A peer-reviewed version of this preprint was published in PeerJ on 16 May 2017. View the peer-reviewed version (peerj.com/articles/3235), which is the preferred citable publication unless you specifically need to cite this preprint. Brown T, Otero C, Grajales A, Rodriguez E, Rodriguez-Lanetty M. 2017. Worldwide exploration of the microbiome harbored by the cnidarian model, Exaiptasia pallida (Agassiz in Verrill, 1864) indicates a lack of bacterial association specificity at a lower taxonomic rank. PeerJ 5:e3235 https://doi.org/10.7717/peerj.3235 1! Worldwide exploration of the microbiome harbored by the cnidarian model, Exaiptasia 2! pallida indicates a lack of bacterial association specificity at a lower taxonomic rank. 3! 4! Tanya Brown1 5! Christopher Otero1 6! Alejandro Grajales2 7! Estefanía Rodríguez2 8! Mauricio Rodriguez-Lanetty1* 9! 10! 1 Department of Biological Sciences, Florida International University, Miami, FL, 33199 11! 2 Division of Invertebrate Zoology, American Museum of Natural History, New York, NY 10024 12! 13! * corresponding author 14! Email address: [email protected] 15! Phone: 305-348-4922 16! Fax: 305-348-1986 17! 18! Keywords 19! Cnidaria, microbiome, host-microbe interaction, coral, sea anemone. 20! 21! 22! 23! 24! 25! ! 1! PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2338v1 | CC BY 4.0 Open Access | rec: 4 Aug 2016, publ: 4 Aug 2016 26! Abstract 27! Examination of host-microbe interactions in basal metazoans, such as cnidarians is of great 28! interest from an evolutionary perspective to understand how host-microbial consortia have 29! evolved. To address this problem, we analyzed whether the bacterial community associated with 30! the cosmopolitan and model sea anemone Exaiptasia pallida shows specific patterns across 31! worldwide populations ranging from the Caribbean Sea, and the Atlantic and Pacific oceans. By 32! comparing sequences of the V1-V4 hypervariable regions of the bacterial 16S rRNA gene, we 33! revealed that anemones host a complex and diverse microbial community. When examined at the 34! phylum level, bacterial diversity and abundance associated with E. pallida are broadly conserved 35! across geographic space with samples, containing largely Proteobacteria and Bacteroides. 36! However, the species-level makeup within these phyla differs drastically across space suggesting 37! a high-level core microbiome with local adaptation of the constituents. Indeed, no bacterial OTU 38! was ubiquitously found in all anemones samples. We also revealed changes in the microbial 39! community structure after rearing anemone specimens in captivity within a period of four 40! months. These results contrast with the postulation that cnidarian hosts might actively select and 41! maintain species-specific microbial communities that could have resulted from an intimate co- 42! evolution process. Instead, our findings suggest that environmental settings, not host specificity 43! seem to dictate bacterial community structure associated with this sea anemone. More than 44! maintaining a specific composition of bacterial species some cnidarians associate with a wide 45! range of bacterial species as long as they provide the same physiological benefits towards the 46! maintenance of a healthy host. The examination of the previously uncharacterized bacterial 47! community associated with the cnidarian sea anemone model E. pallida is the first global-scale 48! study of its kind. ! 2! 49! Introduction 50! Insights into the microbiome diversity of metazoan hosts have triggered a considerable 51! interest in uncovering the regulatory principles underlying host/microbe interactions across 52! multicellular organisms. Over the last several years, microbial symbionts living with vertebrates 53! have been clearly shown to influence disease, physiological and developmental phenotypes in 54! their host (Tremaroli & Backhed 2012; Blaser et al. 2013; Le Chatelier et al. 2013; Lozupone et 55! al. 2013; Ridaura et al. 2013). In many marine invertebrates, bacteria associated with host 56! epithelium have also been shown to play a pivotal role in host development (McFall-Ngai et al. 57! 2013). For instance, in the bobtail squid, the bioluminescent bacteria, Allivibrio fisheri 58! (Beijerinck 1889; Urbanczyk et al. 2007) are required symbionts from early host developmental 59! stages so that a functional and healthy light organ can develop (McFall-Ngai 1994; Nyholm & 60! McFall-Ngai 2004). Similar profound effects have been documented for more basal metazoans 61! such as cnidarians. In the case of Hydra viridis (Medusozoa: Hydrozoa), induced absence of a 62! microbial community in host polyps causes strong developmental defects and reduces asexual 63! reproduction via budding (Rahat & Dimentman 1982). This suggests that the evolution of 64! microbes and host interactions dates back to earlier diverging metazoan lineages (i.e. cnidarians), 65! which has triggered an imperative interest to understand whether bacterial cores comprised of 66! specific species have evolved in intimate association with their hosts since the early times of 67! metazoan evolution (Bosch and Miller 2016). 68! 69! Despite the simple body plans in cnidarians molecular analyses of the microbiota 70! associated with these early-diverging organisms, predominantly corals (Anthozoa: Scleractinia), 71! have uncovered an unprecedented bacterial diversity (Rohwer et al. 2001; Bourne & Munn 2005; ! 3! 72! Sunagawa et al. 2009; Rodriguez-Lanetty et al. 2013). Additionally, the species composition and 73! structure of these microbial partnerships are complex and dynamic. The association between 74! coral host and the consortia of these microorganisms, including bacteria, fungi, viruses and the 75! intracellular microalgae Symbiodinium, has been referred to as the coral holobiont (Rohwer et al. 76! 2001). Several studies demonstrate that certain bacterial groups associate specifically with some 77! coral species (Rohwer et al. 2001; Morrow et al. 2012; Speck & Donachie 2012; Bayer et al. 78! 2013; Rodriguez-Lanetty et al. 2013), implicating the effects of coevolution between coral 79! lineages and certain bacterial strains. However, other studies have revealed that the dominant 80! bacterial genera differ between geographically-spaced hosts of the same coral species (Klaus et 81! al. 2007; Kvennefors et al. 2010; Littman et al. 2010) and even locally within reefs (Kvennefors 82! et al. 2010), which suggests that environmental factors are largely responsible in shaping coral- 83! associated microbial community diversity. 84! 85! The elucidation of the mechanisms that mediate the complex interactions between 86! microbial communities and anthozoans may be facilitated by studying a tractable model system 87! that can be cultured and manipulated in laboratory conditions. For this purpose, the sea anemone 88! (Actiniaria) Exaiptasia pallida, previously known as Aiptasia pallida (see Grajales & Rodriguez 89! 2014), has been proposed as a model organism to study various aspects of the cell biology and 90! physiology of anthozoan-Symbiodinium symbiosis (Weis et al. 2008) (Fig. 1). The use of this 91! cnidarian model system has advanced our understanding of the molecular and cellular 92! mechanism underlying anthozan/Symbiodinium regulation (Davy et al. 2012). Likewise, the E. 93! pallida model system could benefit investigations regarding the influence of the associated 94! microbiota on physiological, developmental, and disease-resistant host cnidarian phenotypes. ! 4! 95! However, we still lack baseline knowledge about the bacterial diversity and assemblages 96! associated with this sea anemone. In the current study, we characterized the composition, 97! structure and specificity patterns of microbial communities associated with the sea anemone E. 98! pallida from worldwide populations using samples from the Caribbean Sea, and the Atlantic and 99! the Pacific oceans. We then compared the microbial composition of natural versus specimens 100! reared in the laboratory over several periods of time as means to document the effect of aquarium 101! conditions in the composition and structure of microbial taxa. 102! 103! Material and Methods 104! Sample collection, DNA extraction and V1-V4 16S rRNA pyrosequencing: Specimens of 105! Exaiptasia pallida were collected from 10 wild populations from different ocean basins 106! worldwide (Table 1) including the Caribbean Sea; Northeastern and Western Atlantic; and 107! Eastern, Central and Northwestern Pacific. Ethanol-preserved samples from the populations 108! above were obtained from the invertebrate collection at the American Museum of Natural 109! History (AMNH). Four additional groups of samples of E. pallida were added to the study. One 110! group was obtained from a commercial pet store, a second group from an outdoor flow-through 111! sea water system at the Keys Marine Laboratory (KML, Florida) and the other two were reared 112! in the lab for different time periods: one from a six-year laboratory reared clonal population 113! (CC7) originally obtained from a reef in the upper Florida Keys and the second from anemones 114! more recently collected from the KML (Florida) and maintained in the lab for four months. Total 115! DNA was extracted from the entire body of the collected sea anemone samples (3-4 per 116! population site) using the DNeasy Plant Mini Kit DNA (Promega, Madison, WI) following the 117! standard protocol recommended by the manufacturer.!!! ! 5! 118! To assess DNA quality and lack of PCR