Microbial Bioﬁlms Facilitate Adhesion in Biofouling Invertebrates JOHN D. ZARDUS*, BRIAN T. NEDVED, YING HUANG, CAWA TRAN, AND MICHAEL G. HADFIELD† Kewalo Marine Laboratory, 41 Ahui Street, Honolulu, Hawaii 96813 Abstract. Much interest has focused on the role of mi- crobial layers— bioﬁlms—in stimulating attachment of in- vertebrates and algae to submerged marine surfaces. We investigated the inﬂuence of bioﬁlms on the adhesion strength of settling invertebrates. Larvae of four species of biofouling invertebrate were allowed to attach to test sur- faces that were either clean or coated with a natural bioﬁlm. Measuring larval removal under precisely controlled ﬂow forces, we found that bioﬁlms signiﬁcantly increased adhe- sion strength in the ascidian Phallusia nigra, the polychaete tubeworm Hydroides elegans, and the barnacle Balanus amphitrite at one or more developmental stages. Attach- ment strength in a fourth species, the bryozoan Bugula neritina, was neither facilitated nor inhibited by the pres- ence of a bioﬁlm. These results suggest that adhesive strength and perhaps composition may vary across different invertebrate taxa at various recruitment stages, and mark a new path of inquiry for biofouling research. Introduction Marine biofouling refers to the accumulation of organ- isms and biogenic structures on ship hulls and other sub- merged surfaces. Permanently ﬁxed to a substratum, sessile marine invertebrates in particular compose the framework of many fouling assemblages. Animals such as barnacles and tubeworms also produce shells or other ﬁrm structures as they grow, allowing for attachment of additional organ- isms after primary substratum has been preempted, resulting in multilayered fouling communities (Scheer, 1945). This biological buildup increases frictional drag on ships, smoth- ers oceanographic equipment, adds bulk to ﬂoating struc- tures, clogs seawater lines to power plants, and promotes structural deterioration (Thompson et al., 1988). For the global shipping industry alone, biofouling costs billions of dollars per year in prevention, maintenance, and fuel con- sumption (Alliance for Coastal Technologies, 2004). Biofouling begins with adsorption of organic molecules onto newly submerged surfaces, followed by the accrual of bacteria, diatoms, and other microorganisms bound together in a ﬁlm of extracellular polymeric substances (Zobell and Allen, 1935). Such bioﬁlms develop on surfaces within hours of immersion, increasing in density and structural complexity over time (Donlan, 2002). Subsequent interac- tions of macrobiota with these microbial ﬁlms lead, within days and weeks, to the attachment and growth of inverte- brates and algae, which account for most of the hydrody- namic drag associated with biofouling (Schultz, 2007). In colonizing a surface, many invertebrates rely on a swimming larval stage to make contact with and attach to the substratum. The larvae then metamorphose to a seden- tary stage, in some taxa growing in size and in others dividing asexually to form a spreading colony. Critical links in the biofouling process include detection of appropriate substrata and secure adhesion by larvae. Chemical cues play a pivotal role in invertebrate settlement (Hadﬁeld and Paul, 2001), and microbial bioﬁlms in particular provide bio- chemical signals that larvae employ in selecting a settlement site, attaching to it, and undergoing metamorphosis (Mead- ows and Williams, 1963; Hadﬁeld et al., 1994; Keough and Raimondi, 1994; Carpizo-Ituarte and Hadﬁeld, 1998; Unabia and Hadﬁeld, 1999; Huang and Hadﬁeld, 2003; Dahms et al., 2004). Bioﬁlms can also modulate physical surface properties such as wettability or texture (Crisp and Ryland, 1960; Gray et al., 2002), which are important to Received 22 September 2006; accepted 27 August 2007. * Current address, The Citadel, Dept. of Biology, 171 Moultrie St., Charleston, SC 29409 † To whom correspondence should be addressed. E-mail: hadﬁeld@ hawaii.edu Reference: Biol. Bull. 214: 91–98. (February 2008) © 2008 Marine Biological Laboratory 91