Functional chloroplasts in metazoan cells - a unique evolutionary strategy in animal life

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.     

Short-term retention of kleptoplasty from a green alga (Bryopsis) in the sea slug Placida sp. YS001

The capacity of sea slugs (sacoglossans) for retaining chloroplasts from food algae provides important insights into endosymbiotic relationships and kleptoplasty. A sea slug species was captured accidentally in the Yellow Sea and identified as Placida sp. YS001 based on phylogenetic analyses of the COX1 and 16S gene sequence. Its life cycle was recorded using microscope. Photosynthetic analysis by pulse amplitude modulated fluorometry during starvation revealed shortterm functional kleptoplasty. An ultrastructural comparison of the slug and alga showed that a change in the chloroplast structure and the phagosome might correspond to short-term endosymbiosis. The horizontally transferred genes, psbO and lectin, were not cloned in the adults or eggs. This study demonstrates the morphological adaptation that occurs during short-term endosymbiotic relationships and provides fresh insights.     

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.     

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.     

Molecular determination of kleptoplast origins from the sea slug Plakobranchus ocellatus (Sacoglossa,Gastropoda) reveals cryptic bryopsidalean (Chlorophyta) diversity in the Hawaiian Islands

The sacoglossan sea slug species complex Plakobranchus ocellatus is a common algivore throughout the tropical Pacific, including the Hawaiian Islands. Plakobranchus ocellatus is kleptoplastic—it sequesters and retains algal chloroplasts—a characteristic that can be exploited to molecularly characterize diminutive bryopsidalean algae that are typically difficult to locate, collect, and identify. Previous DNA barcode analyses of both P. ocellatus and its kleptoplasts have been conducted primarily in the western Pacific and have only minimally sampled the most eastern populations in the Hawaiian Islands. Using two chloroplast markers, rbcL and tufA, kleptoplast samples from an Oahu population of P. ocellatus were amplified and cloned to identify their algal sources. Plakobranchus ocellatus sequester chloroplasts from up to 11 bryopsidalean algal species, all but one being diminutive in thallus size. Notably, eight of the detected algal species were new records to the Hawaiian Islands. A sequestration preference study demonstrated that the O‘ahu population of P. ocellatus preferentially sequesters chloroplasts from diminutive, epilithic taxa. Using coxI barcoding of P. ocellatus, we showed the O‘ahu population to be part of a clade that includes sequences from the neighboring island Maui, Australia, and the Philippines. The use of P. ocellatus as a novel sampling tool allows the exploration of the green algal community diversity and composition at a fine scale.     

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.     

Polymorphic adaptations in metazoans to establish and maintain photosymbioses

Mutualistic symbioses are common throughout the animal kingdom. Rather unusual is a form of symbiosis, photosymbiosis, where animals are symbiotic with photoautotrophic organisms. Photosymbiosis is found among sponges, cnidarians, flatworms, molluscs, ascidians and even some amphibians. Generally the animal host harbours a phototrophic partner, usually a cyanobacteria or a unicellular alga. An exception to this rule is found in some sea slugs, which only retain the chloroplasts of the algal food source and maintain them photosynthetically active in their own cytosol – a phenomenon called ‘functional kleptoplasty’. Research has focused largely on the biodiversity of photosymbiotic species across a range of taxa. However, many questions with regard to the evolution of the ability to establish and maintain a photosymbiosis are still unanswered. To date, attempts to understand genome adaptations which could potentially lead to the evolution of photosymbioses have only been performed in cnidarians. This knowledge gap for other systems is mainly due to a lack of genetic information, both for non‐symbiotic and symbiotic species. Considering non‐photosymbiotic species is, however, important to understand the factors that make symbiotic species so unique. Herein we provide an overview of the diversity of photosymbioses across the animal kingdom and discuss potential scenarios for the evolution of this association in different lineages. We stress that the evolution of photosymbiosis is probably based on genome adaptations, which (i) lead to recognition of the symbiont to establish the symbiosis, and (ii) are needed to maintain the symbiosis. We hope to stimulate research involving sequencing the genomes of various key taxa to increase the genomic resources needed to understand the most fundamental question: how have animals evolved the ability to establish and maintain a photosymbiosis?     

Digestive system of the sacoglossan Plakobranchus ocellatus (Gastropoda: Opisthobranchia): light- and electron-microscopic observations with remarks on chloroplast retention

The sacoglossan Plakobranchus ocellatus feeds by sucking the cytoplasmic contents from algae and retains intact algal chloroplasts within the cells of the digestive gland. Morphology of the entire digestive system of this species was firstly described by means of a combination of histology and electron microscopy (both SEM and TEM). The short alimentary canal is confined to the head, and the anus opens at the anterior right corner of the pericardial swelling, as is the case in many non-shelled sacoglossans. The alimentary canal of the specimens examined rarely contained ingesta, suggesting that the retained chloroplasts provide sufficient nourishment to the sacoglossan hosts and that sea slugs with empty stomachs survive well in the field. The digestive gland, with the retained chloroplasts, branches from the stomach and is sparsely distributed throughout the body, including the head region, but is aggregated mainly in the dorsal lamellae. Chloroplasts were occasionally found in the epithelial cells in the transitional region from the stomach wall to the digestive gland, which may be a site at which chloroplasts are incorporated into the animal cells by endocytosis. Numerous microvilli filling the lumen of the digestive gland suggest that molecules are actively transferred within the gland. The sea slug thus apparently provides a favorable environment to support the long-term retention and function of chloroplasts.     

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.     

Mollusc/algal chloroplast symbiosis: how can isolated chloroplasts continue to function for months in the cytosol of a sea slug in the absence of an algal nucleus?

A marine sea slug, Elysia chlorotica, has acquired the ability to carry out photosynthesis as a result of forming an intracellular symbiotic association with chloroplasts of the chromophytic alga, Vaucheria litorea. The symbiont chloroplasts (kleptoplasts) are functional, i.e. they evolve oxygen and fix CO₂ and actively transcribe and translate proteins for several months in the sea slug cytosol. Considering the dependency of plastid function on nuclear genes, the level of kleptoplast activity observed in the animal cell is quite remarkable. Possible factors contributing to this long-lasting functional association that are considered here include: the presence of an algal nuclear genome in the sea slug, autonomous chloroplasts, unusual chloroplast/protein stability, re-directing of animal proteins to the kleptoplast, and lateral gene transfer. Based on our current understanding, the acquisition and incorporation of intact algal plastids by E. chlorotica is aided by the robustness of the plastids and the long-term functional activity of the kleptoplasts appears to be supported by both plastid and protein stability and contributions from the sea slug.     

Acquired Phototrophy through Retention of Functional Chloroplasts Increases Growth Efficiency of the Sea Slug Elysia viridis

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.     

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.     

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.     

Interspecific evolution: microbial symbiosis,endosymbiosis and gene transfer

Microbial symbioses are interesting in their own right and also serve as exemplary models to help biologists to understand two important symbioses in the evolutionary past of eukaryotic cells: the origins of chloroplasts and mitochondria. Most, if not all, microbial symbioses have a chemical basis: compounds produced by one partner are useful for the other. But symbioses can also entail the transfer of genes from one partner to the other, which in some cases cements two cells into a bipartite, co-evolving unit. Here, we discuss some microbial symbioses in which progress is being made in uncovering the nature of symbiotic interactions: anaerobic methane-oxidizing consortia, marine worms that possess endosymbionts instead of a digestive tract, amino acid-producing endosymbionts of aphids, prokaryotic endosymbionts living within a prokaryotic host within mealybugs, endosymbionts of an insect vector of human disease and a photosynthetic sea slug that steals chloroplasts from algae. In the case of chloroplasts and mitochondria, examples of recent and ancient gene transfer to the chromosomes of their host cell illustrate the process of genetic merger in the wake of organelle origins.     

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.     

Ultrastructure of sequestered chloroplasts in sacoglossan gastropods with differing abilities for plastid uptake and maintenance

Abstract. Many sacoglossan sea slugs incorporate intact, functional chloroplasts from their algal food sources into specialized cells lining the digestive diverticulum. The chloroplasts in adults of Elysia clarki are photosynthetically functional for many months. Members of this species feed on algae in the Ulvophyceae, including species of Penicillus and Bryopsis. However, other sacoglossans (Elysia patina, Elysia rufescens, and Placida kingstoni) use similar algal food sources as do adults of E. clarki, but are unable to maintain the chloroplasts for more than a week, with individuals of P. kingstoni apparently being unable to maintain chloroplasts for >24 h. We have examined chloroplast sequestering cells of these species looking for morphological differences that may help explain the variation in chloroplast sequestration and maintenance among them. Our results indicate that P. kingstoni does not actively sequester chloroplasts at all, digesting them instead. However, the plastid sequestering mechanisms of individuals of E. patina and E. rufescens are similar to those of E. clarki, and the degradation of chloroplasts by specimens of E. patina is ultrastructurally similar to the same process in E. clarki, although chloroplast degradation occurs much more slowly in individuals of E. clarki. Our results suggest that species-level differences in the digestive capability of the phagosomes involved in the uptake of chloroplasts account for variation in the length of these kleptoplastic associations.     

The chloroplast genome of Phacus orbicularis (Euglenophyceae): an initial datum point for the phacaceae

The Euglenophyceae chloroplast was acquired when a heterotrophic euglenoid engulfed a green alga and subsequently retained the algal chloroplast, in a process known as secondary endosymbiosis. Since this event, Euglenophyceae have diverged widely and their chloroplast genomes (cpGenomes) have as well. Changes to the cpGenome include extensive gene rearrangement and the proliferation of introns, the analyses of which have proven to be useful in examining cpGenome changes throughout the Euglenophyceae. The Euglenales fall into two families, Euglenaceae and Phacaceae. Euglenaceae contains eight genera and at least one cpGenome has been published for each genus. Phacaceae, on the other hand, contains three genera, none of which have had a representative chloroplast genome sequenced. Members of this family have many small disk‐shaped chloroplasts that lack pyrenoids. We sequenced and annotated the cpGenome of Phacus orbicularis in order to fill in the large gap in our understanding of Euglenophyceae cpGenome evolution, especially in regard to intron number and gene order. We compared this cpGenome to those of species from both the Euglenaceae and Eutreptiales of the Euglenophyceae phylogenetic tree. The cpGenome showed characteristics that were more derived than that of the basal species Eutreptia viridis, with extensive gene rearrangements and nearly three times as many introns. In contrast, it contained fewer introns than all but one of the previously reported Euglenaceae cpGenomes, had a smaller estimated genome size, and shared greater synteny with two main branches of that family.     

Algivore or Phototroph? Plakobranchus ocellatus (Gastropoda) Continuously Acquires Kleptoplasts and Nutrition from Multiple Algal Species in Nature

The sea slug Plakobranchus ocellatus (Sacoglossa, Gastropoda) retains photosynthetically active chloroplasts from ingested algae (functional kleptoplasts) in the epithelial cells of its digestive gland for up to 10 months. While its feeding behavior has not been observed in natural habitats, two hypotheses have been proposed: 1) adult P. ocellatus uses kleptoplasts to obtain photosynthates and nutritionally behaves as a photoautotroph without replenishing the kleptoplasts; or 2) it behaves as a mixotroph (photoautotroph and herbivorous consumer) and replenishes kleptoplasts continually or periodically. To address the question of which hypothesis is more likely, we examined the source algae for kleptoplasts and temporal changes in kleptoplast composition and nutritional contribution. By characterizing the temporal diversity of P. ocellatus kleptoplasts using rbcL sequences, we found that P. ocellatus harvests kleptoplasts from at least 8 different siphonous green algal species, that kleptoplasts from more than one species are present in each individual sea slug, and that the kleptoplast composition differs temporally. These results suggest that wild P. ocellatus often feed on multiple species of siphonous algae from which they continually obtain fresh chloroplasts. By estimating the trophic position of wild and starved P. ocellatus using the stable nitrogen isotopic composition of amino acids, we showed that despite the abundance of kleptoplasts, their photosynthates do not contribute greatly to the nutrition of wild P. ocellatus, but that kleptoplast photosynthates form a significant source of nutrition for starved sea slugs. The herbivorous nature of wild P. ocellatus is consistent with insights from molecular analyses indicating that kleptoplasts are frequently replenished from ingested algae, leading to the conclusion that natural populations of P. ocellatus do not rely on photosynthesis but mainly on the digestion of ingested algae.