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Home , Elysia chlorotica, Elysia timida, Elysia viridis, Kleptoplasty, Plakobranchoidea, Sacoglossa

© 2021 The Japan Mendel Society Cytologia 86(2): 103–107

Cytologia Focus:

A Photosynthetic : A Sacoglossan Sea that Steals

Ryota Aoki1 and Sachihiro Matsunaga2*

1 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278–8510, Japan 2 Laboratory of Integrated Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, 5–1–5 Kashiwanoha, Kashiwa, Chiba 277–8561, Japan

Received April 8, 2021; accepted April 15, 2021

Summary Sacoglossan sea are able to steal chloroplasts from their algal prey and acquire photosynthetic capacity (termed ). These ‘stolen’ provide sea slugs with a long-term supply of organic car- bon and energy. This augmented nutrient supply brings many benefits in terms of survival, body planning, repro- ductive traits, and body regeneration. However, the mechanisms of maintenance of chloroplasts and photosynthe- sis in sea slugs are poorly understood. Here, we introduce this mysterious phenomenon, including recent research findings, and consider its feasibility for synthetic biology, e.g., construction of artificial photosynthetic animal cells.

Keywords Kleptoplasty, Sacoglossan , , Alga, Synthetic biology.

Research on has rapidly diversified into various photosynthesise using these ‘stolen’ plastids and assimi- fields, including biology (Mine et al. 2018, Takano late photosynthate for periods ranging from a few days et al. 2018, Miyamura et al. 2019, Kuroiwa et al. 2020, to several months. A particular puzzle is that the sea Yoshida et al. 2020), molecular biology (Uchida et al. slugs are able to maintain photosynthetic activity despite 2018, Ota et al. 2019, Miyagishima and Fujiwara 2020), the algal nuclei having been digested. Because some and biotechnology (Hayashi et al. 2018). Recent prod- photosynthetic are encoded in the algal nuclear ucts derived from algae include bioethanol and so-called , photosynthesis should be impaired. Here, we ‘functional ’ (Hosokawa and Kawano 2020). Het- introduce this mysterious phenomenon about sea slags. erotrophic and some have symbiotic re- Kleptoplasty was first noted in viridis (De lationships with unicellular alga or cyanobacteria (Venn Negri and De Negri 1876) and has subsequently been et al. 2008). Examples of such relationships are found observed in the closely related , E. atroviridis in several phyla, e.g., (giant clams and nudi- (Kawaguchi et al. 1965) and E. chlorotica (Trench et al. branchs), Porifera (sponges), Cnidaria (corals, anemones, 1969). There have been several physiological studies and hydra), Acoelomorpha (flatworms) (Van Steenkiste of kleptoplasty (Hinde and Smith 1972, Jensen 1986, et al. 2019), and Chordata (ascidians). In these cases, Clark et al. 1990). can be grown in a the host obtains oxygen and organic carbon via their laboratory and the kleptoplasts are retained for a long intercellular symbiont’s photosynthesis. Although a large period (10–12 months). It feeds on particular species of number of studies have been made on symbioses be- algae, including vaucheria, V. litorea, and tween animals or heterotrophic protists and algae, little V. compacta, which belong to a coenocytic is known about their underlying molecular mechanisms. genus (West 1979). The sea slug is unable to complete A more mysterious and somewhat controversial sym- metamorphosis and development to the adult without biotic phenomenon is observed in several sacoglossan uptake from the algal prey (Rumpho et al. 2011). molluscs (sea slugs) within the , par- Vaucheria species are coenocytic filamentous algae, i.e., ticularly in the genus Elysia (Rumpho et al. 2000, 2011). they consist of a single multinucleate cell (Pelletreau These slugs have evolved mechanisms for the capture et al. 2011). Sea slugs use their radular teeth to break the of algae prey and selective retention of functional chlo- cell wall and suck out the cell contents, including plas- roplasts (called kleptoplasty). They are, thereby, able to tids. The plastids are then incorporated into the tubular cells of their digestive diverticula, where they carry out * Corresponding author, e-mail: [email protected] photosynthesis (Rumpho et al. 2011) (Fig. 1). DOI: 10.1508/cytologia.86.103 Two main hypotheses are proposed to explain this 104 R. Aoki and S. Matsunaga Cytologia 86(2)

Fig. 1. Origin of kleptoplasty in the sea slug: Elysia chlorotica. Hatched mature sea-slug larvae change their prey from to algae and V. compacta. The sea slug sucks out the algal cell contents using its radular teeth; the algal nuclei are digested, but the chloroplasts are incorporated into tubular cells of the diverticula, without being digested. There is no clear evidence for . Sea slugs are unable to complete metamorphosis to develop into a juvenile and an adult in the absence of their algal prey and uptake. Photosynthesis from kleptoplasts could contribute to reproductive traits, body plan, and regeneration of the host. phenomenon. First, horizontal gene transfer (HGT) ac- spatio-temporal analysis has demonstrated uptake of companies kleptoplasty. Alternatively, the mechanism kleptoplasty-derived carbon and nitrogen into sea slugs is intrinsic to the chloroplasts themselves. In the first (Cruz et al. 2020). Using radioisotopes, it was shown hypothesis, after the digestion of the algal nuclei, the al- that the uptake of carbon and nitrogen reached the repro- gal genome should be directly incorporated into the host ductive organs. Light was required not only for energy without being decomposed. However, no supply but also affected body size and the number of nuclear-encoded algal-derived HGT to the germline was offspring in E. atroviridis (Shiroyama et al. 2020). That observed in E. chlorotica (Wägele et al. 2011, Pierce study showed that light intensity and availability et al. 2012, Bhattacharya et al. 2013). Similarly, there were correlated with shell height and the total number was no evidence of HGT between E. timida and Plako- of . These data suggest that kleptoplasty serves branchus ocellatus. In several transcriptomic analyses as a photosynthetic device that supplies nutrients that of sea slugs, algal nuclear-encoded mRNA was not de- strengthen the individual, and also supports specific life- tected (Wägele 2011, Pierce et al. 2012). These studies styles, body-plans and reproductive traits. tend to refute the first hypothesis. The second hypothesis An advantage of kleptoplasty is that, in the presence implies that the chloroplasts of Vaucheria species are of light, sea slugs are able to survive without algal food less dependent on the algal genome than in other algae for at least 10 months (Rumpho et al. 2006). Sea slugs and embryophytes (Rumpho et al. 2006); therefore, the starved in the dark, lost weight much more rapidly than kleptoplasts may be genetically autonomous. Approxi- those starved in the light (Hinde and Smith 1975). Ad- mately 60% of chloroplasts isolated from V. litorea con- ditionally, survival and growth rate were greater in the tinued to evolve oxygen for 2 days, whereas fewer than light than in the dark for P. ocellatus (functional chlo- 30% of chloroplasts isolated from spinach did so after roplasts were retained for >17 days) (Yamamoto et al. 1 day (Green et al. 2005). In addition, RuBisCo 2013, Akimoto et al. 2014). Conversely, in a similar continued to be synthesised in kleptoplasts, 3 days after experiment using E. trisinuata, there were no significant extraction, as in the wild chloroplasts. Moreover, it is differences in different light levels (functional chloro- known that V. litorea chloroplasts are resistant to osmot- plasts were retained <4 days). These experiments dem- ic stress (Gallop et al. 1980, Green et al. 2005). These onstrated that dependence on photosynthesis is related observations imply that kleptoplasty relies on specific to the duration of retention of chloroplasts. Nevertheless, properties of the chloroplasts of Vaucheria species. some sea slugs with short-term functional kleptoplasty The greatest benefit of the phenomenon of kleptoplas- did obtain supplementary nutrition and energy via pho- ty is probably not related to the conferment of crypsis tosynthesis. Conversely, it has also been reported that but to the supply of nutrients. Photosynthetic carbon is photosynthesis may not be important for the survival supplied to sea slugs by the kleptoplasts (Trench et al. of sea slugs (Christa et al. 2014). In that report, there 1974, Kopp et al. 2015, LeKieffre et al. 2018). Recent were no significant differences in weight loss among 2021 A Sacoglossan Sea Slug that Steals Chloroplasts 105 animals that survived several months of starvation in search and development, is the use of long-coding DNA, complete darkness, or in the light in the presence of the involving synthesis on the genome-scale, construction photosynthesis inhibitor monolinuron, or in animals of artificial , and their insertion into artificial with no treatment. These results support the hypothesis cells. A few laboratories are attempting to create arti- that besides being a source of solar power, kleptoplasts ficial photosynthetic animal cells based on the concept can serve as a food reserve. Indeed, kleptoplasts are a of (Matsunaga 2018, Puri et al. 2021). We source of lipids, and it is known that starved sea slugs may be able to create photosynthetic animal cells that, decompose them as a nutrient source in the dark (Pelle- like sea slugs, are capable of photosynthesis for several treau et al. 2014, Cartaxana et al. 2017, Rey et al. 2017). months using the following strategies: firstly, transfer Thus, there are two aspects of nutrient supply by klepto- of genes related to algal photosynthesis into the nucleus plasts: as a direct source and via photosynthesis. of cultured cells by HGT; secondly, use of microinjec- Reliance on photosynthesis also bring disadvantages. tion technology to insert chloroplasts into cultured cells, There is an optimal light intensity for sea slugs; too ‘imposed’ chloroplasts. Conveniently, cultured cells can strong or too weak light both adversely affect the growth be genetically preserved and amplified into the next of sea slugs (Donohoo et al. 2020). Reactive oxygen spe- generation by cell division. The feasibility of construct- cies (ROS) formed by the addition of electrons to oxygen ing artificial photosynthetic animal cells is increased by through photosynthesis can seriously damage animal using sequencing tools with high speed, low cost, and membranes and intracellular proteins, which, ultimately high throughput, and genome modification tools such as may lead to cell death. However, in contrast to the chlo- CRISPR-Cas9 systems. Artificial photosynthetic animal roplasts of green algae, the kleptoplasts of E. timida pos- cells are expected to contribute to medical fields with sess a mechanism that preferentially accepts electrons; applications in cancer treatment and photosynthetic ther- plastoquinone maintains an oxidised state that inhibits apy using oxygen (Wang et al. 2019, Chávez the formation of harmful ROS (Christa et al. 2018, Car- et al. 2020). taxana et al. 2019, Havurinne and Tyystjärvi 2020). In Although kleptoplasty still leaves many open ques- this way, several sea slugs have apparently adapted and tions in relation to HGT, retention of kleptoplasts, and optimised their photosynthetic activity which seems like its relationship to reproduction and regeneration, it is a a very short-term evolution. fascinating phenomenon in phylogenetics and evolution. Are sea slugs genetically adapted to kleptoplasty? Further elucidation could lead to a new understanding of RNA-seq analysis was performed to identify genetic the symbiotic phenomenon and create new possibilities interaction between host and kleptoplast during E. chlo- from the viewpoint of synthetic biology. rotica development (Chan et al. 2018). That research showed that during incorporation of V. litorea plastids, Acknowledgements genes involved in microbe-associated molecular pat- terns and oxidative stress-response mechanisms were This research was supported by grants from MXT/ significantly up-regulated. This implies that functional JSPS KAKENHI (19H03259 and 20H03297) and JST, kleptoplasty belongs to a category of animal–algal sym- CREST Grant Number JPMJCR20S6 to SM. We thank biotic interactions resembling that between corals and Harry Taylor, PhD, from Edanz Group (https://en- dinoflagellates. author-services.edanz.com/ac) for editing a draft of this Recently, it was reported that a sea slug’s detached manuscript. head was able to survive and regenerate a whole new body (Mitoh and Yusa 2021). Although sacoglossan sea References slugs are able to autotomize, known as regeneration of only a part of the body, such as the tail (Fleming et al. Akimoto, A., Hirano, Y. M., Sakai, A. and Yusa, Y. 2014. Relative 2007), surprisingly, this study found that two species of importance and interactive effects of photosynthesis and food in two solar-powered sea slugs. Mar. Biol. 161: 1095–1102. sacoglossan sea slug, E. marginata and E. atroviridis Bhattacharya, D., Pelletreau, K. N., Price, D. 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