Data_Sheet_2_Effects of Experimental Terrestrialization on the Skin Mucus Proteome of African Lungfish (Protopterus dolloi).XLSX

Animal mucosal barriers constantly interact with the external environment, and this interaction is markedly different in aquatic and terrestrial environments. Transitioning from water to land was a critical step in vertebrate evolution, but the immune adaptations that mucosal barriers such as the skin underwent during that process are essentially unknown. Vertebrate animals such as the African lungfish have a bimodal life, switching from freshwater to terrestrial habitats when environmental conditions are not favorable. African lungfish skin mucus secretions contribute to the terrestrialization process by forming a cocoon that surrounds and protects the lungfish body. The goal of this study was to characterize the skin mucus immunoproteome of African lungfish, Protopterus dolloi, before and during the induction phase of terrestrialization as well as the immunoproteome of the gill mucus during the terrestrialization induction phase. Using LC-MS/MS, we identified a total of 974 proteins using a lungfish Illumina RNA-seq database, 1,256 proteins from previously published lungfish sequence read archive and 880 proteins using a lungfish 454 RNA-seq database for annotation in the three samples analyzed (free-swimming skin mucus, terrestrialized skin mucus, and terrestrialized gill mucus). The terrestrialized skin mucus proteome was enriched in proteins with known antimicrobial functions such as histones and S100 proteins compared to free-swimming skin mucus. In support, gene ontology analyses showed that the terrestrialized skin mucus proteome has predicted functions in processes such as viral process, defense response to Gram-negative bacterium, and tumor necrosis factor-mediated signaling. Importantly, we observed a switch in immunoglobulin heavy chain secretion upon terrestrialization, with IgW1 long form (IgW1L) and IgM1 present in free-swimming skin mucus and IgW1L, IgM1, and IgM2 in terrestrialized skin mucus. Combined, these results indicate an increase in investment in the production of unique immune molecules in P. dolloi skin mucus in response to terrestrialization that likely better protects lungfish against external aggressors found in land.

动物黏膜屏障持续与外界环境发生交互作用，且该交互在水生与陆生环境中存在显著差异。从水生向陆生的转变是脊椎动物演化历程中的关键节点，但皮肤等黏膜屏障在此过程中发生的免疫适应性变化，目前仍基本未知。以非洲肺鱼为代表的脊椎动物具有双模式生活史，当环境条件不适宜时，它们会从淡水生境切换至陆生生境。非洲肺鱼的皮肤黏液分泌物可形成包裹并保护自身的茧状结构，助力其陆生化进程。本研究旨在解析多洛肺鱼（Protopterus dolloi）在陆生化诱导阶段前后的皮肤黏液免疫蛋白质组（immunoproteome），以及陆生化诱导阶段的鳃黏液免疫蛋白质组。本研究借助液相色谱-串联质谱（LC-MS/MS），依托肺鱼Illumina RNA测序（RNA-seq）数据库共鉴定出974种蛋白质，从已发表的肺鱼序列读取档案中获得1256种蛋白质，并通过肺鱼454 RNA-seq数据库完成注释；本次分析共涵盖三类样本：自由游泳状态下的皮肤黏液、陆生化状态下的皮肤黏液以及陆生化状态下的鳃黏液。与自由游泳状态下的皮肤黏液相比，陆生化状态下的皮肤黏液蛋白质组中，组蛋白、S100蛋白等已知具备抗菌功能的蛋白质显著富集。作为佐证，基因本体（GO）分析显示，陆生化状态下的皮肤黏液蛋白质组所预测的功能涉及病毒增殖过程、革兰氏阴性菌防御反应以及肿瘤坏死因子介导的信号通路等。尤为重要的是，本研究观察到陆生化过程中免疫球蛋白重链的分泌模式发生转变：自由游泳状态下的皮肤黏液中仅存在长型IgW1（IgW1L）与IgM1，而陆生化状态下的皮肤黏液中则包含IgW1L、IgM1与IgM2。综合以上结果，本研究表明多洛肺鱼会通过增加皮肤黏液中独特免疫分子的合成以响应陆生化过程，该变化或可更好地保护肺鱼免受陆生环境中外界侵害因子的威胁。