2.2.1. Saponin Composition

Identification of the most prominent saponins in the crude extracts of the nine sea cucumber species (peak areas > 10,000 mu) yielded 102 different saponin-like molecules (Table S2). However, several of the saponins showed the same exact molecular mass, but different retention times, indicating unknown isomers of potentially known saponin compounds (Table S3).

A hierarchical cluster analysis was performed to explore the similarity of saponin compositions between the different holothurian species. Except for *H. edulis*, we observed that all sea cucumber species cluster with species from the same genus (using the Kelley-Gardner-Sutcliffe (KGS) penalty function for identifying significant clusters, Figure 4). Note, that all species from the genus *Bohadschia* formed a clear separated cluster compared to *Actinopyga* and *Holothuria*.

**Figure 4.** Cluster dendrogram of sea cucumber species based on their studied saponin and sapogenin compositions ("average" distance type, log-transformed data, R version 1.2.5019).

More detailed analysis of the various saponin compounds revealed that compound M1104T11.1 (abbreviation indicates molecular mass (M) and retention time (T)) was present in all nine sea cucumber species, M1118T8.9 in eight and M600T9.3 and M1374T9 in seven species (*cf.* Table S3). Composition and relative intensities of both saponins and sapogenins, which are visualized for each sea cucumber species (Figure 5A,B), showed that *Bohadschia* species contained the highest number of known saponins, as well as the highest intensities, whereas signal intensities of sapogenins were especially high in the genus *Holothuria*. Interestingly, the three investigated *Bohadschia* species, which were among the most active in inhibiting *C. closterium* growth (Figure 5A), were the only ones containing M1426T10.3 (*m*/*z* 1426.698; C67 H110 O32), M1410T11.3 (m/z 1410.703; C67 H110 O31), and M1424T9.8 ( *m*/*z* 1424.6823; C67 H108 O32), which represent analogous molecular formulas to the known saponins *bivittoside D-like, bivittoside C-like,* and *marmoratoside A-like*, respectively (Figures S2–S4).

**Figure 5.** Major saponin compounds detected in the studied sea cucumbers (peak area ≥ 104). (**A**) saponin diversity and relative intensity and (**B**) sapogenin (aglycon) diversity and relative intensity. Sample codes represent exact mass (M in Da), and retention time (T in min). Di fferent colors represent the presence of sulphate groups (in blue), non-sulphate groups (in black) and pure compounds (in purple and red). Bubble size correlates with di fferences in relative peak areas of the respective molecules.

### 2.2.2. Total Saponin Concentration

The total triterpene glycoside concentration of the crude extracts was assessed using the vanillin-sulfuric acid colorimetric assay (Figure 6). *H. atra* (0.456 mg mL−<sup>1</sup> ± 0.08) and *H. whitmaei* (0.496 mg mL−<sup>1</sup> ± 0.08)had thelowest saponin concentration, whereas*A. echinites*(2.106 mg mL−<sup>1</sup> ± 0. 16), *A. mauritiana* (1.880 mg mL−<sup>1</sup> ±0.15),*B. vitiensis*(1.181 mg mL−<sup>1</sup> ±0.01), and*B. argus*(1.130 mg mL−<sup>1</sup> ± 0.01) contained the highest concentrations of saponins. Saponin concentration in the genus *Actinopyga* was significantly higher than within *Holothuria* and *Bohadschia* (Kruskal-Wallis test; *p* < 0.05).

**Figure 6.** Absolute saponin concentration of the tested crude extracts. (**<sup>a</sup>**–**<sup>c</sup>**) indicate significant differences between different sea cucumber crude extracts. Kruskal–Wallis, Dunn's method as a multiple comparison test. Significance level at *p* < 0.05 was applied.

### *2.3. Anti-Fouling E*ff*ects of Purified Saponin Fractions and Pure Compounds*

### 2.3.1. AF Assay with an Emphasis on Saponins

Based on the LRR of *Chl a* calculated for 1.5 μg mL−<sup>1</sup> of different fractions, the Kruskal-Wallis test revealed that fraction 3 and 4 had a significant negative effect on the growth of *C. closterium* (*p* < 0.05). The first two fractions, on the other hand, had a significant positive effect on the growth of the diatom species (Figure 7A,B).

**Figure 7.** Logarithmic response ratio (LRR) of *C. closterium* following exposure to *B. argus* extract fractions in suspended cells in the water (**A**) and attached to the substrate (**B**). Fr.1 and Fr.2: impure, Fr. 3: semi-pure, Fr. 4: pure singular saponin species (*bivittoside D-like*). a–d represent result of Kruskal-Wallis test; *p* < 0.05.

### 2.3.2. Saponin Profile of the Fractions

The most abundant saponin compounds in *B. argus* (i.e., C67 H110 O32, *bivittoside D-like* and C67 H108 O32, *marmoratoside A-like*; Figure S5, Table S4) were isolated to examine whether saponins are responsible for the observed anti-fouling activities. As shown in Figure S6, the mixed fraction 3, containing the saponin species M1426T10.3 (*bivittoside D-like*), M1454T10.7 (*stichoposide D-like*) and 1424T10.4 (*marmoratoside A-like*), as well as the relatively pure fraction 4 with mainly saponin M1426T10.3 (*bivittoside D-like*) strongly inhibited growth as well as attachment of the diatom. Fraction 1 and 2, on the other hand, contained a mixture of saponins (except *bivittoside D-like*), which did not affect the growth and attachment of *C. closterium*. Currently, the main three saponin species from fraction 3 are further purified and their molecular structure is being elucidated via NMR spectroscopy.
