Photoprotective mechanisms in Elysia species hosting Acetabularia chloroplasts shed light on host-donor compatibility in photosynthetic sea slugs.

Sacoglossa sea slugs have garnered attention due to their ability to retain intracellular functional chloroplasts from algae, while degrading other algal cell components. While protective mechanisms that limit oxidative damage under excessive light are well documented in plants and algae, the photoprotective strategies employed by these photosynthetic sea slugs remain unresolved. Species within the genus Elysia are known to retain chloroplasts from various algal sources, but the extent to which the metabolic processes from the donor algae can be sustained by the sea slugs is unclear. By comparing responses to high-light conditions through kinetic analyses, molecular techniques, and biochemical assays, this study shows significant differences between two photosynthetic Elysia species with chloroplasts derived from the green alga Acetabularia acetabulum. Notably, Elysia timida displayed remarkable tolerance to high-light stress and sophisticated photoprotective mechanisms such as an active xanthophyll cycle, efficient D1 protein recycling, accumulation of heat-shock proteins and α-tocopherol. In contrast, Elysia crispata exhibited absence or limitations in these photoprotective strategies. Our findings emphasize the intricate relationship between the host animal and the stolen chloroplasts, highlighting different capacities to protect the photosynthetic organelle from oxidative damage.

Proteomic analysis of the mucus of the photosynthetic sea slug Elysia crispata.

Elysia crispata is a tropical sea slug that can retain intracellular functional chloroplasts from its algae prey, a mechanism termed kleptoplasty. This sea slug, like other gastropods, secretes mucus, a viscous secretion with multiple functions, including lubrication, protection, and locomotion. This study presents the first comprehensive analysis of the mucus proteome of the sea slug E. crispata using gel electrophoresis and HPLC-MS/MS. We identified 306 proteins in the mucus secretions of this animal, despite the limited entries for E. crispata in the Uniprot database. The functional annotation of the mucus proteome using Gene Ontology identified proteins involved in different functions such as hydrolase activity (molecular function), carbohydrate-derived metabolic processes (biological processes) and cytoskeletal organization (cell component). Moreover, a high proportion of proteins with enzymatic activity in the mucus of E. crispata suggests potential biotechnological applications including antimicrobial and antitumor activities. Putative antimicrobial properties are reinforced by the high abundance of hydrolases. This study also identified proteins common in mucus samples from various species, supporting a common mechanism of mucus in protecting cells and tissues while facilitating animal movement. SIGNIFICANCE: Marine species are increasingly drawing the interest of researchers for their role in discovering new bioactive compounds. The study "Proteomic Analysis of the Mucus of the Photosynthetic Sea Slug Elysia crispata" is a pioneering effort that uncovers the complex protein content in this fascinating sea slug's mucus. This detailed proteomic study has revealed proteins with potential use in biotechnology, particularly for antimicrobial and antitumor purposes. This research is a first step in exploring the possibilities within the mucus of Elysia crispata, suggesting the potential for new drug discoveries. These findings could be crucial in developing treatments for severe diseases, especially those caused by multidrug-resistant bacteria, and may lead to significant advances in medical research.

Prey species and abundance affect growth and photosynthetic performance of the polyphagous sea slug Elysia crispata.

Some sacoglossan sea slugs steal functional macroalgal chloroplasts (kleptoplasts). In this study, we investigated the effects of algal prey species and abundance on the growth and photosynthetic capacity of the tropical polyphagous sea slug Elysia crispata. Recently hatched sea slugs fed and acquired chloroplasts from the macroalga Bryopsis plumosa, but not from Acetabularia acetabulum. However, adult sea slugs were able to switch diet to A. acetabulum, rapidly replacing the great majority of the original kleptoplasts. When fed with B. plumosa, higher feeding frequency resulted in significantly higher growth and kleptoplast photosynthetic yield, as well as a slower relative decrease in these parameters upon starvation. Longevity of A. acetabulum-derived chloroplasts in E. crispata was over twofold that of B. plumosa. Furthermore, significantly lower relative weight loss under starvation was observed in sea slugs previously fed on A. acetabulum than on B. plumosa. This study shows that functionality and longevity of kleptoplasts in photosynthetic sea slugs depend on the origin of the plastids. Furthermore, we have identified A. acetabulum as a donor of photosynthetically efficient chloroplasts common to highly specialized monophagous and polyphagous sea slugs capable of long-term retention, which opens new experimental routes to unravel the unsolved mysteries of kleptoplasty.

Revealing the polar lipidome, pigment profiles, and antioxidant activity of the giant unicellular green alga, Acetabularia acetabulum.

Marine algae are one of the most important sources of high-value compounds such as polar lipids, omega-3 fatty acids, photosynthetic pigments, or secondary metabolites with interesting features for different niche markets. Acetabularia acetabulum is a macroscopic green single-celled alga, with a single nucleus hosted in the rhizoid. This alga is one of the most studied dasycladalean species and represents an important model system in cell biology studies. However, its lipidome and pigment profile have been overlooked. Total lipid extracts were analyzed using hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS), tandem mass spectrometry (MS/MS), and high-performance liquid chromatography (HPLC). The antioxidant capacity of lipid extracts was tested using DPPH and ABTS assays. Lipidomics identified 16 polar lipid classes, corresponding to glycolipids, betaine lipids, phospholipids, and sphingolipids, with a total of 191 lipid species, some of them recognized by their bioactivities. The most abundant polar lipids were glycolipids. Lipid classes less studied in algae were identified, such as diacylglyceryl-carboxyhydroxymethylcholine (DGCC) or hexosylceramide (HexCer). The pigment profile of A. acetabulum comprised carotenoids (17.19%), namely cis-neoxanthin, violaxanthin, lutein and ß,ß-carotene, and chlorophylls a and b (82.81%). A. acetabulum lipid extracts showed high antioxidant activity promoting a 50% inhibition (IC50 ) with concentrations of 57.91 ± 1.20 µg · mL-1 (438.18 ± 8.95 µmol Trolox · g-1 lipid) in DPPH and 20.55 ± 0.60 µg · mL-1 in ABTS assays (918.56 ± 27.55 µmol Trolox · g-1 lipid). This study demonstrates the potential of A. acetabulum as a source of natural bioactive molecules and antioxidant compounds.

Photosynthetic Pigment and Carbohydrate Profiling of Fucus vesiculosus from an Iberian Coastal Lagoon.

Fucus vesiculosus is a brown seaweed with applications in the food, pharmaceutic, and cosmetic industries. Among its most valuable bioactive compounds are the pigment fucoxanthin and polysaccharides (e.g., fucoidans). In this study, we profiled the photosynthetic pigments and carbohydrates of F. vesiculosus from six locations along the Ílhavo Channel in the Iberian coastal lagoon of Ria de Aveiro, Portugal. Photosynthetic performance (Fv/Fm), pigment, and carbohydrate concentrations were similar between locations, despite differences in environmental factors, such as salinity and periods of exposure to desiccation. Concentration of total carbohydrates (neutral sugars + uronic acids) averaged 418 mg g-1 dw. Fucose was the second most abundant neutral sugar, with an average concentration of 60.7 mg g-1 dw, indicating a high content of fucoidans. Photosynthetic pigments included chlorophylls a and c, ß,ß-carotene, and the xanthophylls fucoxanthin, violaxanthin, antheraxanthin, and zeaxanthin. Concentrations of fucoxanthin were higher than those reported for most brown macroalgae, averaging 0.58 mg g-1 dw (65% of total carotenoids). This study indicates that F. vesiculosus from Ria de Aveiro is a valuable macroalgal resource for aquaculture companies operating in the region, with considerable potential to yield high-value bioactive compounds.

Assessing the Trophic Impact of Bleaching: The Model Pair Berghia stephanieae/Exaiptasia diaphana.

Bleaching events associated with climate change are increasing worldwide, being a major threat to tropical coral reefs. Nonetheless, the indirect impacts promoted by the bleaching of organisms hosting photosynthetic endosymbionts, such as those impacting trophic interactions, have received considerably less attention by the scientific community. Bleaching significantly affects the nutritional quality of bleached organisms. The consequences promoted by such shifts remain largely overlooked, namely on specialized predators that have evolved to prey upon organisms hosting photosynthetic endosymbionts and benefit nutritionally, either directly or indirectly, from the available pool of photosynthates. In the present study, we advocate the use of the model predator-prey pair featuring the stenophagous nudibranch sea slug Berghia stephanieae that preys upon the photosymbiotic glass anemone Exaiptasia diaphana to study the impacts of bleaching on trophic interactions. These model organisms are already used in other research fields, and one may benefit from knowledge available on their physiology, omics, and culture protocols under controlled laboratory conditions. Moreover, B. stephanieae can thrive on either photosymbiotic or aposymbiotic (bleached) glass anemones, which can be easily maintained over long periods in the laboratory (unlike photosymbiotic corals). As such, one can investigate if and how nutritional shifts induced by bleaching impact highly specialized predators (stenophagous species), as well as if and how such effects cascade over consecutive generations. Overall, by using this model predator-prey pair one can start to truly unravel the trophic effects of bleaching events impacting coral reef communities, as well as their prevalence over time.

Light modulates the lipidome of the photosynthetic sea slug Elysia timida.

Long-term kleptoplasty, the capability to retain functional stolen chloroplasts (kleptoplasts) for several weeks to months, has been shown in a handful of Sacoglossa sea slugs. One of these sea slugs is Elysia timida, endemic to the Mediterranean, which retains functional chloroplasts of the macroalga Acetabularia acetabulum. To understand how light modulates the lipidome of E. timida, sea slug specimens were subjected to two different 4-week light treatments: regular light and quasi-dark conditions. Lipidomic analyses were performed by HILIC-HR-ESI-MS and MS/MS. Quasi-dark conditions caused a reduction in the amount of essential lipids for photosynthetic membranes, such as glycolipids, indicating high level of kleptoplast degradation under sub-optimal light conditions. However, maximum photosynthetic capacities (Fv/Fm) were identical in both light treatments (≈0.75), showing similar kleptoplast functionality and suggesting that older kleptoplasts were targeted for degradation. Although more stable, the phospholipidome showed differences between light treatments: the amount of certain lipid species of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylglycerol (PG) decreased under quasi-dark conditions, while other lipid species of phosphatidylcholine (PC), PE and lyso-PE (LPE) increased. Quasi-dark conditions promoted a decrease in the relative abundance of polyunsaturated fatty acids. These results suggest a light-driven remodelling of the lipidome according to the functions of the different lipids and highlight the plasticity of polar lipids in the photosynthetic sea slug E. timida.

Food shaped photosynthesis: Photophysiology of the sea slug Elysia viridis fed with two alternative chloroplast donors.

Background: Some Sacoglossa sea slugs steal and integrate chloroplasts derived from the algae they feed on into their cells where they continue to function photosynthetically, a process termed kleptoplasty. The stolen chloroplasts - kleptoplasts - can maintain their functionality up to several months and support animal metabolism. However, chloroplast longevity can vary depending on sea slug species and algal donor. In this study, we focused on Elysia viridis, a polyphagous species that is mostly found associated with the macroalga Codium tomentosum, but that was reported to eat other macroalgae, including Chaetomorpha sp. Methods: We have investigated the changes in E. viridis physiology when provided with the two different food sources to evaluate to which extent the photosynthetic and photoprotective mechanisms of the algae chloroplasts matched those of the plastids once in the animal cells. To perform the study, we rely on the evaluation of chlorophyll a variable fluorescence to study the photophysiological state of the integrated kleptoplasts and high-performance liquid chromatography (HPLC) to study variations in the photosynthetic pigments. Results: We observed that the photosynthetic efficiency of E. viridis is lower when fed with Chaetomorpha. Also, significant differences were observed in the non-photochemical quenching (NPQ) abilities of the sea slugs. While sea slugs fed with C. tomentosum react similarly to high-light stress as the alga, E. viridis hosting Chaetomorpha chloroplasts were unable to properly recover from photoinhibition or perform a functional xanthophyll cycle (XC). Conclusions: Our results showed that, even if the sea slugs fed with the two algae show photosynthetic activities like the respective algal donors, not all the photoprotective mechanisms present in Chaetomorpha can be maintained in E. viridis. This indicates that the functionality of the kleptoplasts does not depend solely on their origin but also on the degree of compatibility with the animal species integrating them.

Sea slugs known as Sacoglossa (also called "solar-powered sea slugs") have a fascinating ability to steal and use chloroplasts from the algae they eat. This process is called kleptoplasty. These stolen chloroplasts, also called kleptoplasts, can remain functional for several months and help the sea slugs with their metabolism by performing photosynthesis like plants. However, the time of chloroplast maintenance can vary depending on the species of sea slug and the type of algae they feed on. In this study, we focused on a species called Elysia viridis, which eats various types of algae, including Codium tomentosum and Chaetomorpha sp. These two algae have different characteristics when it comes to photosynthesis and protection against excessive light. We investigated how the physiology of E. viridis changed when it was given these two different food sources. Our results show that sea slugs had similar photosynthetic activities to their respective food alga. However, not all photoprotective mechanisms of Chaetomorpha algae could be maintained in E. viridis. This suggests that the functionality of the stolen chloroplasts depends not only on their source but also on how well they match the sea slug species that incorporate them.

Kleptoplasty: Getting away with stolen chloroplasts.

Kleptoplasty, the process by which a host organism sequesters and retains algal chloroplasts, is relatively common in protists. The origin of the plastid varies, as do the length of time it is retained in the host and the functionality of the association. In metazoa, the capacity for long-term (several weeks to months) maintenance of photosynthetically active chloroplasts is a unique characteristic of a handful of sacoglossan sea slugs. This capability has earned these slugs the epithets "crawling leaves" and "solar-powered sea slugs." This Unsolved Mystery explores the basis of chloroplast maintenance and function and attempts to clarify contradictory results in the published literature. We address some of the mysteries of this remarkable association. Why are functional chloroplasts retained? And how is the function of stolen chloroplasts maintained without the support of the algal nucleus?

Aposymbiotic Specimen of the Photosynthetic Sea Slug Elysia crispata.

Elysia crispata is a sacoglossan sea slug that retains intracellular, functional chloroplasts stolen from their macroalgal food sources. Elysia crispata juveniles start feeding on the algae following metamorphosis, engulfing chloroplasts and turning green. In laboratory-reared animals, we report one juvenile "albino" specimen unable to retain chloroplasts. Within 6 weeks post-metamorphosis, the aposymbiotic sea slug was significantly smaller than its chloroplast-bearing siblings. This evidence highlights that chloroplast acquisition is required for the normal development of E. crispata.

Sea Slug Mucus Production Is Supported by Photosynthesis of Stolen Chloroplasts.

A handful of sea slugs of the order Sacoglossa are able to steal chloroplasts-kleptoplasts-from their algal food sources and maintain them functionally for periods ranging from several weeks to a few months. In this study, we investigated the role of kleptoplast photosynthesis on mucus production by the tropical sea slug Elysia crispata. Animals reared for 5 weeks in quasi dark (5 µmol photons m-2 s-1) showed similar growth to those under regular light (60-90 µmol photons m-2 s-1), showing that kleptoplast photosynthesis was not relevant for growth when sea slugs were fed ad libitum. However, when subjected to short-term desiccation stress, animals reared under regular light produced significantly more mucus. Furthermore, the carbohydrate content of secreted mucus was significantly lower in slugs limited in the photosynthetic activity of their kleptoplasts by quasi-dark conditions. This study indicates that photosynthesis supports the synthesis of protective mucus in kleptoplast-bearing sea slugs.

Pigment and Fatty Acid Heterogeneity in the Sea Slug Elysia crispata Is Not Shaped by Habitat Depth.

Long-term retention of functional chloroplasts in animal cells occurs only in sacoglossan sea slugs. Analysis of molecules related to the maintenance of these organelles can provide valuable information on this trait (kleptoplasty). The goal of our research was to characterize the pigment and fatty acid (FA) composition of the sea slug Elysia crispata and their associated chloroplasts that are kept functional for a long time, and to quantify total lipid, glycolipid and phospholipid contents, identifying differences between habitats: shallow (0-4 m) and deeper (8-12 m) waters. Specimens were sampled and analyzed after a month of food deprivation, through HPLC, GC-MS and colorimetric methods, to ensure an assessment of long-term kleptoplasty in relation to depth. Pigment signatures indicate that individuals retain chloroplasts from different macroalgal sources. FA classes, phospholipid and glycolipid contents displayed dissimilarities between depths. However, heterogeneities in pigment and FA profiles, as well as total lipid, glycolipid and phospholipid amounts in E. crispata were not related to habitat depth. The high content of chloroplast origin molecules, such as Chl a and glycolipids after a month of starvation, confirms that E. crispata retains chloroplasts in good biochemical condition. This characterization fills a knowledge gap of an animal model commonly employed to study kleptoplasty.

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.

Prevalence and Photobiology of Photosynthetic Dinoflagellate Endosymbionts in the Nudibranch Berghia stephanieae.

Berghia stephanieae is a stenophagous sea slug that preys upon glass anemones, such as Exaiptasia diaphana. Glass anemones host photosynthetic dinoflagellate endosymbionts that sea slugs ingest when consuming E. diaphana. However, the prevalence of these photosynthetic dinoflagellate endosymbionts in sea slugs appears to be short-lived, particularly if B.stephanieae is deprived of prey that host these microalgae (e.g., during bleaching events impacting glass anemones). In the present study, we investigated this scenario, along with food deprivation, and validated the use of a non-invasive and non-destructive approach employing chlorophyll fluorescence as a proxy to monitor the persistence of the association between sea slugs and endosymbiotic photosynthetic dinoflagellates acquired through the consumption of glass anemones. Berghia stephanieae deprived of a trophic source hosting photosynthetic dinoflagellate endosymbionts (e.g., through food deprivation or by feeding on bleached E. diaphana) showed a rapid decrease in minimum fluorescence (Fo) and photosynthetic efficiency (Fv/Fm) when compared to sea slugs fed with symbiotic anemones. A complete loss of endosymbionts was observed within 8 days, confirming that no true symbiotic association was established. The present work opens a new window of opportunity to rapidly monitor in vivo and over time the prevalence of associations between sea slugs and photosynthetic dinoflagellate endosymbionts, particularly during bleaching events that prevent sea slugs from incorporating new microalgae through trophic interactions.

Recovering wasted nutrients from shrimp farming through the combined culture of polychaetes and halophytes.

The bioremediation and biomass production of organic extractive organisms (polychaetes Arenicola marina, Hediste diversicolor and halophyte Salicornia ramosissima) was assessed in an integrated multi-trophic aquaculture (IMTA) framework. Culture trials were performed outdoors using the nutient rich effluent from a shrimp farm employing recirculated aquaculture systems. Similar bioremediation efficiencies were obtained in cultures using a single polyculture tank (1 T) or two trophic levels separated tanks (2 T; ≈ 0.3 and 0.6 m2 operational area, respectively), with a reduction of 74-87% for particulate organic matter (POM), 56-64% for dissolved inorganic nitrogen (DIN) and 60-65% for dissolved inorganic phosphorus (DIP). Hediste diversicolor adapted well to culture conditions, reaching densities up to 5.000 ind. m-2 (≈ 78-98 g m-2). Arenicola marina failed to cope with water temperature that exceeded the species thermal limits, displaying a survival < 10% (20 °C often pointed as the maximum thermal threshold for this species). Productivity of S. ramosissima with 1 T was about twice that obtained with 2 T (≈ 150-170 and ≈ 60-90 g FW m-2 edible aboveground biomass, respectively). The yellowish coloration of cultured plants was likely due to the chemical oxidation and rapid sand filtration pre-treatment applied to the brackish groundwater used in the aquaculture facility, that removed iron (and probably other essential elements). Overall, 1 T design combining H. diversicolor and S. ramosissima displayed the best bioremediation performance and biomass production, while also allowing reducing in half the operational area required to implement this IMTA framework.

On the art of stealing chloroplasts.

Sea slugs increase the longevity of the chloroplasts they steal from algae by limiting the harmful side-effects of photosynthesis.

Photoprotective Role of Neoxanthin in Plants and Algae.

Light is a paramount parameter driving photosynthesis. However, excessive irradiance leads to the formation of reactive oxygen species that cause cell damage and hamper the growth of photosynthetic organisms. Xanthophylls are key pigments involved in the photoprotective response of plants and algae to excessive light. Of particular relevance is the operation of xanthophyll cycles (XC) leading to the formation of de-epoxidized molecules with energy dissipating capacities. Neoxanthin, found in plants and algae in two different isomeric forms, is involved in the light stress response at different levels. This xanthophyll is not directly involved in XCs and the molecular mechanisms behind its photoprotective activity are yet to be fully resolved. This review comprehensively addresses the photoprotective role of 9'-cis-neoxanthin, the most abundant neoxanthin isomer, and one of the major xanthophyll components in plants' photosystems. The light-dependent accumulation of all-trans-neoxanthin in photosynthetic cells was identified exclusively in algae of the order Bryopsidales (Chlorophyta), that lack a functional XC. A putative photoprotective model involving all-trans-neoxanthin is discussed.

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.

Coping with Starvation: Contrasting Lipidomic Dynamics in the Cells of Two Sacoglossan Sea Slugs Incorporating Stolen Plastids from the Same Macroalga.

Several species of sacoglossan sea slugs are able to sequester chloroplasts from algae and incorporate them into their cells. However, the ability to maintain functional "stolen" plastids (kleptoplasts) can vary significantly within the Sacoglossa, giving species different capacities to withstand periods of food shortage. The present study provides an insight on the comparative shifts experienced by the lipidome of two sacoglossan sea slug species, Elysia viridis (long-term retention of functional chloroplasts) and Placida dendritica (retention of non-functional chloroplasts). A hydrophilic interaction liquid chromatography-mass spectrometry approach was employed to screen the lipidome of specimens from both species feeding on the macroalga Codium tomentosum and after 1-week of starvation. The lipidome of E. viridis was generally unaffected by the absence of food, while that of P. dendritica varied significantly. The retention of functional chloroplasts by E. viridis cells allows this species to endure periods of food shortage, while in P. dendritica a significant reduction in the amount of main lipids was the consequence of the consumption of its own mass to endure starvation. The large proportion of ether phospholipids (plasmalogens) in both sea slug species suggests that these compounds may play a key role in chloroplast incorporation in sea slug cells and/or be involved in the reduction of the oxidative stress resulting from the presence of kleptoplasts.