Isolation, culturing, and cryopreservation of Endozoicomonas (Gammaproteobacteria: Oceanospirillales: Endozoicomonadaceae) from reef-building corals v1
Authors: Pogoreutz Claudia , R Voolstra Christian 
Published: September 2018

Endozoicomonas are gram-negative bacteria widely and often abundantly associated with marine invertebrates and fish (Yang et al., 2010; Bayer et al., 2013; Nishijima et al., 2013; Hyun et al., 2014; Katharios et al., 2015; Ding et al., 2016; Neave et al., 2017a; Schreiber et al., 2016; Pogoreutz et al., 2018). Despite their ubiquitous distribution, only few cultured strains are available, as Endozoicomonasare supposedly difficult to isolate and to maintain in pure cultures (Neave et al., 2017b). Here we detail a protocol that allowed us to reproducibly isolate Endozoicomonasfrom stony corals (Acropora humilis) from the Red Sea. This protocol should be useful in isolating abundant Endozoicomonasfrom other corals or marine invertebrates. References Bayer, T., Neave, M.J., Alsheikh-Hussain, A., Aranda, M., Yum, L.K., Mincer, T., et al. (2013) The microbiome of the Red Sea coral Stylophora pistillatais dominated by tissue-associated Endozoicomonasbacteria. Appl. Environ. Microbiol.79: 4759–4762. Ding, J.Y., Shiu, J.H., Chen, W.M., Chiang, Y.R., and Tang, S.L. (2016) Genomic insight into the host-endosymbiont relationship of Endozoicomonas montiporaeCL-33T with its coral host. Front. Microbiol.7: 251. Hyun, D.W., Shin, N.R., Kim, M.S., Oh, S.J., Kim, P.S., Whon, T.W., and Bae, J.W. (2014) Endozoicomonas atrinaesp. nov., isolated from the intestine of a comb pen shell Atrina pectinata. Int. J. Syst. Evol. Microbiol.64: 2312–2318. Katharios, P., Seth-Smith, H.M.B., Fehr, A., Mateos, J.M., Qi, W., Richter, D., et al. (2015) Environmental marine pathogen isolation using mesocosm culture of sharpsnout seabream: Striking genomic and morphological features of novel Endozoicomonassp. Sci. Rep.5: 1–13. Neave, M.J., Michell, C.T., Apprill, A., and Voolstra, C.R. (2017a) Endozoicomonasgenomes reveal functional adaptation and plasticity in bacterial strains symbiotically associated with diverse marine hosts. Sci. Rep.7: 40579. Neave, M. J., Apprill, A., Ferrier-Pagès, C., & Voolstra, C. R. (2016) Diversity and function of prevalent symbiotic marine bacteria in the genus Endozoicomonas. App. Microbiol. Biotech., 100:8315-8324. Neave, M.J., Rachmawati, R., Xun, L., Michell, C.T., Bourne, D.G., Apprill, A., and Voolstra, C.R. (2017b) Differential specificity between closely related corals and abundant Endozoicomonasendosymbionts across global scales. ISMEJ 11: 186-200. Nishijima, M., Adachi, K., Katsuta, A., Shizuri, Y., and Yamasato, K. (2013) Endozoicomonas numazuensissp. nov., a gammaproteobacterium isolated from marine sponges, and emended description of the genus EndozoicomonasKurahashi and Yokota 2007. Int. J. Syst. Evol. Microbiol.63: 709–714. Pogoreutz, C., Rädecker, N., Cárdenas, A., Gärdes, A., Wild, C., and Voolstra, C.R. (2018) Dominance of Endozoicomonasbacteria throughout coral bleaching and mortality suggests structural inflexibility of the Pocillopora verrucosamicrobiome.Ecol. Evol.8: 2240–2252. Schreiber, L., Kjeldsen, K.U., Funch, P., Jensen, J., Obst, M., Lopez-Legentil, S., and Schramm, A. (2016) Endozoicomonasare specific, facultative symbionts of sea squirts. Front. Microbiol. 7: 1–15. Yang, C.-S., Chen, M.-H., Arun, A.B., Chen, C.A., Wang, J.-T., and Chen, W.-M. (2010) Endozoicomonas montiporaesp. nov., isolated from the encrusting pore coral Montipora aequituberculata. Int. J. Syst. Evol. Microbiol.60: 1158–1162.