*4.1. Experimental Setup*

### 4.1.1. Cylindrotheca Closterium Culture

The AF assays were conducted using the diatom species *C. closterium* as the test organism. The diatom cultures were kept in climate chamber with constant temperature of 18 ◦C with a light and dark cycle of 12 h and a light intensity of 90 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup>1. The initial stock culture (strain number CCAP-1017/8) was obtained from Culture Collection of Algae and Protozoa (CCAP), and was prepared

in 250 mL polystyrene culture flasks, filled with sterile artificial seawater enriched with F⁄2 nutrients, which has shown to be an optimal nutrient supply for this algal species [86,87].

### 4.1.2. Preparation of Sea Cucumber Crude Extracts

Extractions of sea cucumbers were performed by freeze dried material. For each extraction a 1:10 ratio (w/v) of freeze-dried sea cucumber tissue (in g) and organic solvent mixture (in mL) was used. In brief, the ground tissue samples were extracted twice with a 1:1 mixture (v/v) of methanol (MeOH) and ethyl acetate (EtOAc) and a third and final time with 100% MeOH. Samples were shaken for at least 3 h during each subsequent extraction. After filtering through filter paper (Diameter: 150 mm, Grade: 3 hw, Sartorius GmbH, 37979, Goettingen, Germany), extracts were dried by rotary evaporation (Rotavapor RII, BUCHI, Flawil, Switzerland) and finally transferred and dried using a centrifugal vacuum concentrator (Speedvac, Christ RVC 2–25 Co plus; Freeze dryer: Christ Alpha 2–4 LD plus). The dried crude extracts were weighted and stored at −20 ◦C until further usage.

### 4.1.3. Anti-Fouling Assay: Experimental Design

The e ffect of the sea cucumber crude extracts on the growth and settlement behavior of *C. closterium* was tested by monitoring the biomass of the diatom after 24 and 72 h incubation, based on chlorophyll a (*Chl a*) concentration of suspended cells in the water as well as attached cells on the flask surface. The AF assays were performed in 40 mL culture flasks (TC Flask T25, SARSTEDT AG & Co. KG, 51588, Nümbrecht, Germany). All crude extracts were dissolved in MeOH and added in triplicates to empty the cell culture flasks in order to obtain three di fferent final concentrations of the crude extracts (150, 15 and 1.5 μg mL−1, Figure 8). After the MeOH evaporated, 10% of the diatom stock (i.e., 1.5 mL of algae inoculated in 15 mL F/2 medium; OD442 = 0.46 ± 0.01) was inoculated to the culture flask pre-filled with 18 mL of sterile artificial seawater. For three days, the flasks were stored horizontally in a growth chamber under the above-mentioned culturing conditions (Section 4.1.1) to perform the diatom surface attachment experiment. Treatments with only MeOH and no sea cucumber crude extract served as control experiment.

The potential AF e ffects of particular saponin species were assessed using fractions isolated from *B. argus*. The assay with the purified saponin fraction and pure saponin compounds were conducted with only the lowest concentrations of 1.5 μg mL−1.

**A B C** 

**Figure 8.** (**A**–**C**). The test organism *C. closterium* under the microscope ( **A**), culture flasks demonstrating high growth rates (left, not-inhibited), medium growth rates (middle) and low growth rates (inhibited) of *C. closterium* (**B**), growth curve of *C. closterium* in 7 days in the stock solution ( **C**).

### 4.1.4. Diatom Growth and Settlement Analyses

*Chlorophyll a measurements:* To assess diatom biomass, *Chl a* was extracted from the water samples after 24 and 72 h of inoculation. Furthermore, to study the attachment behavior of the diatom, the *Chl a* content of *C. closterium* attached to the substrate was extracted after 72 h (end of the experiment), except for the highest concentration. Since we observed that algae biomass was dramatically reduced

in most of the extracts exposed to the highest extract concentration (150 μg mL−1), *Chl a* concentrations in both, suspended in water and attached to substrate, were measured only after 24 h of inoculation. Experimental procedure included filtering water samples through a combusted and acid-washed glass microfiber filter (GF/C, Whatman, GE Healthcare life sciences, Pittsburg, PA 15264-3065, USA) and storing at −80 ◦C until extraction. For extraction, ethanol (90%) was added to the samples, vortexed, and then placed in an ultrasonic bath filled with ice for 30 min. Before measuring pigment concentrations, all samples were stored for 24 h at 4 ◦C. Measurements were conducted with a microplate reader (BioTek, SYNERGY H1, Winooski, VT, USA) to determine the *Chl a* concentration using a fluorescence excitation (Ex) wavelength of 395 nm and emission (Em) wavelength of 680 nm. *Chl a* concentrations were obtained by converting fluorescence data to concentrations using a *Chl a* standard from *Anacystis nidulans* algae (Product Number C 6144, Sigma-Aldrich, St. Louis, MO, USA).

### 4.1.5. Anti-Fouling Effects: Data and Statistical Analyses

Statistical analyses were performed with R (version 1.1.423, R Foundation for Statistical Computing, Vienna, Austria), and SPSS (Version 26, IBM, NY 10504, USA). We assessed the effect of different sea cucumber extracts and concentrations on diatom settlement, as well as cell density of the diatom *C. closterium.* After testing for normality and homoscedastity, Kruskal-Wallis test was conducted for each extract concentration, followed by Kruskal–Wallis post hoc test. The same method was applied for the purified fractions and pure compounds (Section 4.3). Differences were considered significant at a 95% confidence level. The logarithmic response ration (LRR; Equation (1)) was calculated as the ratio of *Chl a* concentration affected by crude extracts to the controls. LRR > 0 illustrates higher *Chl a* concentration and thus a positive effect in extract treatments, while LRR < 0 identifies decreased *Chl a* concentrations, and thus a negative effect compared to control samples.

$$\text{LRR} = \ln(\frac{treatment}{control}) \tag{1}$$

### *4.2. Saponins as Potential Bioactive Compounds A*ff*ecting the Fouling Organism C. closterium*

### 4.2.1. Dereplication of Saponins

To analyze the content of the most abundant saponin species within the different sea cucumber crude extracts (dissolved in MeOH), an aliquot was analyzed using ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS; Tables S2 and S3). Chromatographic separation was achieved on a Waters Acquity BEH C18 column (1.7 μm, 2.1 mm × 50 mm) with an ACQUITY ultra performance liquid chromatography (UPLC) H-Class System (Waters Co., Milford, MA, USA) coupled to a Synapt G2-Si HDMS high-resolution Q-ToF-MS (Waters Co., Manchester, UK) equipped with a LockSpray dual electrospray ion source operated in positive (POS) ionization modes. The Q-ToF-MS was calibrated in resolution mode over a mass-to-charge (*m*/*z*) ranging from 50 to 2000 Dalton by using a 0.5 mmol L−<sup>1</sup> sodium formate solution. For each run leucine enkephalin was used as the lock mass, generating a reference ion for POS mode ([*m*/*z* 556.277 M + H]+) to ensure a mass tolerance for all LC-MS or LC-MS/MS experiments of less than one ppm. Mass spectral data were collected using the MSe data acquisition function to simultaneously obtain information on the intact molecule (no collision energy applied) as well as their fragmentation data (collision energy ramp reaching from 15 to 75 eV). Analytes were eluted at a flow rate of 0.6 mL min−<sup>1</sup> using a linear gradient of milliQ water (H2O, 100%, eluent A) to acetonitrile (ACN, 100%, eluent B) both with 0.1% formic acid. The initial condition was 100% A held for 0.5 min, followed by a linear gradient to 100% B in 19 min. The column was then washed with 100% B for 9.5 min and subsequently returned and held for 2.9 min to the initial conditions (100% eluent A) to equilibrate the column for the following run. The column temperature was set to 40 ◦C.

*Data treatment:* To identify different saponin compounds in the holothurian extracts we compared the molecular masses of known saponins to the here-analyzed mass data (MS1) and by confirmation the saponin nature (Figure 1) by identifying their diagnostic key fragments. Therefore, we used different diagnostic key fragments corresponding to oligosaccharides residues [88], and the sapogenin molecule (aglycone) part (Table 1). Unknown saponin molecules (with different molecular formulas than previously reported) were not considered in this analysis. Given that we identified several saponins with the same exact mass (probably isomers), we retained the following information for compound identification: (1) retention time (RT), (2) molecular weight and (3) the integrated area of the respective peak (Table S3).

**Table 1.** Key diagnostic fragments of saponins detected via the MS/MS analysis of the studied sea cucumbers.


### 4.2.2. Saponin Compounds Composition: Data and Statistical Analyses

The integrated areas have been log transformed to reduce the skewness. Principal component analysis (PCA) was used to evaluate the differences between saponin compositions of the studied sea cucumbers. In order to identify the saponin similarity among different sea cucumber species, a hierarchical cluster analysis (function *hclust*, using packages ape for R) was used. After choosing the best cluster method using cophenetic correlation distances (pearson correlation), the penalty function of Kelley Gardner Sutcliffe (*KGS*; package maptree in R) was used to trim the dendrogram. Compounds with integration values higher than 10,000 were then selected to further study the saponin composition of each of the sea cucumber species.

### 4.2.3. Total Saponin Concentration within the Examined Sea Cucumber Species

Since only known saponins could be identified by the LC-MS/MS data, we also quantified total saponin concentration of different sea cucumbers using a spectrophotometric method with vanillin-sulfuric acid, which was adapted after Hiai and colleagues [98]. Based on their method, sulfuric acid oxidizes saponins and transformes glycone chains to furfural. The free hydroxyl group at the C-3 position of the agylcone part reacts with vanillin and produces a distinctive yellow-brown color [41]. According to this methodology, we prepared 8% vanillin solution (w/v) dissolved in ethanol (analytical grade), and sulfuric acid 72% (v/v) dissolved in distilled water. Crude extracts as well as double distilled water (used as blanks), were mixed with vanillin (8%; AppliChem GmbH, Germany) and sulfuric acid (72%) in a 1:1:10 (v/v/v) proportion in an ice bath. Next, we incubated the obtained solution at 60 ◦C in a water bath for 10 min. To stop the reaction, samples were cooled down on ice. A standard curve was measured, using a concentration gradient of Quillaja bark saponin (AppliChem GmbH, 64291, Darmstadt, Germany), diluted in distilled water. Finally, the absorbance was measured at 540 nm using a microplate reader.

### *4.3. Anti-Fouling E*ff*ects of Purified Saponin Fractions*

We further fractionated the crude extract of *B. argus*, since it had exhibited one of the highest AF activity among the tested organic extracts. The aim was the identification of one or multiple saponin compounds responsible for the anti-fouling activity observed in the crude extract.

### *4.4. Sample Fractionation and Purification*

*Liquid*/*liquid partitioning:* The crude extracts of *B. argus* were first partitioned using (1) EtOAc:H2O (1:1) followed by partitioning of the H2O fraction with (2) n-BuOH:H2O (1:1).

*Solid Phase Extraction (SPE) chromatography:* The BuOH fraction which contained the saponins was further fractionated by SPE chromatography [99]. Therefore, the SPE column (SUPELCLEAN LC18, 60 mL/10 g; Supleco Park, USA) was desalted/washed with 60 mL MeOH and preconditioned with 120 mL distilled water. Then, the concentrated BuOH fraction was added to the column and washed with five elution gradients: (1) Elution with H2O (Fraction A, 120 mL), (2) MeOH:H2O (Fraction B, 50:50, 180 mL), (3) ACN: H2O (Fraction C, 70:30, 180 mL), (4) ACN 100% (Fraction D, 180 mL) and (5) CH2Cl2: MeOH (fraction E, 90:10, 180 mL; Figure 9).

*Preparative HPLC:* Preliminary biological and chemical screening of each SPE fraction showed that fractions B (MeOH:H2O 50:50) and C (CH3CN:H2O 70:30) contained not only diverse and high amounts of saponins, they also had high activities against the fungi *Rhodotorula glutinis* and *Candida albicans* (unpublished data). Therefore, these fractions were selected for further purification by semi-preparative HPLC (Agilent Technologies, 1260 Infinity) with a PDA detector (Agilent, G4212-60008, CA, USA). Chromatographic separation was achieved using a C18 column (Pursuit XRs 5 μm, 250 mm × 10 mm, Agilent, CA, USA) with a pre-column (2.7 μm, 2.1 mm × 5 mm, Agilent, CA, USA) and applying a linear gradient: initial 50% A/50% B, 0–4 min 50% A/50% B; 4–36 min 38% A/62% B; 36–39 min 100% B, and a column reconditioning phase for 39–59 min 100% B, and 8 min to 50% A/50% B. (flow rate 1.5 mL min−1; eluent **A:** 95% H2O and 0.1% of formic acid 98% (Roth); eluent **B:** ACN and 0.1% formic acid). Several fractions were collected by peak picking at specific retention times. In order to determine the saponin composition of the obtained fractions and pure compounds, the fractions and compounds were dissolved in HPLC-grade MeOH, filtered through a 0.2 μm syringe filter, and injected into the HPLC-DAD-MS system, as previously described in Section 4.2.1. The peak integration of saponins in the final fractions has been assessed (Table S4), and these fractions have been used for AF assay.

**Figure 9.** Flow chart showing the applied procedure for isolating the bioactive saponin compounds (Cutignano et al., 2015; Ebada et al., 2008 [99,100]). Sample set 1 and 2 refers to the samples that were tested for anti-fouling (AF) activity in this study.

**Supplementary Materials:** Figure S1A–F: *Chl a* concentrations in the suspended cells in the water after incubation of *C. closterium* with di fferent concentrations of sea cucumbers extracts (A = 150 μg mL−1; C = 15 μg mL−1; E = 1.5 μg mL−1) and of *C. closterium* attached to the flask surface (B = 150 μg mL−1; D = 15 μg mL−1; F = 1.5 μg mL−1). Dashed lines separate di fferent genera of sea cucumbers (*Holothuria, Bohadschia, Actinopyga*). CT = Control. (a–e) indicate significance levels according to post hoc test. Figure S2: LC/MS spectra of the crude extracts of genus *Holothuria* (Y-axis relative intensity in % of maximum peak, x-axis retention time in minutes). Figure S3: LC/MS spectra of the crude extracts of genus *Bohadschia* (Y-axis relative intensity in % of maximum peak, x-axis retention time in minutes). Figure S4: LC/MS spectra of the crude extracts of genus *Actinopyga* (Y-axis relative intensity in % of maximum peak, x-axis retention time in minutes). Figure S5: LC/MS spectra of fractions isolated from *B. argus* (see Table S4; Y-axis relative intensity in % of maximum peak, x-axis retention time in minutes). Figure S6: Identified saponins species presented in di fferent fractions isolated from *B. argus.* The red color referred to the presence of a semi-purified saponin species (*bivittoside D-like* at *m*/*z* 1426.698). Size of bubbles represented the peak area of the molecules obtained from LC/MS analysis. Table S1. Significant di fferences (reported as *p-values*) of sea cucumber crude extracts compared to control experiments using the Kruskal–Wallis test. Table S2: Saponins reported, and found in studied species. Table S3: Exact mass ( *m*/*z*), molecular formula, retention time (RT), and intensity signal (IntSig) of saponins, and sapogenins (aglycone parts) presented in the three sea cucumber genera *Holothuria*, *Bohadschia* and *Actinopyga*. Table S4: Exact mass ( *m*/*z*), molecular formula, retention time (RT in minutes) and intensity signal of saponins presented in isolated fractions of *B. argus.*

**Author Contributions:** E.K., M.Y.K., M.S., S.R., and P.J.S. conceived and designed the experiments; E.K., N.G., M.Y.K. performed the experiments; E.K., N.G., M.S., M.Y.K., M.R., S.R. analyzed the data; E.K., M.Y.K., M.R., M.S., P.J.S. wrote the paper; E.K., N.G., M.Y.K., S.R., M.R., M.S., P.J.S. reviewed and edited the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors acknowledge funding by the Federal Ministry of Education and Research (BMBF) via the Germany-Indonesia Anti-infective Cooperation (GINAICO) gran<sup>t</sup> number 16GW0106 and Deutsche Forschungsgemeinschaft (DFG) funding INST 1841147.1FUGG for the high-resolution mass spectrometer Waters Synapt G2-Si.

**Acknowledgments:** We would like to thank Sabine Flöder and Christian Spindler for their support in phytoplankton cultivation and media preparation, also Pedro Martinez Arbizu for his advices in developing the R codes. The authors acknowledge funding by the BMBF via the GINAICO gran<sup>t</sup> (16GW0106) and DFG funding (INST 1841147.1FUGG). We also thank anonymous reviewers for valuable comments, and their time which helped to improve the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
