Chemical Control of Marine Associated Microbial Communities in Sessile Antarctic Invertebrates

1 Chemical control of marine associated microbial communities 2 in sessile Antarctic invertebrates 3 4 Carlos Angulo-Preckler1,2*, Eva García-Lopez3, Blanca Figuerola4, Conxita Avila1,2, 5 Cristina Cid3 6 7 1Department of Evolutionary Biology Ecology and Environmental Sciences, Faculty of 8 Biology, University of Barcelona, 08028 Barcelona, Catalonia, Spain. 9 2Biodiversity Research Institute (IrBIO), 08028 Barcelona, Catalonia, Spain. 10 3Microbial Evolution Laboratory, Center of Astrobiology (CSIC-INTA), 28850 Madrid. 11 Spain. 12 4 Institute of Marine Sciences (ICM-CSIC), Pg. Marítim de la Barceloneta 37-49, 08003 13 Barcelona, Catalonia, Spain 14 *Corresponding author; Carlos Angulo-Preckler, [email protected] 15 (+34 639947626) 16 ORCID: orcid.org/0000-0001-9028-274X 17 18 Short tittle: Pontential antifouling activity of Antarctic invertebrates 19 20 21 22 23 24 25 26 27 28 Keywords: Marine benthos; Porifera; Bryozoa; bacteria; eukaryotes; antifouling; 29 antimicrobial activity. 30 31 32 1 33 Abstract 34 Organisms living in the sea are exposed to fouling by other organisms that may try to 35 settle on top of them. Many benthic marine invertebrates, including sponges and 36 bryozoans, contain natural products with antimicrobial properties, since microbes 37 usually constitute the first stages of fouling. Extracts from four Antarctic sponges 38 (Myxilla (Myxilla) mollis, Mycale tylotornota, Rossella nuda, and Anoxycalyx 39 (Scolymastra) joubini), and two bryozoan species (Cornucopina pectogemma and 40 Nematoflustra flagellata), were tested separately for antifouling properties in field 41 experiments. The different crude extracts from these invertebrates were incorporated 42 into a substratum gel at natural concentrations for an ecological approach. Treatments 43 were tested by submerging plates covered by these substratum gels under water in situ 44 during one lunar cycle (28 days) at Deception Island (South Shetland Islands, 45 Antarctica). Remarkably, the butanolic extracts of M. tylotornota and C. pectogemma 46 showed complete growth inhibition of microscopic eukaryotic organisms, one of the 47 succession stages involved in biofouling. Our results suggest that different chemical 48 strategies may exist to avoid fouling, although the role of chemical defenses is often 49 species-specific. Thus, the high specificity of the microbial community attached to the 50 coated plates seems to be modulated by the chemical cues of the crude extracts of the 51 invertebrates tested. 52 53 54 55 56 57 58 59 60 1. Introduction 61 Submerged substrates are potentially exposed to a myriad of fouling organisms, and 62 thus, competition is very intense, including many chemically mediated interactions 63 (Buss, 1990; Steinberg & de Nys 2002). The colonization of a substratum (biofouling) 64 in the sea is a highly dynamic and complex process involving 1) adsorption on the new 65 surface of dissolved organic molecules, forming a conditioning film, 2) colonization by 66 bacteria with specific cell-surface, cell-cell, and interpopulation interactions shaping the 67 structure, composition, and functions of surface-associated microbial communities 68 (Dang & Lovell 2016), 3) colonization by microscopic, unicellular eukaryotes, such as 69 diatoms, fungi, and heterotrophic eukaryotes, and 4) settlement and growth of 70 multicellular eukaryonts, such as invertebrate larvae and algal spores (Maki & Mitchell, 71 2002; Wahl, 1997; Lema et al. 2019). 72 Several physicochemical properties (e.g. surface hydrophobicity, wetability, and/or 73 surface molecular topography) may determine the adhesion of different bacterial species 74 and microbial community assembly in the biofilms (Wiencek & Fletcher 1997). 75 Bacterial adhesion on submerged surfaces is a highly complex proccess, not only 76 controled by surface properties of the substrate, but also by surface properties of the 77 bacterium itself (Harder & Yee 2009). 78 Being sessile organisms, sponges and bryozoans rely mainly on bioactive compounds 79 for defense against predators, competition for space, and overgrowth by fouling 80 (Proksch et al. 2002). Moreover, their associated microorganisms (symbionts) may also 81 be involved in chemical protection against fouling (Dobretsov et al., 2005; Ortlepp et 82 al., 2008). Most invertebrates may remain relatively free from macrofouling while they 3 83 often present some degree of microfouling (Richmond & Seed 1991; Dobretsov et al. 84 2006). 85 From an ecological perspective, there are only few reports which analyze the inhibition 86 activity of the organism extracts under field conditions. Only in some of them has been 87 possible to measure the fouling settlement (Henrikson & Pawlik 1995; 1998, Angulo- 88 Preckler et al. 2015; Dobretsov & Rittschof 2020). 89 For Antarctic marine benthic organisms, very few studies have evaluated invertebrate 90 antifouling defenses (Angulo-Preckler et al., 2015; Slattery et al., 1995; Cano et al. 91 2018). As mentioned above, potential colonization and overgrowth could be a potent 92 selective pressure on marine benthic organisms, favoring the development of chemical 93 defenses against fouling. Thus, it is ecologically relevant to perform in situ experiments 94 to establish the activity of the organisms’ extracts against environmental 95 microorganisms under real environmental conditions. A field assay method for testing 96 the antifouling activity of crude organic extracts of marine organisms was developed by 97 Henrikson and Pawlik (1995). Accordingly, the main advantages of this methodology 98 are (i) the extract is incorporated into the medium simulating natural situations, where 99 the products are located within the organism, (ii) antifouling substances are liberated 100 slowly, as it presumably occurs in living organisms and (iii) the physical characteristics 101 of the settlement surface remain unchanged (Henrikson & Pawlik 1995). 102 This study aims to evaluate the potential antimicrofouling activity of sessile marine 103 Antarctic invertebrates by comparing their crude extracts under real conditions. We 104 hypothesize that these benthic invertebrates use chemical defence to regulate surface- 105 associated microorganisms colonization as an efficient way to control fouling pressure. 106 Therefore, we used extracts (lipophilic and less hydrophobic extracts) from these 107 invertebrates incorporated into artificial substrata that were submerged under in situ 108 conditions. The biofilms (surface-associated communities developing on these 109 substrata) were analyzed in terms of species composition and relative abundances 110 (bacteria and eukaryotes) and used to infer the antifouling activity of each invertebrate 111 species tested. 112 113 2. Material and Methods 114 2.1. Sample collection and processing 115 Sessile Antarctic invertebrate fauna from two common phyla (Porifera and Bryozoa) 116 were selected to evaluate their potential antifouling activity. Four common species of 117 sponges, two demosponges (Myxilla (Myxilla) mollis Ridley & Dendy, 1886, Mycale 118 tylotornota Koltun, 1964), and two hexactinellids (Rossella nuda Topsent, 1901, 119 Anoxycalyx (Scolymastra) joubini (Topsent, 1916)), altogether with two abundant 120 bryozoan species (Cornucopina pectogemma (Goldstein, 1882), and Nematoflustra 121 flagellata (Waters, 1904)), were selected for the experiment (Table 1). The sponges 122 were collected in the Eastern Weddell Sea during the ANT/XXI-2 cruise of R/V 123 Polarstern (AWI, Germany), during the austral summer of 2003/2004, through Bottom 124 and Agassiz Trawls. Bryozoans were collected by scuba diving in the vicinity of 125 Livingston Island (South Shetland Islands) during the austral summer of 2012. A 126 portion of each sample was conserved for further taxonomical identification at the 127 University of Barcelona (UB). The remaining material was frozen at -20°C until it was 128 needed for the experiments. 129 Each organism was disgregated into small pieces and ground with a mortar and a pestle 130 homogenizing it in acetone to collect the crude extracts. Then, crude extracts were 131 fractionated by polarity, separating the most polar compounds by extraction in butanol 132 (BuOH) from the less polar lipophilic compounds by extraction in diethylether (Et2O). 5 133 The extraction procedure has been extensively described in previous works of our team 134 (e.g., Avila et al. 2000). Natural concentrations of each extract compounds (Et2O or 135 BuOH) were calculated as the total dry weight (DWT) of each sample (Total Dry 136 Weight = dry weight of the solid residue + dry weight of the aqueous residue + dry 137 weight of the Et2O extract + dry weight of the BuOH extract; see Table 1). Fractioning 138 by polarity is important to understand which kind of compounds are responsible for the 139 activity, if any. 140 2.2. Experimental design 141 Gels were prepare by dissolving 1.57 g of PhytagelTM (Sigma Chemical) per 100 ml 142 distilled water and stiring for 10 s. After heated until boiling, the gel was allowed to 143 cool down before an aliquot of extract dissolved in 3 ml solvent (ether or methanol) was 144 added and shaked to obtain the treatments. Each treatment combines one species and 145 one type of extract. The amount of tissue extracted was equivalent to the amount of gel 146 prepared, so that each experimental dish would have a natural concentration of 147 metabolites as in the extracted organism (Angulo-Preckler et al. 2015). 148 Three replicates of each extract treatment were

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