Kleptoplasty.

Gregor Christa introduces kleptoplasty - the process by which heterotrophs steal chloroplasts from algae and incorporate them into their cytosol.

The complete mitochondrial genome of the photosymbiotic sea slug Berghia stephanieae (Valdés, 2005) (Gastropoda, Nudibranchia).

Berghia stephanieae (Nudibranchia, Cladobranchia) is a photosymbiotic sea slug that feeds exclusively on sea anemones from the genus Exaiptasia. It then specifically incorporates dinoflagellates belonging to the Symbiodiniaceae obtained from their prey. Here, we present the complete mitochondrial genome sequence of B. stephanieae combining Oxford Nanopore long read and Illumina short-read sequencing data. The mitochondrial genome has a total length of 14,786 bp, it contains the 13 protein-encoding genes, 23 tRNAs, and two rRNAs and is similar to other nudibranchs except for the presence of a duplicated tRNA-Ser 1.

Identification of scavenger receptors and thrombospondin-type-1 repeat proteins potentially relevant for plastid recognition in Sacoglossa.

Functional kleptoplasty is a photosymbiotic relationship, in which photosynthetically active chloroplasts serve as an intracellular symbiont for a heterotrophic host. Among Metazoa, functional kleptoplasty is only found in marine sea slugs belonging to the Sacoglossa and recently described in Rhabdocoela worms. Although functional kleptoplasty has been intensively studied in Sacoglossa, the fundamentals of the specific recognition of the chloroplasts and their subsequent incorporation are unknown. The key to ensure the initiation of any symbiosis is the ability to specifically recognize the symbiont and to differentiate a symbiont from a pathogen. For instance, in photosymbiotic cnidarians, several studies have shown that the host innate immune system, in particular scavenger receptors (SRs) and thrombospondin-type-1 repeat (TSR) protein superfamily, is playing a major role in the process of recognizing and differentiating symbionts from pathogens. In the present study, SRs and TSRs of three Sacoglossa sea slugs, Elysia cornigera, Elysia timida, and Elysia chlorotica, were identified by translating available transcriptomes into potential proteins and searching for receptor specific protein and/or transmembrane domains. Both receptors classes are highly diverse in the slugs, and many new domain arrangements for each receptor class were found. The analyses of the gene expression of these three species provided a set of species-specific candidate genes, that is, SR-Bs, SR-Es, C-type lectins, and TSRs, that are potentially relevant for the recognition of kleptoplasts. The results set the base for future experimental studies to understand if and how these candidate receptors are indeed involved in chloroplast recognition.

An LED-based multi-actinic illumination system for the high throughput study of photosynthetic light responses.

The responses of photosynthetic organisms to light stress are of interest for both fundamental and applied research. Functional traits related to the photoinhibition, the light-induced loss of photosynthetic efficiency, are particularly interesting as this process is a key limiting factor of photosynthetic productivity in algae and plants. The quantitative characterization of light responses is often time-consuming and calls for cost-effective high throughput approaches that enable the fast screening of multiple samples. Here we present a novel illumination system based on the concept of 'multi-actinic imaging' of in vivo chlorophyll fluorescence. The system is based on the combination of an array of individually addressable low power RGBW LEDs and custom-designed well plates, allowing for the independent illumination of 64 samples through the digital manipulation of both exposure duration and light intensity. The illumination system is inexpensive and easily fabricated, based on open source electronics, off-the-shelf components, and 3D-printed parts, and is optimized for imaging of chlorophyll fluorescence. The high-throughput potential of the system is illustrated by assessing the functional diversity in light responses of marine macroalgal species, through the fast and simultaneous determination of kinetic parameters characterizing the response to light stress of multiple samples. Although the presented illumination system was primarily designed for the measurement of phenotypic traits related to photosynthetic activity and photoinhibition, it can be potentially used for a number of alternative applications, including the measurement of chloroplast phototaxis and action spectra, or as the basis for microphotobioreactors.

Searching for antigenotoxic properties of marine macroalgae dietary supplementation against endogenous and exogenous challenges.

The functional characterization of marine macroalgae toward their potential to strength genome protection is still scarce. Hence, the aim of this study was to assess the antigenotoxic potential of Ulva rigida, Fucus vesiculosus, and Gracilaria species in Drosophila melanogaster following dietary exposure and adopting the somatic mutation and recombination test (SMART). All macroalgae displayed a genoprotection activity, namely against an exogenous challenge (streptonigrin). The action against subtler endogenous pressures was also noted indicating that supplementation level is a critical factor. Gracilaria species provided ambivalent indications, since 10% of G. vermiculophylla inhibited the egg laying and/or larvae development, while 10% of G. gracilis promoted spontaneous genotoxicity. The effects of U. rigida were modulated (in intensity) by the growing conditions, demonstrating higher genoprotection against streptonigrin-induced damage when grown in an aquaculture-controlled system, while the effectiveness against spontaneous genotoxicity was more apparent in specimens grown under wild conditions. In contrast, F. vesiculosus did not produce significant differences in its potential under varying growing conditions. Overall, these findings shed some light on the macroalgae ability toward genome protection, contributing to the development of algaculture industry, and reinforcing the concept of functional food and its benefits.

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?

Mitochondrial Genome Assemblies of Elysia timida and Elysia cornigera and the Response of Mitochondrion-Associated Metabolism during Starvation.

Some sacoglossan sea slugs sequester functional plastids (kleptoplasts) from their food, which continue to fix CO2 in a light dependent manner inside the animals. In plants and algae, plastid and mitochondrial metabolism are linked in ways that reach beyond the provision of energy-rich carbon compounds through photosynthesis, but how slug mitochondria respond to starvation or alterations in plastid biochemistry has not been explored. We assembled the mitochondrial genomes of the plastid-sequestering sea slugs Elysia timida and Elysia cornigera from RNA-Seq data that was complemented with standard sequencing of mitochondrial DNA through primer walking. Our data confirm the sister species relationship of the two Sacoglossa and from the analysis of changes in mitochondrial-associated metabolism during starvation we speculate that kleptoplasts might aid in the rerouting or recycling of reducing power independent of, yet maybe improved by, photosynthesis.

Photoprotection in a monophyletic branch of chlorophyte algae is independent of energy-dependent quenching (qE).

Phototrophic organisms need to ensure high photosynthetic performance whilst suppressing reactive oxygen species (ROS)-induced stress occurring under excess light conditions. The xanthophyll cycle (XC), related to the high-energy quenching component (qE) of the nonphotochemical quenching (NPQ) of excitation energy, is considered to be an obligatory component of photoprotective mechanisms. The pigment composition of at least one representative of each major clade of Ulvophyceae (Chlorophyta) was investigated. We searched for a light-dependent conversion of pigments and investigated the NPQ capacity with regard to the contribution of XC and the qE component when grown under different light conditions. A XC was found to be absent in a monophyletic group of Ulvophyceae, the Bryopsidales, when cultivated under low light, but was triggered in one of the 10 investigated bryopsidalean species, Caulerpa cf. taxifolia, when cultivated under high light. Although Bryopsidales accumulate zeaxanthin (Zea) under high-light (HL) conditions, NPQ formation is independent of a XC and not related to qE. qE- and XC-independent NPQ in the Bryopsidales contradicts the common perception regarding its ubiquitous occurrence in Chloroplastida. Zea accumulation in HL-acclimated Bryopsidales most probably represents a remnant of a functional XC. The existence of a monophyletic algal taxon that lacks qE highlights the need for broad biodiversity studies on photoprotective mechanisms.

Why It Is Time to Look Beyond Algal Genes in Photosynthetic Slugs.

Eukaryotic organelles depend on nuclear genes to perpetuate their biochemical integrity. This is true for mitochondria in all eukaryotes and plastids in plants and algae. Then how do kleptoplasts, plastids that are sequestered by some sacoglossan sea slugs, survive in the animals' digestive gland cells in the absence of the algal nucleus encoding the vast majority of organellar proteins? For almost two decades, lateral gene transfer (LGT) from algae to slugs appeared to offer a solution, but RNA-seq analysis, later supported by genome sequencing of slug DNA, failed to find any evidence for such LGT events. Yet, isolated reports continue to be published and are readily discussed by the popular press and social media, making the data on LGT and its support for kleptoplast longevity appear controversial. However, when we take a sober look at the methods used, we realize that caution is warranted in how the results are interpreted. There is no evidence that the evolution of kleptoplasty in sea slugs involves LGT events. Based on what we know about photosystem maintenance in embryophyte plastids, we assume kleptoplasts depend on nuclear genes. However, studies have shown that some isolated algal plastids are, by nature, more robust than those of land plants. The evolution of kleptoplasty in green sea slugs involves many promising and unexplored phenomena, but there is no evidence that any of these require the expression of slug genes of algal origin.

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.

The Cylindrobulla/Ascobulla complex--unraveling problems in identification and adding to Cylindrobulla diversity (Gastropoda, Heterobranchia, Sacoglossa) by describing a new species.

Sacoglossa (Gastropoda: Heterobranchia) are generally considered a monophyletic group, previously associated within the now defunct "Opisthobranchia", but now basally located within Panpulmonata. In the light of this new phylogenetic hypothesis, detailed knowledge of the most basal groups within Sacoglossa is of paramount importance. This study focuses on the genus Cylindrobulla, which is usually considered the most basal group within the Sacoglossa from a morphological point of view, because it does not share the typical elongate radula teeth of all other Sacoglossa. We describe a new species, Cylindrobulla schuppi sp. nov., and provide data on its food. We reexamined and clarify the radula of the type species C. beauii, review the genus with all other valid species, provide new characters to aid in the proper identification of species within this genus, compare it to the very similar genus Ascobulla, present a determination key using external characters to ensure proper identification of the two similar genera, and discuss phylogenetic relationships within the shelled sacoglossan, the Oxynoacea.

Switching off photosynthesis: The dark side of sacoglossan slugs.

Sometimes the elementary experiment can lead to the most surprising result. This was recently the case when we had to learn that so-called "photosynthetic slugs" survive just fine in the dark and with chemically inhibited photosynthesis. Sacoglossan sea slugs feed on large siphonaceous, often single-celled algae by ingesting their cytosolic content including the organelles. A few species of the sacoglossan clade fascinate researcher from many disciplines, as they can survive starvation periods of many months through the plastids they sequestered, but not immediately digested - a process known as kleptoplasty. Ever since the term "leaves that crawl" was coined in the 1970s, the course was set in regard to how the subject was studied, but the topics of how slugs survive starvation and what for instance mediates kleptoplast longevity have often been conflated. It was generally assumed that slugs become photoautotrophic upon plastid sequestration, but most recent results challenge that view and the predominant role of the kleptoplasts in sacoglossan sea slugs.

Plastid survival in the cytosol of animal cells.

Some marine slugs sequester plastids from their algae food, which can remain photosynthetically functional in the animal's digestive gland cells in the absence of algal nuclei. The sequestered plastids (kleptoplasts) appear to maintain functional photosystems through a greater autonomy than land plant plastids. If so, kleptoplast robustness is a plastid-intrinsic property, and it depends on the animal to manage an alien organelle on the loose in order to maintain it long term.

Identification of sequestered chloroplasts in photosynthetic and non-photosynthetic sacoglossan sea slugs (Mollusca, Gastropoda).

BACKGROUND: Sacoglossan sea slugs are well known for their unique ability among metazoans to incorporate functional chloroplasts (kleptoplasty) in digestive glandular cells, enabling the slugs to use these as energy source when starved for weeks and months. However, members assigned to the shelled Oxynoacea and Limapontioidea (often with dorsal processes) are in general not able to keep the incorporated chloroplasts functional. Since obviously no algal genes are present within three (out of six known) species with chloroplast retention of several months, other factors enabling functional kleptoplasty have to be considered. Certainly, the origin of the chloroplasts is important, however, food source of most of the about 300 described species is not known so far. Therefore, a deduction of specific algal food source as a factor to perform functional kleptoplasty was still missing. RESULTS: We investigated the food sources of 26 sacoglossan species, freshly collected from the field, by applying the chloroplast marker genes tufA and rbcL and compared our results with literature data of species known for their retention capability. For the majority of the investigated species, especially for the genus Thuridilla, we were able to identify food sources for the first time. Furthermore, published data based on feeding observations were confirmed and enlarged by the molecular methods. We also found that certain chloroplasts are most likely essential for establishing functional kleptoplasty. CONCLUSIONS: Applying DNA-Barcoding appeared to be very efficient and allowed a detailed insight into sacoglossan food sources. We favor rbcL for future analyses, but tufA might be used additionally in ambiguous cases. We narrowed down the algal species that seem to be essential for long-term-functional photosynthesis: Halimeda, Caulerpa, Penicillus, Avrainvillea, Acetabularia and Vaucheria. None of these were found in Thuridilla, the only plakobranchoidean genus without long-term retention forms. The chloroplast type, however, does not solely determine functional kleptoplasty; members of no-retention genera, such as Cylindrobulla or Volvatella, feed on the same algae as e.g., the long-term-retention forms Plakobranchus ocellatus or Elysia crispata, respectively. Evolutionary benefits of functional kleptoplasty are still questionable, since a polyphagous life style would render slugs more independent of specific food sources and their abundance.

Plastid-bearing sea slugs fix CO2 in the light but do not require photosynthesis to survive.

Several sacoglossan sea slugs (Plakobranchoidea) feed upon plastids of large unicellular algae. Four species--called long-term retention (LtR) species--are known to sequester ingested plastids within specialized cells of the digestive gland. There, the stolen plastids (kleptoplasts) remain photosynthetically active for several months, during which time LtR species can survive without additional food uptake. Kleptoplast longevity has long been puzzling, because the slugs do not sequester algal nuclei that could support photosystem maintenance. It is widely assumed that the slugs survive starvation by means of kleptoplast photosynthesis, yet direct evidence to support that view is lacking. We show that two LtR plakobranchids, Elysia timida and Plakobranchus ocellatus, incorporate (14)CO2 into acid-stable products 60- and 64-fold more rapidly in the light than in the dark, respectively. Despite this light-dependent CO2 fixation ability, light is, surprisingly, not essential for the slugs to survive starvation. LtR animals survived several months of starvation (i) in complete darkness and (ii) in the light in the presence of the photosynthesis inhibitor monolinuron, all while not losing weight faster than the control animals. Contrary to current views, sacoglossan kleptoplasts seem to be slowly digested food reserves, not a source of solar power.

Is ftsH the key to plastid longevity in sacoglossan slugs?

Plastids sequestered by sacoglossan sea slugs have long been a puzzle. Some sacoglossans feed on siphonaceous algae and can retain the plastids in the cytosol of their digestive gland cells. There, the stolen plastids (kleptoplasts) can remain photosynthetically active in some cases for months. Kleptoplast longevity itself challenges current paradigms concerning photosystem turnover, because kleptoplast photosystems remain active in the absence of nuclear algal genes. In higher plants, nuclear genes are essential for plastid maintenance, in particular, for the constant repair of the D1 protein of photosystem II. Lateral gene transfer was long suspected to underpin slug kleptoplast longevity, but recent transcriptomic and genomic analyses show that no algal nuclear genes are expressed from the slug nucleus. Kleptoplast genomes themselves, however, appear expressed in the sequestered state. Here we present sequence data for the chloroplast genome of Acetabularia acetabulum, the food source of the sacoglossan Elysia timida, which can maintain Acetabularia kleptoplasts in an active state for months. The data reveal what might be the key to sacoglossan kleptoplast longevity: plastids that remain photosynthetically active within slugs for periods of months share the property of encoding ftsH, a D1 quality control protease that is essential for photosystem II repair. In land plants, ftsH is always nuclear encoded, it was transferred to the nucleus from the plastid genome when Charophyta and Embryophyta split. A replenishable supply of ftsH could, in principle, rescue kleptoplasts from D1 photodamage, thereby influencing plastid longevity in sacoglossan slugs.

What remains after 2 months of starvation? Analysis of sequestered algae in a photosynthetic slug, Plakobranchus ocellatus (Sacoglossa, Opisthobranchia), by barcoding.

The sacoglossan sea slug, Plakobranchus ocellatus, is a so-called long-term retention form that incorporates chloroplasts for several months and thus is able to starve while maintaining photosynthetic activity. Little is known regarding the taxonomy and food sources of this sacoglossan, but it is suggested that P. ocellatus is a species complex and feeds on a broad variety of Ulvophyceae. In particular, we analysed specimens from the Philippines and starved them under various light conditions (high light, low light and darkness) and identified the species of algal food sources depending on starvation time and light treatment by means of DNA-barcoding using for the first time the combination of two algal chloroplast markers, rbcL and tufA. Comparison of available CO1 and 16S sequences of specimens from various localities indicate a species complex with likely four distinct clades, but food analyses do not indicate an ecological separation of the investigated clades into differing foraging strategies. The combined results from both algal markers suggest that, in general, P. ocellatus has a broad food spectrum, including members of the genera Halimeda, Caulerpa, Udotea, Acetabularia and further unidentified algae, with an emphasis on H. macroloba. Independent of the duration of starvation and light exposure, this algal species and a further unidentified Halimeda species seem to be the main food source of P. ocellatus from the Philippines. It is shown here that at least two (or possibly three) barcode markers are required to cover the entire food spectrum in future analyses of Sacoglossa.

Transcriptomic evidence that longevity of acquired plastids in the photosynthetic slugs Elysia timida and Plakobranchus ocellatus does not entail lateral transfer of algal nuclear genes.

Sacoglossan sea slugs are unique in the animal kingdom in that they sequester and maintain active plastids that they acquire from the siphonaceous algae upon which they feed, making the animals photosynthetic. Although most sacoglossan species digest their freshly ingested plastids within hours, four species from the family Plakobranchidae retain their stolen plastids (kleptoplasts) in a photosynthetically active state on timescales of weeks to months. The molecular basis of plastid maintenance within the cytosol of digestive gland cells in these photosynthetic metazoans is yet unknown but is widely thought to involve gene transfer from the algal food source to the slugs based upon previous investigations of single genes. Indeed, normal plastid development requires hundreds of nuclear-encoded proteins, with protein turnover in photosystem II in particular known to be rapid under various conditions. Moreover, only algal plastids, not the algal nuclei, are sequestered by the animals during feeding. If algal nuclear genes are transferred to the animal either during feeding or in the germ line, and if they are expressed, then they should be readily detectable with deep-sequencing methods. We have sequenced expressed mRNAs from actively photosynthesizing, starved individuals of two photosynthetic sea slug species, Plakobranchus ocellatus Van Hasselt, 1824 and Elysia timida Risso, 1818. We find that nuclear-encoded, algal-derived genes specific to photosynthetic function are expressed neither in P. ocellatus nor in E. timida. Despite their dramatic plastid longevity, these photosynthetic sacoglossan slugs do not express genes acquired from algal nuclei in order to maintain plastid function.