The effects of spatial variance , temperature and pH on bacterial community composition in the sea anemone , Anemonia viridis

Marine organisms are well known to have associations with microorganisms and little is known about the specific nature of the microorganism host interactions. This study investigates the bacterial community composition in the sea anemone Anemonia viridis using Denaturing Gradient Gel Electrophoresis; bacterial community composition was assessed for spatial variance. The results show that there was no significant variation within a location or between the locations. These results are concordant with previous studies and suggest that bacterial populations are stable in Anemonia viridis. The Plymouth Student Scientist, 2014, 7, (1), 3-13 [4] Introduction Marine invertebrates are ubiquitous throughout the world, as are the microorganisms that live symbiotically within them. Marine organisms are well known to have specific relationships with numerous microorganisms (Thakur & Müller, 2005) and little is known about the microbial relationship and diversity associated with such marine organisms (Menezes et al, 2010). What is known however, is that in many ecosystems these complex microbial communities are extremely important components (Ainsworth, Thurber, & Gates, 2010; Menezes et al, 2010). Even though interactions between bacteria and invertebrates are common in the marine environment, the bacterial mode of association (mutualism, commensalism or parasitism) and particular physiological functions are often unidentified (Schuett & Doepke, 2009). Sea anemones, corals, jellyfish and hydras are all part of the phylum Cnidaria (Sabourault et al, 2009), Cnidaria can be found globally and in a multitude of depths and environmental conditions. The characteristics of Cnidaria include a sac-like body with a single oral opening surrounded by tentacles (Sabourault et al, 2009). Apart from the distinct characteristics of their anatomy they all exhibit a mutual relationship with numerous microorganisms. It has been suggested that functions of microorganisms to the host involve autotrophy, nitrogen fixation, nitrification, nutrient cycling in a modified version of the marine microbial loop and the participation in the host chemical defence system against predators and epibionts (Thakur & Müller 2005; Kellogg, 2004). Investigations by Barott et al, (2011); Olson & Kellogg, (2010) and Ritchie, (2006) have suggested that microorganisms in corals are host-specific, and Wegley et al, (2007) hypothesised that organisms can adapt to differing ecological niches by ‘switching’ their microbial associates. Rohwer et al, (2002) describe in their investigation how they found that the microorganisms are not only host-specific in corals but also stable and would be maintained over space and time. Hentschel et al, (2006) confirmed this in sponges and described temporal and biogeographical variation to be minor. Sea anemones possess endobiotic bacteria which harbour within the column, tentacles and the mucus surface layer (Schuett & Doepke, 2009). Investigations into the diversity of microorganisms found within sea anemones have reported a wide variety of heterotrophic bacteria (Du et al, 2010). Schuett & Doepke (2009) conducted an investigation where their aim was to find the pathogenic potential of endobiotic bacteria, and they found that eighty one percent of the species or groups were host-specific. With the search for new sources of natural products increasing (Larsen et al, 2005; Menezes et al, 2010), it is vital to fully understand how these microorganisms vary within the organisms and between geographical location, how stable they are with environmental changes and the roles they exhibit to the host organisms. In this investigation the bacterial community composition was analysed in Anemonia viridis. Variation within and between different geographical/ spatial destinations were related to the following hypothesis; bacterial community composition will not vary within a site or between spatial destinations. The Plymouth Student Scientist, 2014, 7, (1), 3-13 [5] Materials & Methods Sample Locations There were five sample locations all within the British Isles, UK. Three were on the south coast (one Cornwall & two Devon) and two sites were on the north coast of Cornwall as shown in Fig. 1 Fig. 1: The five sample site destinations 1: Hannafore Point, Looe (50°20’39.5”N, 4°27’20.23”W) 2: Mount Batten Bay, Plymouth (50°21’33.0”N, 4°7’53.0”W) 3: Bigbury-OnSea (50°16’53.1”N, 3°53’56.72”W) 4: Hayle Bay, New Polzeath (50°34’47.41”N, 4°55’18.50”W) 5: Bude Bay, Bude (50°50’15.83”N, 4°33’22.9”W) (http://digimap.edina.ac.uk/marineclassic/map.action? scale=general). Collection and process of samples Before collection all of the sampling equipment (forceps, scissors and Micro centrifuge tubes (MCC), an example of a set for one collection site) was sterilised in an autoclave at 121°C for 15 min at 15 psi. All sampling was conducted during September – October 2011 and samples were taken from a lower shore transect across the length of the beach, from different rock pools ranging in size 0.5 – 1.0 m. From each site three samples of A.viridis were taken. Only brown morphs of the species were collected from the sample locations. Five tentacles from each of the sample organisms were cut and placed into sterile, labelled MCC tubes. The samples were kept on ice until returning to the laboratory (within four hours of collection), where they were flash frozen in liquid nitrogen and stored at -20°C. Temperature, salinity, light intensity and O2 were all measured from each of the rock pools where samples were collected, the weather remained the same and the tidal range varied within 0.5 m at low tide on all of the collection days and times. 1 4 5