Phytoplankton Virus Production Negatively Affected by Iron Limitation

Fe-limited monocultures of the ubiquitous algae Micromonas pusilla and Phaeocystis globosa were infected with their respective viruses (MpV and PgV) to ascertain the effect of Fe-limitation on phytoplankton host-virus dynamics. The effect of the viral shunt on Fe concentrations and bioavailability is starting to gain attention, since not only is Fe released through lysis, but also its solubility is increased by the simultaneous release of Fe-binding dissolved organic ligands. However, the effect of Fe-limitation on the process of viral lysis itself is poorly understood. In this study fine adjustment of a seawater-based culture medium including the use of ultra-clean trace metal conditions and protocols allowed for Fe-limited growth at nanomolar amounts as opposed to micromolar amounts typically employed in culturing. Viral lysates derived from Fe-limited and Fe-replete (for comparison) hosts were cross-inoculated in hosts of both Fe treatments, to judge the quality of the resulting lysate as well as the effect of Fe introduction after initial infection. For both phytoplankton host-virus systems, the virus burst size reduced strongly under Fe stress, i.e. on average 28 ±1% of replete. Moreover, the MpV virus progeny showed highly reduced infectivity of 30±7%, whereas PgV infectivity was not affected. A small addition of Fe to Fe-limited cultures coming from the Fe-replete lysate counteracted the negative effect of Fe-limitation on phytoplankton virus production to some extent (but still half of replete), implying that the physiological history of the host at the moment of infection was an important underlying factor. These results indicate that Fe-limitation has the strong potential to reduce the loss of phytoplankton due to virus infection, thereby affecting the extent of Fe-cycling through the viral shunt. To what extent this affects the contribution of viral lysis-induced organic ligand release needs further study.


Abstract
Fe-limited monocultures of the ubiquitous algae Micromonas pusilla and Phaeocystis globosa were infected with their respective viruses (MpV and PgV) to ascertain the effect of Fe-limitation on phytoplankton host-virus dynamics.
The effect of the viral shunt on Fe concentrations and bioavailability is starting to gain attention, since not only is Fe released through lysis, but also its solubility is increased by the simultaneous release of Fe-binding dissolved organic ligands. However, the effect of Fe-limitation on the process of viral lysis itself is poorly understood. In this study fine adjustment of a seawater-based culture medium including the use of ultra-clean trace metal conditions and protocols allowed for Fe-limited growth at nanomolar amounts as opposed to micromolar amounts typically employed in culturing. Viral lysates derived from Fe-limited and Fe-replete (for comparison) hosts were cross-inoculated in hosts of both Fe treatments, to judge the quality of the resulting lysate as well as the effect of Fe introduction after initial infection. For both phytoplankton host-virus systems, the virus burst size reduced strongly under Fe stress, i.e. on average 28 ±1% of replete. Moreover, the MpV virus progeny showed highly reduced infectivity of 30±7%, whereas PgV infectivity was not affected. A small addition of Fe to Fe-limited cultures coming from the Fe-replete lysate counteracted the negative effect of Fe-limitation on phytoplankton virus production to some extent (but still half of replete), implying that the physiological history of the host at the moment of infection was an important underlying factor. These results indicate that Fe-limitation has the strong potential to reduce the loss of phytoplankton due to virus infection, thereby affecting the extent of Fe-cycling through the viral shunt. To what extent this affects the contribution of viral lysisinduced organic ligand release needs further study.

Introduction
Phytoplankton form the base of most marine pelagic food webs and are important in sequestering atmospheric carbon dioxide (CO2) through photosynthesis. The production of phytoplankton is controlled by physicochemical variables (bottom-up) as well as by biological factors (topdown). Main bottom-up controls of phytoplankton are light and nutrient availability (Behrenfeld et al., 2006). The latter can be subdivided into major (nitrate, phosphate and silicate) and micro-nutrients (e.g. iron) (de Baar et al. 1990;Martin et al. 1990). Top-down factors, e.g. grazing and viral infection, influence the organic matter flux differently (Wilhelm and Suttle, 1999;Weitz and Wilhelm, 2012). While grazing transfers photosynthetically fixed carbon and organic nutrients up the food chain (Calbet and Landry, 2004), viral lysis results in the release of the hosts' cellular content into the surrounding water (Gobler et al., 1997;Wilhelm and Suttle, 1999). Thereupon, the flow of nutrients through the microbial food web is stimulated by bacterial recycling of the dissolved and dead particulate matter (Suttle 2005;Brussaard et al. 2005;Brussaard and Martínez 2008). Virally-induced mortality of various different natural phytoplankton groups was found to be at least an equally important loss factor as microzooplankton grazing (Baudoux et al., 2007;Mojica et al., 2016).
In order to understand and predict changes in phytoplankton community composition, it is important to elucidate how bottom-up and top-down factors interact and affect phytoplankton population dynamics. Several studies using phytoplankton host-virus culture systems showed that major nutrient availability influences viral production (Maat and Brussaard 2016 and see review by Mojica and Brussaard 2014). For example, phosphorus (P) limitation of the virally infected phytoplankton host results in a prolonged latent period, i.e. the time between infection and the initial release of progeny viruses from the host cell, for the infecting viruses (Maat et al., 2014). Moreover, P-stress resulted in reduced viral burst size, i.e. the number of newly formed viruses released per lysed host cell (Bratbak et al., 1998;Maat et al., 2014). These studies proposed shortage of phosphorus as a viral production substrate as well as possible host energy deficiency as reasons for the lower and delayed viral particle yield. There is, however, virtually nothing is known on the effect of micronutrient limitation on phytoplankton host-virus interactions.
Furthermore, as iron (Fe) solubility in seawater is low (Millero, 1998;Liu and Millero, 2002), marine phytoplankton depend on Fe-binding ligands to increase solubility and therefore bioavailability (Gledhill and van den Berg, 1994;Rue and Bruland, 1995). The redox state of Fe is an important factor in Fe bioavailability. The oxidized Fe(III) state is the more stable and thus prevalent state in marine conditions, while the reduced Fe(II) state is the more bioavailable (Breitbarth et al., 2010;Shaked and Lis, 2012). Release of reactive oxygen species during phytoplankton growth has been shown to contribute to bioavailability of Fe by facilitating reduction of Fe(III) (Kustka et al., 2005;Garg et al., 2007). Furthermore, organic exudates have been connected to lowered Fe(II) oxidation rates experimentally (González et al., 2014). Part of the Febinding ligand pool is thought to be of marine biological origin. Strong Fe-binding organic ligands called siderophores are purposefully produced by bacteria (Butler, 2005;Mawji et al., 2011). Humic acids and polysaccharide excretions are other recognized Fe chelators with a biological origin (Laglera et al. 2011;Hassler et al. 2011). The highest Fe-binding ligand concentrations generally correlate with biological activity (Rue and Bruland, 1995;Gerringa et al., 2006;Ibisanmi et al., 2011). Viral lysis, releasing organic substances in seawater, may well be an important contributor to the ligand pool (Gobler et al. 1997;Poorvin et al. 2004;Poorvin et al. 2011). In these studies by Poorvin and others, it was found that bacterial and cyanobacterial lysates provided organically bound Fe in a form more bioavailable than supplied inorganic, ethylenediaminetetraacetic acid (EDTA) bound or desferrioxamine B (DFB) bound Fe. In comparison to the studied bacteria's self-produced siderophores, lysates were also found to contain more bioavailable Fe.
Fe-limitation negatively affects phytoplankton physiology and growth (Behrenfeld et al. 1996;de Baar et al. 1990;Martin et al. 1990;Timmermans, Gerringa, et al. 2001). Besides energetic consequences of Fe-limitation in terms of the cell's ability to harvest light energy (Geider and La Roche, 1994), Fe is also found to be an essential micronutrient for DNA replication (Netz et al., 2012;Zhang, 2014). As parasites viruses are dependent on the metabolism of their host for the production of their progeny. We hypothesize that viral production depends on the degree of Fe-stress of the host. Viral lysis in turn affects the production of Fe-binding organic ligands and thus the solubility of the limiting Fe. Thus far, in terms of impact on Fe cycling, studies focussed solely on the release of Fe or Fe-binding organic ligands upon viral lysis and not on the virus growth cycle (Gobler et al. 1997;Poorvin et al. 2004;Poorvin et al. 2011). Here we examine virus production characteristics under Fe-limitation for two key ecologically relevant phytoplankton hosts: the nanoeukaryotic bloomforming Prymnesiophyte Phaeocystis globosa and the picoeukaryotic Prasinophyte Micromonas pusilla. Phaeocystis is a globally occurring, bloomforming genus (Vaulot et al., 1994), with P. globosa ecologically relevant in temperate marine waters (Schoemann et al., 2005). Viruses have been found to drive P. globosa bloom decline (Brussaard, 2004a;Brussaard et al., 2005;Baudoux et al., 2006). M. pusilla is a common species that is distributed globally (Not et al., 2004(Not et al., , 2005Vaulot et al., 2008). It has been speculated that viral control of this species is continuous (Cottrell et al., 1995). As model species for diverse regions and ecological niches, these species were chosen in this study to offer a broad insight in the response to Fe-limitation of phytoplankton hostvirus systems in world oceans subject to changing conditions. Limiting concentrations were of ecological relevance to represent a natural context.

Experimental design and sampling
Steady state exponentially growing phytoplankton cultures were subdivided per treatment (Fe-limited and Fe-replete) in 6 replicate 500 mL culture flasks. Two days later the viral infection experiment started 3 h into the light period. For each treatment, 2 replicate cultures received viruses produced on Fe-limited host culture (VL), 2 replicates received viruses produced on Fe-replete host culture (VR), and 2 replicates did not receive viruses and served as non-infected controls (C). Fe-limited lysates were added not only to the respective Fe-limited host cultures, but also to the Fe-replete host in order to test for the reduced infectivity we observed under Fe-limitation (see Results). For this reason and to still guarantee a one-step infection cycle we aimed to add 20-25 viruses per algal cell for P. globosa. Given lower yields for M. pusilla, we endeavoured to add at least 5-10 viruses per algal cell, while still maintaining a ~10% v/v addition. Similarly, Fe-replete lysate was also added to Fe-limited host cultures.
This caused a Fe-spike of about 0.9 µM (10% v/v of Fe-replete medium containing lysate), which allows testing whether a spike of Fe influences virus proliferation.
At steady state, i.e. after at least 8 volume changes and consistent phytoplankton counts (2.1±0.4 × 10 6 and 2.1±0.7 × 10 6 for P. globosa and M. pusilla, respectively), samples were collected for dissolved macronutrients (nitrogen and phosphorus) and Fe, as well as pigment composition. Nutrient samples (5 mL after washing of filter and tube) were 0.2 µm filtered (25 mm diameter Acrodisk, Pall) and frozen at -20°C until analysis. GF/C filtered (1.2 µm nominal pore size, 25 mm diameter, Whatman) algal pigment samples of 50 mL were frozen at -80°C until analysis. The number of infective phytoplankton viruses was determined using the most probable number (MPN) endpoint dilution assay according to Suttle (1993). In short, 10-fold dilution series were set up in 5 replicate tubes using a dilute Fe-replete phytoplankton culture at a density of ~10 6 cells mL -1 . A row of uninfected control tubes was added to each analysis. Cell lysis was regularly scored by eye and the final score after 14 days globosa (4.8 x 10 -12 g cell -1 ), while 19'-butanoyloxyfucoxanthin and fucoxanthin concentrations were 0 and 89% of Fe-replete, respectively. The photoprotective xanthophyll derivatives in Fe-limited P. globosa are increased relative to Chlorophyll-a, i.e. the diadinoxanthin concentration over Chlorophyll-a is 162% of Fe-replete (0.58 and 0.57 x 10 -11 g x cell -1 , respectively) and the diatoxanthin concentration is 181% of Fe-replete (0.08 and 0.07 x 10 -11 g x cell -1 , respectively). Fe-limited M. pusilla cultures (

Viral infection characteristics
Infection of both phytoplankton species resulted in one-step infection cycles with full lysis of the cultures whereas the non-infected controls grew or maintained constant cell number ( Figures 1A,B and 2A,B). Cell growth in the non-infected Fe-replete cultures reflects the synchronized cell division during the dark period (Brussaard et al., 1999). In the Fe-limited cultures growth of P. globosa halted, which was due to stress from the frequent sampling, since the Fe-limited subcultures that were sampled only once a day did show some growth (data not  Table 3). In comparison to P.
globosa, the reduced growth rate of Fe-limited steady state M. pusilla did not significantly affect the extent of reduction in virus burst size. Nonetheless, M.
pusilla required a higher cellular Fe concentration. This implies that Fe-limited P. globosa, able to grow at the lower Fe concentration, displays a more efficient virus proliferation (despite the low Fv/Fm at the start of infection).
Noteworthy, the higher Fe concentration needed to allow for sustainable growth of M. pusilla under Fe-limitation did not prevent the loss of infectivity of the virus progeny. Infectivity of the Fe-limited MpV lysate was reduced to 30±7%, while in contrast PgV did not show decreased infectivity for Fe-limited hosts.
Our results signify that a stronger Fe-stress experienced by the Fe-limited M.
pusilla (expressed in reduced growth rate) is more likely responsible for the production of impaired virus progeny than a changed photosynthetic capacity (Fv/Fm 0.2 for P. globosa compared to 0.6 for M. pusilla). The fact that total virus abundance, as measured after staining with a nucleic acid dye, was higher than the infective abundance indicates that ( in Fv/Fm for the VR-and VL-infected cultures were largely comparable ( Figure   1D and 2D). However, the Fe-treatment history of the virus (VL or VR) did matter in combination with Fe-limited P. globosa cells, i.e. infection with VR did delay the decline in Fv/Fm with more than a day ( Figure 1C). Infection with a VR lysate is analogous to a relief in Fe-limitation at the time of infection. When the Fe-limited P. globosa cultures were infected with a VR lysate, they were effectively spiked with an Fe increase of ~10% relative to Fe-replete conditions (0.9 µM). The 100-to 300-fold increase of Fe with the addition of an Fe-replete lysate (0.9 µM vs. 1-3 nM) takes the culture Fe concentration well out of limitation ranges which are generally considered to be in nano-to picomolar ranges (de Baar et al. 1990;Martin et al. 1990;Brand 1991 pusilla is more capable of mobilizing the Fe added for viral production. Utilization of the limiting macronutrient P when added post infection was also found to stimulate virus production of M. pusilla (Maat et al., 2016). Although our results indicate that an infected host is capable of mobilizing the limiting Fe for viral production upon addition post infection, it did not lead to a complete recovery of virus production as compared to Fe-replete conditions (around 50% of the replete treatment; Table 2). Mp limited + VL 20 ± 0 24 ± 5.2 Mp limited + VR 46 ± 2 54 ± 0.5  In conclusion, viral infection of both phytoplankton species is distinctly influenced by Fe-limitation. The differences in sensitivity of the host to Felimitation subsequently affected the progeny virus growth properties.
Phaeocystis and Micromonas occur in Fe-limited and Fe-replete conditions alike (Not et al. 2004(Not et al. , 2005Schoemann et al., 2005). Viral lysis has been shown to be an important mortality term for P. globosa under natural conditions, and also M. pusilla is readily infected (Cottrell et al., 1995;Brussaard, 2004a;Brussaard et al., 2005;Baudoux et al., 2006;Martínez Martínez et al., 2015). Further experimental and in-situ study of phytoplankton host-virus dynamics under Fe-limitation is essential to elucidate the level of response specificity, but also the effects on the viral shunt in terms of nutrient cycling in general as well as Fe speciation.