Chemical Defense Against Fouling in the Solitary Ascidian Phallusia nigra BOAZ MAYZEL*, MARKUS HABER, AND MICHA ILAN Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 6997801, Israel Abstract. The solitary ascidian Phallusia nigra is rarely fouled by epibionts. Here, we tested the antifouling activity of its crude extracts in laboratory and field assays. P. nigra extracts inhibited the growth of all eight tested environmen- tal bacteria and two of four laboratory bacteria. Extracts of the sympatric, but fouled solitary ascidian Herdmania mo- mus inhibited only one test bacterium. Scanning electron microscopy confirmed that the tunic surface of P. nigra is largely bacteria-free. Both ascidian extracts significantly inhibited the larval metamorphosis of the bryozoan Bugula neritina at the tested concentration range of 0.05–2 mg ml -1 . Both crude extracts were toxic to larvae of the brine shrimp Artemia salina at natural volumetric whole-tissue concentrations, but only P. nigra showed activity at 2 mg ml -1 and below (LC 50 = 1.11 mg ml -1 ). P. nigra crude extracts also significantly reduced the settlement of barna- cles, polychaetes, and algae in Mediterranean field assays and barnacle settlement in Red Sea trials. Comparisons between control experiments and pH values monitored in all experiments indicate that the observed effects were not due to acidity of the organic extracts. Our results show that P. nigra secondary metabolites have antifouling activities, which may act in synergy with previously proposed physi- ological antifouling mechanisms. Introduction In marine benthic environments, any exposed, unde- fended, long-lived surface becomes fouled (Wahl, 1989). The fouling process consists of multiple steps starting with the development of an organic film by adhesion of macro- molecules, followed by the settlement of microorganisms and the development of a microbial biofilm. The settlement of unicellular and multicellular eukaryotes establishes the mature fouling community composed of prokaryotes, fungi, diatoms, algae, protists, and invertebrates (Krug, 2006). Fouling also occurs on the surfaces of living organisms, and sessile marine invertebrates are particularly susceptible to fouling (Wahl, 1989). Although fouling organisms can be beneficial to their hosts, for example by offering camouflage and by providing protection from predators by their second- ary metabolites (Laudien and Wahl, 1999), the interaction is largely considered harmful. Among the negative effects are increased hydrodynamic drag on the host, which can lead to dislodgment of sessile hosts; and disruption of the host’s feeding, either by direct competition for food particles or by changing the water flow to the host, which is especially important for filter-feeding organisms (Stoecker, 1978; Wahl, 1989; Krug, 2006). Ascidians (phylum: Chordata, subphylum: Tunicata) are soft-bodied, sessile, filter-feeding organisms. They occur in all oceans in colonial and solitary forms. Fouling on ascid- ians can lead to the obstruction of the inhalant and exhalant siphons as well as to the clogging of the branchial basket, important to both respiration and feeding (Lambert, 1968; Koplovitz et al., 2011). Both naturally fouled and non- fouled ascidian species have been reported from various geographical regions (e.g., Stoecker, 1980; Uriz et al., 1991; Davis and White, 1994). Fouled ascidian species might use behavioral responses (e.g., actively closing the siphons or squirting water and particles from the branchial basket through apertures [Hoyle, 1953]) and physical responses (e.g., regular tissue sloughing [Goodbody, 1962] and mucus secretion [Wahl et al., 1998]) to overcome fouling. Short- lived ascidians might be able to grow and reproduce before becoming fouled (Stoecker, 1980). Among the taxa reported as epibionts on ascidians are bacteria, algae, bryozoans, Received 25 September 2013; accepted 5 September 2014. * To whom correspondence should be addressed. E-mail: bmayzel@ gmail.com Reference: Biol. Bull. 227: 232–241. (December 2014) © 2014 Marine Biological Laboratory 232