Correction: Individual-based model highlights the importance of trade-offs for virus-host population dynamics and long-term co-existence.

[This corrects the article DOI: 10.1371/journal.pcbi.1010228.].

Molecular evidence of parallel evolution in a cyanophage.

Antagonistic interactions between bacteriophage (phage) and its bacterial host drives the continual selection for resistance and counter-defence. To date, much remains unknown about the genomic evolution that occurs as part of the underlying mechanisms. Such is the case for the marine cyanobacteria Synechococcus and viruses (cyanophages) that infect them. Here, we monitored host and phage abundances, alongside genomic changes to the phage populations, in a 500-day (~55 bacterial generations) infection experiment between Synechococcus sp. WH7803 and the T4-type cyanophage S-PM2d, run parallel in three replicate chemostats (plus one control chemostat). Flow cytometric count of total abundances revealed relatively similar host-phage population dynamics across the chemostats, starting with a cycle of host population collapse and recovery that led to phases of host-phage coexistence. Whole-genome analysis of the S-PM2d populations detected an assemblage of strongly selected and repeatable genomic changes, and therefore parallel evolution in the phage populations, early in the experiment (sampled on day 39). These consisted mostly of non-synonymous single-nucleotide-polymorphisms and a few instances of indel, altogether affecting 18 open-reading-frames, the majority of which were predicted to encode virion structures including those involved in phage adsorption onto host (i.e., baseplate wedge, short tail fibre, adhesin component). Mutations that emerged later (sampled on day 500), on the other hand, were found at a larger range of frequencies, with many lacking repeatability across the chemostats. This is indicative of some degree of between-population divergence in the phage evolutionary trajectory over time. A few of the early and late mutations were detected within putative auxiliary metabolic genes, but these generally occurred in only one or two of the chemostats. Less repeatable mutations may have higher fitness costs, thus drawing our attention onto the role of trade-offs in modulating the trajectory of a host-phage coevolution.

Differential toxicity of bioorthogonal non-canonical amino acids (BONCAT) in Escherichia coli.

Single-cell methods allow studying the activity of single bacterial cells, potentially shedding light on regulatory mechanisms involved in services like biochemical cycling. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising method for studying bacterial activity in natural communities, using the methionine analogues L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG) to track protein production in single cells. Both AHA and HPG have been deemed non-toxic, but recent findings suggest that HPG affects bacterial metabolism. In this study we examined the effect of AHA and HPG on Escherichia coli with respect to acute toxicity and growth. E. coli exposed to 5.6-90 µM HPG showed no growth, and the growth rate was significantly reduced at >0.35 µM HPG, compared to the HPG-free control. In contrast, E. coli showed growth at concentrations up to 9 mM AHA. In assays where AHA or HPG were added during the exponential growth phase, the growth sustained but the growth rate was immediately reduced at the highest concentrations (90 µM HPG and 10 mM AHA). Prolonged incubations (20h) with apparently non-toxic concentrations suggest that the cells incorporating NCAAs fail to divide and do not contribute to the next generation resulting in the relative abundance of labelled cells to decrease over time. These results show that HPG and AHA have different impact on the growth of E. coli. Both concentration and incubation time affect the results and need to be considered when designing BONCAT experiments and evaluating results. Time course incubations are suggested as a possible way to obtain more reliable results.

Individual-based model highlights the importance of trade-offs for virus-host population dynamics and long-term co-existence.

Viruses play diverse and important roles in ecosystems. In recent years, trade-offs between host and virus traits have gained increasing attention in viral ecology and evolution. However, microbial organism traits, and viral population parameters in particular, are challenging to monitor. Mathematical and individual-based models are useful tools for predicting virus-host dynamics. We have developed an individual-based evolutionary model to study ecological interactions and evolution between bacteria and viruses, with emphasis on the impacts of trade-offs between competitive and defensive host traits on bacteria-phage population dynamics and trait diversification. Host dynamics are validated with lab results for different initial virus to host ratios (VHR). We show that trade-off based, as opposed to random bacteria-virus interactions, result in biologically plausible evolutionary outcomes, thus highlighting the importance of trade-offs in shaping biodiversity. The effects of nutrient concentration and other environmental and organismal parameters on the virus-host dynamics are also investigated. Despite its simplicity, our model serves as a powerful tool to study bacteria-phage interactions and mechanisms for evolutionary diversification under various environmental conditions.

Adaptive evolution of viruses infecting marine microalgae (haptophytes), from acute infections to stable coexistence.

Collectively known as phytoplankton, photosynthetic microbes form the base of the marine food web, and account for up to half of the primary production on Earth. Haptophytes are key components of this phytoplankton community, playing important roles both as primary producers and as mixotrophs that graze on bacteria and protists. Viruses influence the ecology and diversity of phytoplankton in the ocean, with the majority of microalgae-virus interactions described as 'boom and bust' dynamics, which are characteristic of acute virus-host systems. Most haptophytes are, however, part of highly diverse communities and occur at low densities, decreasing their chance of being infected by viruses with high host specificity. Viruses infecting these microalgae have been isolated in the laboratory, and there are several characteristics that distinguish them from acute viruses infecting bloom-forming haptophytes. Herein we synthesise what is known of viruses infecting haptophyte hosts in the ocean, discuss the adaptive evolution of haptophyte-infecting viruses -from those that cause acute infections to those that stably coexist with their host - and identify traits of importance for successful survival in the ocean.

Temperate infection in a virus-host system previously known for virulent dynamics.

The blooming cosmopolitan coccolithophore Emiliania huxleyi and its viruses (EhVs) are a model for density-dependent virulent dynamics. EhVs commonly exhibit rapid viral reproduction and drive host death in high-density laboratory cultures and mesocosms that simulate blooms. Here we show that this system exhibits physiology-dependent temperate dynamics at environmentally relevant E. huxleyi host densities rather than virulent dynamics, with viruses switching from a long-term non-lethal temperate phase in healthy hosts to a lethal lytic stage as host cells become physiologically stressed. Using this system as a model for temperate infection dynamics, we present a template to diagnose temperate infection in other virus-host systems by integrating experimental, theoretical, and environmental approaches. Finding temperate dynamics in such an established virulent host-virus model system indicates that temperateness may be more pervasive than previously considered, and that the role of viruses in bloom formation and decline may be governed by host physiology rather than by host-virus densities.

Host-virus-predator coexistence in a grey-box model with dynamic optimization of host fitness.

Lytic viruses are believed to affect both flow patterns and host diversity in microbial food webs. Models resolving host and virus communities into subgroups can represent both aspects. However, when flow pattern is the prime interest, such models may seem unnecessary complex. This has led to proposals of black-box models using only total community sizes as state variables. This simplification creates a coexistence problem, however, since predator and virus communities then compete for the same, shared, prey = host community. Mathematically, this problem can be solved by introducing feedbacks allowing community-level properties to adapt. The different mathematical alternatives for such feedback represent different ecological assumptions and thus different hypotheses for how the balance between predators and viruses is controlled in nature. We here explore a model where the feedback works through an increase in host community resistance in response to high virus abundances, thereby reducing virus production. We use a dynamic "strategy" index S to describe the balance between defensive and competitive abilities in the host community, and assume the rate of change in S to be proportional to the local slope of the per capita fitness gradient for the host. We explore how such a "grey-box" model can allow stable coexistence of viruses and predators, and how equilibrium food web structure, virus-to-host ratio, and partitioning of host production varies; both as functions of host community traits, and as functions of external bottom-up and top-down drivers.

Simple models combining competition, defence and resource availability have broad implications in pelagic microbial food webs.

In food webs, interactions between competition and defence control the partitioning of limiting resources. As a result, simple models of these interactions contain links between biogeochemistry, diversity, food web structure and ecosystem function. Working at hierarchical levels, these mechanisms also produce self-similarity and therefore suggest how complexity can be generated from repeated application of simple underlying principles. Reviewing theoretical and experimental literature relevant to the marine photic zone, we argue that there is a wide spectrum of phenomena, including single cell activity of prokaryotes, microbial biodiversity at different levels of resolution, ecosystem functioning, regional biogeochemical features and evolution at different timescales; that all can be understood as variations over a common principle, summarised in what has been termed the 'Killing-the-Winner' (KtW) motif. Considering food webs as assemblages of such motifs may thus allow for a more integrated approach to aquatic microbial ecology.

The Response of Heterotrophic Prokaryote and Viral Communities to Labile Organic Carbon Inputs Is Controlled by the Predator Food Chain Structure.

Factors controlling the community composition of marine heterotrophic prokaryotes include organic-C, mineral nutrients, predation, and viral lysis. Two mesocosm experiments, performed at an Arctic location and bottom-up manipulated with organic-C, had very different results in community composition for both prokaryotes and viruses. Previously, we showed how a simple mathematical model could reproduce food web level dynamics observed in these mesocosms, demonstrating strong top-down control through the predator chain from copepods via ciliates and heterotrophic nanoflagellates. Here, we use a steady-state analysis to connect ciliate biomass to bacterial carbon demand. This gives a coupling of top-down and bottom-up factors whereby low initial densities of ciliates are associated with mineral nutrient-limited heterotrophic prokaryotes that do not respond to external supply of labile organic-C. In contrast, high initial densities of ciliates give carbon-limited growth and high responsiveness to organic-C. The differences observed in ciliate abundance, and in prokaryote abundance and community composition in the two experiments were in accordance with these predictions. Responsiveness in the viral community followed a pattern similar to that of prokaryotes. Our study provides a unique link between the structure of the predator chain in the microbial food web and viral abundance and diversity.

Linking internal and external bacterial community control gives mechanistic framework for pelagic virus-to-bacteria ratios.

For more than 25 years, virus-to-bacteria ratios (VBR) have been measured and interpreted as indicators of the importance of viruses in aquatic ecosystems, yet a generally accepted theory for understanding mechanisms controlling VBR is still lacking. Assuming that the denominator (total bacterial abundance) is primarily predator controlled, while viral lysis compensates for host growth rates exceeding this grazing loss, the numerator (viral abundance) reflects activity differences between prokaryotic hosts. VBR is then a ratio between mechanisms generating structure within the bacterial community and interactions between different plankton functional types controlling bacterial community size. We here show how these arguments can be formalized by combining a recently published model for co-evolutionary host-virus interactions, with a previously published "minimum" model for the microbial food web. The result is a framework where viral lysis links bacterial diversity to microbial food web structure and function, creating relationships between different levels of organization that are strongly modified by organism-level properties such as cost of resistance.

Defining Planktonic Protist Functional Groups on Mechanisms for Energy and Nutrient Acquisition: Incorporation of Diverse Mixotrophic Strategies.

Arranging organisms into functional groups aids ecological research by grouping organisms (irrespective of phylogenetic origin) that interact with environmental factors in similar ways. Planktonic protists traditionally have been split between photoautotrophic "phytoplankton" and phagotrophic "microzooplankton". However, there is a growing recognition of the importance of mixotrophy in euphotic aquatic systems, where many protists often combine photoautotrophic and phagotrophic modes of nutrition. Such organisms do not align with the traditional dichotomy of phytoplankton and microzooplankton. To reflect this understanding, we propose a new functional grouping of planktonic protists in an eco-physiological context: (i) phagoheterotrophs lacking phototrophic capacity, (ii) photoautotrophs lacking phagotrophic capacity, (iii) constitutive mixotrophs (CMs) as phagotrophs with an inherent capacity for phototrophy, and (iv) non-constitutive mixotrophs (NCMs) that acquire their phototrophic capacity by ingesting specific (SNCM) or general non-specific (GNCM) prey. For the first time, we incorporate these functional groups within a foodweb structure and show, using model outputs, that there is scope for significant changes in trophic dynamics depending on the protist functional type description. Accordingly, to better reflect the role of mixotrophy, we recommend that as important tools for explanatory and predictive research, aquatic food-web and biogeochemical models need to redefine the protist groups within their frameworks.

Fractal Hypothesis of the Pelagic Microbial Ecosystem-Can Simple Ecological Principles Lead to Self-Similar Complexity in the Pelagic Microbial Food Web?

Trophic interactions are highly complex and modern sequencing techniques reveal enormous biodiversity across multiple scales in marine microbial communities. Within the chemically and physically relatively homogeneous pelagic environment, this calls for an explanation beyond spatial and temporal heterogeneity. Based on observations of simple parasite-host and predator-prey interactions occurring at different trophic levels and levels of phylogenetic resolution, we present a theoretical perspective on this enormous biodiversity, discussing in particular self-similar aspects of pelagic microbial food web organization. Fractal methods have been used to describe a variety of natural phenomena, with studies of habitat structures being an application in ecology. In contrast to mathematical fractals where pattern generating rules are readily known, however, identifying mechanisms that lead to natural fractals is not straight-forward. Here we put forward the hypothesis that trophic interactions between pelagic microbes may be organized in a fractal-like manner, with the emergent network resembling the structure of the Sierpinski triangle. We discuss a mechanism that could be underlying the formation of repeated patterns at different trophic levels and discuss how this may help understand characteristic biomass size-spectra that hint at scale-invariant properties of the pelagic environment. If the idea of simple underlying principles leading to a fractal-like organization of the pelagic food web could be formalized, this would extend an ecologists mindset on how biological complexity could be accounted for. It may furthermore benefit ecosystem modeling by facilitating adequate model resolution across multiple scales.

What difference does it make if viruses are strain-, rather than species-specific?

Theoretical work has suggested an important role of lytic viruses in controlling the diversity of their prokaryotic hosts. Yet, providing strong experimental or observational support (or refutation) for this has proven evasive. Such models have usually assumed "host groups" to correspond to the "species" level, typically delimited by 16S rRNA gene sequence data. Recent model developments take into account the resolution of species into strains with differences in their susceptibility to viral attack. With strains as the host groups, the models will have explicit viral control of abundance at strain level, combined with explicit predator or resource control at community level, but the direct viral control at species level then disappears. Abundance of a species therefore emerges as the combination of how many strains, and at what abundance, this species can establish in competition with other species from a seeding community. We here discuss how species diversification and strain diversification may introduce competitors and defenders, respectively, and that the balance between the two may be a factor in the control of species diversity in mature natural communities. These models can also give a dominance of individuals from strains with high cost of resistance; suggesting that the high proportion of "dormant" cells among pelagic heterotrophic prokaryotes may reflect their need for expensive defense rather than the lack of suitable growth substrates in their environment.

Optimal defense strategies in an idealized microbial food web under trade-off between competition and defense.

Trophic mechanisms that can generate biodiversity in food webs include bottom-up (growth rate regulating) and top-down (biomass regulating) factors. The top-down control has traditionally been analyzed using the concepts of "Keystone Predation" (KP) and "Killing-the-Winner" (KtW), predominately occuring in discussions of macro- and micro-biological ecology, respectively. Here we combine the classical diamond-shaped food web structure frequently discussed in KP analyses and the KtW concept by introducing a defense strategist capable of partial defense. A formalized description of a trade-off between the defense-strategist's competitive and defensive ability is included. The analysis reveals a complex topology of the steady state solution with strong relationships between food web structure and the combination of trade-off, defense strategy and the system's nutrient content. Among the results is a difference in defense strategies corresponding to maximum biomass, production, or net growth rate of invading individuals. The analysis thus summons awareness that biomass or production, parameters typically measured in field studies to infer success of particular biota, are not directly acted upon by natural selection. Under coexistence with a competition specialist, a balance of competitive and defensive ability of the defense strategist was found to be evolutionarily stable, whereas stronger defense was optimal under increased nutrient levels in the absence of the pure competition specialist. The findings of success of different defense strategies are discussed with respect to SAR11, a highly successful bacterial clade in the pelagic ocean.

A theoretical analysis of how strain-specific viruses can control microbial species diversity.

Pelagic prokaryote communities are often dominated by the SAR11 clade. The recent discovery of viruses infecting this clade led to the suggestion that such dominance could not be explained by assuming SAR11 to be a defense specialist and that the explanation therefore should be sought in its competitive abilities. The issue is complicated by the fact that prokaryotes may develop strains differing in their balance between competition and viral defense, a situation not really captured by present idealized models that operate only with virus-controlled "host groups." We here develop a theoretical framework where abundance within species emerges as the sum over virus-controlled strains and show that high abundance then is likely to occur for species able to use defense mechanisms with a low trade-off between competition and defense, rather than by extreme investment in one strategy or the other. The J-shaped activity-abundance community distribution derived from this analysis explains the high proportion low-active prokaryotes as a consequence of extreme defense as an alternative to explanations based on dormancy or death due to nutrient starvation.

SAR11 viruses and defensive host strains.

Arising from Y. Zhao et al. Nature 494, 357­360 (2013). The recent findings of abundant SAR11 viruses by Zhao et al. are intriguing, and add new insight into the on-going discussion of why SAR11 bacteria are highly successful in the pelagic ocean. On the basis of high SAR11 virus abundance, Zhao et al. claim that SAR11 bacteria are competition specialists. Alternatively, we show here how their findings could be consistent with a dominance of defensive SAR11 strains. Considering their high abundance, understanding why SAR11 bacteria are so successful has important implications for the study of the pelagic ecosystem. There is a Reply to this Brief Communication Arising by Giovannoni, S., Temperton, B. & Zhao, Y. Nature 499, http://dx.doi.org/10.1038/nature12388 (2013).

Adding a cost of resistance description extends the ability of virus-host model to explain observed patterns in structure and function of pelagic microbial communities.

By adding a generic description of cost of resistance (COR) to the existing 'killing-the-winner' model, we show how this expands the model's explanatory power to include rank-abundance relationships in the host population. The model can predict a counter-intuitive relationship previously suggested in the literature, where abundant viruses are associated with rare hosts and vice versa. The model explains the observed dominance of slow-growing prokaryotes as the result of successful defence strategies, rather than as dormancy of hosts lacking essential substrates. In addition to these important conceptual aspects, the model is able to reproduce realistic values for virus : host ratios and partitioning of bacterial production between predatory loss and viral lysis. A high COR is also shown to increase the community's richness and Shannon diversity index. This model thus not only couples life strategies at the cellular level with system properties, but it also links the two system level properties of biogeochemical flows and diversity to each other. The model operates with host groups, and consequences for biodiversity when interpreting these groups in terms of species and strains are discussed.