Sacoglossan Mollusc Research Articles

The polyketide to fatty acid transition in the evolution of animal lipid metabolism

Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized metabolites. The evolutionary origin of the animal FAS and its relationship to the diversity of PKSs remain unclear despite the critical role of lipid synthesis in cellular metabolism. Recently, an animal FAS-like PKS (AFPK) was identified in sacoglossan molluscs. Here, we explore the phylogenetic distribution of AFPKs and other PKS and FAS enzymes across the tree of life. We found AFPKs widely distributed in arthropods and molluscs (>6300 newly described AFPK sequences). The AFPKs form a clade with the animal FAS, providing an evolutionary link bridging the type I PKSs and the animal FAS. We found molluscan AFPK diversification correlated with shell loss, suggesting AFPKs provide a chemical defense. Arthropods have few or no PKSs, but our results indicate AFPKs contributed to their ecological and evolutionary success by facilitating branched hydrocarbon and pheromone biosynthesis. Although animal metabolism is well studied, surprising new metabolic enzyme classes such as AFPKs await discovery.

Photochemical activity of membrane-localised polyketide derived marine natural products

Marine organisms rely upon complex bio-chemical systems to manage photo-induced oxidative damage as part of a sophisticated chemical defense. Using a combination of synthetic chemistry and biophysical techniques, we provide evidence to support the notion that marine derived polyene γ-pyrone metabolites of sacoglossan molluscs become localised at cell membranes where they influence the total absorption of the harmful radiation by the lipid membrane, thereby functioning as sunscreen agents. We also report two new hyperperoxide analogues of the putative polyene biosynthetic precursors that themselves are likely candidate natural products.

First chemical study of the sacoglossan Elysia patagonica: Isolation of a γ-pyrone propionate hydroperoxide

• The chemistry of the sacoglossan mollusc Elysia patagonica has been investigated for the first time.

Sea Slug Kleptoplasty and Plastid Maintenance in a Metazoan

[Trench (1969)][1] was the first to characterize the kleptoplastic (i.e. “stolen plastid”) relationship between the sacoglossan mollusc Elysia chlorotica and its algal prey ( Vaucheria litorea ). In contrast to E. chlorotica , which retains only the plastids of the alga in densely packed

The making of a photosynthetic animal.

Symbiotic animals containing green photobionts challenge the common perception that only plants are capable of capturing the sun's rays and converting them into biological energy through photoautotrophic CO(2) fixation (photosynthesis). 'Solar-powered' sacoglossan molluscs, or sea slugs, have taken this type of symbiotic association one step further by solely harboring the photosynthetic organelle, the plastid (=chloroplast). One such sea slug, Elysia chlorotica, lives as a 'plant' when provided with only light and air as a result of acquiring plastids during feeding on its algal prey Vaucheria litorea. The captured plastids (kleptoplasts) are retained intracellularly in cells lining the digestive diverticula of the sea slug, a phenomenon sometimes referred to as kleptoplasty. Photosynthesis by the plastids provides E. chlorotica with energy and fixed carbon for its entire lifespan of ~10 months. The plastids are not transmitted vertically (i.e. are absent in eggs) and do not undergo division in the sea slug. However, de novo protein synthesis continues, including plastid- and nuclear-encoded plastid-targeted proteins, despite the apparent absence of algal nuclei. Here we discuss current data and provide hypotheses to explain how long-term photosynthetic activity is maintained by the kleptoplasts. This fascinating 'green animal' provides a unique model to study the evolution of photosynthesis in a multicellular heterotrophic organism.

In vivo quantification of kleptoplastic chlorophyll a content in the “solar-powered” sea slug Elysia viridis using optical methods: spectral reflectance analysis and PAM fluorometry

Kleptoplasty is a particularly remarkable type of symbiosis, consisting of the presence of functional chloroplasts in the tissues of a host of another species. One of the most well-studied types of kleptoplasty is the association between sacoglossan molluscs (sea slugs) and algal chloroplasts. After ingestion, the chloroplasts remain photosynthetically functional and provide photosynthates to the host, therefore named as "solar-powered" sea slugs. This study evaluated the use of two optical methods, spectral reflectance analysis and in vivo Chl fluorescence, as measured by pulse amplitude modulated (PAM) fluorometry, for the in vivo quantification of kleptoplastic chlorophyll (Chl) a content in the sacoglossan Elysia viridis (Montagu, 1804) bearing chloroplasts of the macroalgae Codium tomentosum var. mucronatum (G. Hamel) Ardré. The Chl a content of E. viridis specimens was compared to a number of reflectance-based indices and to the dark-level fluorescence, F(o). Most reflectance-based indices varied linearly with the symbiosis Chl a content over the whole range of pigment content variation. Most significant correlations (P < 0.001) were found between indices using as reference the reflectance at 750 nm, with the proportion of pigment content explained by the indices varying between 63.5% and 85.9%. F(o) varied linearly with the Chl a content only for low pigment levels (below 4-6 microg Chl a per individual), above which it followed a saturation-like pattern. The use of optical methods was illustrated by monitoring the changes in Chl a content of specimens during periods of starvation and subsequent recovery. The results of this study suggest that, if basic requirements of signal detection and reproducible measuring geometry are verified, these optical methods may be readily applied to other photosynthetic symbioses.

Feeding preferences and host associations of specialist marine herbivores align with quantitative variation in seaweed secondary metabolites

Consequences of congeneric quantitative variation in secondary metabolites of seaweeds on diet specificity and host association in specialist marine herbivores have received little attention. We investigated quantitative variation in caulerpenyne and oxytoxin 1 in 7 species of green seaweeds from the genus Caulerpa, along with the feeding preferences and host associations of 4 co-occurring sacoglossan molluscs. C. taxifolia and C. sertularioides contained high concentrations of metabolites and were preferred least by all herbivores. Algae with intermediate metabolite concentrations (C. racemosa, C. serrulata, and C. cupressoides) were preferred by Elysia tomentosa and Lobiger viridis. Oxynoe viridis and Stiliger smaragdinus had strong preferences for different low concentration Caulerpa species (C. racemosa var. laetevirens and C. lentillifera), suggesting not all feeding preferences are based exclusively on the major metabolites. In situ host associations of L. viridis and S. smaragdinus mirrored their feeding preferences, but this was not the case for E. tomentosa. Furthermore, those algal species with the highest and lowest metabolite concentrations had the lowest overall densities of sacoglossans. The results imply that the direct influence of quantitative variation in Caulerpa chemistry may only be limited to host associations in some sacoglossans. However, feeding pressure from multiple herbivore species with unique preferences could still contribute to variation in chemical defence amongst congeneric algae.

Grazing rates of Elysia tomentosa on native and introduced Caulerpa taxifolia

The marine alga Caulerpa taxifolia Vahl (C. Agardh), recognized globally as one of the most prolific non-native species introductions, has been introduced to several temperate locations from where it has since rapidly expanded. C. taxifolia is protected by a toxin (terpenoid) in its tissues that limits grazing by native herbivores. Sacoglossan molluscs of the genus Elysia are among the few organisms that graze C. taxifolia; however, little is known about their feeding ecology. In the current study, we quantified the grazing rates of Elysia tomentosa on native C. taxifolia (Moreton Bay, Queensland) and introduced C. taxifolia (Botany Bay and Lake Conjola, New South Wales). Grazing rates were similar at Moreton Bay sites and Botany Bay; however, they were significantly lower in Lake Conjola. At the maximum observed grazing rate, slugs ate their body weight in C. taxifolia (dry weight) every 18-24 h. Differences in grazing rates between locations may be explained by differences in C. taxifolia morphology rather than native or introduced origin.

Effects of light exposure on the retention of kleptoplastic photosynthetic activity in the sacoglossan mollusc Elysia viridis

The effects of light exposure on the photosynthetic activity of kleptoplasts were studied in the sacoglossan mollusc Elysia viridis. The photosynthetic activity of ingested chloroplasts was assessed in vivo by non-destructively measuring photophysiological parameters using pulse amplitude modulation (PAM) fluorometry. Animals kept under starvation were exposed to two contrasting light conditions, 30 μmol photons m−2 s−1 (low light, LL), and 140 μmol photons m−2 s−1 (high light, HL), and changes in photosynthetic activity were monitored by measuring the maximum quantum yield of photosystem II (PSII), Fv/Fm, the minimum fluorescence, Fo, related to chlorophyll a content, and by measuring rapid light-response curves (RLC) of relative electron transport rate (rETR). RLCs were characterised by the initial slope of the curve, αRLC, related to efficiency of light capture, and the maximum rETR level, rETRm,RLC, determined by the carbon-fixation metabolism. Starvation induced the decrease of all photophysiological parameters. However, the retention of photosynthetic activity (number of days for Fv/Fm > 0), as well as the rate and the patterns of its decrease over time, varied markedly with light exposure. Under HL conditions, a rapid, exponential decrease was observed for Fv/Fm, αRLC and rETRm,RLC, Fo not showing any consistent trend of variation, and retention times ranged between 6 and 15 days. These results suggested that the retention of chloroplast functionality is limited by photoinactivation of PSII reaction center protein D1. In contrast, under LL conditions, a slower decrease in all parameters was found, with retention times varying from 15 to 57 days. Fv/Fm, αRLC and rETRm,RLC exhibited a bi-phasic pattern composed by a long phase of slow decrease in values followed by a rapid decline, whilst Fo decayed exponentially. These results were interpreted as resulting from lower rates of D1 photoinactivation under low light and from the gradual decrease in carbon provided by photosynthesis due to reduction of functional photosynthetic units.

In vivo and in vitro differences in chloroplast functionality in the two north Atlantic sacoglossans (Gastropoda, Opisthobranchia) Placida dendritica and Elysia viridis

The photosynthetic functionality in chloroplasts in the two sacoglossan molluscs Placida dendritica and Elysia viridis from the Trondheim fjord in Norway was studied. P. dendritica and E. viridis with no functional chloroplasts in their digestive system were introduced to the green macroalgae Codium fragile. Our results showed that P. dendritica was not able to retain functional (photosynthetic) chloroplasts. Transmission electron microscopy (TEM) showed that chloroplasts were directly digested when phagocytosed into the digestive cells. Four stages of chloroplast degradation were observed. A corresponding operational quantum yield of chl a fluorescence (ΦPSII ~ 0) indicated autofluorescence, and the presence of highly degraded chl a supported these observations. In contrast, E. viridis was able to retain functional chloroplasts. For this species it took only 1 week for the chloroplasts inside the digestive cells to acquire the same ΦPSII and light utilisation coefficient (α) as C. fragile kept under the same light conditions. Data for 8 days showed a 2–6-fold increase in the maximum photosynthetic rate (Pmax) and light saturation index (Ek) relative to C. fragile. This increase in available light was probably caused by a reduced package effect in the digestive gland of E. viridis relative to C. fragile, resulting in a partial photoacclimation response by reducing the turnover time of electrons (τ). Isolated pigments from C. fragile compared to E. viridis showed the same levels of photosynthetic pigments (chl a and b, neoxanthin, violaxanthin, siphonaxanthin, siphonein and β,e-carotene) relative to μg chl a (w:w), indicating that the chloroplasts in E. viridis did not synthesise any new pigments. After 73 days of starvation, it was estimated that chloroplasts in E. viridis were able to stay photosynthetic 5–9 months relative to the size of the slugs, corresponding to an RFC of level 8 (a retention ability to retain functional chloroplasts (RFC) for more than 3 months). The reduction in ΦPSII, Pmax and α as a function of time was caused by a reduction in chloroplast health and number (chloroplast thylakoid membranes and PSII are degraded). These observations therefore conclude that chloroplasts from C. fragile cannot divide or synthesise new pigments when retained by E. viridis, but are able to partially photoacclimate by decreasing τ as a response to more light. This study also points to the importance of siphonaxanthin and siphonein as chemotaxonomic markers for the identification of algal sources of functional chloroplasts.

New Caulerpenyne-derived Metabolites of an Elysia Sacoglossan from the South Indian Coast

Chemical analysis of the secondary metabolite pattern of the sacoglossan mollusc Elysia cf. expansa, collected along South Indian coasts, showed the presence of the typical Caulerpa-derived sesquiterpene caulerpenyne (1) and two new minor co- occurring metabolites, the compounds dihydrocaulerpenyne (4) and expansinol (5). The chemical characterization of these molecules, structurally related to 1, is reported.

Genetic differentiation of Elysia timida (Risso, 1818) populations in the Southwest Mediterranean and Mar Menor coastal lagoon

Genetic variation at 10 enzyme loci was analysed in Elysia timida sacoglossan mollusc samples, originating from both coastal lagoon and marine sites. The observed heterozygosity ranged from 0.390 (Los Urrutias) to 0.277 (Tabarca). Marine and coastal lagoon populations were characterised by exclusive alleles. Examination of the spatial structure was performed using Nei's distances and F-statistics, and indicated significant genetic differences between marine groups and coastal lagoon populations. Multivariate analysis of allele frequencies also suggested that different arrays of genotypes prevail in coastal lagoon samples compared to marine ones.

Synthesis and structure determination of kahalalide F (1,2).

Kahalalide F, the only member of the family of peptides called kahalalides, isolated from the sacoglossan mollusc Elysia rufescens and the green alga Bryopsis sp., with important bioactivity, is in clinical trials for treatment of prostate cancer. An efficient solid-phase synthetic approach is reported. Kahalalide F presents several synthetic difficulties: (i) an ester bond between two beta-branched and sterically hindered amino acids; (ii) a didehydroamino acid; and (iii) a rather hydrophobic sequence with two fragments containing several beta-branched amino acids in a row, one of them terminated with a saturated aliphatic acid. The cornerstones of our strategy were (i) a quasiorthogonal protecting system with allyl, tert-butyl, fluorenyl, and trityl-based groups, (ii) azabenzotriazole coupling reagents, (iii) formation of the didehydroamino acid residue on the solid phase, and (iv) cyclization and final purification in solution. HPLC, high-field NMR, and biological activity studies showed that the correct stereochemistry of the natural product is that proposed by Rinehart et al. whereas the stereochemistry proposed by Scheuer et al. is that of a biologically less active diastereoisomer.

New Polypropionate Pyrones from the Philippine Sacoglossan Mollusc Placobranchus ocellatus

Six new propionate-derived metabolites, 4– 9, have been isolated along with the known 9,10-deoxytridachione ( 1) from the sacoglossan mollusc Placobranchus ocellatus collected in the Philippines. The structures of the new compounds were determined by spectrometric methods.

Caulerpenyne‐Amine Reacting System as a Model for in vivo Interactions of Ecotoxicologically Relevant Sesquiterpenoids of the mediterranean‐adapted tropical green seaweed caulerpa taxifolia

AbstractCaulerpenyne (1), the most abundant of the ecotoxicologically relevant sesquiterpenoids of the Mediterranean‐adapted tropical green seaweed Caulerpa taxifolia, was found to react with Et3N or pyridine in MeOH by initial deprotection of C(1)HO to give oxytoxin 1 (2a), previously isolated from the sacoglossan mollusc Oxynoe olivacea. With BuNH2, without any precaution to exclude light, 1 gave the series of racemic 3 and 4, and achiral (4E,6E)‐5, (4E,6Z)‐5, (4Z,6E)‐5, and (4Z,6Z)‐5 pyrrole compounds, corresponding to formal C(4) substitution, 4,5‐β‐elimination, and (E/Z)‐isomerization at the C(4)C(5) and C(6)C(7) bonds. Changing to CDCl3 as solvent in the dark, 1 gave cleanly, via 2a as an intermediate, 3 and (4E,6E)‐5. The latter proved to be prone to (E/Z)‐photoisomerization. Under standard acetylation conditions, 3 gave (4E,6E)‐5 via acetamide 7 as an intermediate. Particular notice is warranted by selective deprotection of 1 at C(1), mimicking enzyme reactions, and unprecedented formation of pyrrole compounds from freely‐rotating, protected 1,4‐dialdehyde systems.

Isolation of functional chloroplasts from the sacoglossan mollusc Elysia viridis Montague

SUMMARYA novel technique has been developed in which functional chloroplasts have been isolated and purified from the symbiotic sacoglossan mollusc Elysia viridis, Montague. This entailed the progressive filtration and gel‐filtration of a hyaluronidase‐incubated homogenate. Isopycnic centrifugation of the resultant crude chloroplast suspension reduced the cytoplasmic/mitochondrial and microbody contamination by approx. 70 and 90% respectively. Purified chloroplast suspensions were capable of linear rates of 14CO2 fixation for at least 25 min at 50 μmol m2 s−1 (PPFD), even though approx. 54% of assimilate was being released into the bathing medium. Values for CO2 fixation and assimilate release were similar to those obtained for the intact mollusc.

ChemInform Abstract: Tridachiapyrones: Propionate‐Derived Metabolites from the Sacoglossan Mollusc Tridachia crispata.

Chemischer InformationsdienstVolume 17, Issue 27 Natural Products ChemInform Abstract: Tridachiapyrones: Propionate-Derived Metabolites from the Sacoglossan Mollusc Tridachia crispata. M. B. KSEBATI, M. B. KSEBATISearch for more papers by this authorF. J. SCHMITZ, F. J. SCHMITZSearch for more papers by this author M. B. KSEBATI, M. B. KSEBATISearch for more papers by this authorF. J. SCHMITZ, F. J. SCHMITZSearch for more papers by this author First published: July 8, 1986 https://doi.org/10.1002/chin.198627319AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume17, Issue27July 8, 1986 RelatedInformation

Persistence of functional chloroplasts in Elysia viridis (Opisthobranchia, Sacoglossa).

SEVERAL species of sacoglossan mollusc retain in the cells of their digestive diverticula large numbers of chloroplasts derived from their food plants (mostly siphonaceous algae). These associations are capable of photosynthesis1–4. In freshly collected Elysia viridis (Montagu) (which obtains chloroplasts from Codium fragile (Sur.) Hariot) the net rate of fixation is approximately 40% of that in the intact seaweed and the chlorophyll content, g−1 fresh weight, is similar in animal derived material and seaweed (Trench, Boyle and Smith, in preparation). This report describes an experiment showing that E. viridis can retain functional chloroplasts for at least 3 months when starved in the light and at least one month when starved in the dark.