Kleptoplasty does not promote major shifts in the lipidome of macroalgal chloroplasts sequestered by the sacoglossan sea slug Elysia viridis

Sacoglossan sea slugs, also known as crawling leaves due to their photosynthetic activity, are highly selective feeders that incorporate chloroplasts from specific macroalgae. These “stolen” plastids - kleptoplasts - are kept functional inside animal cells and likely provide an alternative source of energy to their host. The mechanisms supporting the retention and functionality of kleptoplasts remain unknown. A lipidomic mass spectrometry-based analysis was performed to study kleptoplasty of the sacoglossan sea slug Elysia viridis fed with Codium tomentosum. Total lipid extract of both organisms was fractionated. The fraction rich in glycolipids, exclusive lipids from chloroplasts, and the fraction rich in betaine lipids, characteristic of algae, were analysed using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC-MS). This approach allowed the identification of 81 molecular species, namely galactolipids (8 in both organisms), sulfolipids (17 in C. tomentosum and 13 in E. viridis) and betaine lipids (51 in C. tomentosum and 41 in E. viridis). These lipid classes presented similar lipidomic profiles in C. tomentosum and E. viridis, indicating that the necessary mechanisms to perform photosynthesis are preserved during the process of endosymbiosis. The present study shows that there are no major shifts in the lipidome of C. tomentosum chloroplasts sequestered by E. viridis.

of endosymbiosis in other marine biological interactions [8][9][10] . These studies have identified significant changes in the host lipidome during the establishment of symbionts 8,11 , suggesting an active role of lipids in this process.
Endosymbiosis is a rare process in nature because it requires the integration of host and symbiont membranes and their common evolution 12 . While kleptoplasty is a general phenomenon in protists 13,14 , to date, sacoglossans are the only metazoans known to maintain this type of association 15 . This mechanism of endosymbiosis could have some similar features to the interaction between eukaryotic cells and endosymbiotic cyanobacteria, which gave rise to chloroplast-containing eukaryotes 12 . Despite the evolution from cyanobacteria to vascular plants, molecular composition of chloroplasts has been highly conserved 16 . Chloroplast membranes are characterized by the occurrence of high proportions of glycolipids, being monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) the most abundant 16 , and sulfolipids (sulfoquinovosyl diacylglycerol, SQDG), which are not found in extraplastidial membranes. These classes of specific lipids are biosynthesized within the chloroplast [17][18][19] (Fig. 1). A conserved proportion of these molecules integrates the complexes of photosynthetic machinery of chloroplasts, being critical to sustain an optimal rate of photosynthesis 20 . Inner chloroplast envelope and thylakoid membranes show similar relative proportions of lipids, highlighting the role of the former in the biogenesis of thylakoid membranes 20 . Additionally, betaine lipids (diacylglyceryl-N,N,N-trimethyl homoserine, DGTS) are a class of lipids found in algae and that can be translocated to the chloroplast 21 . DGTS is considered as a more ancient membrane lipid, which has been progressively replaced by phosphatidylcholine (PC) during the evolution of vascular plants 22 . Although algae have preserved the DGTS biosynthetic pathway 23 , DGTS competes with PC and therefore the levels of both lipid classes are reciprocal 22 .
Lipidomic analyses applied to marine samples can provide information at the molecular level, being successfully used to explain biological and molecular processes [24][25][26] . In this sense, we selected the ecological model E. viridis (Montagu, 1804) and Codium tomentosum (Stackhouse, 1797) to investigate the association of functional macroalgal chloroplasts inside animal cells, by using lipidomic tools. Elysia viridis collected in the coast of Portugal has a narrow feeding preference, mostly retaining functional plastids from genus Codium 27 , thus limiting the origin of plastid lipids. Furthermore, E. viridis displays a relatively long-term retention of kleptoplasts 3,4 , which allows researchers to perform experiments on individuals that already display well established functional kleptoplasts inside their animal cells.
Glycolipids and betaine lipids isolated from total lipid extracts of E. viridis and C. tomentosum were studied using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC-MS). The objective of the Lipids can be synthesized entirely within the chloroplast -prokaryotic lipid synthesis -or in collaboration with the endoplasmic reticulum (ER) -eukaryotic lipid synthesis. De novo synthesis of fatty acids occurs inside the chloroplast and can follow two pathways: (i) prokaryotic pathway: fatty acids are transported to envelope membranes and used for glycolipid synthesis (galactolipids and sulfolipids); (ii) eukaryotic pathway: fatty acids are exported as free fatty acids to envelope membranes and their corresponding CoA thioesters are transferred to the ER to integrate lipid structure. Lipid precursors assembled at the ER are exported to the outer envelope membrane for glycolipid synthesis. Glycolipids are incorporated into thylakoid membranes. Although lipid trafficking between membranes occurs through unknown mechanisms (dash lines), it is believed that transport between the ER and the outer envelope membrane occurs by direct contact sites between both membranes or via vesicles; while transport between inner envelope membrane and thylakoid membrane is mediated by vesicles. Under phosphate starvation (Pi), DGDG can be transported to ER and then to the plasma membrane to replace phospholipids. Figure adapted from Dörmann 19 .
present study was to investigate if kleptoplasty promoted any major shifts in the lipidome of C. tomentosum plastids sequestered by the sacoglossan sea slug E. viridis. Hence, we focused the analysis on lipid classes known to be exclusive of chloroplast membranes (i.e., glycolipids), as well as on betaine lipids.

Results
HILIC-LC-MS and MS/MS allowed the identification of glycolipids (in fraction 3, see Methods) and betaine lipids (in fraction 4) in the lipid extracts obtained from the marine species C. tomentosum and E. viridis. The information gathered with the high resolution HILIC-LC-MS and MS/MS analyses provided the detailed structural information to identify the different lipid classes and their molecular species profiles (see Supplementary  Table S1). Overall, seventy-six molecular species were identified in C. tomentosum and sixty-two molecular species in E. viridis samples, as glycolipids and betaine lipids.
Profile of glycolipids. Glycolipids were identified in fraction 3 for both C. tomentosum and E. viridis, distributed between galactolipid (MGDG and DGDG) and sulfolipid classes. Twenty-five molecular species were identified in C. tomentosum and twenty-one molecular species in E. viridis samples.   (Fig. 7). Moreover, DGTS lipidomic profile showed fourteen molecular species that were only identified in C. tomentosum samples, while three DGTS molecular species were only identified in E. viridis samples (Fig. 7). This profile of betaine lipids was confirmed in E. viridis samples being exposed to starvation conditions (see Supplementary Fig. S1 and Supplementary Table S2).

Discussion
The lipidome of chloroplast membranes of C. tomentosum was reflected in the sea slug E. viridis. Indeed, the most abundant lipid classes in chloroplast membranes, MGDG, DGDG and SQDG presented similar lipidomic profiles in C. tomentosum and E. viridis samples. The presence of these exclusive lipid classes of chloroplast membranes in E. viridis indicates that there are no major shifts in the lipidome promoted by kleptoplasty and suggests that the mechanisms necessary to perform photosynthesis are preserved during the process of endosymbiosis. This finding confirms the robustness of C. tomentosum chloroplasts, which do not experience major changes throughout ingestion and establishment in the digestive epithelial cells of the host 28 .
The lipidomic profile of C. tomentosum is in concordance with that previously described by da Costa et al. 25 . The glycolipids MGDG and DGDG are known to occur in all organisms performing oxygenic photosynthesis 16 . They are synthesized in the chloroplast envelope membranes and redistributed to the thylakoid membranes 19 , where, along with phosphatidylglycerol (PG), perform an important role in the stability and activity of photosynthetic complexes 16 . Moreover, MGDG and DGDG contribute to the stabilization of plastid membranes and lipid trafficking with extraplastidial membranes 20,29 . Therefore, the conservation of MGDG and DGDG molecular species, along with fatty acyl composition, evidences the preservation of plastid membrane composition and function during kleptoplasty.
The lipidomic profile of sulfolipids shows the absence of several molecular species of SQDG in E. viridis when compared to C. tomentosum samples. These differences may be due to either extremely low concentration, likely below the detection limits, in sea slug samples or replacement during the process of endosymbiosis. Nevertheless, the most abundant molecular species were the same in both marine organisms. SQDG plays an important role in chloroplast development and regeneration 30 , thus the loss of these molecular species may be related with the process of establishment of the stolen plastid in cells of its new host. Although SQDG is associated with photosystem II 31 , the presence of SQDG does not always correlate with photosynthetic capacity 32 . However, SQDG performs relevant functions in chloroplasts and recently it has been discovered that in microalgae cells SQDG plays the role of sulphur storage lipids that is used for protein synthesis in early phases of sulphur deprivation 33 . On the other hand, under phosphate-limited conditions, phospholipids are replaced with non-phosphate containing lipids such as MGDG, DGDG and SQDG 32 . Under phosphate deprivation, DGDG levels increase and its excess is transferred to extraplastidial membranes to replace phospholipids (Fig. 1), limiting the consumption of phosphate for membrane lipid synthesis 32 . Furthermore, since SQDG and PG are anionic lipids, SQDG partially replaces PG to maintain the anionic surface charge of thylakoid membranes 32 . Although glycolipids can replace phospholipids under phosphate deprivation, lipid trafficking with extraplastidial membranes is limited to DGDG, since SQDG and MGDG were never detected outside plastids 34,35 . The location of glycolipids in the membrane of chloroplasts and thylakoids has been related with signalling and coordination functions, regulating chloroplast lipids and cytosolic partners 16,20 . Since the glycolipid profile was preserved in kleptoplasts, these functions are likely retained during the process of chloroplast sequestration in sea slug cells.
MGTS and DGTS belong to a less studied class of lipids, whose metabolic pathways and functions are still not well characterized in marine algae 36 . DGTS has been substituted by PC during the evolution of vascular plants 37 , thus their distribution is limited to algae, lower plants and fungi 22 . A study in freshwater algae has suggested that DGTS acts as a donor of diacylglycerol (DAG), as well as polyunsaturated fatty acids, which will be used in the biosynthesis of galactolipids 38 . In accordance, rhythmic fluctuation of DGTS with time was inversely correlated with the levels of MGDG 39   lipidomic profile of DGTS between the macroalgae and the sea slug point towards the occurrence of a minor remodelling during the process of endosymbiosis. Interestingly, some SQDG and DGTS molecular species identified in C. tomentosum, but not in E. viridis samples, display a similar fatty acyl composition. The absence of these molecular species in E. viridis may be related to chloroplast membrane adjustments during the integration in their new host cells 11 , or, as it was suggested above, to an extremely low concentration of these molecules in the sea slug.
The lipidomic approach used herein has opened a new perspective in the study of kleptoplasty. The minor differences recorded in the lipidome of plastid membranes in C. tomentosum and E. viridis indicate that the integrity of chloroplast membranes is conserved during the process of endosymbiosis.

Methods
Sampling. Samples of C. tomentosum and specimens of E. viridis were collected in September 2015 on the intertidal rocky shore of Praia de Labruge (41°16′28.9″N; 8°43′45.3″W), Vila do Conde (Portugal). Macroalga and sea slug samples were rinsed with freshwater purified through reverse osmosis, frozen, freeze-dried and stored individually at −20 °C for biochemical analysis. A total of five samples of C. tomentosum (n = 5) and E. viridis (n = 5) were analysed individually. Additionally, four specimens of E. viridis (n = 4) were placed under starvation during one week (a time frame that allows sea slugs to completely empty their guts) and subsequently analysed individually. The analysis of starved sea slugs was performed to validate the results presented in this study, as any algal lipid species detected in these specimens could only originate from kleptoplasts.  Fractionation was performed by solid-phase extraction using a Supelclean ™ LC-Si SPE Tube (bed wt. 500 mg, volume 3 mL cartridges, SUPELCO). Column was activated with 6 mL of n-hexane and then the sample of lipid extract (110 and 4 mg of macroalgae and sea slug, respectively) was applied after to be dissolved on 300 μL of chloroform. Subsequently, the following sequential elution was performed to separate lipid fractions: 5 mL (C. tomentosum)/4 mL (E. viridis) of chloroform, 8 mL (C. tomentosum)/3 mL (E. viridis) of diethyl ether:acetic acid (98:2, v/v), 5 mL of acetone:methanol (9:1, v/v) and 6 mL of methanol. Total lipid extracts were fractionated in four lipid fractions: fraction 1 (rich in neutral lipids), fraction 2 (rich in pigments), fraction 3 (rich in glycolipids) and fraction 4 (rich in betaine lipids and phospholipids). Fractions 3 and 4 were recovered separated and dried under nitrogen stream and stored at −20 °C prior to analysis by HILIC-LC-MS.

Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC-MS). A high
performance LC (HPLC) system (Thermo scientific Accela TM ) with an autosampler coupled online to the Q-Exactive ® mass spectrometer with Orbitrap ® technology was used. The solvent system consisted of two mobile phases as follows: mobile phase A (acetonitrile:methanol:water 50:25:25, v/v/v with 1 mM ammonium acetate) and mobile phase B (acetonitrile:methanol 60:40, v/v, with 1 mM ammonium acetate). Initially, 0% of mobile phase A was held isocratically for 8 min, followed by a linear increase to 60% of A within 7 min and a maintenance period of 15 min, returning to the initial conditions in 10 min. A volume of 5 µL of each sample containing 5 µg of lipid extract and 95 µL of eluent B was introduced into the Ascentis ® Si column (15 cm × 1 mm, 3 µm, Sigma-Aldrich) with a flow rate of 40 µL min −1 and at 30 °C. The mass spectrometer with Orbitrap ® technology was operated in simultaneous positive (electrospray voltage 3.0 kV) and negative (electrospray voltage −2.7 kV) modes with high resolution with 70.000 and automatic gain control (AGC) target of 1e6, the capillary temperature was 250 °C and the sheath gas flow was 15 U. In MS/MS experiments, a resolution of 17.500 and AGC target of 1e5 was used and the cycles consisted in one full scan mass spectrum and ten data-dependent MS/MS scans were repeated continuously throughout the experiments with the dynamic exclusion of 60 seconds and intensity threshold of 1e4. Normalized collision energy ™ (CE) ranged between 25, 30 and 35 eV. Data acquisition was carried out using the Xcalibur data system (V3.3, Thermo Fisher Scientific, USA). All the analyses were performed in analytical triplicates. The identification of molecular species of polar lipids was based on the assignment of the molecular ions observed in LC-MS/MS spectra. A mass accuracy (Qual Browser) of ≤5 ppm was used in order to uniquely identify the molecular species.