Altered growth and death in dilution-based viral predation assays

Ben Knowles; Juan A. Bonachela; Nick Cieslik; Alice Della Penna; Ben Diaz; Nick Baetge; Micheal J. Behrenfeld; Karen Naumovitz; Emmanuel Boss; Jason R. Graff; Kimberly H. Halsey; Liti Haramaty; Lee Karp-Boss; Kay D. Bidle

doi:10.1371/journal.pone.0288114

Altered growth and death in dilution-based viral predation assays

Ben Knowles, Juan A. Bonachela, Nick Cieslik, Alice Della Penna, Ben Diaz, Nick Baetge, Micheal J. Behrenfeld, Karen Naumovitz, Emmanuel Boss, Jason R. Graff, Kimberly H. Halsey, Liti Haramaty, Lee Karp-Boss, Kay D. Bidle

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English (US)
Article number	e0288114
Journal	PLoS One
Volume	18
Issue number	7 JULY
DOIs	https://doi.org/10.1371/journal.pone.0288114
State	Published - Jul 2023
Externally published	Yes

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@article{6cd89d2473074cf0aa6cb9783a77b4b0,

title = "Altered growth and death in dilution-based viral predation assays",

abstract = "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 {\textquoteleft}plate effect{\textquoteright} 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.",

author = "Ben Knowles and Bonachela, {Juan A.} and Nick Cieslik and Penna, {Alice Della} and Ben Diaz and Nick Baetge and Behrenfeld, {Micheal J.} and Karen Naumovitz and Emmanuel Boss and Graff, {Jason R.} and Halsey, {Kimberly H.} and Liti Haramaty and Lee Karp-Boss and Bidle, {Kay D.}",

note = "Publisher Copyright: Copyright: {\textcopyright} 2023 Knowles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",

year = "2023",

month = jul,

doi = "10.1371/journal.pone.0288114",

language = "English (US)",

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journal = "PLoS One",

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T1 - Altered growth and death in dilution-based viral predation assays

AU - Knowles, Ben

AU - Bonachela, Juan A.

AU - Cieslik, Nick

AU - Penna, Alice Della

AU - Diaz, Ben

AU - Baetge, Nick

AU - Behrenfeld, Micheal J.

AU - Naumovitz, Karen

AU - Boss, Emmanuel

AU - Graff, Jason R.

AU - Halsey, Kimberly H.

AU - Haramaty, Liti

AU - Karp-Boss, Lee

AU - Bidle, Kay D.

N1 - Publisher Copyright: Copyright: © 2023 Knowles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2023/7

Y1 - 2023/7

N2 - 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.

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Altered growth and death in dilution-based viral predation assays

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