Department of Biology, University of North Florida, Jacksonville, Florida, United States of America.
Smithsonian Environmental Research Center, Edgewater, Maryland, United States of America.
PLoS One. 2020 Mar 13;15(3):e0230108. doi: 10.1371/journal.pone.0230108. eCollection 2020.
Recent trends suggest that marine disease outbreaks caused by opportunistic pathogens are increasing in frequency and severity. One such malady is seagrass wasting disease, caused by pathogens in the genus Labyrinthula. It is suspected that pathogenicity is intimately linked to the ability of the host to initiate defense responses; however, supportive evidence is lacking. To address this, we developed two techniques, including 1) a new qPCR-based pathogen detection method, and 2) an immune profiling panel via four host-biomarker assays (measuring peroxidase, exochitinase, polyphenol oxidase, and lysozyme activities). These techniques were then used to experimentally investigate the impact of environmental stressors (namely, elevated temperature and salinity) on host immunity and how immune status might affect susceptibility to Labyrinthula infection. In the first experiment, we subjected individual turtlegrass (Thalassia testudinum) shoots to short-term (7 d) abiotic stressors alone. In a second experiment, the same abiotic stressor conditions were followed by pathogen exposure (7 additional d), simulating a scenario where we attempt to isolate the impact of environmental stressors on the host seagrass species by removing the stressor as the pathogen is introduced. The qPCR assay successfully quantified the abundance of Labyrinthula spp. cells from both pure cultures and seagrass tissues across a broad range of predominately pathogenic strains, with high sensitivity. Immune enzyme assays revealed that all four biomarkers were constitutively active in turtlegrass individuals, but specific activities were largely unaffected by the chosen abiotic stressor conditions. We also identified positive correlations between pathogen load and two biomarkers (peroxidase, exochitinase), regardless of abiotic stress treatment, further demonstrating the potential utility of these biomarkers in future applications.
最近的趋势表明，由机会性病原体引起的海洋疾病爆发的频率和严重程度正在增加。其中一种疾病是海草草叶退化病，由 Labyrinthula 属的病原体引起。人们怀疑病原体的致病性与宿主启动防御反应的能力密切相关；然而，缺乏支持性证据。为了解决这个问题，我们开发了两种技术，包括 1）一种新的基于 qPCR 的病原体检测方法，和 2）通过四种宿主生物标志物测定法（测量过氧化物酶、外几丁质酶、多酚氧化酶和溶菌酶活性）的免疫分析面板。然后，我们使用这些技术来实验性地研究环境胁迫（即，高温和高盐度）对宿主免疫的影响，以及免疫状态如何影响对 Labyrinthula 感染的易感性。在第一个实验中，我们将单个海龟草（Thalassia testudinum）枝条单独暴露于短期（7 天）非生物胁迫下。在第二个实验中，在相同的非生物胁迫条件下，随后进行病原体暴露（再持续 7 天），模拟了一种试图通过在引入病原体的同时去除胁迫源来隔离环境胁迫对宿主海草草种影响的情况。qPCR 检测成功地定量了广泛存在的致病性菌株的 Labyrinthula spp.细胞的丰度，具有很高的灵敏度。免疫酶测定显示，四种生物标志物在海龟草个体中均保持着组成型活性，但特定的活性在很大程度上不受所选非生物胁迫条件的影响。我们还发现，无论是否存在非生物胁迫处理，病原体负荷与两种生物标志物（过氧化物酶、外几丁质酶）之间都存在正相关关系，这进一步证明了这些生物标志物在未来应用中的潜力。
