Department of Marine and Coastal Science, Rutgers University, New Brunswick, New Jersey, United States of America.
Department of Ecology and Evolutionary Biology, University of California, Los Angeles, Los Angeles, California, United States of America.
PLoS One. 2023 Jul 7;18(7):e0288114. doi: 10.1371/journal.pone.0288114. eCollection 2023.
Viral lysis of phytoplankton is one of the most common forms of death on Earth. Building on an assay used extensively to assess rates of phytoplankton loss to predation by grazers, lysis rates are increasingly quantified through dilution-based techniques. In this approach, dilution of viruses and hosts are expected to reduce infection rates and thus increase host net growth rates (i.e., accumulation rates). The difference between diluted and undiluted host growth rates is interpreted as a measurable proxy for the rate of viral lytic death. These assays are usually conducted in volumes ≥ 1 L. To increase throughput, we implemented a miniaturized, high-throughput, high-replication, flow cytometric microplate dilution assay to measure viral lysis in environmental samples sourced from a suburban pond and the North Atlantic Ocean. The most notable outcome we observed was a decline in phytoplankton densities that was exacerbated by dilution, instead of the increased growth rates expected from lowered virus-phytoplankton encounters. We sought to explain this counterintuitive outcome using theoretical, environmental, and experimental analyses. Our study shows that, while die-offs could be partly explained by a 'plate effect' due to small incubation volumes and cells adhering to walls, the declines in phytoplankton densities are not volume-dependent. Rather, they are driven by many density- and physiology-dependent effects of dilution on predation pressure, nutrient limitation, and growth, all of which violate the original assumptions of dilution assays. As these effects are volume-independent, these processes likely occur in all dilution assays that our analyses show to be remarkably sensitive to dilution-altered phytoplankton growth and insensitive to actual predation pressure. Incorporating altered growth as well as predation, we present a logical framework that categorizes locations by the relative dominance of these mechanisms, with general applicability to dilution-based assays.
浮游植物病毒裂解是地球上最常见的死亡形式之一。在广泛用于评估被食草动物捕食的浮游植物损失率的测定方法的基础上，裂解率越来越多地通过基于稀释的技术来量化。在这种方法中，稀释病毒和宿主预计会降低感染率，从而提高宿主净增长率（即积累率）。稀释和未稀释宿主增长率之间的差异被解释为衡量病毒裂解死亡速率的可测量指标。这些测定通常在体积≥1L 的情况下进行。为了提高通量，我们实施了一种小型化、高通量、高复制、流式细胞术微板稀释测定法，以测量来自郊区池塘和北大西洋的环境样本中的病毒裂解。我们观察到的最显著结果是浮游植物密度下降，而不是预期的由于病毒-浮游植物相遇减少而导致的增长率增加，稀释会加剧这种下降。我们试图通过理论、环境和实验分析来解释这种违反直觉的结果。我们的研究表明，虽然由于小培养体积和细胞附着在壁上而导致的“平板效应”，死亡可能部分得到解释，但浮游植物密度的下降与体积无关。相反，它们是由稀释对捕食压力、营养限制和生长的许多密度和生理依赖效应驱动的，所有这些都违反了稀释测定的原始假设。由于这些效应与体积无关，因此这些过程可能发生在所有稀释测定中，我们的分析表明这些过程对稀释改变的浮游植物生长非常敏感，而对实际捕食压力不敏感。我们提出了一个逻辑框架，将这些过程分为改变生长和捕食的过程，并根据这些机制的相对主导地位对地点进行分类，该框架具有普遍适用于基于稀释的测定的适用性。
