The fish skin microbiome serves as a protective barrier, influencing host health and facilitating interactions between the host and its environment. While there have been studies on the roles and composition of the fish skin microbiome, there remains a paucity of data on how environmental variation influences these microbes in natural populations. Here, we used culture-dependent and -independent approaches to characterise the skin microbiome of wild three-spined stickleback populations and examine how environmental factors influence microbial diversity and community composition across 17 freshwater lochs on the island of North Uist, Scotland. Culture-dependent methods involve plating a skin microbiome sample on an array of agar followed by sequencing the 16S rRNA gene, whereas culture-independent methods involve sequencing the 16S rRNA gene directly from skin swabs of individual stickleback fish. Analysis of 300 swabs and 450 cultured samples showed that microbial community composition was primarily driven by environmental variables, especially host habitat, water pH, salinity and metal concentrations, while host traits (e.g., sex, length) contributed minimally. A set of dominant bacterial genera—Janthinobacterium, Pseudomonas, Acinetobacter, and Psychrobacter—constituted a core skin microbiota across lochs, suggesting that these taxa may play important roles in this microbial community. Culture-based sequencing recovered an average of 87.4% of the amplicon sequence variants (ASVs), representing 99.7% of the relative abundance detected by direct swab sequencing, and additionally identified 51.9% more ASVs not detected in the swab samples. Future investigations of our cultured isolates will explore the functional traits and interactions among these dominant taxa.

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