**1. Introduction**

Biofouling is the colonization process of micro- (i.e., protozoa, bacteria, fungi and diatoms) or macro-organisms (i.e., algae and invertebrates) on either living (known as epibiosis) or artificial substrates [1,2]. Mckenzie and Grigolava (1996) described that epibiosis decreased host fitness, their survival rate, and species abundance, as well as affects their community composition [3]. Fouling on living surfaces of marine invertebrates can increase their friction as well as their body weight, and thus reduces speed, elasticity, and flexibility of the fouled organism, which in turn may lead to reduced viability and death [4,5]. Shading by fouling organisms can also impact negatively on the

growth rate of the fouled organisms due to a reduced photosynthetic rate of macroalgae [6]. Biofouling processes are however not only relevant from an ecological perspective, but they have also important economic implications [4]. Marine biofouling shortens the lifespan and increases the maintenance costs of underwater constructions like ship hulls and aquaculture cages [7,8]. It also increases the weight and the friction of a ship, which in turn decreases the maximum cruising speed as well as increases fuel consumption [7,9]. In order to counteract the biofilm production on these structures, various synthetic anti-fouling (AF) paints that contain toxicants such as mercury (mercuric oxide (HgO), mercuric arsenate (AsO4Hg3)), arsenic (arsenic trioxide (As2O3)), copper (cuprous arsenite (AsO3Cu3)), as well as organotins (mainly tributyltin (TBT) based compounds) and rosin-based paint, have been applied in the past [7,9]. However, there are numerous studies showing that all the latter paints are hazardous for the environment and negatively a ffect the growth rate and reproduction of both fouling and non-fouling marine organisms [7,8,10–12]. As a result of such studies, production and application of TBT-based AF paints was internationally banned in the 1990s [7,13], and substituted with copper-based and booster biocides. However, recent research showed that these compounds still display toxic effects on marine organisms. The use of natural products as biological-based AF biocides in coating has been suggested as a new sustainable alternative, since they generally show biocompatibility, biodegradability, and thus their toxicity e ffects (if any) will not accumulate and generate long-lasting perturbations in the environment [14–18]. Engineering of an e ffective biological-based AF coating may not only protect the marine environment, but could also have substantial economic benefits by increasing the lifespan of underwater structures and by reducing the fuel consumption rate in the shipping industry (i.e., 60\$ billion per annum). Furthermore, reduced fuel consumption also decreases carbon dioxide and sulfur dioxide emissions to the atmosphere [19–21] and therefore mitigate the effects of world-wide shipping to climate change.

In order to avoid the negative e ffects from unwanted epibioses, organisms can either accept and tolerate the presence of the fouling organisms (i.e., by developing a symbiosis) or avoid them, by either changing their habitat or developing chemical defenses [3,22–24]. AF defenses of marine organisms include mucus secretion (e.g., in sea star *Marthasterias glacialis*; [25]), shedding, microroughness, burrowing, scraping, and cleaning their body wall as well as chemical defense [4,26–28]. Chemical anti-fouling compounds can have various modes of action. They can be toxic to epibionts [29–31], inhibit settlement of larvae from fouling organisms [5,32,33] or prevent development of bacterial biofilms by disrupting bacterial communication via inhibition of the bacterial acylated homoserine lactone (AHL) signaling pathways [34]. Until 2017, almost 200 di fferent AF compounds were described from marine invertebrates such as echinoderms, sponges, gorgonians and soft corals [35]. These AF compounds belong to various groups of terpenoids (i.e., triterpenes, sesquiterpenes and diterpenes), alkaloids, steroids, triterpene glycosides (saponins), polyacetylenes, butenolides, peptides and phenol derivatives [30,35,36]. More recently, the AF activities of compounds isolated from sea cucumbers such as *Holothuria atra* [37,38], *H. nobilis* [38], *H. edulis* [39], *H. glaberrima* [40], *H. tubulosa* and *H. polii* [41] were reported, as these sea cucumbers keep their body surfaces conspicuously free of fouling organisms [3]. Echinoderms, and especially sea cucumbers, are known to produce a wide variety of triterpene glycosides or saponins [42]. Saponins are composed of a hydrophilic glycone and a hydrophobic aglycone (i.e., sapogenin; Figure 1) that, depending on the holothuroids (*cf.* Figure 2), are located in the Cuverian Tubules (CT), in its body wall and its viscera [43]. Because of the membranolytic activities of saponin, a wide range of bioactivities such as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory, ichthyotoxic, as well as anti-fouling properties have been reported [42,44,45].

The colonization process of fouling organisms starts after the first contact of the respective surface to sea water [4]. After "biochemical conditioning," which is initiated by adsorption of macromolecules to the surface, a bacterial biofilm develops. This is followed by the colonization of unicellular eukaryotes and algae such as diatoms [4]. One such algae is the meroplanktonic diatom *Cylindrotheca closterium*, that showed rapid growth particularly on surfaces [46,47]. This diatom species is also known to produce different types of hydrophilic and carbohydrate-rich extracellular polymeric substances (EPS) that often represent the major component of the extracellular aggregative matrix [46]. EPS plays a crucial role in the biofilm formation, and the microbial and physicochemical defenses of the diatom [48,49], their motility [50], cell to cell and cell to substratum adhesion [51], as well as in the settlement success and post larval growth of other organisms [52–54]. Thus, *C. closterium* has been used as a model organism in the past for early stage fouling studies [50,52,55].

**Figure 1.** Structure of the saponin molecule "*bivittoside D*", *m*/*z* 1449.687 [M + Na]+ [56], consisting of the glycone and aglycone moieties (produced with ChemDraw, version 16.0.1.4 (77)).

The aim of this study is to determine AF activities of different crude extracts of tropical Indo-Pacific sea cucumber species against the fouling diatom species *C. closterium*. To identify phylogenetic differences in AF activities of sea cucumber species, we choose nine species from three different genera (i.e., *Holothuria*, *Bohadschia*, *Actinopyga*). Also, we tested purified fractions enriched in saponins as well as pure saponin compounds to verify whether these sea cucumber characteristic compounds were involved in the observed AF activities of sea cucumbers.

**Figure 2.** Phylogeny tree of the here studied sea cucumbers (CT=cuvierian tubule; adapted from [57–60].
