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Molecules
2020 Oct 19;2520:. doi: 10.3390/molecules25204808.
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Chemical Defense Mechanisms and Ecological Implications of Indo-Pacific Holothurians.
Kamyab E
,
Rohde S
,
Kellermann MY
,
Schupp PJ
.
Abstract
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.
Figure 1. Phylogeny tree and presence/absence of Cuvierian Tubules (CTs) in studied sea cucumbers (n = number of tested specimens; adapted from [32,44,45,46]).
Figure 2. Percent feeding deterrent activity of sea cucumber crude extracts. The line at 40% indicates significant deterrence (p < 0.05, Fisher’s exact test, 1-tailed). Results express average values + standard error for the four replicated sea cucumber species (i.e., H. atra, A. echinites, S. chloronotus and B. argus).
Figure 3. Percentages of brine shrimp mortality following exposure to sea cucumber crude extracts at a concentrations of 10, 100 and 1000 µg mL−1 after 48 h. Results express average values ± standard deviation. A, B and C indicate significant differences between species at each concentration (Kruskal-Wallis post-hoc test, p < 0.05).
Figure 4. Calculated LC50 concentrations from the brine shrimp mortality assay for the 14 different sea cucumber crude extracts after 48 h. Results express average values + standard error.
Figure 5. Total antibacterial activities of sea cucumber crude extracts against pathogenic (a) and non-pathogenic (b) environmental bacteria.
Figure 6. Saponin concentration of the 14 tested sea cucumber crude extracts. Results express average values + standard error. (A–C) indicate significant differences between sea cucumber crude extracts. The Kruskal-Wallis post-hoc method for multiple comparisons was applied (p < 0.05).
Figure 7. Overall biological activities of the tested sea cucumber crude extracts. Sizes of bubble plots represent the percentage of each bioactivity for the four different bioassays. Color intensities represent the overlap of the replicates (duplicates; cf. Figure 1). Abbreviations: FA = fish feeding assay (% deterrence), ADA = agar diffusion assay against environmental pathogenic and non-pathogenic bacteria.
Figure 8. Major saponin compounds detected in the butanol fractions from the crude extracts of the three most active sea cucumber species (peak area ≥103). Compound code represent the exact mass (M in Da), and retention time (T in min). The compounds code of M1426T10.3 related to bivittoside D, and M1410T11.3 related to bivittoside C.
Figure 9. Structure of the two saponin molecules isolated from B. argus (Produced with ChemDraw, version 16.0.1.1(77)).
Figure 10. Percent deterrent activity of the butanol fractions from the three most active sea cucumber extracts, as well as two purified compounds (i.e., bivittoside D and bivittoside C) from B. argus. The line at 40% indicates significant deterrence (p < 0.05, Fisher’s exact test, 1-tailed).
Figure 11. LC50 concentration of the sea cucumber crude extracts against brine shrimp larvae after 48 h. Results express mean values ± standard error.
Figure 12. Workflow applied for isolating the bioactive saponin compounds from three most active sea cucumbers, B. argus, H. fuscopunctata and S. chloronotus. Compounds 1 (i.e., bivittoside D) and 2 (i.e., bivittoside C) were both isolated from “Fraction B” and “Fraction C” of B. argus.
