TITLE: Isolation of bacteria associated with mucus on shark skin
AUTHORS: Claudia Pogoreutz, Gabriela Perna, Mauvis A. Gore, Rupert F. Ormond, Christopher R. Clarke, Christian Voolstra

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 (</span><span style = "font-style:italic;">Carcharhinus melanopterus</span><span>) 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.</span></div><div class = "text-block"><span style = "font-weight:bold;">References</span></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>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?</span><span style = "font-style:italic;">Zoology</span><span style = "font-style:italic;font-style:italic;">,</span><span style = "font-style:italic;">127</span><span>, 1-19. https://doi.org/10.1016/j.zool.2018.02.004.</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Doane, M. P., Haggerty, J. M., Kacev, D., Papudeshi, B., & Dinsdale, E. A. (2017). The skin microbiome of the common thresher shark (</span><span style = "font-style:italic;">Alopias vulpinus</span><span>) has low taxonomic and gene functionβ‐diversity. </span><span style = "font-style:italic;">Environmental Microbiology Reports</span><span>, </span><span style = "font-style:italic;">9</span><span>, 357-373. https://doi.org/10.1111/1758-2229.12537.</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">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</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">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.</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Pogoreutz, C., and C.R. Voolstra. 2018. Isolation, culturing, and cryopreservation of</span><span style = "font-style:italic;">Endozoicomonas </span><span>(Gammaproteobacteria: Oceanospirillales: Endozoicomonadaceae) from reef-building corals. Protocols.iohttps://doi.org/10.17504/protocols.io.t2aeqae</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>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. </span><span style = "font-style:italic;">BMC Animal Microbiome</span><span>, </span><span style = "font-style:italic;">1</span><span>, 9. https://doi.org/10.1186/s42523-019-0011-5.</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>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. </span><span style = "font-style:italic;">Frontiers in Microbiology</span><span>, </span><span style = "font-style:italic;">8</span><span>, 1050. https://doi.org/10.3389/fmicb.2017.01050.</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Rohwer, F., Seguritan, V., Azam, F., & Knowlton, N. (2002). Diversity and distribution of coral-associated bacteria.</span><span style = "font-style:italic;">Marine Ecology Progress Series</span><span style = "font-style:italic;font-style:italic;">,</span><span style = "font-style:italic;">243</span><span>, 1-10. doi:10.3354/meps243001.</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Sanders, E. R. (2012). Aseptic laboratory techniques: plating methods. </span><span style = "font-style:italic;">Journal of Visualized Experiments</span><span style = "font-style:italic;font-style:italic;">,</span><span style = "font-style:italic;">63</span><span>, e3064.</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Tsutsui, S., Yamaguchi, M., Hirasawa, A., Nakamura, O., & Watanabe, T. (2009). Common skate (</span><span style = "font-style:italic;">Raja kenojei</span><span>) secretes pentraxin into the cutaneous secretion: the first skin mucus lectin in cartilaginous fish.</span><span style = "font-style:italic;">Journal of Biochemistry</span><span>, 146, 295-306.https://doi.org/10.1093/jb/mvp069.</span></div><style>
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[STEPS]
?. Sampling: Collect replicate swab samples to potentially increase diversity of isolates. If sampling is vessel-based, and circumstances permit, let the skin of the shark first dry for a couple of seconds, and then swab the desired skin area (approx. 8 – 10 strokes, if feasible) using a sterile cotton swab. Use a new swab for each replicate for each location on skin. Put each swab individually back into its collection tube, and close the tube. Wrap top in parafilm if needed.
?. We did not use a specific transport medium, but kept the samples cool and minimized time spent between sampling and inoculation (
?. Take the swab samples back to the lab as soon as possible and use to immediately inoculate medium of choice in a sterile environment (depending on facilities available: laminar flow cabinet, or open on a clean bench using candles/Bunsen burner, if possible in a closed room and try to limit airflow) using standard aseptic techniques. Minimize the time passed between sample collection and inoculation to minimize stress for the sampled bacterial cells.
?. The media of choice will depend on the research question, i.e. specifically whether the aim is to maximize diversity or whether a specific bacterial functional group is targeted (i.e., a ‘general’ rich media vs. selective media).
?. Incubate plates at a slightly lower temperature than the environment they were collected/isolated from to inhibit overgrowth by fast-growing bacteria.
?. Incubate plates overnight, or until colony formation on the agar becomes apparent.
?. For the next step, do not wait until individual colonies merge, or pronounced smears/biofilms form. If colony formation is slow, keep monitoring for new growth over the course of several days.
?. Pick individual bacterial colonies with a clean inoculation loop (1 to 10 ul, as preferred). Streak out colony on a new agar plate using the standard Four Quadrant Streak method (Sanders 2012). Carry out at least 2 purification steps per isolate.
?. After purification from a single colony (min. 2 clean passages), confirm identity with Sanger Sequencing using a universal bacterial full-length 16S rRNA primer pair (e.g., 27F 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R 5'-GGTTACCTTGTTACGACTT-3'; Lane 1991).
?. For long-term storage, consider standard cryopreservation in glycerol stocks (e.g., 25% - 40% glycerol in broth) at -80°C or -140°C (e.g., as described here https://www.protocols.io/view/isolation-culturing-and-cryopreservation-of-endozo-t2aeqae).