Identifying coral microbiome needles in the microbial haystack: sampling and analysis considerations for prokarya in planulae and polyps

Bacteria are fundamental to coral health and resilience, though a dearth of knowledge exists on the formation of the coral microbiome in early life history stages. Although this initial microbial consortium is thought to hold key roles throughout development, its sparsity is easily obscured by environmental background microbiota. This opacity renders accurate studies of the early coral microbiome difficult, and limits application in breeding and restoration techniques. Therefore, accounting for the presence of non-host-associated bacteria in the background environment is imperative for the accurate identification of early coral-bacteria associations. Here we outline a workflow that both minimizes and accounts for the environmental background microbiome (EBM) in sampling, downstream data cleaning, and statistical analysis. Utilizing this workflow, we examined the bacterial communities associated with planktonic coral larvae and primary polyps of two scleractinian corals, Orbicella faveolata and Colpophyllia natans, with consideration for two EBMs, sampling rinse water and the settlement substrate biofilm. 16S rRNA amplicon sequencing revealed substantial mixing of coral-associated bacterial communities with those of the underlying rinse water and biofilms. Nonetheless, the coral larval microbiome could be statistically delineated from the rinse water background, which displayed species specificity as early as four days after fertilization. Paired-sample statistical analysis yielded similar results, with several putative coral primary polyp-associated bacteria found to be distinct from the biofilm background microbiome. These results suggest that (a) the presence of non-host-associated bacteria in early life history coral samples is inevitable and necessitates careful consideration when studying this life stage, and (b) the coral microbiome signal can be distinguished from the EBM with strategic sampling and subsequent downstream analysis.