title: B64_seawater_seawater
design: Sampling Ten individuals of A. arnoldorum, P. labrovittatus, and B. paula, were caught in Pago Bay, Guam, in May 18 and 19, 2023. Fish were caught in the intertidal zone using hand-held nets and transferred into new zip-lock bags, each containing 50 mL (A. arnoldorum and P. labrovittatus) or 100 mL (B. paula) of autoclaved seawater. Fish were left in the bags for 30 seconds to rinse the external surface that could have been contaminated during fishing and handling. After rinsing, water was removed, and fish mucus was obtained by swabbing the fish skin (both ventral and dorsal areas). To assess the relative contribution of the environment to the fish skin microbiome, we sampled the microbial biofilm of the substrate where the three species were found: supratidal rocks for A. arnoldorum, rockpools for P. labrovittatus, and subtidal rock holes for B. paula. In the case of B. paula, for each fish specimen, we sampled the exact substrate on which the fish was residing; thus, the number of fish skin samples equals the number of fish substrates. For the other two fish species, which are more mobile, we sampled substrates, either rock or rockpools, where the fish were found, but the number of fish skin samples in these cases does not correspond to the same number of fish on the substrate. For comparison, we also collected 3 L of seawater samples from our general sampling area. Specifically, we sampled nearshore surface water near the A. arnoldorum and P. labrovittattus habitat and again approximately 10 m from shore, corresponding to the B. paula habitat. All seawater samples were collected in triplicate on different days: May 18 and May 20, 2023. Seawater samples were filtered using a vacuum pump (GAST) through either a 1.2 m or 0.45 m pore filter (Whatman). Fish skin swabs and filters were placed into 2 mL tubes and stored at -80 C until further processing.  Microbiome profiles  DNA was extracted using ZymoBIOMICS DNA extraction kit (Zymo Research) following the manufacturer's protocol. The V4 region of the 16S rRNA gene was amplified using modified forward 515F (Parada et al., 2016) and reverse 806R (Apprill et al., 2015) primers. The product from the first PCR was used in the second PCR, which was designed to complete the addition of Illumina adapters and the 6-mer dual indices as previously described by (Kozich et al., 2013). Both PCRs were generated using Takara PCR buffer and Hot Start polymerase (Takara). The first PCR was carried out in 30 L volume reactions with the following final concentrations: dNTPs at 0.8 mM, each primer at 0.5 M, and 30 to 100 ng of total DNA. The first PCR cycling conditions were as follows: a hot-start activation step for 2 min at 95 C, followed by 30 cycles of 95 C for 40 s, 58 C for 2 min, and 72 C for 1 min, with a final extension of 5 min at 72 C. The PCR was purified using GeneJet (ThermoFisher). The second PCR was performed in a 20-L reaction volume using the same components and final concentrations as above, with 30 ng of the first PCR product serving as the template. The second PCR cycling condition was as follows: a hot start activation at 95 C for 2 min, followed by five cycles of 95 C for 40 s, 59 C for 2 min, and 72 C for 1 min, and concluded with a final extension at 72 C for 7 min. All samples were pooled and gel-purified using the Qiagen gel purification kit. The V4-16S library pool was sent to Michigan State University Research Technology Support Facility Genomics Core for Illumina MiSeq sequencing, using a 2 x 250 bp cycling kit.