TITLE: No Title
AUTHORS: 

[DESCRIPTION]
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.


[GUIDELINES]
For the Isolation of Endozoicomonas, several factors are important to consider: homogenization, dilution, incubation temperature, replication, and colony morphology:

(1)	Homogenization: Thoroughly homogenize the coral tissue to make sure the Endozoicomonas aggregates are 'set free' from the host tissue (e.g., for 30 seconds at medium speed with an UltraTurrax or comparable tool). We didn't do that in a first attempt and all we isolated was Vibrios. Once we implemented the tissue homogenization, overall taxonomic diversity of isolates increased.

(2)	Dilution: if the Endozoicomonas make up a high proportion of the microbiome (for instance, 60 - 90 % in Red Sea Acropora humilis), the dilution will help to select for abundant organisms. We have plated tissue slurry "undiluted" and in 1:10, 1:100, 1:1000 dilutions. The Endozoicomonas grew on the Marine Agar plates inoculated with 'undiluted' and 1:10 diluted slurries.

(3)	Temperature: we settled for an incubation temperature low compared to ambient seawater temperatures (23°C in our case; the central Red Sea has an annual temperature average of 29°C). At this temperature, Vibrios, Alteromonas, and other fast-growing taxa would form colonies within the first 24 h, while the Endozociomonas started to appear on day 4 post-inoculation, but would form many more colonies than other taxa.

(4)	Replication: we had an agar plate replication of n = 3 for each dilution of slurry and type of media (other media than Marine Agar were used, too, but no Endozociomonas grew on them). This was not entirely necessary in our case because we had hundreds of Endozoicomonas colonies growing, but it might increase the chance of isolating Endozoicomonas strains (or other bacteria) that are not quite as abundant.

(5)	Colony morphology: Endozoicomonas will form tiny round convex creamy colonies with entire margins. When plating the picked colonies, you will see that the average Endozoicomonas colony is rather sticky and will strongly adhere to loops or needles. These bacteria are certainly easier to handle in suspension culture than on plates. Once you have successfully isolated Endozoicomonas on plates and confirmed their identity with Sanger sequencing, you may transfer them in liquid culture (Difco2216 Marine Broth). 

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.