Abstract
Field collections of marine invertebrates are often accompanied by delays in preservation, which may impact microbiome composition. Here, we tested the effects of delayed preservation and relaxation methods on microbiome diversity and composition in the colonial ascidian Trididemnum solidum using 16S rRNA amplicon sequencing. Replicate samples collected from Belizean reefs were either (1) immediately preserved in ethanol ("control"), (2) held in ambient seawater for 3 h before preservation ("SW"), or (3) held in ambient seawater with menthol (a common pre-preservation relaxation technique for ascidian identification) for 3 h before preservation ("SW + M"). All T. solidum microbiomes were different from ambient seawater bacterioplankton and dominated by the same microbial taxa, including the genera Thalassobaculum, Tistrella, and Synechocystis. However, the 3-h delay in sample preservation (SW) significantly reduced microbiome richness compared to controls (p = 0.028), while menthol treatment (SW + M) mitigated this diversity loss (p = 0.208). Microbial composition at the community level did not differ significantly for either delayed preservation method compared to controls (SW p = 0.054, SW + M p = 0.052). Taxon-level shifts were rare but did occur, most notably a bloom of the facultatively anaerobic gammaproteobacterium Catenococcus that was 37x (SW) and 197x (SW + M) more abundant in delayed preservations. After a 3-h preservation delay (SW), only 122 microbial taxa (1.85% of total) exhibited significantly differential abundances with controls, with menthol treatment (SW + M) reducing taxon-level shifts to 65 taxa (0.98%). Our results showed that brief delays in preservation did not significantly alter community-level microbiome composition and dominant taxa, with menthol exposure counteracting minor microbiome shifts associated with preservation delays.