Photosynthesis from stolen chloroplasts can support sea slug reproductive fitness.

Some sea slugs are able to steal functional chloroplasts (kleptoplasts) from their algal food sources, but the role and relevance of photosynthesis to the animal host remain controversial. While some researchers claim that kleptoplasts are slowly digestible 'snacks', others advocate that they enhance the overall fitness of sea slugs much more profoundly. Our analysis shows light-dependent incorporation of 13C and 15N in the albumen gland and gonadal follicles of the sea slug Elysia timida, representing translocation of photosynthates to kleptoplast-free reproductive organs. Long-chain polyunsaturated fatty acids with reported roles in reproduction were produced in the sea slug cells using labelled precursors translocated from the kleptoplasts. Finally, we report reduced fecundity of E. timida by limiting kleptoplast photosynthesis. The present study indicates that photosynthesis enhances the reproductive fitness of kleptoplast-bearing sea slugs, confirming the biological relevance of this remarkable association between a metazoan and an algal-derived organelle.

The photon menace: kleptoplast protection in the photosynthetic sea slug Elysia timida.

Absorption of excessive light by photosymbiotic organisms leads to the production of reactive oxygen species that can damage both symbiont and host. This is highly relevant in sacoglossan sea slugs that host functional chloroplasts 'stolen' from their algal foods (kleptoplasts), because of limited repair capacities resulting from the absence of algal nuclear genes. Here, we experimentally demonstrate (i) a host-mediated photoprotection mechanism in the photosynthetic sea slug Elysia timida, characterized by the closure of the parapodia under high irradiance and the reduction of kleptoplast light exposure; and (ii) the activation of a reversible xanthophyll cycle in kleptoplasts, which allows excessive energy to be dissipated. The described mechanisms reduce photoinactivation under high irradiance. We conclude that both host-mediated behavioural and plastid-based physiological photoprotective mechanisms can mitigate oxidative stress induced by high light in E. timida These mechanisms may play an important role in the establishment of long-term photosynthetically active kleptoplasts.

Kleptoplast photoacclimation state modulates the photobehaviour of the solar-powered sea slug Elysia viridis.

Some sacoglossan sea slugs incorporate intracellular functional algal chloroplasts (kleptoplasty) for periods ranging from a few days to several months. Whether this association modulates the photobehaviour of solar-powered sea slugs is unknown. In this study, the long-term kleptoplast retention species Elysia viridis showed avoidance of dark independently of light acclimation state. In contrast, Placida dendritica, which shows non-functional retention of kleptoplasts, showed no preference over dark, low or high light. High light-acclimated (HLac) E. viridis showed a higher preference for high light than low light-acclimated (LLac) conspecifics. The position of the lateral folds (parapodia) was modulated by irradiance, with increasing light levels leading to a closure of parapodia and protection of kleptoplasts from high light exposure. Furthermore, closure of parapodia occurred at higher irradiance in HLacE. viridis Our results strongly indicate that kleptoplast photoacclimation state modulates the photobehaviour of the solar-powered sea slug E. viridis.

Profiling of Heterobranchia Sea Slugs from Portuguese Coastal Waters as Producers of Anti-Cancer and Anti-Inflammatory Agents.

Bioprospection of marine invertebrates has been predominantly biased by the biological richness of tropical regions, thus neglecting macro-organisms from temperate ecosystems. Species that were not the object of studies on their biochemical composition include the Heterobranchia gastropods Armina maculata, Armina tigrina and Aglaja tricolorata, inhabitants of the Portuguese Atlantic coastal waters. Here, we present for the first time the fatty acid profile of neutral lipids and homarine content of these three species. Qualitative and quantitative differences in the fatty acid content among species points to the existence of a fatty acid profile of neutral lipids, particularly of each genus. The results from cytotoxicity assays, using the acetonic extracts of the gastropods on human gastric adenocarcinoma (AGS) and human lung adenocarcinoma (A549) cell lines, revealed a pronounced cytotoxic effect of the A. tigrina extract on both cell lines (IC50 values of 68.75 and 69.77 µg mL−1 for AGS and A549, respectively). It is worth noting the significant reduction of NO levels in LPS-challenged RAW 264.7 macrophages exposed to A. tricolorata extract, at concentrations as low as 125 µg mL−1.

Functional morphology of the glandular esophageal pouches of chitons (Mollusca, Polyplacophora).

The esophageal pouches of Chaetopleura angulata and Acanthochitona fascicularis were investigated using light and transmission electron microscopy. These pouches linked to the posterior region of the esophagus are known as sugar glands as they contain a fluid rich in polysaccharide digesting enzymes. They are the second largest glands in the digestive system of chitons, just after the digestive gland. In both species, the pouches contain a dense array of finger-shaped villi. The villi epithelium includes absorptive cells, basophilic secretory cells, mucus-secreting cells, and basal cells. Some absorptive cells were bordered by a dense cover of long microvilli, whereas other absorptive cells had short and sparse microvilli. Absorptive cells contain several lysosomes, mitochondria, peroxisomes, a few small Golgi stacks, some lipid droplets, and large amounts of glycogen. The basophilic secretory cells are characterized by the presence of many electron-dense vesicles, with a glycoprotein content, a large number of rough endoplasmic reticulum cisternae, and a highly developed Golgi apparatus. Mucus-secreting cells are characterized by large vesicles containing acid polysaccharides and wide Golgi stacks. Basal cells that were found at the base of the epithelium in contact with the basal lamina exhibit histological and ultrastructural features of enteroendocrine cells. We suggest that these glandular pouches are involved in extracellular and intracellular digestion, and accumulate lipid and glycogen reserves.

A new cytotoxic tambjamine alkaloid from the Azorean nudibranch Tambja ceutae.

The chemical investigation of Azorean nudibranch mollusk Tambja ceutae led us to isolate a new member of the tambjamine family, tambjamine K (1). The bryozoan Bugula dentata, prey of the nudibranch, was also analyzed and found to contain compound 1 in very small amounts together with known blue pigment 2 and tambjamines A (3) and B (4). The structure of tambjamine 1 was elucidated by the interpretation of the spectroscopic data as well as by the comparison with related compounds. Compounds 1 and 2 possess antiproliferative activity, in particular, tambjamine K (1) displayed high cytotoxicity against both tumor and non-tumor mammalian cells.

Histochemical and ultrastructural characterization of the posterior esophagus of Bulla striata (Mollusca, Opisthobranchia).

The posterior esophagus of Bulla striata, running from the gizzard to the stomach, was investigated with light and electron microscopy to obtain new data for a comparative analysis of the digestive system in cephalaspidean opisthobranchs. In this species, the posterior esophagus can be divided into two regions. In the first, the epithelium is formed by columnar cells with apical microvilli embedded in a cuticle. Many epithelial and subepithelial secretory cells are present in this region. In both, electron-lucent secretory vesicles containing filaments and a peripheral round mass of secretory material fill the cytoplasm. These acid mucus-secreting cells may also contain a few dense secretory vesicles. In the second part of the posterior esophagus, the cuticle is absent and the epithelium is ciliated. In this region, epithelial cells may contain larger lipid droplets and glycogen reserves. Subepithelial secretory cells are not present, and in epithelial secretory cells the number of dense vesicles increases, but most secretory cells still contain some electron-lucent vesicles. These cells secrete a mixture of proteins and acid polysaccharides and should be considered seromucous. The secretory cells of the posterior esophagus are significantly different from those previously reported in the anterior esophagus of this herbivorous species.

Biosynthetic evidence supporting the generation of terpene chemodiversity in marine mollusks of the genus Doriopsilla.

This paper reports the study of terpene biosynthesis in the marine nudibranch Doriopsilla pelseneeri. In vivo feeding experiments with 13C-glucose proved the de novo origin of the terpene metabolites via a mevalonate pathway. Characterization of the stereochemical relationship of the new acetyl pelseneeriols (7 and 8) suggests a two-step mechanism for the sesquiterpene cyclization, leading to a slight refinement of terpene tailoring in this family of marine invertebrates.

Mannitol oxidase and polyol dehydrogenases in the digestive gland of gastropods: Correlations with phylogeny and diet.

Mannitol oxidase and polyol dehydrogenases are enzymes that convert polyalcohols into sugars. Mannitol oxidase was previously investigated in terrestrial snails and slugs, being also present in a few aquatic gastropods. However, the overall distribution of this enzyme in the Gastropoda was not known. Polyol dehydrogenases are also poorly studied in gastropods and other mollusks. In this study, polyalcohol oxidase and dehydrogenase activities were assayed in the digestive gland of 26 species of gastropods, representing the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, including herbivores and carnivores were analyzed. Ultrastructural observations were undertake in species possessing mannitol oxidase, in order to investigate the correlation between this enzyme and the presence of tubular structures known to be associated with it. Mannitol oxidase activity was detected in the digestive gland of herbivores from the clades Caenogastropoda and Heterobranchia, but not in any carnivores or in herbivores from the clades Patellogastropoda, Neritimorpha and Vetigastropoda. In most of the species used in this study, dehydrogenase activities were detected using both D-mannitol and D-sorbitol as substrates. Nevertheless, in some carnivores these activities were not detected with both polyalcohols. Ultrastructural observations revealed tubular structures in digestive gland cells of some species having mannitol oxidase activity, but they were not observed in others. Based on our results, we suggest that mannitol oxidase first occurred in a herbivorous or omnivorous ancestor of Apogastropoda, the clade formed by caenogastropods and heterobranchs, being subsequently lost in those species that shifted towards a carnivorous diet.

High unexpected genetic diversity of a narrow endemic terrestrial mollusc.

The Iberian Peninsula has an extensive record of species displaying strong genetic structure as a result of their survival in isolated pockets throughout the Pleistocene ice ages. We used mitochondrial and nuclear sequence data to analyze phylogeographic patterns in endemic land snails from a valley of central Portugal (Vale da Couda), putatively assigned to Candidula coudensis, that show an exceptionally narrow distributional range. The genetic survey presented here shows the existence of five main mitochondrial lineages in Vale da Couda that do not cluster together suggesting independent evolutionary histories. Our results also indicate a departure from the expectation that species with restricted distributions have low genetic variability. The putative past and contemporary models of geographic distribution of Vale da Couda lineages are compatible with a scenario of species co-existence in more southern locations during the last glacial maximum (LGM) followed by a post-LGM northern dispersal tracking the species optimal thermal, humidity and soil physical conditions.

With a little help from DNA barcoding: investigating the diversity of Gastropoda from the Portuguese coast.

The Gastropoda is one of the best studied classes of marine invertebrates. Yet, most species have been delimited based on morphology only. The application of DNA barcodes has shown to be greatly useful to help delimiting species. Therefore, sequences of the cytochrome c oxidase I gene from 108 specimens of 34 morpho-species were used to investigate the molecular diversity within the gastropods from the Portuguese coast. To the above dataset, we added available COI-5P sequences of taxonomically close species, in a total of 58 morpho-species examined. There was a good match between ours and sequences from independent studies, in public repositories. We found 32 concordant (91.4%) out of the 35 Barcode Index Numbers (BINs) generated from our sequences. The application of a ranking system to the barcodes yield over 70% with top taxonomic congruence, while 14.2% of the species barcodes had insufficient data. In the majority of the cases, there was a good concordance between morphological identification and DNA barcodes. Nonetheless, the discordance between morphological and molecular data is a reminder that even the comparatively well-known European marine gastropods can benefit from being probed using the DNA barcode approach. Discordant cases should be reviewed with more integrative studies.

Conservation Status of a Recently Described Endemic Land Snail, Candidula coudensis, from the Iberian Peninsula.

We assessed the distribution, population size and conservation status of Candidula coudensis, a recently described endemic land snail from Portugal. From March 2013 to April 2014, surveys were carried out in the region where the species was described. We found an extent of occurrence larger than originally described, but still quite small (13.5 km2). The species was found mainly in olive groves, although it occurred in a variety of other habitats with limestone soils, including grasslands, scrublands and stone walls. Minimum population estimate ranged from 110,000-311,000 individuals. The main identified potential threats to the species include wildfires, pesticides and quarrying. Following the application of IUCN criteria, we advise a conservation status of either "Least Concern" or "Near-threatened" under criterion D (restricted population).

Coloration and defense in the nudibranch gastropod Hypselodoris fontandraui.

The striking color patterns of chromodorid (and other) nudibranchs appear to be indicative of aposematism. In Müllerian mimicry, all the mimic species have a defense mechanism. It has been proposed that a group of blue, white, and yellow Mediterranean and northeastern Atlantic species of the genus Hypselodoris form a Müllerian mimetic circle. One of these, H. fontandraui, lacks the mantle dermal formations (repugnatorial glands) that are typically found in other members of this circle and are reservoirs of feeding deterrent compounds. It therefore seemed possible that this animal lacks chemical defense and acts like a Batesian mimic. Within this study, we found that this nudibranch contains the furanosesquiterpenoid tavacpallescensin, most probably derived from sponges of the genus Dysidea, upon which it probably feeds. The metabolite concentrations were measured from samples of the mantle rim, other external parts, and internal organs. Concentrations were about 4 times higher in the mantle rim than in the other external parts, and more than 20 times higher in the mantle rim than in the internal organs, considerably exceeding the threshold value of concentration showing the maximum dose effect as feeding deterrent against the crustacean Palaemon elegans. In conclusion, the reported data clearly demonstrate that H. fontandraui is chemically defended in much the same way as its aposematic, co-occurring, and blue-colored congeners within the Müllerian mimetic circle and is not a Batesian mimic.

Light and electron microscopy study of the salivary glands of the carnivorous opisthobranch Philinopsis depicta (Mollusca, Gastropoda).

Cephalaspideans are a group of opisthobranch gastropods that comprises carnivorous and herbivorous species, allowing an investigation of the relationship between these diets and the morphofunctional features of the salivary glands. In this study, the salivary glands of the carnivorous cephalaspidean Philinopsis depicta were observed by light and electron microscopy. The secretory epithelium of these ribbon-shaped glands is formed by ciliated cells, granular cells and cells with apical vacuole. In ciliated cells the nucleus and most cytoplasmic organelles are located in the wider apical region and a very thin stalk reaches the base of the epithelium. These cells possess significant amounts of glycogen. Granular cells are packed with electron-dense secretory granules and also contain several cisternae of rough endoplasmic reticulum and Golgi stacks. The other type of secretory cell is mainly characterized by the presence of a large apical vacuole containing secretion. These cells possess high amounts of rough endoplasmic reticulum cisternae and several Golgi stacks. Vesicles with peripheral electron-dense material are also abundant, and seem to fuse to form the apical vacuole. The available data point out to a significant difference between the salivary glands of carnivorous and herbivorous cephalaspidean opisthobranchs, with an intensification of protein secretion in carnivorous species.