Douwe S. Maat

Elevated CO2 and Phosphate Limitation Favor Micromonas pusilla through Stimulated Growth and Reduced Viral Impact

ABSTRACT Growth and viral infection of the marine picoeukaryote Micromonas pusilla was studied under a future-ocean scenario of elevated partial CO2 (pCO2; 750 μatm versus the present-day 370 μatm) and simultaneous limitation of phosphorus (P). Independent of the pCO2 level, the ratios of M. pusilla cellular carbon (C) to nitrogen (N), C:P and N:P, increased with increasing P stress. Furthermore, in the P-limited chemostats at growth rates of 0.32 and 0.97 of the maximum growth rate (μmax), the supply of elevated pCO2 led to an additional rise in cellular C:N and C:P ratios, as well as a 1.4-fold increase in M. pusilla abundance. Viral lysis was not affected by pCO2, but P limitation led to a 150% prolongation of the latent period (6 to 12 h) and an 80% reduction in viral burst sizes (63 viruses per cell) compared to P-replete conditions (4 to 8 h latent period and burst size of 320). Growth at 0.32 μmax further prolonged the latent period by another 150% (12 to 18 h). Thus, enhanced P stress due to climate change-induced strengthened vertical stratification can be expected to lead to reduced and delayed virus production in picoeukaryotes. This effect is tempered, but likely not counteracted, by the increase in cell abundance under elevated pCO2. Although the influence of potential P-limitation-relieving factors, such as the uptake of organic P and P utilization during infection, is unclear, our current results suggest that when P limitation prevails in future oceans, picoeukaryotes and grazing will be favored over larger-sized phytoplankton and viral lysis, with increased matter and nutrient flow to higher trophic levels.

Springtime dynamics, productivity and activity of prokaryotes in two Arctic fjords

In the Kongsfjorden–Krossfjorden system (Spitsbergen), increasing temperatures enhance glacier melting and concomitant intrusion of freshwater. These altered conditions affect the timing, intensity, and composition of the phytoplankton spring bloom in Kongsfjorden; yet, the effects on prokaryotes (bacteria and archaea) are not well understood. The aim of this study was to examine springtime prokaryote communities in both fjords as a function of hydrographic and phytoplankton variability. Prokaryote community composition was studied in two consecutive years by molecular fingerprinting of the 16S rRNA gene. In addition, we measured bacterial abundance, productivity (3H-Leucine uptake), and single-cell activity using catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography. Differences in bacterial and archaeal communities were found between Kongsfjorden and Krossfjorden. Furthermore, an increase in productivity, abundance, and proportion of active bacterial cells was observed during the course of spring. Bacteroidetes were the most abundant bacterial group among the assessed taxa in both Kongsfjorden and Krossfjorden. Multivariate analysis of the microbial community fingerprints revealed a strong temporal shaping of both the bacterial and archaeal communities in addition to a spatial separation between the two fjords. A significant part of the observed bacterial variation could be explained by cyanobacterial biomass, as deduced from pigment analysis, and by phosphate concentration. Archaea were mainly controlled by abiotic factors. We speculate that the bacterial response to hydrographic changes and glacier meltwater is mediated through shifts in phytoplankton abundance and composition, whereas archaea are directly influenced by abiotic environmental variables.

Combined Phosphorus Limitation and Light Stress Prevent Viral Proliferation in the Phytoplankton Species Phaeocystis globosa, but Not in Micromonas pusilla

Under natural conditions phytoplankton are often simultaneously subjected to phosphorus (P) limitation and suboptimal light levels. Potential interacting effects of P-limitation and light availability on phytoplankton virus-host interactions have thus far not been reported. We studied the influence of three environmentally relevant light levels (low; 25, medium; 100 and high; 250 µmol quanta m-2 s-1) in combination with P-limitation (vs. P-replete conditions) on virus proliferation in the key phytoplankton species Micromonas pusilla and Phaeocystis globosa. Cultures were acclimated to balanced P-limited growth at 3 light levels by semi-continuous culturing, before one-step infection experiments were carried out in batch mode. Under optimal conditions (medium light, P-replete), the latent period (time until first release of progeny viruses) was 6-9 h and 9-12 h, and the burst size (number of viruses released per lysed host cell) was 241±5 and 690±28 for M. pusilla virus MpV and P. globosa virus PgV, respectively. Low light intensity under P-replete conditions prolonged the latent period of PgV (with maximally 3 h). The PgV burst size was 2.8-fold reduced under low light and 2.2-fold reduced under high light. The 10-fold range in light intensity did not affect viral latent period or burst size in P-replete M. pusilla. However, P-limitation (under optimal light) also led to elongated latent periods (with maximally 3 h compared to P-replete) and the viral burst sizes decreased by 2.7-fold for MpV and 3.5-fold for PgV. Finally, infectivity assays showed that PgV progeny from the P-limited high and low light cultures largely lost their infectivity, reducing their infective burst sizes to only 2-4 infective viruses per lysed host cell. Our study demonstrates that the effects of specific light and P-availability on virus-phytoplankton interaction are not only species specific, but can also strengthen each other’s effects. Relatively small differences in environmental conditions with depth, geography or time have the potential to drastically affect viral infection of phytoplankton, with consequent effects on host species composition and biogeochemical fluxes.

Atlantic Advection Driven Changes in Glacial Meltwater: Effects on Phytoplankton Chlorophyll-a and Taxonomic Composition in Kongsfjorden, Spitsbergen

Phytoplankton biomass and composition was investigated in a high Arctic fjord (Kongsfjorden, 79˚N, 11˚40’E) using year round weekly pigment samples collected from October 2013 to December 2014. In addition, phytoplankton dynamics supplemented with physical and chemical characteristics of the 2014 spring bloom (April –June 2014) were assessed in two locations in Kongsfjorden. The goal was to elucidate effects of Atlantic advection on spatial phytoplankton chlorophyll-a (chl-a) and taxonomic composition. Chl-a declined during the polar night to a minimum of 0.01 mg m-3, followed by a 1000-fold increase until May 28. Atlantic advection prevented sea ice formation and increased springtime melting of marine terminating glaciers. This coincided with spatial and temporal differences in abundances of flagellates (prasinophytes, haptophytes, cryptophytes, and chrysophytes) and diatoms in early spring. More flagellated phytoplankton were observed in the non-stratified central Kongsfjorden, whereas diatoms were more abundant in the stratified inner fjord. Contrasting conditions between locations were reduced when glacial melt water stratification expanded towards the mouth of the fjord, mediating a diatom dominated surface bloom at both locations. We suggest that glacial melt water governs spring bloom spatial timing and composition in the absence of sea ice driven stratification. The spring bloom exhausted surface nutrient concentrations by the end of May. The nutrient limited post bloom period (June-October) was characterized by reduced biomass and pigments of flagellated phytoplankton, consisting of prasinophytes, haptophytes, chrysophytes and to a lesser extent cryptophytes and peridinin-containing dinoflagellates.

Virus production in phosphorus-limited Micromonas pusilla stimulated by a supply of naturally low concentrations of different phosphorus sources, far into the lytic cycle

Earlier studies show that the proliferation of phytoplankton viruses can be inhibited by depletion of soluble reactive phosphorus (SRP; orthophosphate). In natural marine waters, phytoplankton phosphorus (P) availability is, however, largely determined by the supply rate of SRP (e.g. through remineralization) and potentially by the source of P as well (i.e. the utilization of soluble non-reactive P; SNP). Here we show how a steady low supply of P (mimicking natural P recycling) to virally infected P-limited Micromonas pusilla stimulates virus proliferation. Independent of the degree of P limitation prior to infection (0.32 and 0.97μmax chemostat cultures), SRP supply resulted in 2-fold higher viral burst sizes (viruses lysed per host cell) as compared with no addition (P starvation). Delaying these spikes during the infection cycle showed that the added SRP was utilized for extra M. pusilla virus (MpV) production far into the lytic cycle (18 h post-infection). Moreover, P-limited M. pusilla utilized several SNP compounds with high efficiency and with the same extent of burst size stimulation as for SRP. Finally, addition of virus-free MpV lysate (representing a complex SNP mixture) to newly infected cells enhanced MpV production, implicating host-associated alkaline phosphatase activity, and highlighting its important role in oligotrophic environments.

Characterization and Temperature Dependence of Arctic Micromonas polaris Viruses

Global climate change-induced warming of the Artic seas is predicted to shift the phytoplankton community towards dominance of smaller-sized species due to global warming. Yet, little is known about their viral mortality agents despite the ecological importance of viruses regulating phytoplankton host dynamics and diversity. Here we report the isolation and basic characterization of four prasinoviruses infectious to the common Arctic picophytoplankter Micromonas. We furthermore assessed how temperature influenced viral infectivity and production. Phylogenetic analysis indicated that the putative double-stranded DNA (dsDNA) Micromonas polaris viruses (MpoVs) are prasinoviruses (Phycodnaviridae) of approximately 120 nm in particle size. One MpoV showed intrinsic differences to the other three viruses, i.e., larger genome size (205 ± 2 vs. 191 ± 3 Kb), broader host range, and longer latent period (39 vs. 18 h). Temperature increase shortened the latent periods (up to 50%), increased the burst size (up to 40%), and affected viral infectivity. However, the variability in response to temperature was high for the different viruses and host strains assessed, likely affecting the Arctic picoeukaryote community structure both in the short term (seasonal cycles) and long term (global warming).