Hutchings Brenna, López-Legentil Susanna, Stefaniak Lauren, Nydam Marie, Erwin Patrick M
Department of Biology & Marine Biology, Center for Marine Science, University of North Carolina Wilmington, Wilmington, North Carolina, USA.
Department of Marine Science, Coastal Carolina University, Conway, South Carolina, USA.
Microbiologyopen. 2025 Apr;14(2):e70019. doi: 10.1002/mbo3.70019.
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.
海洋无脊椎动物的野外采集常常伴随着保存延迟，这可能会影响微生物群落组成。在此，我们使用16S rRNA扩增子测序技术，测试了保存延迟和放松方法对群体海鞘Trididemnum solidum微生物群落多样性和组成的影响。从伯利兹珊瑚礁采集的重复样本，要么（1）立即保存在乙醇中（“对照”），（2）在保存前于环境海水中放置3小时（“SW”），要么（3）在保存前于添加薄荷醇（一种用于海鞘鉴定的常见保存前放松技术）的环境海水中放置3小时（“SW + M”）。所有Trididemnum solidum的微生物群落均与环境海水浮游细菌不同，且由相同的微生物分类群主导，包括Thalassobaculum属、Tistrella属和集胞藻属。然而，与对照相比，样本保存延迟3小时（SW）显著降低了微生物群落丰富度（p = 0.028），而薄荷醇处理（SW + M）减轻了这种多样性损失（p = 0.208）。与对照相比，两种延迟保存方法在群落水平上的微生物组成均无显著差异（SW p = 0.054，SW + M p = 0.052）。分类水平的变化很少见但确实发生了，最显著的是兼性厌氧γ-变形菌Catenococcus的大量繁殖，在延迟保存中其丰度分别是对照的37倍（SW）和197倍（SW + M）。在保存延迟3小时（SW）后，只有122个微生物分类群（占总数的1.85%）与对照相比表现出显著的丰度差异，薄荷醇处理（SW + M）将分类水平的变化减少到65个分类群（0.98%）。我们的结果表明，短暂的保存延迟不会显著改变群落水平的微生物群落组成和优势分类群，薄荷醇处理可抵消与保存延迟相关的微小微生物群落变化。
