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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.

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 13 C and 15 N 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.

A new sacoglossan species of the genus Elysia Risso, 1818 from southern Italy is described based on its external morphology, color pattern, radular teeth, and reproductive system. Specimens of this new species were previously attributed to Elysia viridis (Montagu, 1804) because of morphological similarity, but maximum-likelihood and Bayesian analyses based on four genes (mitochondrial cytochrome c oxidase subunit I, 16S rRNA, nuclear 28S rRNA, Histone 3), including 28 additional species of Elysia from the Atlantic, showed that they are distinct. In addition, to determine the identity of these specimens, different species delimitation analyses [Automatic Barcode Gap Discovery (ABGD), General Mixed Yule Coalescent (GMYC), Bayesian Poisson Tree Processes, (bPTP)] were carried out. The molecular results support the validity of this new species of Elysia from the Mediterranean Sea, named Elysia rubeni sp. nov. This new species is characterized by a bright light greenish surface color due to the abundance of kleptoplasts. It can be distinguished by its bright blue specks and minute sparkling yellow dots scattered along the body, as well as its smooth and greyish rhinophores.

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.

Solar-powered sea slugs (Sacoglossa: Gastropoda) have long captured the attention of laymen and scientists alike due to their remarkable ability to steal functional chloroplasts from their algal food, enslaving them to withstand long starvation periods. Recently, a wealth of data has shed insight into this remarkable relationship; however, the cellular mechanisms governing this process are still completely unknown. This study explores these mechanisms, providing insight into the chloroplast retention and delayed digestion, occurring within the slug’s digestive gland. We examine the relationships between functional chloroplast and lysosome abundances during starvation, in live material, for the long-term retaining species Elysia timida, the ambiguous long/short-term retaining Elysia viridis, and the short-term retaining Thuridilla hopei, to elucidate digestive differences that contribute to the development of functional kleptoplasty. Functional chloroplast and lysosome abundance are measured using chlorophyll a autofluorescence and the pH-dependent stain acridine orange. In each species, the number of chloroplasts and lysosomes is indirectly proportional, with the plastid density decreasing when starvation begins. We also present a new FIJI/Image J Plugin, the 3D—Accounting and Measuring Plugin, 3D-AMP, which enables the reliable analysis of large image sets.

The enigmatic association of photosynthetically active chloroplasts from algae and some sacoglossan sea slugs, called functional kleptoplasty, is a functional unique system of photosymbioses observed in metazoans. Besides the specific adaptations of the slugs necessary to incorporate and maintain the plastids, the organelles need to ensure optimal photosynthesis. Photoprotective mechanisms in the plastids, namely the xanthophyll cycle (XC) and the high-energy dependent quenching (qE) part of the non-photochemical quenching (NPQ), and of the repair of the D1 protein of Photosystem II (PSII) are considered crucial for kleptoplast longevity in the slugs. Here, we studied the sea slugs Elysia viridis fed with the naturally occurring XC and qE-deficient Bryopsis hypnoides, and E. timida fed on Acetabularia acetabulum, an alga that possesses both mechanisms. The aim of the study was to understand (i) whether qE remains active after ingestion of kleptoplasts by E. timida, (ii) how different light intensities affect the photosynthetic activity of kleptoplasts with and without photoprotection mechanisms, and (iii) if the kleptoplasts are able to repair photodamaged D1 protein. With regard to NPQ, freshly incorporated kleptoplasts responded to different light stress in the same manner as the chloroplasts in their native host algae. Even after three weeks of incorporation the qE part of NPQ was present in the kleptoplasts of E. timida. However, the presence of the qE component of NPQ did not prevent the kleptoplasts from significant PSII photoinactivation under high light intensities. This is probably due to the fact that the kleptoplasts have a reduced PSII repair capacity, despite plastid encoded repair mechanisms in every sacoglossan food source. Hence, photoprotective mechanisms are probably not a key factor explaining kleptoplast longevity in Sacoglossa sea slugs.

Some sacoglossan sea slugs incorporate intracellular functional algal chloroplasts (kleptoplasty) for periods ranging from a few days to several months. Whether this association modulates the photobehaviour of solar-powered sea slugs is unknown. In this study, the long-term retention species Elysia viridis showed avoidance of dark independently of light acclimation state. On the contrary, Placida dendritica, which shows non-functional retention of kleptoplasts, showed no preference over dark, low or high light. High light acclimated (HLac) E. viridis showed a higher preference for high light than low light acclimated (LLac) conspecifics. The position of the lateral folds (parapodia) was modulated by irradiance, with increasing light levels leading to a closure of parapodia and protection of kleptoplasts from high light exposure. Furthermore, closure of parapodia occurred at higher irradiances in HLac E. viridis. Our results strongly indicate that kleptoplasts photoacclimation state modulates the photobehaviour of the solar-powered sea slug E. viridis.

Functional kleptoplasty in sacoglossan sea slugs is among the most curious photosynthetic associations known. One member of these marine molluscs, Elysia viridis, is known to incorporate plastids from a variety of different algae food sources, but with apparently different outcomes and differences in the time span of the retention of functional kleptoplasts. While it was previously shown that kleptoplasts that stem from Codium tomentosum are kept functional for several weeks (long-term retention, LtR), those that stem from Bryopsis hypnoides or Cladophora rupestris are thought to be of limited use regarding photosynthetic capacity (short-term retention, StR). This is important, because it touches upon the popular yet controversial question of how important photosynthesis is for the thriving of these slugs. The aim of the present study was to determine to what degree the plastid source determines retention time. We, therefore, compared E. viridis feeding on either Cladophora sp. or B. hypnoides. We show that kleptoplasts of B. hypnoides incorporate 14CO2, but with rapidly declining efficiency throughout the first week of starvation, while the plastids of Cladophora sp. are, surprisingly, not incorporated to begin with. The radulae of the different samples showed adjustment to the food source, and when feeding on Cladophora sp., E. viridis survived under laboratory conditions under both starvation and non-starvation conditions. Our results demonstrate that (i) the ability to incorporate plastids by E. viridis differs between the food sources B. hypnoides and Cladophora sp., and (ii) photosynthetic active kleptoplasts are not an inevitable requirement for survival.

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.

Some sacoglossan sea slugs are capable of retaining functional chloroplasts stolen' from macroalgae (kleptoplasts). The present study surveyed the pigment composition of the sea slug Elysia viridis (Montagu, 1804) and its food source Codium tomentosum from three different locations along the Portuguese coast. The pigments siphonaxanthin, trans and cis-neoxanthin, violaxanthin, siphonaxanthin dodecenoate, chlorophyll (Chl) a and Chl b, {varepsilon},{varepsilon}- and {beta},{varepsilon}-carotenes and an unidentified carotenoid were observed in all E. viridis analysed. With the exception of the unidentified carotenoid, the same pigment profile was recorded for the macroalga C. tomentosum. Pigments characteristic of other macroalgae present in the sampling locations (Ulva sp. or the epiphyte Ceramium sp. present on C. tomentosum) were not detected in the slugs (Chl c, fucoxanthin, lutein, {beta},{beta}-carotene). These results suggest that E. viridis retained chloroplasts exclusively from C. tomentosum. The differentiation between sea slugs and respective food source from different locations indicated that the site of collection was less relevant to the separation of groups than differences between the macroalgae and the sea slugs. In general, the carotenoids to Chl a ratios were significantly higher in E. viridis than in C. tomentosum. Further analysis using starved individuals suggests carotenoid retention over Chls during the digestion of kleptoplasts. Finally, despite a loss of 80% of Chl a in E. viridis starved for 2 weeks, measurements of maximum quantum yield of photosystem II (Fv/Fm) using variable Chl a fluorescence indicated a decrease of only 5% of the photosynthetic capacity of kleptoplasts.

Several sacoglossan sea slug species feed on macroalgae and incorporate chloroplasts into tubular cells of their digestive diverticula. We investigated the role of the “stolen” chloroplasts (kleptoplasts) in the nutrition of the sea slug Elysia viridis and assessed how their abundance, distribution and photosynthetic activity were affected by light and starvation. Elysia viridis individuals feeding on the macroalga Codium tomentosum were compared with starved specimens kept in dark and low light conditions. A combination of variable Chl a fluorescence and hyperspectral imaging, and HPLC pigment analysis was used to evaluate the spatial and temporal variability of photopigments and of the photosynthetic capacity of kleptoplasts. We show increased loss of weight and body length in dark-starved E. viridis as compared to low light-starved sea slugs. A more pronounced decrease in kleptoplast abundance and lower photosynthetic electron transport rates were observed in dark-starved sea slugs than in low light-starved animals. This study presents strong evidence of the importance of kleptoplast photosynthesis for the nutrition of E. viridis in periods of food scarcity. Deprived of photosynthates, E. viridis could accelerate the breakdown of kleptoplasts in the dark to satisfy its’ energy requirements.

The photosynthetic functionality in chloroplasts in the two sacoglossan molluscs Placida dendritica and Elysia viridis from the Trondheim fjord in Norway was studied. P. dendritica and E. viridis with no functional chloroplasts in their digestive system were introduced to the green macroalgae Codium fragile. Our results showed that P. dendritica was not able to retain functional (photosynthetic) chloroplasts. Transmission electron microscopy (TEM) showed that chloroplasts were directly digested when phagocytosed into the digestive cells. Four stages of chloroplast degradation were observed. A corresponding operational quantum yield of chl a fluorescence (ΦPSII ~ 0) indicated autofluorescence, and the presence of highly degraded chl a supported these observations. In contrast, E. viridis was able to retain functional chloroplasts. For this species it took only 1 week for the chloroplasts inside the digestive cells to acquire the same ΦPSII and light utilisation coefficient (α) as C. fragile kept under the same light conditions. Data for 8 days showed a 2–6-fold increase in the maximum photosynthetic rate (Pmax) and light saturation index (Ek) relative to C. fragile. This increase in available light was probably caused by a reduced package effect in the digestive gland of E. viridis relative to C. fragile, resulting in a partial photoacclimation response by reducing the turnover time of electrons (τ). Isolated pigments from C. fragile compared to E. viridis showed the same levels of photosynthetic pigments (chl a and b, neoxanthin, violaxanthin, siphonaxanthin, siphonein and β,e-carotene) relative to μg chl a (w:w), indicating that the chloroplasts in E. viridis did not synthesise any new pigments. After 73 days of starvation, it was estimated that chloroplasts in E. viridis were able to stay photosynthetic 5–9 months relative to the size of the slugs, corresponding to an RFC of level 8 (a retention ability to retain functional chloroplasts (RFC) for more than 3 months). The reduction in ΦPSII, Pmax and α as a function of time was caused by a reduction in chloroplast health and number (chloroplast thylakoid membranes and PSII are degraded). These observations therefore conclude that chloroplasts from C. fragile cannot divide or synthesise new pigments when retained by E. viridis, but are able to partially photoacclimate by decreasing τ as a response to more light. This study also points to the importance of siphonaxanthin and siphonein as chemotaxonomic markers for the identification of algal sources of functional chloroplasts.

Polypropionates that incorporate pyrones are a family of polyketides featuring the chemistry of a few marine molluscs capable of phototrophic CO2 fixation as a result of storing viable symbiotic chloroplasts in their bodies. The role and origin of these molecules is poorly investigated, although the unusual biological ACHTUNGTRENUNGactivities and chemistry of these natural products have recently received renewed interest. Here, we report the results of in vivo studies on production of g-pyrone-containing polypropionates in the Mediterranean mollusc Elysia viridis. Biosynthesis of the metabolites in the sacoglossan is shown to proceed through condensation of eight intact C3 units by polyketide synthase assembly. LC–MS and NMR spectroscopic studies demonstrate that the process involves a pyrone tetraene (10) as key intermediate, whereas the levels of the final polypropionates (6, 7 and 9) are related to each other and show a significant dependence upon light conditions.

The north-eastern Atlantic sacoglossan gastropod Elysia viridis was studied on littoral and sublittoral shores of the British Isles from 2001 to 2007 to evaluate its potential role in the known decline of the invasive green alga Codium fragile ssp. fragile. Across its European range, this sacoglossan associates with eight genera of algal hosts within three algal orders and two divisions; these hosts include native and introduced macroalgae. The ‘specialist’ herbivore was investigated primarily within the rocky shore community at Lough Hyne Marine Reserve, County Cork, south-west Ireland, with comparative information from surveys of 95 other sites in the British Isles. In Lough Hyne, E. viridis associated with the green algae Codium, Cladophora and Chaetomorpha as well as the red alga Griffithsia. The sacoglossan associated with Codium, Cladophora and Chaetomorpha as well as the red alga Halurus in Devon, Isle of Wight and the Channel Islands. Recruitment of E. viridis to Codium spp. in the lough was substantially higher than in most areas of the British Isles with 100% of the Codium hosts attacked during annual September surveys. The strong autumn pulse of sacoglossan recruitment, coupled with the asynchronous population dynamics compared to other shores, indicates retention of planktotrophic larvae within the lough. The previously reported decline in sacoglossan density with tidal level was hypothesized to be caused by sublittoral predators. Although our experiments failed to demonstrate an effect by large predators, the foraging of the abundant fingerling fish and small crabs could not be tested conclusively. Field experiments were consistent with the interpretation of intense sacoglossan herbivory in certain areas of the lough. However, even if the sacoglossans contribute to the local decline of C. fragile in the lough, they cannot account for the regional decline of the alga on the north-eastern Atlantic shores.

The importance of photosynthetic products derived from kleptoplasts in several sacoglossan species is being investigated in different fields, such as chemistry, biomolecular genetics and ecology. This study analyzes the effect of kleptoplasts on the survival rates of Elysia timida by evaluating the development of chlorophyll concentration, total length and survival rates of starved E. timida specimens kept in the light and in the dark. Although chlorophyll concentration values were similar in both cases, after 28 days specimens kept in the dark showed a greater size decrease and a lower survival rate (up to 30% lower) than those kept in the light. It is evident that kleptoplasts provide the mollusc with extra energy at the primary metabolism level to compensate for a shortage in food.

Summary 1 Sacoglossan sea slugs (Gastropoda: Opisthobranchia) can change their highly specialized algal host associations in response to introduced hosts. 2 Adult sacoglossans (Placida dendritica) collected from the introduced green macroalga Codium fragile ssp. tomentosoides in west-coast Scottish sea lochs tended to associate with and consume introduced hosts over the native C. tomentosum in pairwise-choice feeding trials. 3 On west-coast Irish shores, where native congeneric hosts were more common, significantly more P. dendritica attacked C. tomentosum on the shore whereas Elysia viridis disproportionately attacked the exotics. In Lough Hyne, Co. Cork, Ireland, juvenile E. viridis attacked both C. fragile ssp. tomentosoides and the native, sympatric C. vermilara; in pairwise-choice feeding trials, sacoglossans preferred the host species from which they were collected. 4 In the Channel Islands, adult E. viridis were common on C. tomentosum on Guernsey shores. In pairwise-choice feeding trials adults exhibited no preference between native and introduced hosts, indicating that the stenophagous herbivores could change their associations. 5 On temperate Australian shores, the native sacoglossan Placida aoteana attacked the recently introduced C. fragile ssp. tomentosoides as well as native congeners and conspecifics. P. aoteana was common and its herbivory evident in Victoria and Tasmania. Slugs collected from native C. fragile exhibited no preference between algal subspecies in Victoria but a strong preference for the introduced subspecies in Tasmania. 6 Flexibility in host use enables stenophagous marine herbivores to respond within years to the presence of recently introduced hosts. The implicit peril of the host-specificity paradigm − that specialists could change their associations − does occur in stenophagous sacoglossans, and there is considerable intra- and interspecific variation in response. 7 Biological control of invasive green algae using sacoglossan herbivores is fraught with several risks, including taxonomic uncertainty of species’ boundaries, ecological flexibility of feeding, unexplored role of host susceptibility and insufficient evolutionary–developmental information to establish coevolutionary associations.

Some sea slugs are capable of retaining functional sequestered chloroplasts (kleptoplasts) for variable periods of time. The mechanisms supporting the maintenance of these organelles in animal hosts are still largely unknown. Non-photochemical quenching (NPQ) and the occurrence of a xanthophyll cycle were investigated in the sea slugs Elysia viridis and E. chlorotica using chlorophyll fluorescence measurements and pigment analysis. The photoprotective capacity of kleptoplasts was compared to that observed in their respective algal source, Codium tomentosum and Vaucheria litorea. A functional xanthophyll cycle and a rapidly reversible NPQ component were found in V. litorea and E. chlorotica but not in C. tomentosum and E. viridis. To our knowledge, this is the first report of the absence of a functional xanthophyll cycle in a green macroalgae. The absence of a functional xanthophyll cycle in C. tomentosum could contribute to the premature loss of photosynthetic activity and relatively short-term retention of kleptoplasts in E. viridis. On the contrary, E. chlorotica displays one of the longest functional examples of kleptoplasty known so far. We speculate that different efficiencies of photoprotection and repair mechanisms of algal food sources play a role in the longevity of photosynthetic activity in kleptoplasts retained by sea slugs.

Factors determining the degree of dietary generalism versus specialism are central in ecology. Species that are generalists at the population level may in fact be composed of specialized individuals. The optimal diet theory assumes that individuals choose diets that maximize fitness, and individual specialization may occur if individuals' ability to locate, recognize, and handle different food types differ. We investigate if individuals of the marine herbivorous slug Elysia viridis, which co-occur at different densities on several green macroalgal species in the field, are specialized to different algal hosts. Individual slugs were collected from three original algal host species (Cladophora sericea, Cladophora rupestris and Codium fragile) in the field, and short-term habitat choice and consumption, as well as long-term growth (proxy for fitness), on four algal diet species (the original algal host species and Chaetomorpha melagonium) were studied in laboratory experiments. Nutritional (protein, nitrogen, and carbon content) and morphological (dry weight, and cell/utricle volume) algal traits were also measured to investigate if they correlated with the growth value of the different algal diets. E. viridis individuals tended to choose and consume algal species that were similar to their original algal host. Long-term growth of E. viridis, however, was mostly independent of original algal host, as all individuals reached a larger size on the non-host C. melagonium. E. viridis growth was positively correlated to algal cell/utricle volume but not to any of the other measured algal traits. Because E. viridis feeds by piercing individual algal cells, the results indicate that slugs may receive more cytoplasm, and thus more energy per unit time, on algal species with large cells/utricles. We conclude that E. viridis individuals are specialized on different hosts, but host choice in natural E. viridis populations is not determined by the energetic value of seaweed diets as predicted by the ODT.

In the British Isles, oligophagous marine herbivores, particularly the ascoglossan (= sacoglossan) sea slug Elysia viridis, associate with the introduced green macroalga Codium fragile ssp. tomentosoides. Slugs prefer to associate with and consume the introduced C. fragile to the native C. tomentosum. Our investigation of adult, larval, and juvenile E. viridis focused on whether this association is attributable (1) to a host switch or (2) to an expansion from native hosts to the introduced C. fragile ssp. tomentosoides. Growth rates and maximum body sizes of E. viridis on introduced hosts were greater than on natives. Although the native Cladophora rupestris induced a high rate of slug metamorphosis, recently metamorphosed juvenile E. viridis (from Codium fragile-feeding parents) were generally not able to feed or grow on the native alga. In contrast, juveniles from Cladophora-feeding parents could eat Cladophora, although their performance was highly variable. Small, postlarval slugs (

The stenophagous ascoglossan (=sacoglossan) opisthobranch Elysia viridis has long been a model organism for the study of endosymbiosis or kleptoplasty as well as one of the few herbivores to consume the introduced green macroalga Codium fragile on European shores. Larval and post-larval dynamics of the ascoglossan were investigated. Planktotrophic larvae of E. viridis grew at 5–10 μm d−1 (shell length) at 15°C on a unicellular algal diet (the cryptophyte Rhodomonas baltica); larvae became competent one month post-hatching. Effective feeding and chloroplast acquisition typically started within 2–3 d of metamorphosis. Slugs grew about 8 mm in the first month of post-larval life. During this period, juveniles held in the light did not grow faster or survive better than conspecifics held in the dark; thus, functional kleptoplasty did not occur during first three weeks of benthic life. While larval growth rates and the nature of metamorphic cues are consistent with those of many other opisthobranch species with planktotrophic larvae, measures of post-larval growth—particularly as it pertains to kleptoplasty—is a new contribution to opisthobranch biology.

Several congeneric species of intertidal green algae inhabit shady, low-desiccation microhabitats. The N.E. Pacific green alga Codium setchellii occurs on sand-influenced rocky shores, particularly on shady, vertical surfaces on low intertidal rocky benches. The S. Pacific congener C. convolutum shows a similar, though less restrictive, distribution. The algal distributional patterns were inversely related to potential desiccation stress. Thallus abundance and/or size increased lower on the shore and on shady aspects. Because some thalli persist in desiccation-prone habitats, C. setchellii and C. convolutum can persist in alternative areas. Spatial patterns of specialist herbivore abundance indicated that stenophagous sea slugs that fed selectively on Codium were disproportionately more frequent on desiccation-prone algal hosts. On Oregon shores, USA, the sea slug Placida dendritica was more frequent on the alga C. setchellii (i) at the upper end of the alga's tidal distribution than at the lower end and (ii) on desiccation-prone, eastern aspects than on those with more benign, western aspects. Populations of slugs on Oregon shores peaked in spring and early summer during the period of peak desiccation to low intertidal species. The seasonal, habitat-associated pattern of slug distribution on algal hosts was geographically and taxonomically robust. On N.E. New Zealand shores, two species of ascoglossan slugs were significantly more frequent on thalli of C. convolutum at the upper end of the alga's tidal distribution than at the lower end. Increased larval settlement of slugs on desiccation-prone algal hosts contributed to this pattern whereas spatial variation in predator abundance did not. Larval metamorphosis experiments with the ascoglossan Elysia viridis from Scottish shores demonstrated that the frequency of larval metamorphosis was significantly greater in the presence of desiccation-prone algal hosts than unstressed ones. Thus, variable algal susceptibility and differential slug settlement and metamorphosis both contribute to this striking pattern of targeted slug attack. In contrast to the wealth of terrestrial examples, this is one of the few marine examples of prey-stress models.

Understanding the mechanisms involved in resource use by an organism is pivotal to understanding its ecology. A conspecific population that as a whole demonstrates a generalist pattern of resource use may in fact consist of relatively specialized individuals. Elysia viridis, a sacoglossan opisthobranch mollusc, tends to demonstrate this type of sympatric variation in diet, although to differing extents depending on the ontogenetic stage. However, the mechanisms underlying this inter-individual variation are poorly understood. Utilizing the basic framework of optimal diet theory, this thesis investigated the prevalence of individual specialization and its effects on energy assimilation in E. viridis on different algal diets and the mechanisms that underpin or constrain diet selection. This was assessed through a combination of laboratory experiments addressing how E. viridis’ original algal host affected algal diet choice, handling efficiency, growth, and the retention of functional chloroplasts (kleptoplasty) in the lab and relating conclusions from these experiments to observations of abundance and size of the sea slug in the study area. Assessments of abundance and size distributions of E. viridis on different algal hosts demonstrated that the sea slug commonly colonized the co-occuring algal species Codium fragile, Cladophora rupestris, and Cladophora sericea in the field. Abundance was generally highest on Cladophora hosts compared to C. fragile hosts, and C. rupestris tended to accommodate larger individuals compared to the other hosts (paper I). In the lab E. viridis tended to select algal diets that had a similar morphology (filamentous septate vs. planar siphonaceous) to their original host, which related to increased handling efficiency through previous experience of feeding techniques required for different algal morphologies. This indicated that short-term diet selection was influenced by differences in feeding efficiency, suggesting E. viridis were specialised to feed on particular diets. However, diet selection did not correlate to the long-term fitness value of a diet, indicating that factors other than nutrition are important for host/diet selection in E. viridis. However, positive growth by E. viridis on all algal diets irrespective of their original algal host indicated that slugs were capable of effectively switching to non-host algae (paper II). Furthermore, E. viridis derived functional kleptoplasts from three different genera of algae (Chaetomorpha, Codium, and Cladophora), refuting claims that members of Cladophorales were unsuitable sources of functional kleptoplasts to E. viridis (paper III). However, kleptoplast functionality varied within the genus Cladophora. Finally we provided evidence that E. viridis receives a substantial fitness benefit under satiation by retaining functional kleptoplasts through increased growth efficiency via phototrophy (paper IV). Overall this thesis contributes substantially to understanding the fitness trade-offs E. viridis faces through diet selection. Furthermore, it emphasizes that assessing energy assimilation in polyphagous sacoglossans requires not only an understanding of the fitness contributions of nutritional and morphological traits of different algal diets but also their role as a source of functional kleptoplasts to the slug. However, many conclusions reached in the lab did not concur with field patterns, indicating a need for further study on biotic (e.g. predation) and abiotic (e.g. wave force) pressures on E. viridis populations and assessment of the roles algal hosts play in circumventing these pressures.

The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic theory of eukaryote origins.

Photosynthetic sea slugs have the ability to "steal" chloroplasts (kleptoplasts) from marine macroalgae and keep them structurally intact and physiologically func- tional. The photosynthetic activity of these symbioses has been assessed using pulse amplitude modulated (PAM) fluorometry. However, the movement of these sacoglossan slugs can impair specific photobiological studies on klepto- plasts. Thus, immobilizing sacoglossan slugs while not in- terfering with the photosynthetic activity would be a meth- odological advance for research in this field. We evaluated the effect of two anesthetics, eugenol and MS-222, on the photosynthetic activity of kleptoplasts and on the behavior of the kleptoplasts-bearing slug Elysia viridis. Anesthetics promoted relaxation of sea slug muscle with no touch reac- tion in about 6 min. Sea slugs immobilized for 120 min completely recovered after anesthetic removal. No signifi- cant differences were found on photosynthetic parameters measured immediately (0 -1 min) after immobilization. The effective quantum yield of photosystem II of E. viridis after 120 min of immobilization was significantly decreased by 12% in the MS-222 treatment, while eugenol promoted no significant effect. Photosynthetic activity assessed by rapid light-response curves (RLC) of relative electron transport rates (rETR) revealed a significant decrease in both initial response to light (-34%) and maximum rETR (rETRm) (- 60%), after 120 min of immobilization using MS-222. After 120 min of immobilization with eugenol, the initial response to light significantly decreased 15% and rETRm decreased 27%. We conclude that, whenever photobiologi- cal studies employing PAM fluorometry require immobili- zation of photosynthetic sea slugs, eugenol can be used as a powerful anesthetic with little impact on the photosynthetic activity of kleptoplasts.

Three Mediterranean sacoglossans (Elysia translucens, Bosellia mimetica and Elysia viridis) have been submitted to chemical investigations. The chemical structure (diterpenoids and polypropionates) of their potential allomones has been clarified. Some metabolites (diterpenoids) are also present in the preferred algal food of the molluscs. E. viridis is able to construct de novo metabolites (polypropionates) absent in the algae. A more general evolutionary trend of Mediterranean Elysioidea molluscs is suggested by adding the results reported herewith to the previous studies on Elysia timida and Thuridilla hopei and by comparing the metabolic pattern of T. hopei with that of the algae Derbesia tenuissima.

The radular teeth of 55 species of Sacoglossa (= Ascoglossa) (Mollusca: Opisthobranchia) with known diets are classified into three basic groups: triangular, blade-shaped and sabot-shaped. Cell wall composition of the food plant is the single most important factor influencing radular morphology. The algae eaten by sacoglossans have either xylan, mannan or cellulose as their structural wall component. Sabot-shaped teeth are associated with diets of Siphonocladales and Cladophorales, which have cellulose cell walls of a 'crossed fibrillar texture'. Triangular teeth with lateral denticles are associated with diets of Caulerpa or with calcified algae. Most of these have xylan as the major structural polysaccharide. Blade-shaped teeth occur in a large number of species representing a wide variety of diets. It is proposed that the three types of teeth function in different manners. Tooth denticulation is correlated with functional group of the food plant. The radular teeth of Elysia viridis feeding on Codium are longer, wider and have more curved tips than teeth of E. viridis feeding on Chaetomorpha . The teeth of E. viridis transferred from Codium to Chaetomorpha in the laboratory change in shape as well as size.

Background Among metazoans, retention of functional diet-derived chloroplasts (kleptoplasty) is known only from the sea slug taxon Sacoglossa (Gastropoda: Opisthobranchia). Intracellular maintenance of plastids in the slug's digestive epithelium has long attracted interest given its implications for understanding the evolution of endosymbiosis. However, photosynthetic ability varies widely among sacoglossans; some species have no plastid retention while others survive for months solely on photosynthesis. We present a molecular phylogenetic hypothesis for the Sacoglossa and a survey of kleptoplasty from representatives of all major clades. We sought to quantify variation in photosynthetic ability among lineages, identify phylogenetic origins of plastid retention, and assess whether kleptoplasty was a key character in the radiation of the Sacoglossa. Results Three levels of photosynthetic activity were detected: (1) no functional retention; (2) short-term retention lasting about one week; and (3) long-term retention for over a month. Phylogenetic analysis of one nuclear and two mitochondrial loci revealed reciprocal monophyly of the shelled Oxynoacea and shell-less Plakobranchacea, the latter comprising a monophyletic Plakobranchoidea and paraphyletic Limapontioidea. Only species in the Plakobranchoidea expressed short- or long-term kleptoplasty, most belonging to a speciose clade of slugs bearing parapodia (lateral flaps covering the dorsum). Bayesian ancestral character state reconstructions indicated that functional short-term retention arose once in the last common ancestor of Plakobranchoidea, and independently evolved into long-term retention in four derived species. Conclusion We propose a sequential progression from short- to long-term kleptoplasty, with different adaptations involved in each step. Short-term kleptoplasty likely arose as a deficiency in plastid digestion, yielding additional energy via the release of fixed carbon. Functional short-term retention was an apomorphy of the Plakobranchoidea, but the subsequent evolution of parapodia enabled slugs to protect kleptoplasts against high irradiance and further prolong plastid survival. We conclude that functional short-term retention was necessary but not sufficient for an adaptive radiation in the Plakobranchoidea, especially in the genus Elysia which comprises a third of all sacoglossan species. The adaptations necessary for long-term chloroplast survival arose independently in species feeding on different algal hosts, providing a valuable study system for examining the parallel evolution of this unique trophic strategy.

The effects of light exposure on the photosynthetic activity of kleptoplasts were studied in the sacoglossan mollusc Elysia viridis. The photosynthetic activity of ingested chloroplasts was assessed in vivo by non-destructively measuring photophysiological parameters using pulse amplitude modulation (PAM) fluorometry. Animals kept under starvation were exposed to two contrasting light conditions, 30 μmol photons m−2 s−1 (low light, LL), and 140 μmol photons m−2 s−1 (high light, HL), and changes in photosynthetic activity were monitored by measuring the maximum quantum yield of photosystem II (PSII), Fv/Fm, the minimum fluorescence, Fo, related to chlorophyll a content, and by measuring rapid light-response curves (RLC) of relative electron transport rate (rETR). RLCs were characterised by the initial slope of the curve, αRLC, related to efficiency of light capture, and the maximum rETR level, rETRm,RLC, determined by the carbon-fixation metabolism. Starvation induced the decrease of all photophysiological parameters. However, the retention of photosynthetic activity (number of days for Fv/Fm > 0), as well as the rate and the patterns of its decrease over time, varied markedly with light exposure. Under HL conditions, a rapid, exponential decrease was observed for Fv/Fm, αRLC and rETRm,RLC, Fo not showing any consistent trend of variation, and retention times ranged between 6 and 15 days. These results suggested that the retention of chloroplast functionality is limited by photoinactivation of PSII reaction center protein D1. In contrast, under LL conditions, a slower decrease in all parameters was found, with retention times varying from 15 to 57 days. Fv/Fm, αRLC and rETRm,RLC exhibited a bi-phasic pattern composed by a long phase of slow decrease in values followed by a rapid decline, whilst Fo decayed exponentially. These results were interpreted as resulting from lower rates of D1 photoinactivation under low light and from the gradual decrease in carbon provided by photosynthesis due to reduction of functional photosynthetic units.

Photosynthesis is a fundamental process sustaining heterotrophic organisms at all trophic levels. Some mixotrophs can retain functional chloroplasts from food (kleptoplasty), and it is hypothesized that carbon acquired through kleptoplasty may enhance trophic energy transfer through increased host growth efficiency. Sacoglossan sea slugs are the only known metazoans capable of kleptoplasty, but the relative fitness contributions of heterotrophy through grazing, and phototrophy via kleptoplasts, are not well understood. Fitness benefits (i.e. increased survival or growth) of kleptoplasty in sacoglossans are commonly studied in ecologically unrealistic conditions under extended periods of complete darkness and/or starvation. We compared the growth efficiency of the sacoglossan Elysia viridis with access to algal diets providing kleptoplasts of differing functionality under ecologically relevant light conditions. Individuals fed Codium fragile, which provide highly functional kleptoplasts, nearly doubled their growth efficiency under high compared to low light. In contrast, individuals fed Cladophora rupestris, which provided kleptoplasts of limited functionality, showed no difference in growth efficiency between light treatments. Slugs feeding on Codium, but not on Cladophora, showed higher relative electron transport rates (rETR) in high compared to low light. Furthermore, there were no differences in the consumption rates of the slugs between different light treatments, and only small differences in nutritional traits of algal diets, indicating that the increased growth efficiency of E. viridis feeding on Codium was due to retention of functional kleptoplasts. Our results show that functional kleptoplasts from Codium can provide sacoglossan sea slugs with fitness advantages through photosynthesis.

Sacoglossans are specialized marine herbivores that tend to have a close evolutionary relationship with their macroalgal hosts, but the widely distributed species Elysia viridis can associate with several algal species. However, most previous investigations on the field abundance and size distribution of E. viridis have focussed on Codium spp. in the British Isles, and algae from this genus are considered superior hosts for E. viridis. In the present study, we investigated the abundance and size distribution of E. viridis on 6 potential host algae with differing morphologies (the septate species Cladophora sericea, Cladophora rupestris, Chaetomorpha melagonium, and Ceramium virgatum, as well as the siphonaceous species Codium fragile and Bryopsis sp.) at 2 sites on the Swedish west coast over the course of a year. In spring, slugs were almost absent from all algal hosts. In summer and autumn, E. viridis consistently occurred on several of the algal species at both sites. The highest number of small E. viridis were found on C. sericea, intermediate numbers of significantly larger E. viridis were found on C. rupestris, while fewer, intermediate sized animals were found on C. fragile. Throughout the study period, only a few E. viridis individuals were found on C. melagonium, Bryopsis sp., and C. virgatum. Our results indicate that E. viridis is an annual species in Sweden, capable of exploiting co-occurring congeneric and intergeneric algal hosts with differing morphologies. These results corroborate previous findings that E. viridis can exploit several different algal species, but does not indicate that C. fragile is a superior host.

Early in its life cycle, the marine mollusc Elysia chlorotica Gould forms an intracellular endosymbiotic association with chloroplasts of the chromophytic alga Vaucheria litorea C. Agardh. As a result, the dark green sea slug can be sustained in culture solely by photoautotrophic CO2fixation for at least 9 months if provided with only light and a source of CO2. Here we demonstrate that the sea slug symbiont chloroplasts maintain photosynthetic oxygen evolution and electron transport activity through photosystems I and II for several months in the absence of any external algal food supply. This activity is correlated to the maintenance of functional levels of chloroplast-encoded photosystem proteins, due in part at least to de novo protein synthesis of chloroplast proteins in the sea slug. Levels of at least one putative algal nuclear encoded protein, a light-harvesting complex protein homolog, were also maintained throughout the 9-month culture period. The chloroplast genome ofV. litorea was found to be 119.1 kb, similar to that of other chromophytic algae. Southern analysis and polymerase chain reaction did not detect an algal nuclear genome in the slug, in agreement with earlier microscopic observations. Therefore, the maintenance of photosynthetic activity in the captured chloroplasts is regulated solely by the algal chloroplast and animal nuclear genomes.

Solar-powered “leaves that crawl”? This description of photosynthetic sea slugs (adapted from Bill Rudman [[www.austmus.gov.au/seaslugs/solarpow.htm][1]] and Robert Trench [1975]) aptly describes the symbiotic association that occurs between certain molluscan sea slugs and algal chloroplasts.

A local population of the introduced green macroalga Codium fragile ssp. tomentosoides was investigated in Oban in the district of Argyll, western Scotland. Periodic surveys demonstrated that sacoglossan densities (Elysia viridis and Placida dendritica) were unusually high in 1997 and early 1998, slug herbivory noticeably damaged algal thalli, and the local algal population was decimated. These patterns provide indirect evidence of the significance of slug herbivory on the introduced alga.

Criteria for defining “chloroplast symbiosis” are outlined. Investigations are described into the presence or absence of chloroplast symbiosis in Limapontia capitata,“pale” and “dark” forms of L. depressa, Alderia modesta and Elysia viridis. Apart from E. viridis, only “pale” forms of L. depressa showed signs of the phenomenon. All sacoglossans hitherto shown definitely to possess “chloroplast symbiosis” have the twin characteristics of being elysioid and having siphonaceous algae as the source of chloroplasts. It is therefore suggested that the phenomenon may not be very widespread in the order, probably occurring in less than half of all species.

Chloroplasts of Codium fragile , which remain photosynthetically active for long periods after entry into the digestive cells of Elysia viridis , are naturally very ‘robust’ organelles. Their capacity for photosynthetic carbon fixation in vitro declines at a very much slower rate than that of higher plant chloroplasts, with some activity detectable seven days after isolation. They are resistant to mild physical abrasion and to the high osmotic pressure of seawater. Chloroplasts from the tip of Codium plants have much higher rates of photosynthesis than those from the base, and, in laboratory feeding experiments, animals acquired more chloroplasts from the tip than from the base. Accurate measurements of rates of chloroplast turnover in Elysia during feeding proved difficult, but the fastest rate was probably about one-third to one-half of the chloroplast population replaced in a week. Elysia could select Codium , in the presence of other seaweeds, at temperatures of 8-9°, but not 18-19°. The method of selecting Codium appears to be by ‘trial and error’ rather than by chemosensory means. Elysia moved towards light intensities of up to 12 W m -2 and away from intensities above 24 W m -2 , which is approximately the intensity at which maximum rates of photosynthesis occur.

An account is given of the elysiid sacoglossans collected on diving expeditions to the eastern Mediterranean shores of Yugoslavia and Greece during the period 1978– 1986, supplemented by material collected around Naples and some preserved specimens from other Mediterranean localities. In all, 6 species of Elysia and 1 of Bosellia were investigated, including one new to science, E. gordanae from the Yugoslav Istrian Peninsula. The enigmatic Elysia translucens Pruvot-Fol, 1957 was found in the eastern basin for the first time. The presence of the Bermudan, Canary Islands and Jamaican E. flava Verrill, 1901 was confirmed in the Greek Ionian and Aegean Seas, as well as in the Gulf of Corinth. The geographical ranges of the other species were extended by these collections from the eastern basin. The 7 elysiid species were illustrated from life and the radulae compared ; great differences in the size, shape and denticulation of the teeth of the different species were evident

When Elysia viridis or Tridachia crispata were incubated in the light in solutions containing NaH 14 CO 3 and either glucose or galactose, fixed 14 C was released to the medium as glucose or galactose. The effect is selective for although, for example, mannose also causes 14 C release from E. viridis , none occurs if sorbose, fructose or maltose are present. The phenomenon has been reported in other associations between autotrophs and heterotrophs, where it has formed the basis of ‘inhibition techniques'. It is assumed that the external unlabelled compound can enter and exchange with the labelled form at some stage in its movement through the association. In E. viridis , the effect was also found with alanine. Various lines of evidence show that the ‘inhibiting’ compound traps the 14 C as it moves between animal cells. The total amount of fixed 14 C released by glucose ‘inhibition’ in Elysia was approximately 12%. ‘Inhibition techniques’ may be of considerable value in the study of intercellular transport of molecules.

Summary The ultrastructure of chloroplasts in the frond of Codium fragile and in symbiotic association with the digestive cells of Elysia viridis is described. In the hepatopancreas the chloroplasts are always bounded by a host membrane and contaminant algal cytoplasm may be attached to the chloroplast envelope. The possibility that‘butterfly’shaped chloroplasts and chloroplasts with membranous baffles represent different forms of chloroplast division is discussed. The presence of non-Codium chloroplasts in the digestive cells of E. viridis is reported.

Summary The 14C-assimilates after photosynthetic assimilation of H14CO3- by chloroplasts of the green siphonaceous alga Codium fragile symbiotic with the marine opisthobranch Elysia viridis were analysed and compared with the typical 14C-assimilate pattern of intact Codium plants. Most of the characteristic assimilates of symbiotic plastids found as soluble 14C-compounds in Elysia are consistent with the 14C-assimilates extracted from Codium. Remarkable differences are noted in the quantitative distribution of 14C, thus indicating that photosynthates formed by the symbiotic chloroplasts are metabolized by the animal partner. The symbiotic chloroplasts are fully functional entities.

SUMMARY An ultrastructural approach was used to study the effects of light and dark starvation on chloroplasts symbiotic in the mollusc Elysia viridis. Starvation for 27 days in a 16 h light/8 h dark photoperiod resulted in loss of starch grains and a gradual disintegration of the chloroplasts by a progressive increase in the inter-thylakoid spaces, resulting in the separation and degeneration of the photosynthetic membranes. To account for these observations a process of photodestruction is suggested. Chloroplasts in animals starved in total darkness for a corresponding period remained relatively intact, and showed a marked increase in the number of plastoglobuli per chloroplast. This increase in plastoglobuli may be a result of the normal ageing process of chloroplasts with a redundant photosynthetic apparatus.

SUMMARY Tissues of Elysia viridis contain a ‘factor’ which stimulates release of fixed 14C from isolated Codium chloroplasts and also from the symbiotic algae of Anemonia sulcata. This ‘factor’ appears to be thermolabile, non-particulate, and only occurs in effective amounts in the chloroplast-containing tissues of the animal. So far, attempts to mimic the action of Elysia homogenates by varying the pH of the medium, or by adding Codium homogenates, fructose 1,6 di-phosphatase or‘2C-glucose to it, have failed.

The embryonal development and survival of adults of Elysia viridis in lowered salinities was investigated. Adults survived down to 15‰, but produced egg masses only down to 17.5‰. Embryonal development normally took 5–6 days in 30‰, but was prolonged to over 8 days in 17.5‰. The percentage of abnormally developing eggs increased in low salinities, more than 90‰ abnormal in 17.5‰. The larvae hatched in a normal way only within the range 30–20‰.

Chloroplasts of Codium fragile fix carbon photosynthetically at rates comparable to or higher than those of the intact plant for several hours after isolation. When stored in the dark at 5°C in a simple mineral medium containing mannitol as an osmoticum and no added cofactors, the chloroplasts continued to fix carbon photosynthetically for at least 5 days after isolation, although at a progressively declining rate; they retained the ‘halo’ characteristic of an intact outer envelope for the entire period. Patterns of 14 C fixation by isolated chloroplasts and intact Codium were generally similar, except that no incorporation of 14 C into sucrose could be detected in isolated chloroplasts, and incorporation into glycolic acid was higher. Only 2% of the photosynthetically fixed 14 C was released to the medium by isolated chloroplasts. Their Hill reaction was relatively weak, but this may have been due to the impermeability of the outer envelope which could not be ruptured even by the severe osmotic shock of transfer from the suspension medium (osmotic pressure greater than 14 atm) to distilled water. It is suggested that the ‘robustness’ of chloroplasts from siphonaceous algae may be an important factor in their ability to enter and continue functioning inside animal cells.

1. 1. Light microscopic and electron microscopic autoradiography have been used to study the incorporation of tritiated leucine into protein by the sacoglossan opisthobranch mollusc, Elysia viridis and its endosymbiotic chloroplasts acquired from the seaweed, Codium fragile. 2. 2. Light microscopic autoradioagraphy showed that several animal tissues incorporated [3H]-leucine into protein. Among these were the “secretory zones” of the digestive diverticulum, the testes and the pedal mucus gland. It is suggested that uptake of the metabolite was via the general integument. 3. 3. Electron microscopic autoradiography showed that protein in the chloroplasts becomes labelled after a 3-hr incubation, and the silver grain density over the chloroplasts does not change during a 1–12 hr “cold chase”. The possible sources of this protein are considered and evidence suggests that it is synthesized within the chloroplasts. 4. 4. Thus these chloroplasts have some degree of biochemical autonomy outside the algal cells.