soil metagenome water metagenome gut metagenome and skin metagenome

A total of 19 adult Odorrana schmackeri frogs (10 females and 9 males) were captured between July and August 2024 from a 10-meter stream transect in Gaojiayan Town, Hubei Province, China (30.60N, 111.05E; elevation 274 m). The sampling site exhibited stable hydrological conditions, with the water temperature consistently maintained at 22.7 degrees. All specimens were collected nocturnally between 18:00 and 24:00 to control for circadian rhythm effects. Each individual was rinsed three times with 50 mL of sterile water to remove transient environmental microbes. Resident skin microbiota were collected using standardized swabbing procedures (three passes each on dorsal, lateral, and ventral surfaces) with sterile nylon-flocked swabs (SHX1-9 and SHC1-10 represented male and female skin microbial samples, respectively). Following euthanasia with buffered 1percentage of MS-222 (tricaine methanesulfonate; Sigma-Aldrich Corporation), the entire intestinal tracts were aseptically dissected and stored separately (GHX1-9 and GHC1-10 represented male and female gut microbial samples). Four replicate water samples (W1-4) were collected by filtering through 0.22-micrometre membranes (47 mm diameter; Millipore Corporation, Billerica, MA, USA). Quadruplicate soil cores (30 x 30 x 10 cm depth, spaced 10 m apart) were obtained (TR1-4) from riparian zones adjacent to frog microhabitats. All biological and environmental samples were immediately flash-frozen in liquid nitrogen and stored at -80 degrees until DNA extraction.