Subcuticular Bacteria Associated With Two Common New Zealand Echinoderms: Characterization Using 16S rRNA Sequence Analysis and Fluorescence in situ Hybridization SCOTT A. LAWRENCE 1 , RONAN O’TOOLE 1 , MICHAEL W. TAYLOR 2 , AND SIMON K. DAVY 1, * 1 School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand; and 2 School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland, New Zealand Abstract. Many echinoderms contain subcuticular bacte- ria (SCB), symbionts which reside in the lumen between the host’s epidermal cells and outer cuticle. This relationship is common, existing in about 60% of echinoderms studied so far, yet the function of SCB remains largely unknown. In this study, phylogenetic analysis was carried out on 16S rRNA sequences obtained from echinoderm-associated bac- teria, resulting in the identification of four species of puta- tive SCB. All four bacteria were identified from the ho- lothurian Stichopus mollis, and two of the four were also found in the asteroid Patiriella sp. Two of these bacteria belong to the Alphaproteobacteria, and two to the Gamma- proteobacteria. In addition to phylogenetic analysis, fluo- rescence in situ hybridization (FISH) assays were carried out on Patiriella sp., S. mollis, and the asteroid Astrostole scabra. Results showed that Patiriella sp. and S. mollis contain SCB, in agreement with the phylogenetic analysis, while SCB were not detected in A. scabra. Of the bacteria detected using FISH, more than 80% were recognized as belonging to the Alphaproteobacteria in both host species. However, in S. mollis about 20% of the detected SCB successfully hybridized with the Gammaproteobacteria- specific probe, whereas bacteria belonging to this class were never observed in Patiriella sp. This is only the second study to characterize SCB by molecular means, and is the first to identify SCB in situ using FISH. Introduction Symbiosis, the living together of different organisms, is common in the marine environment. Many marine inverte- brates form symbioses with microbes, where the smaller partner (the symbiont) lives either in or on the surface of its invertebrate host. Subcuticular bacteria (SCB) are symbi- onts that live between the epidermis and the cuticle (the subcuticular space) in a range of echinoderms. Despite first being described over 30 years ago (Holland and Nealson, 1978), little is known about echinoderm-SCB symbioses, such as the identity of the bacterial symbionts, their mode of transmission into the subcuticular space, or what benefit, if any, the echinoderms or bacteria gain from the relationship. Previous work has relied mainly on microscopy to charac- terize SCB by morphological means (Feral, 1980; Lesser and Blakemore; 1990, Roberts et al., 1991; McKenzie and Kelly, 1994). Such studies have succeeded in determining which echinoderms contain SCB and how abundant the bacteria are within their hosts, and have gone some way toward approximate identification of SCB based on their morphology. One clear trend that has emerged is the prev- alence of this symbiosis. McKenzie and co-workers (Mc- Kenzie et al., 1998), in their meta-analysis of SCB distri- bution studies, found that more than half of the echinoderm species examined to date contain SCB. SCB have been identified as rod-shaped, gram-negative bacteria, which can be divided into three morphological groups (McKenzie and Kelly, 1994; Kelly and McKenzie, 1995). SCB appear to be abundant within the host and, although varying values have been reported, even the lowest densities are in the order of more than 10 7 SCB cells g -1 ash free dry Received 20 July 2009; accepted 6 November 2009. * To whom correspondence should be addressed. E-mail: simon.davy@ vuw.ac.nz Reference: Biol. Bull. 218: 95–104. (February 2010) © 2010 Marine Biological Laboratory 95