Methodological review and meta‐analysis of dilution assays for estimates of virus‐ and grazer‐mediated phytoplankton mortality

Abstract

This manuscript reviews methodological differences among modified dilution assay studies used to partition phytoplankton mortality into virus‐ and grazer‐mediated fractions, and discusses their implications. A meta‐analysis is also described, based on virus‐ and grazer‐mediated mortality and instantaneous growth rates extracted from these studies. As the α value used to assess the significance of these rates was not consistent across studies, metadata was re‐analyzed (i.e., rates were re‐calculated) using three levels of significance: α = 0.05, α = 0.1, and α‐not‐considered. The average virus‐mediated mortality rate observed for each re‐analysis was 0.04 d−1, 0.10 d−1, and 0.08 d−1, respectively. Further, 80%, 74%, and 56% of virus‐mediated mortality rates were between −0.1 d−1 and 0.1 d−1. Power analysis was used to demonstrate that typical modified dilution assays lack the sensitivity to consistently detect small virus‐mediated mortality rates, and shows how dilution scheme modifications can affect power. Rates of virus‐mediated mortality were also discriminated based on salinity, depth, use of nutrient amendments, and choice of technique for estimating apparent growth rates. For all three re‐analyses, a significant positive correlation was observed between virus‐mediated mortality (d−1) and instantaneous growth rate (d−1) (Pearson r = 0.72, 0.74, 0.32, all p values < 0.01), suggesting that virally mediated mortality is intimately linked to host growth. This manuscript highlights the need for greater standardization in the analysis and presentation of information when using the modified dilution approach for estimating phytoplankton mortality, and provides recommendations for the application of future assays.

Storm-Induced Predator-Prey Decoupling Promotes Springtime Accumulation of North Atlantic Phytoplankton

“…Although the 2-point approach used here has been demonstrated as a valid alternative to a dilution series for estimating grazing rates (Chen, 2015;Morison and Menden-Deuer, 2017), such an approach for viral dilutions remains to be assessed. As demonstrated in a recent meta-analysis, the modified dilution method suffers from a weak statistical power, even when multiple dilutions are used (Staniewski and Short, 2018). Despite these caveats, the calculated accumulation rates presented here matched well with the rates observed in situ, suggesting that viral lysis negligibly contributed to overall phytoplankton mortality.…”

“…To estimate virus-induced mortality, microzooplankton grazing, and phytoplankton growth rates, we used a "modified dilution assay" (Evans et al, 2003;Kimmance and Brussaard, 2010;Staniewski and Short, 2018) using the 2-point method (Worden and Binder, 2003;Chen, 2015;Morison and Menden-Deuer, 2017) in a total of 11 experiments conducted at the station ( Table 1). Water for the experiments was collected from the surface (5 m) using the CTD rosette sampler.…”

“…Both casts were conducted within 2 h in order for the incubations to start before sunrise and to minimize changes in water characteristics. Water from the first cast was used to generate grazer-free filtered seawater (FSW) by direct gravityfiltration of the water from the CTD Niskin bottles through a membrane filter capsule (Pall) with a pore size of 0.45 µm (Mojica et al, 2016), which allows retention of large viruses (Evans et al, 2003;Staniewski and Short, 2018). Some of the FSW was further processed through a 30 kDa tangential flow filtration system (Millipore) to obtain the virus-free diluent (TFF).…”

Seasonal Variability and Drivers of Microzooplankton Grazing and Phytoplankton Growth in a Subtropical Estuary

“…The incubation period used in our dilutions may have been too short to capture viral infection, as often the viral lytic cycle can exceed 24 h (Evans et al, 2003;Jacquet et al, 2005). Lastly, recent meta-analysis revealed that over half of viral-induced mortality rates measured via the modified dilution approach were minimal (−0.1-0.1 day −1 ), which suggests that typical dilution assays may lack the sensitivity needed to detect viral mortality (Staniewski and Short, 2018). Other sources may have contributed to phytoplankton mortality in the estuary, such as consumption by mesozooplankton or benthic invertebrates (e.g., bivalves), though both processes were not measurable via dilution incubations and typically account for less consumption of primary production compared to microzooplankton (Cloern et al, 2014;Steinberg and Landry, 2017).…”

Quantifying spatiotemporal variability in zooplankton dynamics in the Gulf of Mexico with a physical-biogeochemical model

“…viral lysis of phytoplankton or predation by un-modeled higher predators). However, grazing mortality is explicitly modeled in NEMURO and viral mortality is generally not a substantial loss term for bulk phytoplankton (Brum et al, 2014;Staniewski and Short, 2018). Quadratic mortality was retained for PZ, to account for predation pressure of un-modeled planktivorous fish.…”