Isolation of bacteria associated with mucus on shark skin v1
Authors: Pogoreutz Claudia , Perna Gabriela , A. Gore Mauvis , F. Ormond Rupert , R. Clarke Christopher , R Voolstra Christian 
Published: September 2020

Animals and plants are metaorganisms or holobionts associated with prokaryotic and eukaryotic microbes, the diversity and community composition of which is increasingly being characterized thanks to the advent of culture-independent next-generation sequencing applications (Rohwer et al. 2002, Bang et al. 2018). In order to investigate the mechanistic underpinnings of host-microbe interactions, however, traditional culture-based approaches are critical to complement culture-independent -omics techniques. One venue for microbial cell culture-based approaches is the study of microbes associated with large aquatic vertebrates that cannot be readily studied and/or sampled under standardized and controlled conditions in the lab, such as sharks (Pogoreutz et al. 2019). The skin of sharks, rays, and skates is characterized by a mucus layer secreted by specialized secretory cells (mucous cells) in the epidermis (Meyer and Seegers 2012). Rays and skates produce sufficient amounts of epidermal mucus to be scraped off with sterile tools and collected in culture tubes, serial dilutions of which can be used for inoculation in growth media for subsequent bacterial colony picking and isolation (Tsutsui et al. 2009; Ritchie et al. 2020). Sharks however only secrete a thin and inconspicuous epidermal mucus layer in comparison (Meyer and Seegers 2012), rendering the scraping approach unfeasible. Here we detail an alternative and non-invasive protocol that allowed us to reproducibly isolate a diversity of bacteria from the skin mucus of black tip reef sharks (Carcharhinus melanopterus) using a swab sampling approach. Importantly, swabbing may help reduce sampling stress on sharks, as it does not require sharks being lifted onto a vessel. References Bang, C., Dagan, T., Deines, P., Dubilier, N., Duschl, W. J., Fraune, S., Hentschel, U., Hirt, H., Hülter, N., Lachnit, T.,Picazo, D., Pita, L., Pogoreutz, C., Rädecker, N., Saad, M.M., Schmitz, R.A., Schulenburg, H., Voolstra, C.R., Weiland-Bräuer, N., Ziegler, M., Bosch, T.C.G. (2018).Metaorganisms in extreme environments: do microbes play a role in organismal adaptation?Zoology,127, 1-19. https://doi.org/10.1016/j.zool.2018.02.004. Doane, M. P., Haggerty, J. M., Kacev, D., Papudeshi, B., & Dinsdale, E. A. (2017). The skin microbiome of the common thresher shark (Alopias vulpinus) has low taxonomic and gene functionβ‐diversity.Environmental Microbiology Reports,9, 357-373. https://doi.org/10.1111/1758-2229.12537. Meyer, W., and Seegers, U. (2012) Basics of skin structure and function in elasmobranchs: a review. J Fish Biol 80:1940–1967. https://doi.org/10.1111/j.1095-8649.2011.03207.x Lane, D. J. (1991). 16S/23S rRNA sequencing. In: Nucleic acid techniques in bacterial systematics. Stackebrandt, E., Goodfellow, M., eds. John Wiley and Sons, New York, NY, pp. 115-175. Pogoreutz, C., and C.R. Voolstra. 2018. Isolation, culturing, and cryopreservation ofEndozoicomonas (Gammaproteobacteria: Oceanospirillales: Endozoicomonadaceae) from reef-building corals. Protocols.iohttps://doi.org/10.17504/protocols.io.t2aeqae Pogoreutz, C., Gore, M. A., Perna, G., Millar, C., Nestler, R., Ormond, R. F., Clarke, C.R. & Voolstra, C.R. (2019). Similar bacterial communities on healthy and injured skin of black tip reef sharks.BMC Animal Microbiome,1, 9. https://doi.org/10.1186/s42523-019-0011-5. Ritchie, K. B., Schwarz, M., Mueller, J., Lapacek, V. A., Merselis, D., Walsh, C. J., & Luer, C. A. (2017). Survey of antibiotic-producing bacteria associated with the epidermal mucus layers of rays and skates.Frontiers in Microbiology,8, 1050. https://doi.org/10.3389/fmicb.2017.01050. Rohwer, F., Seguritan, V., Azam, F., & Knowlton, N. (2002). Diversity and distribution of coral-associated bacteria.Marine Ecology Progress Series,243, 1-10. doi:10.3354/meps243001. Sanders, E. R. (2012). Aseptic laboratory techniques: plating methods.Journal of Visualized Experiments,63, e3064. Tsutsui, S., Yamaguchi, M., Hirasawa, A., Nakamura, O., & Watanabe, T. (2009). Common skate (Raja kenojei) secretes pentraxin into the cutaneous secretion: the first skin mucus lectin in cartilaginous fish.Journal of Biochemistry, 146, 295-306.https://doi.org/10.1093/jb/mvp069.