Laura Núñez-Pons

Natural products from Antarctic colonial ascidians of the genera Aplidium and Synoicum: variability and defensive role.

Ascidians have developed multiple defensive strategies mostly related to physical, nutritional or chemical properties of the tunic. One of such is chemical defense based on secondary metabolites. We analyzed a series of colonial Antarctic ascidians from deep-water collections belonging to the genera Aplidium and Synoicum to evaluate the incidence of organic deterrents and their variability. The ether fractions from 15 samples including specimens of the species A. falklandicum, A. fuegiense, A. meridianum, A. millari and S. adareanum were subjected to feeding assays towards two relevant sympatric predators: the starfish Odontaster validus, and the amphipod Cheirimedon femoratus. All samples revealed repellency. Nonetheless, some colonies concentrated defensive chemicals in internal body-regions rather than in the tunic. Four ascidian-derived meroterpenoids, rossinones B and the three derivatives 2,3-epoxy-rossinone B, 3-epi-rossinone B, 5,6-epoxy-rossinone B, and the indole alkaloids meridianins A–G, along with other minoritary meridianin compounds were isolated from several samples. Some purified metabolites were tested in feeding assays exhibiting potent unpalatabilities, thus revealing their role in predation avoidance. Ascidian extracts and purified compound-fractions were further assessed in antibacterial tests against a marine Antarctic bacterium. Only the meridianins showed inhibition activity, demonstrating a multifunctional defensive role. According to their occurrence in nature and within our colonial specimens, the possible origin of both types of metabolites is discussed.

Chemical defenses of tunicates of the genus Aplidium from the Weddell Sea (Antarctica)

Predation and competition are important factors structuring Antarctic benthic communities and are expected to promote the production of chemical defenses. Tunicates are subject to little predation, and this is often attributed to chemical compounds, although their defensive activity has been poorly demonstrated against sympatric predators. In fact, these animals, particularly the genus Aplidium, are rich sources of bioactive metabolites. In this study, we report the natural products, distribution and ecological activity of two Aplidium ascidian species from the Weddell Sea (Antarctica). In our investigation, organic extracts obtained from external and internal tissues of specimens of A. falklandicum demonstrated to contain deterrent agents that caused repellency against the Antarctic omnivorous predator, the sea star Odontaster validus. Chemical analysis performed with Antarctic colonial ascidians Aplidium meridianum and Aplidium falklandicum allowed the purification of a group of known bioactive indole alkaloids, meridianins A-G. These isolated compounds proved to be responsible for the deterrent activity.

Illudalane Sesquiterpenoids of the Alcyopterosin Series from the Antarctic Marine Soft Coral Alcyonium grandis

Chemical investigation of the lipophilic extract of the Antarctic soft coral Alcyonium grandis led us to the finding of nine unreported sesquiterpenoids, 2-10. These molecules are members of the illudalane class and in particular belong to the group of alcyopterosins, illudalanes isolated from marine organisms. The structures of 2-10 were determined by interpretation of spectroscopic data. Repellency experiments conducted using the omnivorous Antarctic sea star Odontaster validus revealed a strong activity in the lipophilic extract of A. grandis against predation.

Chemo-ecological studies on hexactinellid sponges from the Southern Ocean

Hexactinellids (glass sponges) are an understudied class with syncytial organization and poor procariotic associations, thought to lack defensive secondary metabolites. Poriferans, though, are outstanding sources of bioactive compounds; nonetheless, a growing suspicion suggests that many of these chemicals could be symbiont-derived. In Polar latitudes, sponges are readily invaded by diatoms, which could provide natural products. Hexactinellids are typical of deep waters; but in Antarctica, they dominate the upper shelf providing shelter and food supply to many opportunistic mesograzers and macroinvertebrates, which exert strong ecological pressures on them. Aiming to examine the incidence of defensive activities of hexactinellids against consumption, feeding experiments were conducted using their lipophilic fractions. Antarctic hexactinellid and demosponge extracts were tested against the asteroid Odontaster validus and the amphipod Cheirimedon femoratus as putative sympatric, omnivorous consumers. Hexactinellids yielded greater unpalatable activities towards the amphipod, while no apparent allocation of lipophilic defenses was noted. After chemical analyses on the lipophilic fractions from these Antarctic glass sponges, quite similar profiles were revealed, and no peculiar secondary metabolites, comparable to those characterizing other poriferans, were found. Instead, the lipidic compounds 5α(H)-cholestan-3-one and two glycoceramides were isolated for their particular outspread presence in our samples. The isolated compounds were further assessed in asteroid feeding assays, and their occurrence was evaluated for chemotaxonomical purposes in all the Antarctic samples as well as in glass sponges from other latitudes by NMR and MS. Characteristic sphingolipids are proposed as chemical markers in Hexactinellida, with possible contributions to the classification of this unsettled class.

Feeding repellence in Antarctic bryozoans

The Antarctic sea star Odontaster validus and the amphipod Cheirimedon femoratus are important predators in benthic communities. Some bryozoans are part of the diet of the asteroid and represent both potential host biosubstrata and prey for this omnivorous lysianassid amphipod. In response to such ecological pressure, bryozoans are expected to develop strategies to deter potential predators, ranging from physical to chemical mechanisms. However, the chemical ecology of Antarctic bryozoans has been scarcely studied. In this study we evaluated the presence of defenses against predation in selected species of Antarctic bryozoans. The sympatric omnivorous consumers O. validus and C. femoratus were selected to perform feeding assays with 16 ether extracts (EE) and 16 butanol extracts (BE) obtained from 16 samples that belonged to 13 different bryozoan species. Most species (9) were active (12 EE and 1 BE) in sea star bioassays. Only 1 BE displayed repellence, indicating that repellents against the sea star are mainly lipophilic. Repellence toward C. femoratus was found in all species in different extracts (10 EE and 12 BE), suggesting that defenses against the amphipod might be both lipophilic and hydrophilic. Interspecific and intraspecific variability of bioactivity was occasionally detected, suggesting possible environmental inductive responses, symbiotic associations, and/or genetic variability. Multivariate analysis revealed similarities among species in relation to bioactivities of EE and/or BE. These findings support the hypothesis that, while in some cases alternative chemical or physical mechanisms may also provide protection, repellent compounds play an important role in Antarctic bryozoans as defenses against predators.

Deterrent activities in the crude lipophilic fractions of Antarctic benthic organisms: chemical defences against keystone predators

Generalist predation constitutes a driving force for the evolution of chemical defences. In the Antarctic benthos, asteroids and omnivore amphipods are keystone opportunistic predators. Sessile organisms are therefore expected to develop defensive mechanisms mainly against such consumers. However, the different habits characterizing each predator may promote variable responses in prey. Feeding-deterrence experiments were performed with the circumpolar asteroid macropredator Odontaster validus to evaluate the presence of defences within the apolar lipophilic fraction of Antarctic invertebrates and macroalgae. A total of 51% of the extracts were repellent, yielding a proportion of 17 defended species out of the 31 assessed. These results are compared with a previous study in which the same fractions were offered to the abundant circum-Antarctic amphipod Cheirimedon femoratus. Overall, less deterrence was reported towards asteroids (51%) than against amphipods (80.8%), principally in sponge and algal extracts. Generalist amphipods, which establish casual host–prey sedentary associations with biosubstrata (preferentially sponges and macroalgae), may exert more localized predation pressure than sea stars on certain sessile prey, which would partly explain these results. The nutritional quality of prey may interact with feeding deterrents, whose production is presumed to be metabolically expensive. Although optimal defence theory posits that chemical defences are managed and distributed as to guarantee protection at the lowest cost, we found that only a few organisms localized feeding deterrents towards most exposed and/or valuable body regions. Lipophilic defensive metabolites are broadly produced in Antarctic communities to deter opportunistic predators, although several species combine different defensive traits.

Lipophilic Defenses From Alcyonium Soft Corals of Antarctica

Alcyonacean soft corals lack physical or skeletal defenses and their nematocyst system is weak, leading to the conclusion that soft corals mainly rely on chemistry for protection from predators and microbes. Defensive chemicals of primary and secondary metabolic origin are exuded in the mucus surface layer, explaining the general lack of heavy fouling and predation in corals. In Antarctic ecosystems, where generalist predation is intense and mainly driven by invertebrate consumers, the genus Alcyonium is represented by eight species. Our goal was to investigate the understudied chemical ecology of Antarctic Alcyonium soft corals. We obtained six samples belonging to five species: A. antarcticum, A. grandis, A. haddoni, A. paucilobulatum, and A. roseum, and assessed the lipid-soluble fractions for the presence of defensive agents in these specimens. Ethyl ether extracts were tested in feeding bioassays with the sea star Odontaster validus and the amphipod Cheirimedon femoratus as putative sympatric predators. Repellent activities were observed towards both consumers in all but one of the samples assessed. Moreover, three of the extracts caused inhibition to a sympatric marine bacterium. The ether extracts afforded characteristic illudalane sesquiterpenoids in two of the samples, as well as particular wax esters subfractions in all the colonies analyzed. Both kinds of metabolites displayed significant deterrent activities demonstrating their likely defensive role. These results suggest that lipophilic chemicals are a first line protection strategy in Antarctic Alcyonium soft corals against predation and bacterial fouling.

Defensive Metabolites from Antarctic Invertebrates: Does Energetic Content Interfere with Feeding Repellence?

Many bioactive products from benthic invertebrates mediating ecological interactions have proved to reduce predation, but their mechanisms of action, and their molecular identities, are usually unknown. It was suggested, yet scarcely investigated, that nutritional quality interferes with defensive metabolites. This means that antifeedants would be less effective when combined with energetically rich prey, and that higher amounts of defensive compounds would be needed for predator avoidance. We evaluated the effects of five types of repellents obtained from Antarctic invertebrates, in combination with diets of different energetic values. The compounds came from soft corals, ascidians and hexactinellid sponges; they included wax esters, alkaloids, a meroterpenoid, a steroid, and the recently described organic acid, glassponsine. Feeding repellency was tested through preference assays by preparing diets (alginate pearls) combining different energetic content and inorganic material. Experimental diets contained various concentrations of each repellent product, and were offered along with control compound-free pearls, to the Antarctic omnivore amphipod Cheirimedon femoratus. Meridianin alkaloids were the most active repellents, and wax esters were the least active when combined with foods of distinct energetic content. Our data show that levels of repellency vary for each compound, and that they perform differently when mixed with distinct assay foods. The natural products that interacted the most with energetic content were those occurring in nature at higher concentrations. The bioactivity of the remaining metabolites tested was found to depend on a threshold concentration, enough to elicit feeding repellence, independently from nutritional quality.

Chemical Interactions in Antarctic Marine Benthic Ecosystems

Antarctic marine ecosystems are immersed in an isolated, relatively constant environment where the organisms inhabiting their benthos are mainly sessile suspension feeders. For these reasons, physical and chemical biotic interactions play an essential role in structuring these marine benthic communities (Dayton et al., 1974; Orejas et al., 2000). These interactions may include diverse strategies to avoid predation (e.g. Iken et al., 2002), competition for space or food (e.g. Bowden et al., 2006) and avoiding fouling (e.g. Rittschof, 2001; Peters et al., 2010). For instance, in the marine benthos, one of the most extended effective strategies among sessile soft-bodied organisms is chemical defense, mediated by several bioactive natural products mostly considered secondary metabolites (e.g. Paul et al., 2011). The study of the “chemical network” (chemical ecology interactions) structuring the communities provides information about the ecology and biology of the involved species, the function and the structure of the community and, simultaneously, it may lead to the discovery of new compounds useful to humans for their pharmacological potential (e.g. Avila, 1995; Bhakuni, 1998; Munro et al., 1999; Faulkner, 2000; Lebar et al., 2007; Avila et al., 2008). In the last three decades, the study of marine chemical ecology has experienced great progress, thanks to the new technological advances for collecting and studying marine samples, and the possibility of identification of molecules with smaller amounts of compounds (e.g. Paul et al., 2006, 2011; Blunt et al., 2011).

Chemo–ecological interactions in Antarctic bryozoans

Abstract The role of bioactive metabolites in ecological interactions involving Antarctic bryozoans has been scarcely studied. Bryozoans are one of the most abundant and diverse members of the Antarctic benthos and are preyed upon by diverse kinds of predators. They seem to be casual food items of the common Antarctic sea urchin Sterechinus neumayeri and the ubiquitous omnivorous amphipod Cheirimedon femoratus. In this study, the cytotoxic activity against embryos and sperm of the sea urchin S. neumayeri and the substrate preferences of the amphipod C. femoratus were assessed using organic extracts from Antarctic bryozoans, in order to determine the presence of chemical defenses. New adapted protocols were designed using a solidifying gel for simulating bryozoans’ surface. We analyzed 32 organic extracts from 16 samples that belonged to 13 different bryozoan species. No cytotoxicity was detected against embryos of S. neumayeri, while 12 of the 13 bryozoan species were cytotoxic to sperm at natural concentrations. In the substrate preference assays, the amphipod C. femoratus was repelled by ten species. The variable bioactivities found in both types of organic partitions of extracts indicated the presence of both lipophilic and hydrophilic defenses. Inter- and intraspecific variability of chemical defenses was detected also, suggesting environmental-induced responses, symbiotic production, and/or genetic variability. Possible alternative defensive mechanisms are also discussed for species with low or no chemical defense. Our results clearly support the fact that chemically mediated bioactivity in Antarctic bryozoans is common, and there is a likely ecological role of cytotoxic and repellent compounds for their protection.