title: Quantification of Viral and Prokaryotic Production Rates in Benthic Ecosystems: A Methods Comparison.
abstract: Viruses profoundly influence benthic marine ecosystems by infecting and subsequently killing their prokaryotic hosts, thereby impacting the cycling of carbon and nutrients. Previously conducted studies, based on different methodologies, have provided widely differing estimates of the relevance of viruses on benthic prokaryotes. There has been no attempt so far to compare these independent approaches, including contextual comparisons among different approaches for sample manipulation (i.e., dilution or not of the sediments during incubations), between methods based on epifluorescence microscopy (EFM) or radiotracers, and between the use of different radiotracers. Therefore, it has been difficult to identify the most suitable methodologies and protocols to be used as standard approaches for the quantification of viral infections of prokaryotes. Here, we compared for the first time different methods for determining viral and prokaryotic production rates in marine sediments collected at two benthic sites, differing in depth and environmental conditions. We used a highly replicated experimental design, testing the potential biases associated to the incubation of sediments as diluted or undiluted. In parallel, we also compared EFM counts with the3H-thymidine incubations for the determination of viral production rates, and the use of3H-thymidine versus3H-leucine radiotracers for the determination of prokaryotic production. We show here that, independent from sediment dilution, EFM-based values of viral production ranged from 1.4 to 4.6 × 107viruses g-1h-1, and were similar but overall less variable compared to those obtained by the3H-thymidine method (0.3 to 9.0 × 107viruses g-1h-1). In addition, the prokaryotic production rates were not affected by sediment dilution, and the use of different radiotracers provided very consistent estimates (10.3-35.1 and 9.3-34.6 ngC g-1h-1using the3H-thymidine or3H-leucine method, respectively). These results indicated that viral lysis was responsible for the abatement of 55-81% of the prokaryotic heterotrophic production, corroborating previous findings of the major role of viruses in benthic deep-sea ecosystems. Moreover, our methodological comparison for the analysis of viral production in marine sediments suggests that microscopy-based approaches are simpler and more cost-effective than those based on radiotracers. These approaches also reduce time to results and overcome issues related to generation of radioactive waste.
authors.affiliation: Department of Life and Environmental Sciences, Polytechnic University of MarcheAncona, Italy; Stazione Zoologica Anton Dohrn, NaplesItaly.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Rastelli
authors.affiliation: Department of Life and Environmental Sciences, Polytechnic University of Marche Ancona, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Dell'Anno
authors.affiliation: Department of Life and Environmental Sciences, Polytechnic University of Marche Ancona, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Corinaldesi
authors.affiliation: Marine Biological Section, Department of Biology, University of Copenhagen Helsingør, Denmark.
authors.forename: Mathias
authors.initials: M
authors.lastname: Middelboe
authors.affiliation: Institute of Marine Sciences, The University of North Carolina at Chapel Hill, Morehead City NC, USA.
authors.forename: Rachel T
authors.initials: RT
authors.lastname: Noble
authors.affiliation: Department of Life and Environmental Sciences, Polytechnic University of MarcheAncona, Italy; Stazione Zoologica Anton Dohrn, NaplesItaly.
authors.forename: Roberto
authors.initials: R
authors.lastname: Danovaro
keywords: deep-sea ecosystem; epifluorescence microscopy; marine sediments; tritiated leucine; tritiated thymidine; viral production; virus-induced prokaryotic mortality