Cleaning of equipment and preparation of chemicals took place in a class 100 ultra-clean laboratory environment (Interflow). Culture handling and sampling were carried out in a 15°C climate chamber within the ultra-clean laboratory. When outside the ultra-clean environment, sample handling took place inside class 100 laminar flow hoods (Interflow). All rinsing and chemical dissolution and dilution was done using 18 MΩ deionized Milli-Q water (Merck Millipore), further referred to as MQ.

Prior to use, general labware of high- and low-density polyethylene (HDPE and LDPE, respectively) and fluorinated ethylene propylene (FEP) was cleaned thoroughly by pre-rinsing (5x) with MQ, followed by soaking in 6 M hydrochloric acid (HCl) for a minimum of 24 h, and a final thorough (5x) MQ rinsing. A sub-boiling quartz distillation apparatus (Savillex) was used to purify nitric acid (HNO₃) in triplicate, yielding Fe free quartz distilled (3QD) HNO₃. A 0.3 M 3QD-HNO₃ solution is used to fill stored bottles.

The polycarbonate (PC) culture flasks (50–500 mL, VWR) and bottles (1–2 L, Nalgene) were acid cleaned with 1 M HCl for a minimum of 24 h, after which they were rinsed with MQ (5x). Finally, bottles were sterilized with a 10% volume of boiling MQ, i.e., the bottle with MQ was microwaved at 900 W to boiling point and left boiling for ~20 s. After vigorous shaking the hot MQ was poured out over the inverted lid. Culture vessels were then left to air-dry in a laminar flow bench for at least 2 h.

Axenic cultures of the nanoeukaryotic Prymnesiophyte P. globosa G(A) (culture collection of the University of Groningen, the Netherlands) and the picoeukaryotic Prasinophyte M. pusilla LAC38 (Marine Research Center culture collection, Göteborg University, Sweden) were maintained under trace metal clean conditions.

A low Fe-containing medium (Low Trace: LT) based on natural seawater (DFe 0.2 nM, collected west of the Bay of Biscay, Atlantic Ocean after Rijkenberg et al., 2012) was designed without metal chelators such as EDTA. Chelators as EDTA are used as metal buffers in culture media, and are added in 10⁻⁶–10⁻³ M concentrations to ascertain that those transition metals added as micronutrients, e.g., Cu, Co, Fe, Mn, Mo, Ni, V, Zn, are kept in the dissolved phase through complexation by EDTA (Sunda and Huntsman, 1995). However, these high concentrations of artificial ligands completely overrule the natural chemistry of the metals, making the study of natural Fe binding organic ligands impossible (Gerringa et al., 2000). The medium was enriched with the macronutrients NaNO₃ (Sigma-Aldrich) and Na₂HPO₄ (Merck Millipore) to final concentrations of 128 and 8 μM, respectively. The following micronutrients were added: KBr (7.1 μM final concentration), NaF (3.0 μM), CaCl₂·2H₂O, (17.9 μM), SrCl₂·6H₂O (0.8 μM), MgCl₂·6H₂O (2.3 μM) and Na₂SeO₃ (10.0 nM). Vitamins H, B1, and B12 were added to final concentrations of 2.0, 296.0, and 0.4 nM, respectively. A combined Tris(hydroxymethyl) aminomethane (Tris) and HCl buffer was added to a 4.0 μM final concentration. Nutrient and buffer stock solutions were cleaned of trace metal contaminations by equilibration with MnO₂ after van den Berg and Kramer (1979). The MnO₂ was removed using a trace metal clean 47 mm diameter 0.2 μm pore size PC filter (Whatman) in a polysulfone (PSU) filter tower (Nalgene) with an electric vacuum pump (Merck Millipore). This cleaning process was performed twice. To maintain constant growth for multiple generations addition of additional trace metals proved essential. These trace metal additions were kept to a minimum in order to avoid influencing Fe speciation, e.g., interactions between Cu and Fe (González et al., 2016). P. globosa received an additional trace solution containing final concentrations of 2.0 nM ZnSO₄·7H₂O, 1.0 nM CoCl₂·6H₂O, 4.6 nM MnCl₂·4H₂O, and 0.6 nM Na₂MoO₄·2H₂O. M. pusilla showed poor physiological condition and growth using this trace solution. Because it was impossible to maintain as steady state under these conditions, a slightly different trace solution was used, containing final concentrations of 4.0 nM ZnSO₄·7H₂O, 5.0 nM CoCl₂·6H₂O, 1.0 nM CuSO₄·5H₂O, 1.0 nM NiSO₄·6H₂O, 1.0 nM Na₃VO₄, 1.0 nM K₂CrO₄, 9.1 nM MnCl₂·4H₂O, 4.1 nM Na₂MoO₄·2H₂O, and 1.0 nM H₂SeO₃. Fe was added from an acidified 3 μM FeCl stock solution made using a 1000 mg L⁻¹ ICP stock (Fluka, Sigma-Aldrich). The Fe-limiting medium contained final concentrations of 1.0 and 3.0 nM Fe for P. globosa and M. pusilla, respectively. In comparison, the Fe-replete (control) medium contained 9.0 μM FeCl for both species.

Culture temperature was 15°C and irradiance was supplied at 90 μmol quanta m⁻² s⁻¹ under a 16:8 h light:dark cycle. Phytoplankton cultures were maintained semi-continuously to obtain and sustain constant and comparable physiology and growth, i.e., diluting the culture daily with new medium whereby the exact volume was determined by the maximum growth rate possible under Fe-limiting culture conditions without wash-out (Maat et al., 2014). The limiting Fe concentration determined the maximum cell abundance, which was determined before and after dilution using flow cytometry (Marie et al., 1999). At steady state, i.e., after at least 8 volume changes and consistent phytoplankton counts (2.1 ± 0.4 × 10⁶ and 2.1 ± 0.7 × 10⁶ 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.

Axenic viral lysate of the double-stranded DNA viruses PgV-07T (Baudoux and Brussaard, 2005) infecting P. globosa G(A) and MpV-08T (Martínez et al., 2014) infecting M. pusilla LAC38 were obtained by 10% v/v inoculation to exponentially growing phytoplankton host and checked for full lysis by flow cytometry (FCM). Fe-limited lysates of both phytoplankton viruses were initiated by 1% v/v inoculations with Fe-replete lysates, after which a minimum of 5 subsequent 10% v/v inoculations followed before use for the experiment. This way the Fe concentration in the Fe-limited lysate was similar to the Fe concentration in the Fe-limited host culture. The number of infective phytoplankton viruses was determined using the most probable number (MPN) endpoint dilution assay according to Suttle (1993). MPN data was analyzed using the University of British Columbia Computer Science department's Assay Analyser software program (Passmore et al., 2000).

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 endeavored 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.

The moment viruses were added, cultures were maintained in-batch. We examined the effect of frequent handling by taking along a control subculture per Fe-limited treatment that was only gently mixed once a day. Samples for phytoplankton and virus abundance as well as photosynthetic capacity (F_v/F_m) were taken every 4 h until full lysis of the cultures (48 h for P. globosa and 96 h for M. pusilla). Phytoplankton abundance and F_v/F_m were determined directly upon sampling, while viral abundance samples were fixed with glutaraldehyde (EM-grade, 0.5% final concentration), flash frozen in liquid nitrogen and stored at −80°C (Brussaard et al., 2010).

High Performance Liquid Chromatography (HPLC) pigment analysis was performed on steady state phytoplankton samples after Zapata et al. (2000). Phytoplankton cells in fresh samples were discriminated and counted based on Chlorophyll-a red autofluorescence using a FACSCanto flow cytometer (Becton Dickinson) equipped with a 17 mW 633 nm HeNe red laser. Viral abundances were also determined by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson) equipped with a 15 mW 488 nm argon-ion blue laser triggered on green fluorescence, following the protocol by Brussaard et al. (2010). In short, samples were diluted 200–1000-fold using a 2 M Tris-HCl buffer at pH 8 and viruses were stained using the nucleic acid-specific fluorescent dye SYBR Green I (Molecular Probes®, Life Technologies, Thermo Fisher). Raw data were analyzed using Cytowin (Vaulot, 1989; Version 4.31 available at http://application.sb-roscoff.fr/Phyto/index.php), whereby PgV and MpV were easily discriminated by plotting green nucleic acid-specific fluorescence vs. side scatter (Baudoux and Brussaard, 2005; Martínez et al., 2015).

The number of infective phytoplankton viruses was determined using the 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 ~10⁶ 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 post-infection was used to calculate the number of infectious viruses. Dividing this number of infectious by the total number of PgV or MpV provided the % infectious viruses.

Photosynthetic capacity (F_v/F_m) measurements were performed using a Chlorophyll Fluorometer with a red emitter-detector unit (Water-PAM, Waltz). Samples were kept in the dark for 30 min at culturing temperature, after which chlorophyll autofluorescence was measured in duplicate in the dark adapted state (F₀) and after a saturation pulse of 2.5 s (F_m). F_v is defined as the difference between F_m and F₀ (Genty et al., 1989). Concentrations of dissolved inorganic macronutrients nitrate and orthophosphate were verified colorimetrically using a trAAcs 800 auto analyser (Murphy and Riley, 1962; Grasshoff, 1983), and were non-limiting at all times (>128 μM nitrate and >8 μM phosphate). Verification of the dissolved Fe concentration was done using flow injection analysis after Klunder et al. (2011), the detection limit of this method was 0.01 nM.

Exponential growth rate (μ_max) for both Fe-limited and Fe-replete P. globosa and Fe-replete M. pusilla was 0.99 ± 0.11 d⁻¹. M. pusilla showed, however, reduced growth under Fe-limitation (0.63 ± 0.07 d⁻¹). Initial difficulties encountered with consistent semi-continuous culturing of M. pusilla required us to increase the Fe concentration to 3 nM as compared to the 1 nM for P. globosa. Still, the lower steady state μ_max found for M. pusilla under Fe-limitation suggests a less efficient Fe-uptake or utilization of M. pusilla as compared to P. globosa. However, the photosynthetic capacity (F_v/F_m) of the Fe-limited M. pusilla remained high around 0.6. Thus, the smaller-sized M. pusilla requires more Fe to grow, albeit at a lower μ_max, while it is capable of retaining F_v/F_m at a value similar to Fe-replete conditions. Reduced F_v/F_m and growth rate under Fe-limitation has been reported for a small diatom species (Timmermans et al., 2001a); however, both variables were reduced for the same species and not one or the other as found in present study. Our result do not support the earlier reports that small phytoplankton (diatoms and cyanobacteria) are growing better under Fe-limitation than larger phytoplankton (Price et al., 1994; Timmermans et al., 2001b, 2004). Whether the differences are due to species-specific or phytoplankton group related responses is currently unclear.

The cellular Chlorophyll-a concentration at steady state was lower under Fe-limitation compared to the Fe-replete control cultures for both algal species (Tables 1, 2). For M. pusilla the reduction was larger, i.e., Chlorophyll-a concentration was only 40% of Fe-replete concentration, compared to 63% for P. globosa. In the Fe-limited P. globosa cultures, cellular Chlorophyll-c concentration was reduced (to a similar extent; Table 1), whereas for M. pusilla the Chlorophyll-b concentration also decreased compared to Fe-replete (Table 2). Furthermore, Fe-limitation led to a different distribution of light-harvesting xanthophylls in P. globosa (Table 1). Cellular 19'-hexafucoxanthin concentration was found 5-fold higher in the Fe-limited cultures as compared to Fe-replete P. globosa (4.8 × 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 × 10⁻¹¹ g × cell ⁻¹, respectively) and the diatoxanthin concentration is 181% of Fe-replete (0.08 and 0.07 × 10⁻¹¹ g × cell⁻¹, respectively). Fe-limited M. pusilla cultures (Table 2) had lower cellular Chlorophyll-b and light-harvesting xanthophyll concentrations. Relative to the Chlorophyll-a concentration, photoprotective xanthophyll derivatives antheraxanthin, zeaxanthin, and lutein were present in higher amounts. Only violaxanthin remains lower both in absolute and relative terms.

Overall, the pigment analysis shows that for both phytoplankton species the photosystem shifted toward a more photoprotective character under Fe-limited conditions. The lower concentrations of chlorophyll and most light-harvesting xanthophylls furthermore indicated that the Fe-limited cells suffered a lower light-harvesting capacity. Fe is essential for all life and earlier studies showed that Fe-deficiency can lead to anemia in mammals, the dysfunction of Fe-dependent enzymes in yeast, the reduction of the amount of electron-transferring complexes and induction of chlorosis in plants, and a decrease in Fe-intensive light harvesting pigment synthesis and increased photoprotective pigments in phytoplankton (Geider et al., 1993; Mengel, 1994; van Leeuwe and Stefels, 2007; van de Poll et al., 2009; Zhang, 2014). In line with the fact that the Fe concentrations in the Fe-limited cultures were always below the limit of detection, these results confirm that both phytoplankton cultures were indeed Fe-limited, despite that M. pusilla showed healthy F_v/F_m. The more pronounced shift to photoprotective pigment production relative to light-harvesting Chlorophyll-a in M. pusilla compared to P. globosa is in agreement with the strong decline in steady state M. pusilla cell abundance with Fe-limitation while F_v/F_m was unaffected. However, M. pusilla was unable to grow at 1 nM Fe while P. globosa grew well. Our results imply that P. globosa was more affected energetically by Fe-limitation, while M. pusilla suffered instead in overall cellular production.

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, 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 shown). The F_v/F_m of the uninfected Fe-replete phytoplankton cultures remained constant (Figures 1C,D, 2C,D), whereby the small variations observed in the Fe-replete P. globosa cultures relate to the light:dark cycle (Figure 1D). The Fe-limited non-infected control cultures showed a decline in F_v/F_m over time as a result of the Fe deprivation, but to a lesser extent than the infected cultures.

When infected with the corresponding Fe-treatment virus (i.e., Fe-limited host with VL and Fe-replete host with VR), Fe-limitation resulted for the infected P. globosa in slightly delayed (about 4 h) and slower cell lysis than for the Fe-replete cultures (full lysis occurring about a day later; Figures 1A,B). F_v/F_m showed similar differences in temporal dynamics between the Fe-limited and Fe-replete infected cultures upon infection with these viruses (Figures 1C,D). At large, the infected M. pusilla cultures showed similar results (Figure 2). The alterations in host physiological condition in the Fe-limited cultures resulted for both phytoplankton species in slower release of virus progeny and reduced virus yield when infected with VL as compared to Fe-replete cultures (Figures 1E,F, 2E,F). The latent period, e.g., the time period to extracellular release of newly produced viruses, was however unaffected by Fe-limitation of the host. Experiments with the same strain of M. pusilla did show prolonged latent periods when under phosphorus (P) and nitrogen (N) limitation (Maat et al., 2014; Maat and Brussaard, 2016). These were suggested to be due to reduced substrate and host energy availability resulting from impaired photophysiology under major nutrient limitation. In our study Chlorophyll-a concentration was comparably reduced but F_v/F_m of Fe-limited steady state M. pusilla was not impaired. Then again, F_v/F_m of P. globosa cells prior to infection was reduced compared to Fe-replete, still not prolonging the latent period. We cannot be sure of the exact underlying mechanism from the here presented data, but the proliferation of DNA viruses is directly dependent on Fe due to the essential role Fe plays in the catalytic center of ribonucleotide reductase, produced early in infection to support dNTPs production needed for viral DNA synthesis (Romeo et al., 2001). As such, it can be speculated that not the time to produce a virus is affected but instead the number of viruses that can be produced before cell lysis occurs. The burst size of MpV and PgV indeed decreased strongly under Fe-limitation, i.e., to 24 and 29% of the burst size produced under Fe-replete conditions (20 and 196 viruses lysed host cell⁻¹; 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 F_v/F_m 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 (F_v/F_m 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 (i) virus particles and (ii) viral nucleic acids were produced. Still, impaired capsid proteins or host receptors may explain a loss of infectivity. Alternatively, processes known to be Fe-intensive are DNA replication and repair (Netz et al., 2012; Zhang, 2014). Failure of DNA repair could indeed explain the result of reduced percentage infective viruses. Future research should not only focus on the causal aspects per se but also study what causes the dissimilarities in loss of infectivity between the algal viruses since different responses will directly regulate community composition. Furthermore, future studies should screen if and how other marine taxa and different viruses are affected by virus proliferation under Fe-limitation. For example, non-marine tailed bacteriophages have been found to contain iron ions in the tail proteins that can utilize the siderophore-bound iron receptors on the host cell membrane for attachment of the phage and subsequent infection of the bacterial host (Bartual et al., 2010). It is likely that similar interactions also exists for marine bacteriophages (Bonnain et al., 2016). Still open questions are whether such phages will obtain fewer iron ions in their tail under Fe-deprivation, and if this will negatively affect their infectivity. Furthermore, the recently proposed “Ferrojan Horse Hypothesis” by Bonnain et al. (2016) posits that introduction of phage-attached Fe may aid the host. More study is needed to test this theory and its potential interference with siderophore-specific uptake mechanisms and effect on Fe cycling.

For all Fe-replete cultures (i.e., high Fe concentration and consequently high F_v/F_m of around 0.6) and the Fe-limited M. pusilla the lysis dynamics and decline in F_v/F_m for the VR- and VL-infected cultures were largely comparable (Figures 1D, 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 F_v/F_m 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 100–300-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). The concentration increase in our experiment is comparatively drastic to assure lifting of Fe-limitation to levels nearer normal replete cultures. This cross-inoculation provides insight in the potential effects of sudden introduction of Fe, e.g., with (seasonal) dust deposition or terrestrial runoff. The effect of this spike with Fe was apparently enough to prevent instant loss of photosynthetic capacity in the Fe-limited P. globosa cells. For both phytoplankton species, the improved physiological condition upon infection of Fe-limited host with VR virus resulted in enhanced viral production compared to Fe-limited infected with VL virus (by 1.6 and 2.3-fold for PgV and MpV, respectively; Table 3). The effect on burst size is thus stronger for the more Fe-sensitive M. pusilla, indicating that M. 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).

In conclusion, viral infection of both phytoplankton species is distinctly influenced by Fe-limitation. The differences in sensitivity of the host to Fe-limitation subsequently affected the progeny virus growth properties. Phaeocystis and Micromonas occur in Fe-limited and Fe-replete conditions alike (Not et al., 2004, 2005; Schoemann 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 and Suttle, 1995; Brussaard, 2004; Brussaard et al., 2005; Baudoux et al., 2006; Martínez et al., 2015). Virus burst sizes became strongly reduced (on average by 70%) under Fe-limiting relative to Fe-replete conditions. Although, addition of Fe at the time of infection was utilized by the infected Fe-limited host to increase virus production, it was not to the level found under Fe-replete conditions. Thus, Fe-limitation irrevocably interfered with viral productivity. Additionally, Fe-limited M. pusilla demonstrated an evident effect on the quality of the viruses produced as only 30% were infective. The lowered virus infectivity and/or virus yield impair viral control of the specific host species under Fe-limitation. However, at the same time, reduced viral lysis may affect the productivity of remaining non-infected cells and of other phytoplankton species, because viral lysis is considered a driver of Fe cycling by releasing ligand-bound dissolved Fe-species back into the dissolved organic matter pool (Gobler et al., 1997; Poorvin et al., 2004, 2011). What relative contributions different ligands have, and in how far their release is facilitated or impaired by viral lysis requires further study. 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.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.