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.     

Light modulates the lipidome of the photosynthetic sea slug Elysia timida

Long-term kleptoplasty, the capability to retain functional stolen chloroplasts (kleptoplasts) for several weeks to months, has been shown in a handful of Sacoglossa sea slugs. One of these sea slugs is Elysia timida, endemic to the Mediterranean, which retains functional chloroplasts of the macroalga Acetabularia acetabulum. To understand how light modulates the lipidome of E. timida, sea slug specimens were subjected to two different 4-week light treatments: regular light and quasi-dark conditions. Lipidomic analyses were performed by HILIC-HR-ESI-MS and MS/MS. Quasi-dark conditions caused a reduction in the amount of essential lipids for photosynthetic membranes, such as glycolipids, indicating high level of kleptoplast degradation under sub-optimal light conditions. However, maximum photosynthetic capacities (F_v/F_m) were identical in both light treatments (≈0.75), showing similar kleptoplast functionality and suggesting that older kleptoplasts were targeted for degradation. Although more stable, the phospholipidome showed differences between light treatments: the amount of certain lipid species of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylglycerol (PG) decreased under quasi-dark conditions, while other lipid species of phosphatidylcholine (PC), PE and lyso-PE (LPE) increased. Quasi-dark conditions promoted a decrease in the relative abundance of polyunsaturated fatty acids. These results suggest a light-driven remodelling of the lipidome according to the functions of the different lipids and highlight the plasticity of polar lipids in the photosynthetic sea slug E. timida.     

Lipid Accumulation during the Establishment of Kleptoplasty in Elysia chlorotica

The establishment of kleptoplasty (retention of “stolen plastids”) in the digestive tissue of the sacoglossan Elysia chlorotica Gould was investigated using transmission electron microscopy. Cellular processes occurring during the initial exposure to plastids were observed in laboratory raised animals ranging from 1–14 days post metamorphosis (dpm). These observations revealed an abundance of lipid droplets (LDs) correlating to plastid abundance. Starvation of animals resulted in LD and plastid decay in animals <5 dpm that had not yet achieved permanent kleptoplasty. Animals allowed to feed on algal prey (Vaucheria litorea C. Agardh) for 7 d or greater retained stable plastids resistant to cellular breakdown. Lipid analysis of algal and animal samples supports that these accumulating LDs may be of plastid origin, as the often algal-derived 20∶5 eicosapentaenoic acid was found in high abundance in the animal tissue. Subsequent culturing of animals in dark conditions revealed a reduced ability to establish permanent kleptoplasty in the absence of photosynthetic processes, coupled with increased mortality. Together, these data support an important role of photosynthetic lipid production in establishing and stabilizing this unique animal kleptoplasty.     

Sacoglossan sea slugs make routine use of photosynthesis by a variety of species‐specific adaptations

The phenomenon of the uptake, intracellular sequestration, and subsequent usage of algal chloroplasts by the digestive cells of many species of sacoglossan sea slugs, currently called kleptoplasty, has been of considerable interest since its discovery in the 1960s. While a large body of literature reported that captured chloroplasts were photosynthetically active inside slug cells and that plastid longevity in some species might be the result of the horizontal transfer of functional algal nuclear genes into the slug genome, a few recent studies have called the older results into question. Here, we have reviewed the literature and showed that while kleptoplasty occurs in many slug species and almost all derive benefit from kleptoplast photosynthesis, the slug adaptations to maintain the chloroplasts differ from species to species. These adaptations range from behavioral to molecular, including gene transfer, in a variety of combinations.     

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.     

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.     

Linking transpiration reduction to rhizosphere salinity using a 3D coupled soil-plant model

Background and aims

In cold biomes, litter decomposition, which controls the nutrient availability for plants and the ecosystem carbon budget, is strongly influenced by climatic conditions. In this study, focused on the early litter decay within snowbed habitats, the magnitude of the short- and long-term influences of climate warming, the direction of the effects of warmer temperature and advanced snowmelt, and the control of microclimatic features and plant traits were compared.

Methods

Combining experimental warming and space-for-time substitution, mass loss and nutrient release of different plant functional types were estimated in different climatic treatments with the litter bag method.

Results

Plant functional types produced a larger variation in the early-decomposition compared to that produced by climatic treatments. Litter decay was not affected by warmer summer temperatures and reduced by advanced snowmelt. Structural-related plant traits exerted the major control over litter decomposition.

Conclusions

Long-term effects of climate warming, resulting from shifts in litter quality due to changes in the abundance of plant functional types, will likely have a stronger impact on plant litter decomposition than short-term variations in microclimatic features. This weaker response of litter decay to short-term climate changes may be partially due to the opposite influences of higher summer temperatures and advanced snowmelt time.     

Chloroplasts as functional organelles in animal tissues

The marine gastropod molluscs Tridachia crispata, Tridachiella diomedea, and Placobranchus ianthobapsus (Sacoglossa, Opisthobranchia) possess free functional chloroplasts within the cells of the digestive diverticula, as determined by observations on ultrastructure, pigment analyses, and experiments on photosynthetic capacity. In the light, the chloroplasts incorporate H¹⁴CO₃^- in situ. Reduced radiocarbon is translocated to various chloroplast-free tissues in the animals. The slugs feed on siphonaceous algae from which the chloroplasts are derived. Pigments from the slugs and from known siphonaceous algae, when separated chromatographically and compared, showed similar components. Absorption spectra of extracts of slugs and algae were very similar. The larvae of the slugs are pigment-free up to the post-veliger stage, suggesting that chloroplasts are acquired de novo. with each new generation.     

Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, <Emphasis Type="Italic">Cuscuta reflexa</Emphasis> and <Emphasis Type="Italic">Cuscuta gronovii</Emphasis>

Background  

The holoparasitic plant genus Cuscuta comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different Cuscuta species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species Cuscuta reflexa and Cuscuta gronovii. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of Epifagus virginiana, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in C. reflexa to a restricted photosynthetic activity and degenerated chloroplasts in C. gronovii to an achlorophyllous state in E. virginiana.     

Potential key characters in Opisthobranchia (Gastropoda, Mollusca) enhancing adaptive radiation

Five potential key characters which might have enhanced species radiation in the Opisthobranchia (Gastropoda) are discussed. These are: 3–4 cuticular plates in the gizzard of Cephalaspidea s.str., kleptoplasty in Sacoglossa, kleptocnides in Aeolidoidea, a symbiotic relationship with unicellular algae in Phyllodesmium Ehrenberg, 1831, and mantle dermal formations in Chromodorididae. Interpretation of the characters as key innovations is based on phylogeny and/or comparison of species numbers in subgroups. Possible adaptive zones are discussed, and alternative interpretations indicated.     

Wildfire and forest harvest disturbances in the boreal forest leave different long-lasting spatial signatures

Aims

Natural disturbances leave long-term legacies that vary among landscapes and ecosystem types, and which become integral parts of successional processes at a given location. As humans change land use, not only are immediate post-disturbance patterns altered, but the processes of recovery themselves are likely altered by the disturbance. We assessed whether short-term effects on soil and vegetation that distinguish wildfire from forest harvest persist over 60 years after disturbance in boreal black spruce forests, or post-disturbance processes of recovery promote convergence of the two disturbance types.

Methods

Using semi-variograms and Principal Coordinates of Neighbour Matrices, we formulated precise, a priori spatial hypotheses to discriminate spatial signatures following wildfire and forest harvest both over the short- (16–18 years) and long-term (62–98 years).

Results

Both over the short- and the long-term, wildfire generated a wide spectrum of responses in soil and vegetation properties at different spatial scales, while logging produced simpler patterns corresponding to the regular linear pattern of harvest trails and to pre-disturbance ericaceous shrub patches that persist between trails.

Conclusions

Disturbance by harvest simplified spatial patterns associated with soil and vegetation properties compared to patterns associated with natural disturbance by fire. The observed differences in these patterns between disturbance types persist for over 60 years. Ecological management strategies inspired by natural disturbances should aim to increase the complexity of patterns associated with harvest interventions.     

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.     

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.     

Morphological diversity of plant barriers does not increase sediment retention in eroded marly gullies under ecological restoration

Aims

Sediment retention by plant barriers initiates common strategies to conserve soil fertility or restore degraded terrains, including gullied ones. Differences in species performance for sediment retention have been studied but little is known about plant performance in retention when upscaling to plurispecific barriers. We investigated the role of morphological diversity of plant barriers in sediment retention in the context of eroded marly gullies.

Methods

Fifteen plant barriers, composed of combinations of four morphologically contrasting species (grass, shrub, dwarf-shrub and juvenile tree) were tested for their sediment retention potential in an innovative life-size artificial concentrated runoff experiment. We studied the net effect of biodiversity and the role of morphological traits on sediment retention.

Results

We found that grass barriers performed best to retain sediment and morphological diversity significantly impaired sediment retention. This negative effect may be due to runoff concentrating in the least flow-resistant areas (shrubs or trees), resulting in a localized increase in flow velocity and thus an overall decrease in sediment deposition.

Conclusion

To initiate gully restoration by increasing sediment retention in their bed, morphologically homogeneous plant barriers should be favored. Plant diversity, useful for mid- and long-term restoration goals, should be considered later in the process.     

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.     

Chloroplast DNA content in Dinophysis (Dinophyceae) from different cell cycle stages is consistent with kleptoplasty

Kleptoplasty is the retention of plastids obtained from ingested algal prey, which can remain temporarily functional and be used for photosynthesis by the predator. With a new approach based on cell cycle analysis, we have addressed the question of whether the toxic, bloom-forming dinoflagellate Dinophysis norvegica practice kleptoplasty or if they replicate their own plastid DNA. Dividing (G2) and non-dividing (G1) D. norvegica cells from a natural population were physically separated with a flow cytometer based on their DNA content. Average numbers of nuclear and plastid rDNA copies were quantified with real-time PCR both in the G1 and G2 group. Cells from the G1 group contained 5800 ± 340 copies of nuclear rDNA and 1300 ± 200 copies of plastid rDNA; cells from the G2 group contained 9700 ± 58 copies of nuclear rDNA and 1400 ± 220 copies of plastid rDNA (mean ± SD, n  = 3). The ratio G2/G1 in average rDNA copies per cell was 1.67 for nuclear DNA and 1.07 for plastid DNA. These ratios show that plastid acquisition in D. norvegica is either uncoupled with the cell cycle, or plastids accumulate rapidly in the beginning of the cell cycle owing to feeding, as would be expected in a protist with kleptoplastic behaviour but not in a protist with own plastid replication. In addition, flow cytometry measurements on cells from the same population used for real-time PCR showed that when kept without plastidic prey, live Dinophysis cells lost on average 36% of their plastid phycoerythrin fluorescence in 24 h. Together these findings strongly suggest that D. norvegica does not possess the ability for plastid replication.     

Synthesis of several light-harvesting complex I polypeptides is blocked by cycloheximide in symbiotic chloroplasts in the sea slug, Elysia chlorotica (Gould): a case for horizontal gene transfer between alga and animal?

The chloroplast symbiosis between the ascoglossan (=Sacoglossa) sea slug Elysia chlorotica and plastids from the chromophytic alga Vaucheria litorea is the longest-lived relationship of its kind known, lasting up to 9 months. During this time, the plastids continue to photosynthesize in the absence of the algal nucleus at rates sufficient to meet the nutritional needs of the slugs. We have previously demonstrated that the synthesis of photosynthetic proteins occurs while the plastids reside within the diverticular cells of the slug. Here, we have identified several of these synthesized proteins as belonging to the nuclear-encoded family of polypeptides known as light-harvesting complex I (LHCI). The synthesis of LHCI is blocked by the cytosolic ribosomal inhibitor cycloheximide and proceeds in the presence of chloramphenicol, a plastid ribosome inhibitor, indicating that the gene encoding LHCI resides in the nuclear DNA of the slug. These results suggest that a horizontal transfer of the LHCI gene from the alga to the slug has taken place.     

Viruses and thyroiditis: an update

Background

Despite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have co-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur.

Results

We have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 × 10^-4 substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Réunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift.

Conclusion

The absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts.     

Admission C – reactive protein after acute ischemic stroke is associated with stroke severity and mortality: The 'Bergen stroke study'

Background

There is growing evidence that inflammation plays an important role in atherogenesis. Previous studies show that C-reactive protein (CRP), an inflammatory marker, is associated with stroke outcomes and future vascular events. It is not clear whether this is due a direct dose-response effect or rather an epiphenomenon. We studied the effect of CRP measured within 24 hours after stroke onset on functional outcome, mortality and future vascular events.

Methods

We prospectively studied 498 patients with ischemic stroke who were admitted within 24 hours after the onset of symptoms. CRP and NIH stroke scale (NIHSS) were measured at the time of admission. Short-term functional outcome was measured by modified Rankin scale (mRS) and Barthel ADL index (BI) 7 days after admission. Patients were followed for up to 2.5 years for long-term mortality and future vascular events data.

Results

The median CRP at admission was 3 mg/L. High CRP was associated with high NIHSS (p = 0.01) and high long-term mortality (p < 0.0001). After adjusting for confounding variables, high CRP remained to be associated with high NIHSS (p = 0.02) and high long-term mortality (p = 0.002). High CRP was associated with poor short-term functional outcomes (mRS > 3; BI < 95) (p = 0.01; p = 0.03). However, the association was not significant after adjusting for confounding variables including stroke severity (p = 0.98; p = 0.88). High CRP was not associated with future vascular events (p = 0.98).

Conclusion

Admission CRP is associated with stroke severity and long-term mortality when measured at least 24 hours after onset. There is a crude association between high CRP and short-term functional outcome which is likely secondary to stroke severity. CRP is an independent predictor of long-term mortality after ischemic stroke.     

Comparison of Alimentary Systems in Shelled and Non-shelled Sacoglossa (Mollusca,Opisthobranchia)

The Sacoglossa (= Ascoglossa) comprise a ‘complete’ evolutionary series from species with a large shell into which the animal can withdraw completely, through species with a reduced shell covering only the visceral mass, to shell-less (‘nudibranchiate’) forms some of which have lateral wing-like extensions, parapodia, others bearing leaf-like or cylindrical dorsal appendages, cerata. The Sacoglossa are all specialized suctorial feeders, and almost all are stenophagous herbivores. Hence many anatomical adaptations to a particular food occur in the alimentary system. However, a number of characters seem to reflect phylogenetic relationships as well. Among these are presence/absence, shape, and position of pharyngeal pouches, basic shape of radular teeth, branching pattern of digestive gland, and position of anus. The classificatory significance of these characters is discussed.