Supplementary file 1_Phylogenetically distinct Vibrio mediterranei lineages confer robust protection under thermal stress against oyster pathogens.docx

IntroductionMarine bivalve mortality events cause substantial economic losses in aquaculture and threaten global food security. While pathogenic Vibrio species are frequently implicated, growing evidence suggests that loss of beneficial microbes can increase host susceptibility to disease. We previously observed that Vibrio mediterranei was consistently isolated from healthy oysters but systematically disappeared prior to mortality events, coinciding with proliferation of pathogenic Vibrio species. Here, we test whether this pattern reflects a protective functional role.
MethodsScreening eleven V. mediterranei strains against eastern oyster (Crassostrea virginica) larvae revealed three distinct virulence phenotypes – avirulent, pathogenic, and intermediate – that correspond to monophyletic phylogenetic clades.
ResultsPre-colonization with the avirulent strain Vm02 increased larval survival from 10–19% (pathogen-only controls) to 94–97% when challenged with V. harveyi or V. coralliilyticus, representing near-complete protection maintained at both ambient (28 °C) and thermal stress (32 °C) temperatures. Protection was rapid, effective even under simultaneous co-inoculation with the pathogen, and durable through >96 hours post-exposure. Fluorescence microscopy confirmed persistent association of fluorescently tagged Vm02 with larval digestive tissues. Phylogenomic analysis of 33 V. mediterranei genomes placed the three phenotypic groups within well-supported clades separated by 97.1–97.8% average nucleotide identity, approaching species-level thresholds. Pangenome analysis revealed that protective-clade strains harbor 230 unique orthogroups encoding regulatory systems, stress tolerance, metabolic versatility, and a conserved bacteriocin biosynthetic operon. These same strains do not encode the Type I secretion system, Type VI secretion system variants, and TCP pathogenicity island found in pathogenic lineages.
DiscussionTogether, these findings demonstrate that beneficial and pathogenic phenotypes are phylogenetically constrained within distinct V. mediterranei lineages, providing a framework for probiotic development and disease forecasting in shellfish aquaculture.

引言：海洋双壳类（marine bivalve）大规模死亡事件给水产养殖业造成了巨额经济损失，同时也威胁着全球粮食安全。尽管致病性弧菌属（Vibrio）物种常被认为是致病元凶，但越来越多的证据表明，有益微生物的缺失会增强宿主对疾病的易感性。我们此前的研究发现，地中海弧菌（Vibrio mediterranei）可从健康牡蛎中稳定分离得到，但在死亡事件发生前会系统性消失，与此同时致病性弧菌属物种则大量增殖。本研究旨在验证这一现象是否体现了该菌的保护性功能作用。

方法：针对东方牡蛎（Crassostrea virginica）幼虫，对11株地中海弧菌菌株开展筛选试验，结果鉴定出三种截然不同的毒力表型——无致病力型、致病力型及中间型，这些表型分别对应单系发育的系统发育支系。

结果：采用无致病力菌株Vm02对幼虫进行预定殖，可使被哈维氏弧菌（V. harveyi）或珊瑚致弧菌（V. coralliilyticus）攻毒的幼虫存活率，从仅接种病原菌的对照组的10%~19%提升至94%~97%；该保护效果在环境温度（28℃）与热应激温度（32℃）下均能维持，实现近乎完全的保护。该保护作用起效迅速，即便与病原菌同时接种也能发挥防护效果，且在攻毒暴露后超过96小时仍可维持持久的保护作用。荧光显微镜（fluorescence microscopy）观察证实，带有荧光标记的Vm02菌株会持续定殖于幼虫的消化组织内。对33株地中海弧菌基因组开展系统基因组学分析，结果显示三种表型菌株分别处于支持度良好的系统发育支系中，各支系间的平均核苷酸一致性（average nucleotide identity）为97.1%~97.8%，接近物种分类的阈值水平。泛基因组分析（pangenome analysis）结果表明，保护性支系的菌株携带230个独特的同源基因簇（orthogroups），这些基因簇编码调控系统、胁迫耐受性、代谢多样性以及保守的细菌素生物合成操纵子。而致病谱系的菌株则不携带致病谱系中常见的I型分泌系统（Type I secretion system）、VI型分泌系统（Type VI secretion system）变体以及TCP致病岛（TCP pathogenicity island）相关基因。

讨论：综上，本研究结果表明，有益与致病表型在地中海弧菌的不同谱系中存在系统发育上的约束性，该发现为贝类水产养殖业的益生菌开发与病害预测提供了理论框架。