**3. Discussion**

Many marine benthic organisms (e.g., sponges, mussels, starfishes, sea urchins, algae) are known to harbor anti-fouling metabolites that protect them from deleterious fouling organisms (e.g., [33,64–67]). Sea cucumbers do not have visible defensive mechanisms, however their surfaces are free of fouling organisms [40]. Several molecules with various biological activities (e.g., anti-bacterial, anti-fungal, ichthyotoxic) are reported from sea cucumbers, including their anti-fouling properties [38,68]. The AF potential was found to be species specific, and saponins were identified as the main bioactive molecules responsible for these activities [69].

This study demonstrated that the AF properties of the crude extracts of nine sea cucumber species were related to the presence of particular chemical compounds. Our results showed a clear dose-e ffect for the genus *Actinopyga* and *Bohadschia*, with minimal growth and settlement inhibition at the lowest concentration. Only two of the four tested *Holothuria* species (*H. atra* and *H. edulis*) inhibited algal growth and settlement at the highest concentration, whereas their lower doses (15 and 1.5 μg mL−1) actually induced diatom growth, which is following the hormetic e ffects described by Stebbing ([70,71]; Figure 3). Similar patterns have been reported for crude extracts of *Holothuria leucospilota* against the diatoms *Nitzschia closterium* and *Navicola subinflata*, where lower concentrations (i.e., < 400 μg mL−1) of *H. leucospilota* crude extract induced diatom settlement, and at higher concentrations (i.e., > 400 μg mL−1) inhibited their growth [69].

Previous studies have shown that steroidal and triterpene glycosides in sponges, gorgonians, sea stars, sea urchins, and sea cucumbers are responsible for the observed anti-fouling activities [15,36,38,41,65,72,73]. Saponins have often been described from holothurians including their various biological activities [74]. For example, studies on *Holothuria glaberrima* [40], *H. atra* and *Holothuria nobilis* [38] showed that saponins were responsible for the observed anti-fouling activities. Also, Selvin and Lipton (2004), and Ozupeck and Cavas (2017) found that the saponin-enriched fraction of di fferent sea cucumbers (i.e., *Holothuria scabra*, *Holothuria polii* and *Holothuria tubulosa*) had pronounced anti-fouling properties [41,75]. In this study, we demonstrated that the composition of saponins is more similar within species of the same genus. For example, the saponin compositions of the genus *Bohadschia* was rather di fferent from the genus *Holothuria* and *Actinopyga* (Figure 4). These observations were in line with the strong AF e ffects of *B. argus* and *Bohadschia sp.* crude extracts (Figure 3). As apparent from our AF assays, not only *Bohadschia*, but also the genus *Actinopyga* showed much stronger activities compared to *H. atra* and *H. edulis* (Figure 3), which may be explained by significantly higher concentrations of total saponins (*cf.* Figure 6).

Looking at the saponin profile of the studied genera, we observed similar patterns as described by Kalinin and his colleagues (2015), that non-sulphated saponins with molecular weights of *m*/*z* 1426.698, and *m*/*z* 1410.703 were found in the highest intensity in the genus *Bohadschia* [76–78]. All these saponins contain six monosaccharide units in their glycone parts, and were present nearly 1–5-fold higher than the tetraosides ( *m*/*z* 1118.551 and *m*/*z* 1102.556 ), which were present in the other sea cucumber species. Whereas, sulphated saponins (e.g., molecular weights of *m*/*z* 1206.510 and *m*/*z* 868.389), putatively annotated as *echinoside A* and *echinoside B* respectively, were observed only in the two genera

of *Actinopyga* and *Holothuria*. This is in accordance with the results from Kitagawa and colleagues (1981; 1989), and Grauso and colleagues (2019), who reported *echinoside A* and *B* from *Holothuria* (i.e., *H. atra;* [79]), and *Actinopyga* species [80]. *Actinopyga* and *Holothuria* extracts also contained mixtures of biosides including *bivittoside A* like compounds (C41 H66 O12; *m*/*z* 750.455), tetraosides such as the saponin *desholothurin A* (C54 H86 O24; *m*/*z* 1118.551) and *pervicoside B* (C54 H86 O22; *m*/*z* 1086.561). Our data indicated that the AF activity may correlate with the amount and/or type of sugar units in their glycone part (in genus *Bohadschia*). A similar result has been observed by Van Dyck and colleagues (2010), who analyzed the saponin profile of *Holothuria forskali* in undisturbed and under predator stress conditions [43]. In the undisturbed state, the body wall of *H. forskali* produced mainly tetraosides (i.e., *holothurinoside C* (*m*/*z* 1102) and *desholothurin A* (*m*/*z* 1118)), while under stressed conditions *holothurinoside C* was converted to the hexaosides *holothurinoside F* (*m*/*z* 1410) and *holothurinoside H* (*m*/*z* 1440) and *desholothurin A* was converted to the hexaosides *holothurinoside* G ( *m*/*z* 1426). However, Van Dyck and colleagues (2010) also pointed out that *m*/*z* 1426 is produced under both environmental states in the tested sea cucumber, suggesting that *m*/*z* 1426 is a "background prevention signal," and other molecules might play more important roles under stressful conditions [81]. Also, Kalinin and colleagues (2015) described a molecule with the same molecular mass, but di fferent side chain (*m*/*z* 1426) as a characteristic saponin of *Bohadschia*, which was identified as "*bivittoside D.*" A remarkable similarity observed between *H. forskali* and genus *Bohadschia* is the presence of chemically defended CTs (*cf*. Figure 1), each containing di fferent saponin mixtures [43,82].

The mechanism of action of many extracted and isolated molecules with anti-fouling e ffects are usually unclear because of multiple possible interactions involved [83]. As mentioned earlier, saponins are amphiphilic molecules with hydrophilic and hydrophobic properties. The amount of monosaccharides attached on the C-3 position (Figure 2) of the steroid a ffects the hydrophilicity of the saponin molecule, which can a ffect the permeability of the cell membranes by inducing curvature and forming pores in the membrane [84]. Therefore, high integration values of hexaosidic saponins, containing lanosterol as the major sterol within the genus *Bohadschia* [85], may explain their strong AF activities [81].

It can be concluded that the AF activity is species-specific in sea cucumbers and related to not only total saponin concentration (e.g., in *Actinopyga*), but also saponin composition (such as shown in *B. argus*). AF activities of the studied crude extracts showed that *B. argus* contained compounds affecting fouling by the diatom *C. closterium*. Consequently, purified fractions and pure compounds of *B. argus* (Figure 7) confirmed that particular saponin compounds (here *m*/*z* 1426.698) had strong inhibitory e ffects on growth and settlement of the diatom *C. closterium*. Furthermore, the here performed anti-fouling assays can be a promising and fast method for identifying compounds with anti-fouling activity and for pre-selecting bioactive extracts and/or compound from various organism to discover ecologically and potentially pharmaceutically active natural products.

### **4. Materials and Methods**

In this study we investigated nine holothurian species from the family Holothuriidae that were collected from Guam in 2016. These nine species were members of three di fferent genera, *Holothuria (H. whitmaei, H. hilla, H. atra, H. edulis)*, *Bohadschia (B. argus, B. vittiensis, Bohadschia sp.)* and *Actinopyga (A. echinites, A. mauritiana;* Figure 2).
