Simultaneous estimation of viral lysis and protozoan grazing on bacterial mortality using a modified virus-dilution method

Abstract

A modified dilution method designed to simultaneously estimate bacterial mortality due to viral infection and protozoan grazing was developed and compared with the standard dilution protocol. Various fractions of original seawater (non-filtered) and 1.0 μm-filtered seawater (grazer-free) collected from coastal waters in Hokkaido, Japan, were diluted with 10 kDa filtered seawater (virusfree) to set up 4 gradients of predator–prey interaction, and monitored every 12 h for bacterial abundance during a 48 h incubation. In more diluted fractions, bacterial abundance increased rapidly, and a good linear fit with a negative slope (as mortality) was found between the apparent growth rate and fractions of original water. The resulting slopes of the regression in samples prepared from the original seawater were significantly higher than grazer-free samples, which denotes both viral lysis and protozoan grazing in the former, and only viral lysis in the latter. Ranges of specific lytic and grazing rate were 0.53 to 0.98 and 0.05 to 0.13 d–1, respectively, and lytic pressure accounted for 87 to 91% of the total mortality. Comparison of our method (using virus-free diluent) with the standard dilution protocol (using 0.2 μm diluent) showed significant differences in slope and y-intercept (potential growth rate without mortality) found at 0.26 d–1 and 0.88 d–1, respectively. The above results suggest that using the standard dilution protocol might underestimate instantaneous growth rate, particularly in environments where lytic pressure is relatively high.