Viral and Flagellate Control of Prokaryotic Production and Community Structure in Offshore Mediterranean Waters

A dilution and size fractionation approach was used to study the separate and combined effects of viruses and flagellates on prokaryotic production ([³H]leucine incorporation) and community composition (16S rRNA gene PCR and denaturing gradient gel electrophoresis [DGGE]) in the upper mixed layer and the deep chlorophyll maximum in the offshore Mediterranean Sea. Four experiments were established using differential filtration: a resource control without predators (C treatment), treatment in the presence of viruses (V treatment), treatment in the presence of flagellates (F treatment), and treatment in the presence of both predators (VF treatment). The V and VF treatments increased prokaryotic abundance (1.4- to 2.3-fold) and the number of DGGE bands (by up to 43%) and decreased prokaryotic production compared to the level for the C treatment (by 22 to 99%). For the F treatment, significant differences compared to the level for the C treatment were found as well, but trends were not consistent across experiments. The relative abundances of the high-nucleic-acid subgroups of prokaryotes with high scatter (HNAhs) in flow cytometer settings were lower in the V and VF treatments than in the C and F treatments. These differences were probably due to lysis of very active HNA prokaryotes in the V and VF treatments. Our results indicate that the presence of viruses or viruses plus flagellates sustains prokaryotic diversity and controls prokaryotic production by regulating the proportion of the highly active members of the community. Our data also suggest that lysis and grazing control influences the relationship between bacterial community composition and prokaryotic production.During the last 3 decades, a wealth of information on the mechanisms controlling prokaryotic production in the ocean has been accumulated (26). It is commonly accepted that inorganic nutrients, composition and bioavailability of dissolved organic matter (DOM), and predation are the major factors shaping the spatiotemporal variability of prokaryotic production. Protistan grazing might result in the formation of grazing-resistant prokaryotic cell types (16, 23) and in a stimulation of specific growth rates (39) by recycling nutrients (45, 48). Viruses, considered here to be another type of predator, cause a mortality of prokaryotes that is highly variable but, on average, as important as protistan grazing (summarized in references 56 and 63).A modeling approach suggested that viral lysis reduces the carbon flow to higher trophic levels and stimulates prokaryotic production (10). Support for this “stimulation of production” hypothesis comes from experimental data showing that viral lysis increases bacterial production and respiration (34) at the food web level. However, in experimental studies with the viral community either present or absent, a negative effect of viruses on bacterial production was typically found (6, 35, 65). It has also been argued that grazing should have a negative effect on viral production, since grazers consume infected cells (5, 58). However, in a set of experiments performed with freshwater systems, viral infection was stimulated in the presence of grazers, suggesting synergistic interactions (41, 54, 55).Prokaryotic community composition can vary during phytoplankton blooms (4, 40), probably due to changing DOM composition and bioavailability, as suggested by the covariation in the types of ß-glucosidases (4). It has also been suggested that phages control competitive dominants for resource acquisition, thus sustaining diversity (10). This idea has been mathematically described in the “killing the winner” hypothesis (53) and is also applicable to flagellates (52). In a general model, diversity shows a hump-shaped distribution along gradients of increasing resource as well as consumer control (64). However, this relationship can vary for different taxonomic groups of bacteria. In an in situ study conducted in the North Sea, the number of bacterial phylotypes was negatively related to viral abundances and prokaryotic production and respiration (42, 62). Others have shown for the open ocean a positive relationship between the number of phylotypes and productivity (18) and predictability of bacterial community structure over annual cycles (11). Differences in such relationships between environments were also detected (19). Moreover, a latitudinal diversity gradient has been demonstrated for bacterioplankton (12). In a study with coastal water, in the presence of viruses, flagellates, or both types of predators, bacterial production decreased and the number of phylotypes detected by a genetic community fingerprint increased compared to the levels for predator-free controls (65). Overall, there is evidence that protistan grazing (e.g., references 24, 48, and 49) and viral lysis (e.g., references 21, 44, and 61) can affect prokaryotic community composition.Experimental tests of the effect of grazing versus that of viral lysis on prokaryotic production are scarce for the open ocean. Moreover, the links between bacterial production and respiration have mostly been studied for freshwater and coastal marine waters (e.g., references 1, 22, 28, 42, and 62). During an offshore experimental study, in the frame of a mesoscale iron fertilization experiment, changes were detectable in bacterial production, bulk ectoenzymatic activity, and types of β-glucosidase expressed, whereas the number of detectable bacterial phylotypes was remarkably stable (3). This suggests that bacterial production parameters and bacterial community composition were only weakly related. Such observations have also been made in experimental freshwater studies (28).In the present study, we investigated the relative and combined effects of viral lysis and protistan grazing on prokaryotic production and community composition in offshore Mediterranean waters and compared the findings to those for predator-free incubations (resource control). The types of predators had distinct effects on community composition and production and altered the resource control of prokaryotic diversity. When both types of predators were present, prokaryotic production was consistently reduced and diversity likely sustained, probably by controlling the more-active members of the community.