Evaluation of incubation-based methods for estimating virioplankton production in estuaries

Abstract

Assessment of the role of viral lysis in aquatic microbial communities requires a robust means of estimating viral production (VP). Here, 3 incubation-based VP methods (fluorescently labeled viruses [FLV], dilution [DIL], and thymidine incorporation [TdR]) were evaluated in water samples from the Delaware and Chesapeake Bays. In Chesapeake Bay samples, average VP rates were 10-fold higher for FLV and DIL (~3 to 25 × 10 10 viruses l -1 d -1 ) than for TdR (~0.2 to 3 × 10 10 viruses l -1 d -1 ). Estimates of viral-mediated bacterial mortality (VMM) indicate that FLV overestimates VP in eutrophic waters, since >100% of bacterial production (BP) would have been consumed through viral lysis. DIL and TdR VP methods gave more realistic estimates of VMM with respect to BP. The FLV method provides estimates of both VP and virus removal rates; however, it requires preparation of a viral tracer and twice as many microscopic enumerations as the DIL method. DIL was the least difficult and most efficient method; however, bacterial loss during vacuum diafiltration resulted in poor replicability. TdR gave similar VP estimates to DIL, but requires use of a large and poorly constrained conversion factor. With methodological improvements in bacterial cell recovery, DIL should be the most widely applicable method for estimation of VP in highly productive estuarine waters. KEY WORDS: Viral production · Bacteriophage · Marine viruses · Marine microbial ecologyResale or republication not permitted without written consent of the publisher

Enhancement of viral production by addition of nitrogen or nitrogen plus carbon in subtropical surface waters of the South Pacific

“…Hoppe et al 2006). Viral production determined from TdR incorporation varied in the range of 0.2 ± 0.1 pmol TdR l -1 d -1 (Stn SX18) and 12.0 ± 1.8 pmol TdR l -1 d -1 (Stn SX22); these values fall within a range of viral incorporation rates of TdR reported in other marine systems, including coastal and high latitude oceanic regions (Steward et al 1996, Helton et al 2005 Table 1. Physical, chemical, and microbiological variables at the sampling stations.…”

“…Although conversion factors may vary depending on viral genome size, G+C content, and isotope dilution factors, little is known about variations of these variables in oceanic environments. Given that viruses can use host nucleic acids and that RNA-containing viruses would not be labeled, viral production determined by the TdR method would be too low (Helton et al 2005). Conversely, the TdR method might overestimate viral production because of the inclusion of non-specifically labeled components in the fraction of viral DNA.…”

“…The TdR method is particularly suitable for nutrient addition studies in which a large number of samples (n = 24 for each experiment in the present study) must be handled promptly. However, conversion factors that relate TdR incorporation rate to viral production have been poorly constrained, especially in oligotrophic environments (Helton et al 2005). Although conversion factors may vary depending on viral genome size, G+C content, and isotope dilution factors, little is known about variations of these variables in oceanic environments.…”

“…We determined viral production using a TdR approach (Noble & Steward 2001), which has advantages in sensitivity and simplicity over other techniques such as the dilution and the fluorescently labeled virus methods (Noble & Steward 2001, Helton et al 2005. The TdR method is particularly suitable for nutrient addition studies in which a large number of samples (n = 24 for each experiment in the present study) must be handled promptly.…”

Tangential flow diafiltration: an improved technique for estimation of virioplankton production

“…viral-mediated mortality (VMM) (Steward et al 1996, Noble & Fuhrman 2000, Wilhelm et al 2002, and to estimate the amount of C, N, and P released into a system by viral cell lysis (Fuhrman 1992, Steward et al 1996, Gobler et al 1997, Weinbauer & Höfle 1998, Wilhelm et al 2002. Approaches to estimation of VP have been numerous and include electron microscopic observation of the frequency of visibly infected cells (FVIC) (Proctor et al 1993, Steward et al 1996, Binder 1999, Guixa-Boixereu et al 1999, Hwang & Cho 2002, Middelboe et al 2002, Choi et al 2003, Weinbauer et al 2003a, contact rates of viruses and bacteria (Murray & Jackson 1992, Suttle & Chan 1994, incorporation of 3 H-thymidine or 32 P into viral DNA (Steward et al 1992a, Fuhrman & Noble 1995, Kepner et al 1998), viral decay rates (Heldal & Bratbak 1991, Bratbak et al 1992, Guixa-Boixereu et al 1999, Tuomi & Kuuppo 1999, fluorescently labeled viruses (FLVs) as tracers of viral decay and VP (Noble & Fuhrman 2000, Helton et al 2005, this issue), and dilution methods (Wilhelm et al 2002, Winter et al 2004a, Helton et al 2005. The assumptions, advantages, and disadvantages of each of these approaches to measuring VP are summarized in Table 1.…”