Anti-Fouling Effects of Saponin-Containing Crude Extracts from Tropical Indo-Pacific Sea Cucumbers.

Sea cucumbers are bottom dwelling invertebrates, which are mostly found on subtropical and tropical sea grass beds, sandy reef flats, or reef slopes. Although constantly exposed to fouling communities in these habitats, many species are surprisingly free of invertebrate epibionts and microfouling algae such as diatoms. In our study, we investigated the anti-fouling (AF) activities of different crude extracts of tropical Indo-Pacific sea cucumber species against the fouling diatom Cylindrotheca closterium. Nine sea cucumber species from three genera (i.e., Holothuria, Bohadschia, Actinopyga) were selected and extracted to assess their AF activities. To verify whether the sea cucumber characteristic triterpene glycosides were responsible for the observed potent AF activities, we tested purified fractions enriched in saponins isolated from Bohadschia argus, representing one of the most active anti-fouling extracts. Saponins were quantified by vanillin-sulfuric acid colorimetric assays and identified by LC-MS and LC-MS/MS analyses. We were able to demonstrate that AF activities in sea cucumber extracts were species-specific, and growth inhibition as well as attachment of the diatom to surfaces is dependent on the saponin concentration (i.e., Actinopyga contained the highest quantities), as well as on the molecular composition and structure of the present saponins (i.e., Bivittoside D derivative was the most bioactive compound). In conclusion, the here performed AF assay represents a promising and fast method for selecting the most promising bioactive organism as well as for identifying novel compounds with potent AF activities for the discovery of potentially novel pharmacologically active natural products.

Chemical Defense Mechanisms and Ecological Implications of Indo-Pacific Holothurians.

Sea cucumbers are slow-moving organisms that use morphological, but also a diverse combination of chemical defenses to improve their overall fitness and chances of survival. Since chemical defense compounds are also of great pharmaceutical interest, we pinpoint the importance of biological screenings that are a relatively fast, informative and inexpensive way to identify the most bioactive organisms prior to further costly and elaborate pharmacological screenings. In this study, we investigated the presence and absence of chemical defenses of 14 different sea cucumber species from three families (Holothuriidae, Stichopodidae and Synaptidae) against ecological factors such as predation and pathogenic attacks. We used the different sea cucumber crude extracts as well as purified fractions and pure saponin compounds in a portfolio of ecological activity tests including fish feeding assays, cytotoxicity tests and antimicrobial assays against environmental pathogenic and non-pathogenic bacteria. Furthermore, we quantified and correlated the concentrations of sea cucumber characteristic saponin compounds as effective chemical defensive compounds in all 14 crude extracts by using the vanillin-sulfuric acid test. The initial results revealed that among all tested sea cucumber species that were defended against at least one ecological threat (predation and/or bacterial attack), Bohadschiaargus, Stichopuscholoronotus and Holothuria fuscopunctata were the three most promising bioactive sea cucumber species. Therefore, following further fractionation and purification attempts, we also tested saponin-containing butanol fractions of the latter, as well as two purified saponin species from B. argus. We could demonstrate that both, the amount of saponin compounds and their structure likely play a significant role in the chemical defense strategy of the sea cucumbers. Our study concludes that the chemical and morphological defense mechanisms (and combinations thereof) differ among the ecological strategies of the investigated holothurian species in order to increase their general fitness and level of survival. Finally, our observations and experiments on the chemical ecology of marine organisms can not only lead to a better understanding of their ecology and environmental roles but also can help in the better selection of bioactive organisms/compounds for the discovery of novel, pharmacologically active secondary metabolites in the near future.

Acclimation capability inferred by metabolic performance in two sea cucumber species from different latitudes.

The notion that thermal specialists from tropical regions live closer to their temperature limits than temperate eurytherms, seems too generalized. Species specific differences in physiological and biochemical stress reactions are linked to key components of organism fitness, like metabolic capacity, which indicates that acclimation potential across latitudes might be highly diverse rather than simplistic. In this study the exposure of a tropical (Holothuria scabra) and a temperate (Holothuria forskali) sea cucumber species to identical cold- and warm-acclimation stress was compared using the key metabolic parameters, respiration rate, enzyme activity (ETS, LDH, IDH), and energy reserve fractions (lipid, carbohydrate and protein). Results show much broader respiratory adjustments, as response to temperature change, in H. scabra (2-30 µgO2*gww-1*h-1) compared to H. forskali (1.5-6.6 µgO2*gww-1*h-1). Moreover, the tropical species showed clearly pronounced up and down regulation of metabolic enzymes and shifts in energy reserves, due to thermal acclimation, while the same metabolic indicators remained consistent in the temperate species. In summary, these findings indicate enhanced metabolic plasticity in H. scabra at the cost of elevated energy expenditures, which seems to favor the tropical stenotherm in terms of thermal acclimation capacity. The comparison of such holistic metabolic analyses between conspecifics and congeners, may help to predict the heterogeneous effects of global temperature changes across latitudinal gradients.

Ecotoxicological Effects of Four Commonly Used Organic Solvents on the Scleractinian Coral Montipora digitata.

Organic solvents are often used in aquatic toxicity tests to facilitate the testing of hydrophobic or poorly water-soluble substances such as ultraviolet (UV) filters, pesticides, or polycyclic aromatic hydrocarbons (PAHs). Knowledge of intrinsic effects (i.e., measured as standardized and non-standardized endpoints) of such carrier solvents in non-standardized organisms (i.e., corals), is critical to regulatory processes. Therefore, we exposed the reef-building coral Montipora digitata to the most commonly used carrier solvents ethanol, methanol, dimethyl sulfoxide, and dimethylformamide in the range of 10-100 µL L-1 for 16 days. The effects on mortality, photobiological, morphological, and oxidative stress markers were evaluated. In our study, all solvents resulted in significant morphological and/or oxidative stress responses, but not in mortality. Moreover, ethanol led to a rapid increase in turbidity, thus questioning its suitability as a carrier solvent in aquatic studies in general. Based on our observations, we could rank the solvent effects as follows: dimethylformamide < dimethyl sulfoxide ≈ methanol ≤ ethanol, with dimethylformamide showing the least and ethanol the most pronounced effects. We conclude that the use of solvents in toxicity studies with corals, particularly by examining non-standardized (e.g., morphological, physiological) endpoints, should be taken with caution and requires further elaboration.

Towards the Development of Standardized Bioassays for Corals: Acute Toxicity of the UV Filter Benzophenone-3 to Scleractinian Coral Larvae.

Coral reefs have been declining globally at a historically unprecedented rate. Ultraviolet (UV) filters used in sunscreens may contribute to this decline at local scales, which has already led to bans on various organic UV filters in some regions. However, the underlying studies for these bans demonstrated significant flaws in the experimental design due to a lack of validated and standardized testing methods for corals. This study aimed to investigate options for the development of a standard acute toxicity test for the larval stage of scleractinian corals. Planula larvae of two brooding (Leptastrea purpurea and Tubastraea faulkneri) and two spawning (Acropora digitifera and A. millepora) species were exposed to the organic UV filter benzophenone-3 (BP3) for 48 h under static conditions. We observed interspecific variations in toxicity, with A. digitifera being the most sensitive (LC50 = 0.75 µg L-1) and T. faulkneri the least sensitive (LC50 = 2951.24 µg L-1) species. Inhibition of settlement was found to be a useful endpoint leading to an EC50 of 1.84 µg L-1 in L. purpurea larvae. Although the analytical challenges of measuring lipophilic substances in small volume test setups remain, the here applied test design and selected endpoints are suitable for further validation and subsequent standardization.

Effects of thermal stress on the immune and oxidative stress responses of juvenile sea cucumber Holothuria scabra.

Holothuria scabra is the most valued and cultured tropical sea cucumber, given the great demand of this species for human consumption. However, despite its ecological and economic relevance, little is known regarding its immune responses under thermal stress. Here, the main goal was to study the response of sea cucumbers to temperature stress, assessing sub-organismal alterations and acclimation capacities of juveniles to temperature changes. After changing temperature (1 °C/day) for 6 days, organisms were exposed to temperature conditions of 21 °C (cold), 27 °C (control), and 33 °C (warm) over a 30 day period. At each 15-day interval (T0, T15, and T30), six replicates per condition were killed for biochemical analysis. Immune responses were addressed by studying the activity of phenoloxidase (PO) and prophenoloxidase (ProPO) in the coelomic fluid. Antioxidant defence responses-catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR) enzymatic activities-were measured in the muscle and respiratory tree tissues, whereas oxidative damage was evaluated by measuring levels of superoxide radicals (ROS), DNA-strand breaks and lipid peroxidation (LPO). Juvenile H. scabra increased SOD and PO activities when temperature was elevated, and revealed low levels of ROS and damage in both cold and warm treatments throughout the experiment, confirming the organism's moderate thermal stress. After the short acclimation period, the immune and antioxidant responses prevented damage and maintained homeostasis. This multi-biomarker approach highlights its usefulness to monitor the health of H. scabra and to gain insight concerning the use of this high-valued species in global-scale aquaculture from different temperature regions.