Gregor Christa

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.

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.

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 14CO2 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.

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.

Plastid survival in the cytosol of animal cells

Some marine slugs sequester plastids from their algae food, which can remain photosynthetically functional in the animals 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.

Phylogenetic evidence for multiple independent origins of functional kleptoplasty in Sacoglossa (Heterobranchia, Gastropoda)

Sacoglossa is a rather small taxon of marine slugs with about 300 described species, yet it is quite fascinating scientists for decades. This is mainly because of the ability of certain species to incorporate photosynthetically active plastids of their algae prey, a phenomenon known as functional kleptoplasty. With the stolen plastids, these slugs endure weeks (short-term retention) or months (long-term retention) of starvation, though contribution of the plastids to the survival and factors enhancing plastid longevity are unknown. Likewise, contrasting hypotheses on evolution of functional kleptoplasty exist and the phylogenetic relationship of Sacoglossa taxa is still under debate. We analyzed the phylogenetic relationship of 105 sacoglossan species to address the question of the origin of functional kleptoplasty. Based on our phylogenetic analysis and the ancestral character state reconstruction, we conclude that functional short-term retention most likely originated two times and long-term retention at least five times. Previous suggestions that functional long-term kleptoplasty is established with specific plastids are supported by our food analyses in Elysia clarki that finally harbors only plastids of certain algae species over a prolonged starvation period.

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

BackgroundSacoglossan 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.ResultsWe 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.ConclusionsApplying 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.

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.

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.

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.