explaining the presence of labeled long-chain FAs which have not been identified in the algal food source: 1) de novo synthesis of FAs at the kleptoplast level, 2) transfer of algal FAs to the animal cell, and 3) recycling in the animal cell (e.g. elongation/desaturation). Considering the short time-frame in which these three processes occurred and the fact that within 12 h there is no obvious kleptoplast digestion in TEM observations (Fig. 2) we further hypothesize that the step (2) on the path described above is due to translocation from intact kleptoplasts, rather than digestion. This hypothesis is further supported by the fact that digestion of kleptoplasts in a different species, E. timida, occurred only after prolonged starvation (at least 21 days according to Laetz et al. 18 ) as detailed in the following paragraphs.
It was recently shown that kleptoplasts in fed Elysia timida retained only small amounts of starch, while when deprived from their food source (starved), kleptoplasts in E. timida started to accumulate starch 18 . The starting point of starch accumulation was variable, occurring after 3, 10 or 21 days of starvation (first stage of starvation condition), depending on the group being tested (different locations and/or different seasons were tested). After www.nature.com/scientificreports www.nature.com/scientificreports/ further periods of starvation (second stage), starch degradation was higher than starch production. Again the time point of this switch was variable and depended of the targeted group, occurring after 21 and 42 to 88 days. Moreover, it was also coincident with the decline in photosynthetic activity. This suggests that, in a first stage starch accumulated while kleptoplasts are mostly functional, while during a second stage of long starvation kleptoplasts were degraded and starch digestion occurred 18 , either by the host and/or by the kleptoplasts to meet their metabolic needs. It is therefore assumed that kleptoplasts are being degraded/digested after long periods (days to weeks) of starvation. Although similar information does not exist for E. viridis, this species has been shown to retain functional kleptoplasts for at least 3 months 3 , so this species could display a similar pattern to that described for E. timida. In the present work, a significantly shorter time frame was used, with TEM-NanoSIMS data showing 13 C-labeled intact starch grains in the kleptoplasts after 12 h of incubation under light (Figs. 3 and  6). Furthermore, after 3 h hours, 13 C-labeling was already observed in different chloroplast-free organs such as the albumin gland (Fig. 5). These observations of a very fast 13 C-translocation to distant animal tissues strongly support an active transfer of photosynthates from the kleptoplasts to host tissues, rather than a 13 C-incorporation by digestion of kleptoplast starch grains. Nevertheless, it cannot be ruled out, that sea slugs with long-term retention of functional kleptoplasts may also acquire C-compounds derived from photosynthates from the digestion of older kleptoplasts, as hypothesized previously 15,18 . Sacoglossan sea slugs may thus adopt different strategies when chloroplasts-replenishment is impaired for long periods of time, with both pathways being concomitant or occurring at different time scales.
The translocation of photosynthetically acquired carbon to animal tissues was previously hypothesized for two other species of sacoglossan sea slugs, Elysia (=Tridachia) crispata and Tridachiella diomedea 21 . Despite the lower resolution histological approach to pinpoint the presence of 14 C in these organisms, these authors suggested that after 15 min of incubation only chloroplast-containing tissues, i.e., digestive tubules, were labeled. Subsequently, 14 C-incorporation was also detected in chloroplast-free organs, such as the renopericardium (after 60 min), the cephalic neural tissue and the mucus secreting pedal gland (after 120 min), and the intestine (after 300-540 min). In T. diomedea, 14 C was still tracked in animal tissues after 6 weeks of food deprivation. In E. viridis, we first observed 13 C-labeling after 1.5 h in starch grains of kleptoplasts present in digestive tubules (Fig. 6). After longer incubation times, 13 C-labeling was detected in all animal tissues surveyed: albumin gland, gland ducts and gonadal follicles (Figs. 4-6 and S1-S3). Therefore, inorganic 13 C was first photosynthetically accumulated into kleptoplasts starch, followed by a C-transfer to sea slug tissues, conceivably by translocation of soluble C-compounds (sugars, organic acids) or fatty acids. In the present work, the absence of 13 C-labeling in lipid droplets does not support the hypothesis of a direct transfer via lipid droplets across kleptoplast membranes, as seen in algae or suggested for kleptoplastidic foraminifera and the sacoglossan Elysia chlorotica 17,36,37 . Instead, the 13 C-labeled electron-lucent cytoplasm surrounding the kleptoplasts could correspond to FAs being transferred and reprocessed in the vicinity of kleptoplasts. Plastoglobuli, lipoprotein particles surrounded by a membrane lipid monolayer, here present in early stages of incubation (Fig. 2a-c), may also be a vessel for photosynthates transport between chloroplasts and the cytoplasm 38 .

Ammonium assimilation.
A pathway for ammonium assimilation by E. viridis feeding on the algae Codium fragile was suggested from incubations of sea slugs with 15 N-labeled nitrogen substrates 22 , showing that part of ammonium assimilation was light-dependent, and decreased significantly when sea slugs were incubated with specific inhibitors of the GS and GOGAT kleptoplast enzymes. Thus, at least part of ammonium assimilation would have taken place through kleptoplastidic GS-GOGAT activity, acknowledging the possibility of an additional cytoplasmic pathway through the GDH enzyme. This enzyme was shown to occur in some marine mollusks 39,40 . Here, using an imaging approach we directly show 15 N-assimilation in E. viridis previously fed with the algae C. tomentosum. A schematic diagram of the inorganic carbon and ammonium assimilation pathways in E. viridis is presented in Fig. 7, where plain lines represent pathways demonstrated by the present study. While no 13 C-enrichment was recorded in FAs or in isotopic images of dark-incubated individuals, some dark-incubated 15 N-assimilation was observed via NanoSIMS imaging. However, when incubated in the presence of light, sea slugs showed a much higher level of 15 N-enrichment in their tissues. This light-dependent activity of kleptoplastidic enzymes might be explained by the fact that photosynthesis provides energy and carbon skeletons necessary for amino acid synthesis in the chloroplast. An external supply of nitrogen would be essential for processes such as de novo synthesis of plastid-encoded proteins, shown to occur in Elysia chlorotica 5 , a photosynthetic sacoglossan sea slug with the ability to retain functional kleptoplasts for extended periods of time (>6 months). Ammonium assimilation recorded in specimens incubated in the dark (albeit significantly reduced) could result either (i) from a cytoplasmic enzymatic pathway (GDH) of the sea slug itself, or (ii) from residual chloroplast activity. As for 13 C-assimilation, we clearly show that the 15 N-metabolites derived from 15 N-ammonium assimilation were also found in the kleptoplasts and digestive tubules cytoplasm, as well as in kleptoplast-free tissues: albumin gland, gland ducts and gonadal follicles (Figs. 4 and 6). TEM-NanoSIMS analysis showed high 15 N-labeling in some unidentified vesicles and in vesicles associated with the Golgi apparatus, thus identified as secretory vesicles (Fig. S6). Golgi apparatus are involved in the maturation of proteins, receiving these molecules from the endoplasmic reticulum and sending them to their next destination through secretory vesicles 41 .

conclusions
We show with unprecedented spatial and temporal resolution the incorporation of C and N in animal cells mediated by functional kleptoplasts. The present imaging of 13 C-and 15 N-enriched organs and tissues involved in reproductive functions (albumin gland and gonadal follicles) incite further research on the importance of photosynthesis for metabolic pathways involved in parental investment and offspring fitness in kleptoplastidic sea slugs. Future studies are required to confirm in other photosynthetic sacoglossan sea slugs the extent to which photosynthesis-derived primary metabolites are made available to the host from functional kleptoplasts.
www.nature.com/scientificreports www.nature.com/scientificreports/ Methods experimental design. The objective of the present study was to determine if the incorporation of inorganic carbon and nitrogen in the sacoglossan sea slug Elysia viridis was dependent on kleptoplast activity. Sea slugs were collected, acclimated to lab conditions, and incubated with and without isotopically labeled bicarbonate and ammonium as described bellow. The fate of carbon and nitrogen was analysed using compound specific isotope analysis (CSIA) of fatty acid methyl esters (FAME) coupled with high-resolution secondary ion mass spectrometry (NanoSIMS). collection and acclimation. Elysia viridis specimens were collected on the intertidal rocky shore of Praia de Labruge (41°16′28.9″N; 8°43′45.3″W), Vila do Conde (Portugal) from their macroalgal food source Codium tomentosum. Specimens and respective food source were randomly distributed between two 10 L tanks filled with filtered natural seawater, at salinity 35, 16 °C and under a photon irradiance (400-700 nm) of 30 µmol photons m -2 s -1 at the water surface level (14 h:10 h light:dark cycle) for two weeks acclimation before isotopic labeling experiments.
Dual isotopic labeling incubations. Isotopic dual labeling pulse experiments were conducted in closed-systems (1 L glass bottles, 3 independent containers per treatment). Enriched artificial seawater (ASW) was made in accordance with Harrison et al. 42 but using NaH 13 CO 3 ( 13 C isotopic abundance of 99%, SIGMA-ALDRICH) and 15 NH 4 Cl ( 15 N isotopic abundance of 98%, SIGMA-ALDRICH) to a final concentration of 2 mM and 20 µM, respectively (enriched-ASW). Control non-enriched-ASW contained NaH 12 CO 3 and 14 NH 4 Cl in the same concentrations as enriched-ASW. The pulse of isotopic dual labeling started 30 min after the onset of the light period. Sacoglossan sea slugs E. viridis with a length between 9 and 12 mm were incubated in the absence of their food source both under an incident photon irradiance of 80 µmol photons m -2 s -1 (400-700 nm; white fluorescence lamps) and in dark conditions. Dark conditions served as a control for bicarbonate and ammonium assimilation that could potentially be incorporated into animal tissue in a light-independent manner via exchange reactions and/or carboxylation steps in animal metabolism. Individuals in enriched-ASW and exposed to light were sampled after 1.5, 3, 9 and 12 h (n = 3), quickly rinsed with distilled water, immediately frozen in liquid nitrogen and stored at -80 °C for further fatty acids (FAs) analysis. Another subset of three individuals at 1.5, 3, 6 and 12 h were collected, rinsed and fixed in 0.2 M cacodylate buffer containing 4% glutaraldehyde and 0.5 M sucrose and stored at 4 °C for 24 h before tissue preparation for further microscopy and secondary ion mass spectrometry (SIMS) imaging analysis (NanoSIMS 50 L). Additional subsets of three individuals from non-enriched-ASW exposed to light and from enriched-ASW kept in darkness (control treatments) were sampled after a 12 h incubation period and frozen or fixed, as described above, for FAs and SIMS analysis. fatty acid composition. Fatty acids (FAs) were extracted following the method described by Meziane et al. 43 Lipids were extracted by sonication (35 kHz, 20 min) using chloroform/methanol/water (2:1:1,v:v:v). Complex lipids such as triglycerides or phospholipids were hydrolized by saponification (90 min, 90 °C) with NaOH:MeOH (1:2, v-v) to release individual FAs. An internal standard (tricosanoic acid: 23:0, 10 μg) was added to each sample before www.nature.com/scientificreports www.nature.com/scientificreports/ extraction. Individual FAs were then derivatized into fatty acids methyl esters (FAMEs) using boron-trifluoride methanol (BF 3 -MeOH) at 90 °C for 10 min. Samples were then dried under N 2 flux and transferred to hexane for injection in a gas chromatograph (GC, VARIAN CP-3800 equipped with flame ionization detector -FID). Most FAMEs were identified by comparing their retention times with those of known standards (SUPELCO 37, PUFA-1 Marine Source, and Bacterial Mix; SUPELCO Inc., Bellefonte, PA, United States). FAMEs were further identified by GC coupled to a mass spectrometer (GC-MS, VARIAN GC450-220MS). For both devices, FAMEs separation was performed using a SUPELCO OMEGAWAX 320 column (30 m × 0.32 mm i.d., 0.25 μm film thickness) with He as carrier gas. After injection of 1 μl of sample at 60 °C, the temperature was raised to 150 °C at 40 °C min -1 , then to 240 °C (held 14 min) at 3 °C min -1 . FAMEs were systematically corrected for the added methyl group and corresponding individual FAs are designated in this study as CX:Y-nZ, where X is the number of carbons, Y the number of double bonds and Z the position of the ultimate double bond from the terminal methyl.
Compound specific isotope analysis (CSIA) of fatty acid methyl ester (FAME). After GC and GC-MS analyses, carbon stable isotopic ratios (expressed in ‰) of individual fatty acids were measured by gas-chromatography-isotope ratio mass spectrometry (GC-IRMS). Measurements were performed at the UC Davis Stable Isotope Facility of the University of California (Davis, CA, United States). FAMEs dissolved in hexane were injected in splitless mode and separated on a VARIAN factorFOUR VF-5ms column (30 m × 0.25 mm ID, 0.25 micron film thickness). Once separated, FAMEs were quantitatively converted to CO 2 in an oxidation reactor at 950 °C. Following water removal through a nafion dryer, CO 2 enters the IRMS. δ 13 C values were corrected using working standards composed of several FAMEs calibrated against NIST standard reference materials. Stable carbon isotope ratios for individual FA were calculated from FAME data by correcting for the one carbon atom in the methyl group that was added during the derivatization process. This correction was made according to Gladyshev et al. 44 by taking into account the isotope ratio of the derivatized methanol (BF 3 -methanol, -39.1‰ in our study), and the fractional carbon contribution of the free FA to the ester. where δ 13 C FA is the isotopic composition of the free FAs, δ 13 C FAME is the isotopic composition of the FA methyl ester, f is the fractional carbon contribution of the free FA to the ester and δ 13 C CH3OH is the isotopic composition of the methanol derivatization reagent. The isotopic composition of the methanol was determined by the same GC-IRMS system. tissue preparation for microscopy and nanoSiMS imaging. Sea

Microscopy.
Overview semi-thin cuts of 1.5 µm thickness were made from the sea slug body part roughly after the pericardium. Semi-thin sections were cut on a Leica UC7 ultramicrotome using a LEICA glass knife and were placed on circular glass cover slips (AGAR SCIENTIFIC, borosilicate, 10 mm diameter, 1½ mm thickness). Histological overviews were documented on an optical light microscope. Thin sections (70 nm) for electron microscopy observations were made on a LEICA ULTRACUT S microtome with a diamond ultra knife (40°) at the electron microscopy facilities of Angers University (SCIAM). The thin-sections were transferred onto formvar-carbon film copper grids and stained for 10 min with uranyless prior observation. The sections were observed with a transmission electron microscope (TEM, Philips 301 CM100, 80 kV) at the electron microscopy facility of Lausanne University (EMF). TEM grids (thin-sections) were transferred on 10 mm aluminum disks with double stick Cu-tape. Before NanoSIMS analysis, both the thin and semi-thin sections were coated with a ca. 15 nm thick gold layer.
nanoSiMS isotopic image acquisition and data processing. Large areas of interest covering a far-reaching portion of digestive tubules, albumin glands, gland ducts, and gonadal follicles were imaged with optical light microscopy or TEM, and were then analyzed with a NanoSIMS 50 L secondary ion mass spectrometer (Laboratory for Biological Geochemistry, EPFL, Lausanne, Switzerland). This allowed imaging and quantification of the subcellular distribution of 13 C and 15 N enrichment in the exact same areas of the imaged sea slugs tissue, enabling direct correlation of structural and isotopic images. All measurements were performed using the following analytical conditions: 16 keV primary ion beam of Cs + focused to a beam spot of ca. 100-150 nm and counting 12 C 12 C − , 13 C 12 C -, 14 N 12 Cand 15 N 12 Cions in electron multipliers at a mass resolution of> 8000 (Cameca definition), enough to resolve potential interferences in the mass spectrum. Images captured with NanoSIMS 50 L were processed using the L'IMAGE PV-WAVE software (version 10.1, Larry R Nittler, Carnegie Institution of Washington, Washington DC, USA, https://sites.google.com/carnegiescience.edu/limagesoftware/home).
Regions of interest selecting individual anatomic structures were defined and 13 C/ 12 C and 15 N/ 14 N ratios distribution maps were obtained by taking the ratio between the drift-corrected 13 C 12 C − and 12 C 12 C − images, and 15 N 12 C − and 14 N 12 C − images, respectively. Five stacked planes were used for each image. 13 C and 15 N enrichment values in the figures were expressed as logarithmic values of the measured ratios, i.e., 13 C 12 C/ 12 C 2 and 15 N 12 C/ 14 N 12 C. These ratios were also measured in the controls (natural values): 13 C 12 C/ 12 C 2 was 0.0210 ± 0.0002, and 15 N 12 C/ 14 N 12 C was 0.0037 ± 0.0001.