Induction of metamorphosis in the Asian shore crab Hemigrapsus sanguineus: Characterization of the cue associated with biofilm from adult habitat Julie A. Anderson, Charles E. Epifanio ⁎ University of Delaware, College of Earth, Ocean, and Environment, 700 Pilottown Road, Lewes, Delaware, 19958, USA abstract article info Article history: Received 7 July 2009 Received in revised form 7 October 2009 Accepted 8 October 2009 Keywords: Biofilm Chemical cue Conspecific Hemigrapsus sanguineus Metamorphosis Recruitment of crabs to nursery habitat requires settlement of the megalopal stage on suitable substratum followed by metamorphosis into the first juvenile stage. Reducing the time to metamorphosis may result in higher recruitment and survival. Previous work has shown that metamorphosis of the Asian shore crab is accelerated by cues from three different sources: (a) water-soluble exudate produced by conspecific adult crabs; (b) biofilm covering rocks in natural habitat for this species; and (c) abiotic rock from natural habitat. The objective of the present investigation was to characterize the metamorphic cue associated with biofilm from rocky intertidal habitat and to compare the three metamorphic cues (exudate from conspecific adults, biofilm from rocky intertidal, and texture of substratum) that have been identified for H. sanguineus. Results of our study show that megalopae of the Asian shore crab respond strongly to biofilm associated with rocky intertidal habitat that has developed for at least 8 days. We also found that megalopae respond to textured rock surfaces from natural habitat, even when those surfaces had been rendered abiotic. The cue remains active after the biofilm has been exposed to -20 ºC for 12 h, but is de-activated by a few minutes exposure to 100 °C. Moreover, the biofilm cue appears to work in synergy with cues from other sources, but requires actual contact with the biofilm. Our findings show that addition of biofilm to an abiotic textured rock surface significantly decreases mean time to metamorphosis, and simultaneous exposure of megalopae to biofilm-covered rock and to exudate from adult H. sanguineus decreases mean time to metamorphosis even further. The response of this species to multiple cues— and particularly to biofilm in the absence of adult conspecifics—provides a clear advantage in the colonization of virgin habitat and helps explain the very rapid spread of this invasive species along the majority of the east coast of the United States in only two decades. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Chemical and physical cues associated with substratum are well known to influence settlement and metamorphosis of invertebrate larvae (e.g. Pawlik, 1992a; Tamburri et al., 1996; Hadfield, 1998; Finelli and Wethey, 2003). These cues facilitate metamorphosis in favorable habitat, which can minimize mortality and maximize the supply of larvae to juvenile nurseries. Cues that induce metamorphosis are often associated with biofilms attached to the substratum. Biofilms are composed of extracellular polymeric substances produced by diatoms, bacteria, protozoa, and fungi (Patil and Anil, 2005). The metamorphic cue can be associated with the surface of the biofilm or with a water- soluble compound produced by microorganisms that constitute the film (Keough and Raimondi, 1996; Wieczorek et al., 1996; Olivier et al., 2000; McClintock and Baker, 2001; Patil and Anil, 2005). Additionally, the physiological status of microorganisms in the biofilm can affect the production of cues (Wieczorek et al., 1996). Therefore, the response to biofilm may change throughout the season. Recruitment of crabs to nursery habitat requires settlement of the megalopal stage on suitable substratum followed by metamorphosis into the first juvenile stage (Jones and Epifanio, 1995; Epifanio and Garvine, 2001; Van Montfrans et al., 2003). A variety of factors may trigger settlement and metamorphosis of crab larvae. These include: the presence of adult conspecifics, the physical texture of the substratum, and the occurrence of natural biofilm (Weber and Epifanio, 1996; Rodriguez and Epifanio, 2000; Steinberg et al., 2008). Previous studies have determined that metamorphosis of the Asian shore crab Hemigrapsus sanguineus is accelerated in the presence of biofilm from rocky intertidal habitat (Kopin et al., 2001; O'Connor, 2007; Steinberg et al., 2007). This response may be important in facilitating the spread of this invasive species (Kopin et al., 2001; Steinberg et al., 2007). H. sanguineus was first reported near the mouth of Delaware Bay in 1988 and now extends over a 1500 km range along the east coast of the USA from North Carolina to Maine (McDermott, 1998; Breton et al., 2002; Jensen et al., 2002). Adult H. sanguineus are typically found on structured hard bottom. In both its native and introduced habitats, the crab occurs throughout the rocky intertidal and often extends into the adjacent subtidal (Lohrer et al., 2000). At many locations along the East Coast, H. sanguineus is now the dominant intertidal species of crab, reaching Journal of Experimental Marine Biology and Ecology 382 (2009) 34–39 ⁎ Corresponding author. Tel.: +1 302 645 4263; fax: +1 302 645 4213. E-mail addresses: julieann@udel.edu (J.A. Anderson), epi@udel.edu (C.E. Epifanio). Contents lists available at ScienceDirect Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe 0022-0981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2009.10.006