The kleptoplastic sea slug Elysia clarki prolongs photosynthesis by synthesizing chlorophyll a and b

Several species of kleptoplastic, sacoglossan sea slug photosynthesize using chloroplasts sequestered inside their digestive cells from algal food sources. However, sequestered chloroplasts alone are not sufficient for months-long, continuous photosynthesis and maintenance of the chloroplasts in absence of the algal nucleus. Some type of plastid maintenance mechanism must be present to help sustain photosynthetic activity in the long term kleptoplastic species, such as Elysia clarki. We demonstrate that E. clarki starved for 2 weeks are able to synthesize chlorophylls, but that slugs starved for 14 weeks no longer synthesize chlorophyll. The subsidence of chlorophyll synthesis is coincident with the cessation of photosynthesis by the starved slugs, but it is not yet known if the cessation of pigment synthesis is the cause or some other aspect of plastid degradation produces a loss of synthetic ability.     

Examining the retention of functional kleptoplasts and digestive activity in sacoglossan sea slugs

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.     

Slugs' last meals: molecular identification of sequestered chloroplasts from different algal origins in Sacoglossa (Opisthobranchia, Gastropoda)

Some sacoglossan sea slugs have become famous for their unique capability to extract and incorporate functional chloroplasts from algal food organisms (mainly Ulvophyceae) into their gut cells. The functional incorporation of the so-called kleptoplasts allows the slugs to rely on photosynthetic products for weeks to months, enabling them to survive long periods of food shortage over most of their life-span. The algal food spectrum providing kleptoplasts as temporary, non-inherited endosymbionts appears to vary among sacoglossan slugs, but detailed knowledge is sketchy or unavailable. Accurate identification of algal donor species, which provide the chloroplasts for long-term retention is of primary importance to elucidate the biochemical mechanisms allowing long-term functionality of the captured chloroplast in the foreign animal cell environment. Whereas some sacoglossans forage on a variety of algal species, (e.g. Elysia crispata and E. viridis) others are more selective. Hence, characterizing the range of functional sacoglossan-chloroplast associations in nature is a prerequisite to understand the basis of this enigmatic endosymbiosis. Here, we present a suitable chloroplast gene (tufA) as a marker, which allows identification of the respective algal kleptoplast donor taxa by analysing DNA from whole animals. This novel approach allows identification of donor algae on genus or even species level, thus providing evidence for the taxonomic range of food organisms. We report molecular evidence that chloroplasts from different algal sources are simultaneously incorporated in some species of Elysia. NeigborNet analyses for species assignments are preferred over tree reconstruction methods because the former allow more reliable statements on species identification via barcoding, or rather visualize alternative allocations not to be seen in the latter.     

Phagocytosis of algal chloroplasts by digestive gland cells in the photosynthesis-capable slug Elysia timida (Mollusca, Opisthobranchia, Sacoglossa)

Parapodia of the sacoglossan slug Elysia timida were preserved by high-pressure cryofixation during feeding experiments and investigated with transmission electron microscopy. This slug has been known for its long-term retention of active chloroplasts and photosynthesis. We observed different stages of phagocytosis of chloroplast components from ingested algal food by slug digestive gland cells. Thylakoid stacks and stroma of chloroplasts were engulfed by the slug cells. In the slug cells thylakoids were surrounded by one membrane only. This membrane is interpreted as having been generated by the mollusk during phagocytosis. It is inferred to be eukaryotic in origin and unlikely, therefore, to be endowed with the translocons system ordinarily regulating import of algal gene-encoded plastid preproteins. Our structural findings suggest that chloroplast components in the slug cells are thylakoid stacks with chloroplast stroma only.     

Comparison of sister species identifies factors underpinning plastid compatibility in green sea slugs

The only animal cells known that can maintain functional plastids (kleptoplasts) in their cytosol occur in the digestive gland epithelia of sacoglossan slugs. Only a few species of the many hundred known can profit from kleptoplasty during starvation long-term, but why is not understood. The two sister taxa Elysia cornigera and Elysia timida sequester plastids from the same algal species, but with a very different outcome: while E. cornigera usually dies within the first two weeks when deprived of food, E. timida can survive for many months to come. Here we compare the responses of the two slugs to starvation, blocked photosynthesis and light stress. The two species respond differently, but in both starvation is the main denominator that alters global gene expression profiles. The kleptoplasts'' ability to fix CO₂ decreases at a similar rate in both slugs during starvation, but only E. cornigera individuals die in the presence of functional kleptoplasts, concomitant with the accumulation of reactive oxygen species (ROS) in the digestive tract. We show that profiting from the acquisition of robust plastids, and key to E. timida''s longer survival, is determined by an increased starvation tolerance that keeps ROS levels at bay.     

Characterization of the Bacterial Community of the Chemically Defended Hawaiian Sacoglossan Elysia rufescens

Sacoglossans are characterized by the ability to sequester functional chloroplasts from their algal diet through a process called kleptoplasty, enabling them to photosynthesize. The bacterial diversity associated with sacoglossans is not well understood. In this study, we coupled traditional cultivation-based methods with 454 pyrosequencing to examine the bacterial communities of the chemically defended Hawaiian sacoglossan Elysia rufescens and its secreted mucus. E. rufescens contains a defense molecule, kahalalide F, that is possibly of bacterial origin and is of interest because of its antifungal and anticancer properties. Our results showed that there is a diverse bacterial assemblage associated with E. rufescens and its mucus, with secreted mucus harboring higher bacterial richness than entire-E. rufescens samples. The most-abundant bacterial groups affiliated with E. rufescens and its mucus are Mycoplasma spp. and Vibrio spp., respectively. Our analyses revealed that the Vibrio spp. that were highly represented in the cultivable assemblage were also abundant in the culture-independent community. Epifluorescence microscopy and matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS) were utilized to detect the chemical defense molecule kahalalide F on a longitudinal section of the sacoglossan.     

The symbiotic chloroplasts in the sacoglossan Elysia clarki are from several algal species

Abstract. The sacoglossan sea slug Elysia clarki feeds on siphonaceous algae, and intracellularly sequesters chloroplasts, which actively photosynthesize for 4 months. We have determined the algal source of chloroplasts in adults of E. clarki from the Florida Keys, using molecular techniques, feeding experiments, and electron microscopy. Our results clearly demonstrate that specimens of E. clarki sequester chloroplasts from four different species of algae, representing two genera: Penicillus lamourouxii, P. capitatus, Halimeda incrassata , and H. monile. In addition, chloroplasts from more than one species of algae are sequestered simultaneously in the same digestive cell.     

ULTRASTRUCTURAL AND ECOLOGICAL ASPECTS OF THE DEVELOPMENT OF CHLOROPLAST RETENTION IN THE SACOGLOSSAN GASTROPOD ELYSIA TIMIDA

Elysia timida is a common and endemic inhabitant of shallowand very well lit waters in the Mediterranean. This sacoglossanslug retains functional symbiotic chloroplasts derived fromits algal food, Acetabularia acetabulum, although the chloroplastsare not transmitted in the spawn. After hatching and until day12, Elysia timida juveniles do not retain these chloroplastsin the digestive gland. However, newly hatched juveniles retainchloroplasts from Cladophora dalmatica. Development varies seasonallybetween direct (December to April) and lecithotrophic (October,November and May), and this variation may be an adaptation toseasonal calcification of the algal food Acetabularia acetabulum. (Received 12 February 1991; accepted 15 July 1992)     

Discovery of a photosynthesizing animal that can survive for months in a light-dependent manner

Recently M. E. Rumpho and coworkers (USA) established that the marine slug Elysia chlorotica, a gastropod mollusk that feeds on the eukaryotic filamentous yellow-green alga Vaucheria litorea, recruits chloroplasts from the alga and transports them from the digestive apparatus into a special organ of the slug that resembles a green leaf and is an approximately 100-fold increased parapodium—an outgrowth of the slug’s body. The chloroplasts survive inside the slug for up to 10 months and perform active photosynthesis accompanied by assimilation of CO₂. Under conditions of starvation, this photosynthesis becomes for the animal the only source of energy and fixed carbon. For functioning, chloroplasts have to constantly import some short-lived proteins that are encoded in the nucleus of the photosynthesizing organism. Therefore, the authors supposed that a transfer of the corresponding genes must have occurred between the algal and mollusk nuclei. This hypothesis was experimentally confirmed for two genes encoding proteins of the photosynthesizing apparatus. The questions arise of what mechanism was responsible for the transfer of these genes and how the slug created its photosynthesizing organ resembling the leaf of a higher plant rather than the primitive filamentous algal structure which was the source of the acquired chloroplasts and the photosynthesis genes.     

Analysis of chloroplast ribosomes by polyacrylamide gel electrophoresis and electron microscopy

Ribosomal particles were isolated from chloroplasts and cytoplasm of eukaryotes, Euglena gracilis and Spinacia oleracea, and from prokaryotes, E. coli and Anacystis nidulans. The ribosomes were analyzed by polyacrylamide gel electrophoresis and by negative staining in the electron microscope. The prokaryote ribosomes show a slight difference in their electrophoretic mobilities between the two species: E. coli ribosomes migrate ahead of the Anacystis ribosomes. In comparison to eukaryote cytoplasmic ribosomes, chloroplast ribosomes of both species demonstrate a higher electrophoretic mobility and significantly smaller dimensions (about 230 × 187 Å compared to about 197 × 162 Å). Some differences in form were noted for Euglena cytoplasmic ribosomes which may contribute to their high S value. In comparison to prokaryote ribosomes, the mobility of chloroplast ribosomes is similar to the mobility of the prokaryote group of ribosomes, and it specifically coincides with the migrating band of ribosomes from the blue-green alga, Anacystis. Subunits of chloroplast and prokaryote ribosomes have similar mobilities and cannot be distinguished in gels. The similarities in size and in electrophoretic mobilities of chloroplast and blue-green algal ribosomes support the hypothesis of a common phylogenetic origin for the two.     

Individual Specialization to Non-Optimal Hosts in a Polyphagous Marine Invertebrate Herbivore

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.     

Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for Illumina genome sequencing

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.     

Persistence of Functional Chloroplasts in <Emphasis Type="Italic">Elysia viridis</Emphasis> (Opisthobranchia,Sacoglossa)

SEVERAL species of sacoglossan mollusc retain in the cells of their digestive diverticula large numbers of chloroplasts derived from their food plants (mostly siphonaceous algae). These associations are capable of photosynthesis^1–4. In freshly collected Elysia viridis (Montagu) (which obtains chloroplasts from Codium fragile (Sur.) Hariot) the net rate of fixation is approximately 40% of that in the intact seaweed and the chlorophyll content, g^?1 fresh weight, is similar in animal derived material and seaweed (Trench, Boyle and Smith, in preparation). This report describes an experiment showing that E. viridis can retain functional chloroplasts for at least 3 months when starved in the light and at least one month when starved in the dark.     

Mollusc-algal chloroplast endosymbiosis. Photosynthesis, thylakoid protein maintenance, and chloroplast gene expression continue for many months in the absence of the algal nucleus

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 CO(2) fixation for at least 9 months if provided with only light and a source of CO(2). 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 of V. 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.     

Feeding by coral reef mesograzers: algae or cyanobacteria?

Marine studies on herbivory have addressed the role of algae as food and shelter for small consumers, but the potential of benthic cyanobacteria to play similar roles is largely unknown. Here, feeding preferences were measured for eight invertebrate consumers from Guam, offered four common macroalgae and two cyanobacteria. The survivorship of another consumer raised on either macroalgae or cyanobacteria was also assessed. From the choices offered, the sacoglossans Elysia rufescens and E. ornata consumed the green macroalga Bryopsis pennata. The crab Menaethius monoceros preferred the red alga Acanthophora spicifera. The amphipods Parhyale hawaiensis and Cymadusa imbroglio consumed macroalgae and cyanobacteria in equivalent amounts, with C. imbroglio showing less selectivity among diets. In contrast to these patterns, in these assays the gastropods Stylocheilus striatus, Haminoea cymbalum, H. ovalis, and Haminoea sp. fed exclusively, or survived only, on cyanobacteria. Preferences for different cyanobacteria varied. Field surveys of cyanobacteria-associated species yielded 34 different invertebrate taxa and suggested different degrees of specificity in these associations. Tropical mesograzers exploit considerably different food resources, with some species adapted to consume cyanobacterial mats. Benthic cyanobacteria may play important roles as food and shelter for marine consumers and may indirectly influence local biodiversity through their associated fauna.     

PHYLOGENETIC ANALYSIS OF THE GENUS EUGLENA (EUGLENOPHYCEAE) WITH PARTICULAR REFERENCE TO THE TYPE SPECIES EUGLENA VIRIDIS1

Euglena viridis (subgenus Euglena) serves as the type species for the genus Euglena. In this study, molecular phylogenetic analyses using a small subunit (SSU) and a combined SSU–partial large subunit rDNA data set for members of the genus Euglena showed that strains identified as E. viridis on the basis of morphology are distributed between two separate nonsister clades. Although all the E. viridis strains examined were morphologically indistinguishable and possessed spherical mucocysts and stellate chloroplasts with one paramylon center, there was a high degree of sequence divergence between the E. viridis strains in different clades, making this a cryptic species. Like E. viridis, all taxa from the subgenus Euglena are characterized by having one or more stellate chloroplasts with paramylon grains clustered around the center of the chloroplast. These additional taxa were divided into four clades in all the molecular analyses. Strains of Euglena stellata formed two nonsister clades whose members had a single aggregate chloroplast with paramylon center and spindle‐shaped mucocysts. A geniculata clade included species with one or two stellate chloroplasts with paramylon centers and spherical mucocysts, and the cantabrica clade had members with one stellate chloroplast with paramylon center and spherical mucocysts often arranged in spiral rows. Interspersed among these were three additional clades bearing taxa from the subgenus Calliglena that contains members with discoid plastids and pyrenoids that may or may not be capped with paramylon. These taxa formed a laciniata clade, mutabilis clade, and gracilis clade. This study demonstrates that E. viridis and E. stellata are cryptic species that can only be distinguished at the molecular level. Because E. viridis is the designated type species for the genus Euglena, we designated an epitype for E. viridis.     

THE OCCURRENCE AND SIGNIFICANCE OF ENDOSYMBIOTIC CHLOROPLASTS IN THE DIGESTIVE GLANDS OF HERBIVOROUS OPISTHOBRANCHS1

Intact algal chloroplasts have been found in the digestive glands of 5 species of Opisthobranchia belonging to the order Saccoglossa. Preliminary studies on 3 of these confirm their endosymbiotic nature. It is suggested that the occurrence of these endosymbiotic organelles may be widespread among related species of Saccoglossa. Their independent functional existence supports the view that chloroplasts possess a system of nonchromosomal inheritance.     

Chloroplast incorporation and long-term photosynthetic performance through the life cycle in laboratory cultures of Elysia timida (Sacoglossa,Heterobranchia)

Introduction

The Mediterranean sacoglossan Elysia timida is one of the few sea slug species with the ability to sequester chloroplasts from its food algae and to subsequently store them in a functional state in the digestive gland cells for more than a month, during which time the plastids retain high photosynthetic activity (= long-term retention). Adult E. timida have been described to feed on the unicellular alga Acetabularia acetabulum in their natural environment. The suitability of E. timida as a laboratory model culture system including its food source was studied.

Results

In contrast to the literature reporting that juvenile E. timida feed on Cladophora dalmatica first, and later on switch to the adult diet A. acetabulum, the juveniles in this study fed directly on A. acetabulum (young, non-calcified stalks); they did not feed on the various Cladophora spp. (collected from the sea or laboratory culture) offered. This could possibly hint to cryptic speciation with no clear morphological differences, but incipient ecological differentiation. Transmission electron microscopy of chloroplasts from A. acetabulum after initial intake by juvenile E. timida showed different states of degradation — in conglomerations or singularly — and fragments of phagosome membranes, but differed from kleptoplast images of C. dalmatica in juvenile E. timida from the literature. Based on the finding that the whole life cycle of E. timida can be completed with A. acetabulum as the sole food source, a laboratory culture system was established. An experiment with PAM-fluorometry showed that cultured E. timida are also able to store chloroplasts in long-term retention from Acetabularia peniculus, which stems from the Indo-Pacific and is not abundant in the natural environment of E. timida. Variations between three experiment groups indicated potential influences of temperature on photosynthetic capacities.

Conclusions

E. timida is a viable laboratory model system to study photosynthesis in incorporated chloroplasts (kleptoplasts). Capacities of chloroplast incorporation in E. timida were investigated in a closed laboratory culture system with two different chloroplast donors and over extended time periods about threefold longer than previously reported.