Infectious titers of Emiliania huxleyi virus 86 are reduced by exposure to millimolar dimethyl sulfide and acrylic acid

We examined the ability of dimethylsulfoniopropionate (DMSP), its cleavage products dimethyl sulfide (DMS) and acrylic acid (AA), and the oxidized form of DMS dimethylsulfoxide (DMSO), to inhibit infection of Emiliania huxleyi virus 86 (EhV-86). Infectivity was assessed by plaque assay of viral stock that had been exposed to these compounds. The initial concentrations of the compounds tested were 250 mmol L21 for DMSP, DMS, and AA, and 14 mmol L21 for DMSO. These are the maximum concentrations thought to occur in E. huxleyi and therefore the highest EhV-86 might encounter. DMSP and DMSO had no effect on EhV-86; however, both DMS and AA diminished viral titers. Further experiments established that both DMS and AA significantly reduced titers from a concentration of 100 mmol L21 and that they had a greater antiviral effect when applied in combination. The DMSP system in algae could function as a chemical defense against viral infection that would benefit the surviving cells in the population by reducing infective titers of progeny viruses and therefore decreasing the probability of infection of further cells. Viruses are abundant in the sea and are considered to influence ecological processes and the major biogeochemical cycles (Suttle 2005). Studies have shown that despite the decimation of algal populations by viruses, algal cells may remain after the population crash (Jacquet et al. 2002), and some species of phytoplankton are able to coexist with their pathogenic viruses (Thyrhaug et al. 2003). Despite indications that defense mechanisms against virus infection may exist in phytoplankton, no compounds have been identified that could fulfill this important role. Emiliania huxleyi is a widely distributed, biogeochemically significant species of coccolithophore that forms large-scale bloom that may be decimated by viral infection (Wilson et al. 2002). A study of six E. huxleyi strains showed that the activity of their dimethylsulfoniopropionate (DMSP) lyase, the enzyme responsible for cleaving DMSP to dimethyl sulfide (DMS) and acrylic acid (AA), varied by more than 6,000-fold, and they were classified as either ‘‘low lyase’’ and ‘‘high lyase’’ strains (Steinke et al. 1998). Despite extensive screening, to date no viruses have been isolated that are capable of infecting the high DMSP lyase activity strains (Evans 2005). This has led to the suggestion that high DMSP lyase activity may be implicated in an antivirus defense mechanism (Schroeder et al. 2002). Previously the DMSP system has been proposed to serve a number of roles including compatible solute, antioxidant, overflow for excess reduced sulfur and energy, and a chemical defense against grazing (Simo 2001). The grazing chemical defense hypothesis was first suggested after it was observed that microzooplankton were able to clear cultures of E. huxleyi with lower DMSP lyase activity much faster than those with high DMSP lyase activity (Wolfe and Steinke 1996). Further studies revealed that when offered a choice of prey, grazers preferentially selected those E. huxleyi strains with lower DMSP lyase activity (Wolfe et al. 1997). This led to the suggestion that the acrylate produced during grazing by mixing the DMSP lyase with its substrate was harmful to the protists (Wolfe et al. 1997), which is in line with the antimicrobial properties of this compound (Sieburth 1960). More recent studies suggest that prey selection may be directed by signaling molecules, possibly DMSP or related compounds, present on the cell surface or in the near-cell dissolved phase (Strom et al. 2003a, 2003b). Studies of Phaeocystis pouchetii have revealed that DMS is produced and DMSP released during viral infection (Malin et al. 1998). Furthermore, our laboratory data have shown that during viral infection of E. huxleyi, AA and dimethylsulfoxide (DMSO), the oxidized form of DMS, are produced in addition to DMS (Evans 2005). This, in combination with the failure to isolate any viruses against high DMSP lyase activity strains, led us to consider whether the DMSP cleavage pathway could be linked to an antivirus defense mechanism within algal cells. Here we report on the effect of DMSP and the related compounds DMS, AA, and DMSO on the infectivity of E. huxleyi viruses. Materials and methods—E. huxleyi CCMP 1516 (Provasoli-Guillard Centre for the Cultivation of Marine Phytoplankton), a low DMSP lyase activity strain (Steinke et al. 1998), was used in this study along with virus strain E. huxleyi virus 86 (EhV-86). All cultures were maintained in e/2 medium at 15uC under a light : dark cycle of 14 : 10 h and at an illumination of 250 mmol photons m22 s21. Prior to use in the experiments, approximately 50 ml of fresh virus stock was dialyzed twice against 1 liter e/2 medium at 4uC for 1 h to reduce the background concentration of DMSP and related compounds using a 10,000 nominal molecular weight cutoff and regenerated cellulose dialysis membrane prepared according to the protocol of Harris and Angal (1990). To determine whether DMSP, DMS, AA, or DMSO had any effect on EhV-86 infectivity, virus stock was incubated with either 250 mmol L21 DMSP, DMS, or AA, or