Indirect interactions among co-infecting parasites and a microbial mutualist impact disease progression.

Interactions among parasites and other microbes within hosts can impact disease progression, yet study of such interactions has been mostly limited to pairwise combinations of microbes. Given the diversity of microbes within hosts, indirect interactions among more than two microbial species may also impact disease. To test this hypothesis, we performed inoculation experiments that investigated interactions among two fungal parasites, Rhizoctonia solani and Colletotrichum cereale, and a systemic fungal endophyte, Epichloë coenophiala, within the grass, tall fescue (Lolium arundinaceum). Both direct and indirect interactions impacted disease progression. While the endophyte did not directly influence R. solani disease progression or C. cereale symptom development, the endophyte modified the interaction between the two parasites. The magnitude of the facilitative effect of C. cereale on the growth of R. solani tended to be greater when the endophyte was present. Moreover, this interaction modification strongly affected leaf mortality. For plants lacking the endophyte, parasite co-inoculation did not increase leaf mortality compared to single-parasite inoculations. By contrast, for endophyte-infected plants, parasite co-inoculation increased leaf mortality compared to inoculation with R. solani or C. cereale alone by 1.9 or 4.9 times, respectively. Together, these results show that disease progression can be strongly impacted by indirect interactions among microbial symbionts.

Parasites, niche modification and the host microbiome: A field survey of multiple parasites.

Parasites can affect and be affected by the host's microbiome, with consequences for host susceptibility, parasite transmission, and host and parasite fitness. Yet, two aspects of the relationship between parasite infection and host microbiota remain little understood: the nature of the relationship under field conditions, and how the relationship varies among parasites. To overcome these limitations, we performed a field survey of the within-leaf fungal community in a tall fescue population. We investigated how diversity and composition of the fungal microbiome associate with natural infection by fungal parasites with different feeding strategies. A parasite's feeding strategy affects both parasite requirements of the host environment and parasite impacts on the host environment. We hypothesized that parasites that more strongly modify niches available within a host will be associated with greater changes in microbiome diversity and composition. Parasites with a feeding strategy that creates necrotic tissue to extract resources (necrotrophs) may not only have different niche requirements, but also act as particularly strong niche modifiers. Barcoded amplicon sequencing of the fungal ITS region revealed that leaf segments symptomatic of necrotrophs had lower fungal diversity and distinct composition compared to segments that were asymptomatic or symptomatic of other parasites. There were no clear differences in fungal diversity or composition between leaf segments that were asymptomatic and segments symptomatic of other parasite feeding strategies. Our results motivate future experimental work to test how the relationship between the microbiome and parasite infection is impacted by parasite feeding strategy and highlight the potential importance of parasite traits.

Eutrophication, biodiversity loss, and species invasions modify the relationship between host and parasite richness during host community assembly.

Host and parasite richness are generally positively correlated, but the stability of this relationship in response to global change remains poorly understood. Rapidly changing biotic and abiotic conditions can alter host community assembly, which in turn, can alter parasite transmission. Consequently, if the relationship between host and parasite richness is sensitive to parasite transmission, then changes in host composition under various global change scenarios could strengthen or weaken the relationship between host and parasite richness. To test the hypothesis that host community assembly can alter the relationship between host and parasite richness in response to global change, we experimentally crossed host diversity (biodiversity loss) and resource supply to hosts (eutrophication), then allowed communities to assemble. As previously shown, initial host diversity and resource supply determined the trajectory of host community assembly, altering post-assembly host species richness, richness-independent host phylogenetic diversity, and colonization by exotic host species. Overall, host richness predicted parasite richness, and as predicted, this effect was moderated by exotic abundance-communities dominated by exotic species exhibited a stronger positive relationship between post-assembly host and parasite richness. Ultimately, these results suggest that, by modulating parasite transmission, community assembly can modify the relationship between host and parasite richness. These results thus provide a novel mechanism to explain how global environmental change can generate contingencies in a fundamental ecological relationship-the positive relationship between host and parasite richness.

Herbivores and nutrients control grassland plant diversity via light limitation.

Human alterations to nutrient cycles and herbivore communities are affecting global biodiversity dramatically. Ecological theory predicts these changes should be strongly counteractive: nutrient addition drives plant species loss through intensified competition for light, whereas herbivores prevent competitive exclusion by increasing ground-level light, particularly in productive systems. Here we use experimental data spanning a globally relevant range of conditions to test the hypothesis that herbaceous plant species losses caused by eutrophication may be offset by increased light availability due to herbivory. This experiment, replicated in 40 grasslands on 6 continents, demonstrates that nutrients and herbivores can serve as counteracting forces to control local plant diversity through light limitation, independent of site productivity, soil nitrogen, herbivore type and climate. Nutrient addition consistently reduced local diversity through light limitation, and herbivory rescued diversity at sites where it alleviated light limitation. Thus, species loss from anthropogenic eutrophication can be ameliorated in grasslands where herbivory increases ground-level light.

Past is prologue: host community assembly and the risk of infectious disease over time.

Infectious disease risk is often influenced by host diversity, but the causes are unresolved. Changes in diversity are associated with changes in community structure, particularly during community assembly; therefore, by incorporating change over time, host community assembly may provide a framework to resolve causation. In turn, community assembly can be driven by many processes, including resource enrichment. To test the hypothesis that community assembly causally links host diversity to future disease, we experimentally manipulated host diversity and resource supply to hosts, then allowed communities to assemble for 2 years (surveyed 2012-2014). Initially, host diversity increased disease. Subsequently, host diversity did not directly alter disease. However, host diversity determined the trajectory of host community assembly, altering colonisation by exotic host species and richness-independent host phylogenetic diversity, which together reversed the initial increase in disease. Ultimately, incorporating the temporal dimension of community assembly revealed novel mechanisms linking host diversity to future disease.

A growth-defense trade-off is general across native and exotic grasses.

High-resource environments typically favor quick-growing, poorly defended plants, while resource-poor environments typically favor slow-growing, well-defended plants. The prevailing hypothesis explaining this pattern states that, as resource availability increases, well-defended, slow-growing species are replaced by poorly defended, fast-growing species. A second hypothesis states that greater resource availability increases allocation to growth at the expense of defense, within species. Regardless of mechanism, if exotic species are released from enemies relative to natives, shifts in allocation to growth and defense both within and among species could differ by geographic provenance. To test whether resource availability alters growth or defense, within and among species, and whether any such effects differ between natives and exotics, we manipulated soil nutrient supply and access of aboveground insect herbivores and fungal pathogens under field conditions to individuals of six native and six exotic grass species that co-occurred in a North Carolina old field. The prevailing hypothesis' prediction-that species-level enemy impact increases with species' nutrient responsiveness-was confirmed. Moreover, this relationship did not differ between native and exotic species. The second hypothesis' prediction-that individual-level enemy impact increases with nutrient supply, after accounting for species-level variation in performance-was not supported. Together, these results support the idea, across native and exotic species, that plant species turnover is the primary mechanism underlying effects of nutrient enrichment on allocation to growth and defense in plant communities.

Graptolite community responses to global climate change and the Late Ordovician mass extinction.

Mass extinctions disrupt ecological communities. Although climate changes produce stress in ecological communities, few paleobiological studies have systematically addressed the impact of global climate changes on the fine details of community structure with a view to understanding how changes in community structure presage, or even cause, biodiversity decline during mass extinctions. Based on a novel Bayesian approach to biotope assessment, we present a study of changes in species abundance distribution patterns of macroplanktonic graptolite faunas (∼447-444 Ma) leading into the Late Ordovician mass extinction. Communities at two contrasting sites exhibit significant decreases in complexity and evenness as a consequence of the preferential decline in abundance of dysaerobic zone specialist species. The observed changes in community complexity and evenness commenced well before the dramatic population depletions that mark the tipping point of the extinction event. Initially, community changes tracked changes in the oceanic water masses, but these relations broke down during the onset of mass extinction. Environmental isotope and biomarker data suggest that sea surface temperature and nutrient cycling in the paleotropical oceans changed sharply during the latest Katian time, with consequent changes in the extent of the oxygen minimum zone and phytoplankton community composition. Although many impacted species persisted in ephemeral populations, increased extinction risk selectively depleted the diversity of paleotropical graptolite species during the latest Katian and early Hirnantian. The effects of long-term climate change on habitats can thus degrade populations in ways that cascade through communities, with effects that culminate in mass extinction.

A host immune hormone modifies parasite species interactions and epidemics: insights from a field manipulation.

Parasite epidemics can depend on priority effects, and parasite priority effects can result from the host immune response to prior infection. Yet we lack experimental evidence that such immune-mediated priority effects influence epidemics. To address this research gap, we manipulated key host immune hormones, then measured the consequences for within-host parasite interactions, and ultimately parasite epidemics in the field. Specifically, we applied plant immune-signalling hormones to sentinel plants, embedded into a wild host population, and tracked foliar infections caused by two common fungal parasites. Within-host individuals, priority effects were altered by the immune-signalling hormone, salicylic acid (SA). Scaling up from within-host interactions, hosts treated with SA experienced a lower prevalence of a less aggressive parasite, increased burden of infection by a more aggressive parasite, and experienced fewer co-infections. Together, these results indicate that by altering within-host priority effects, host immune hormones can drive parasite epidemics. This study therefore experimentally links host immune hormones to within-host priority effects and parasite epidemics, advancing a more mechanistic understanding of how interactions among parasites alter their epidemics.

Interactions among symbionts operate across scales to influence parasite epidemics.

Parasite epidemics may be influenced by interactions among symbionts, which can depend on past events at multiple spatial scales. Within host individuals, interactions can depend on the sequence in which symbionts infect a host, generating priority effects. Across host individuals, interactions can depend on parasite phenology. To test the roles of parasite interactions and phenology in epidemics, we embedded multiple cohorts of sentinel plants, grown from seeds with and without a vertically transmitted symbiont, into a wild host population, and tracked foliar infections caused by three common fungal parasites. Within hosts, parasite growth was influenced by coinfections, but coinfections were often prevented by priority effects among symbionts. Across hosts, parasite phenology altered host susceptibility to secondary infections, symbiont interactions and ultimately the magnitude of parasite epidemics. Together, these results indicate that parasite phenology can influence parasite epidemics by altering the sequence of infection and interactions among symbionts within host individuals.

A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies.

Residual host cell proteins (HCPs) in biopharmaceuticals derived from recombinant DNA technology can present potential safety risks to patients or compromise product stability. Thus, the downstream purification process is designed to demonstrate robust removal of these impurities. ELISA using polyclonal anti-HCP antibodies as reagents for capture, detection, and quantitation purposes is most commonly used to monitor HCP removal during process development, but this technique has limitations. More recently, LC-MS for residual HCP characterization has emerged as a powerful tool to support purification process development. However, mass spectrometry needs to overcome the enormous dynamic range to detect low ppm levels of residual HCPs in biopharmaceutical samples. We describe a simple and powerful methodology to characterize residual HCPs in (monoclonal) antibodies by combining a novel sample preparation procedure using trypsin digestion and a shotgun proteomics approach. Differing from the traditional methodology, the sample preparation approach maintains nearly intact antibody while HCPs are digested. Thus, the dynamic range for HCP detection by MS is 1 to 2 orders of magnitude less than the traditional trypsin digestion sample preparation procedure. HCP spiking experiments demonstrated that our method could detect 0.5 ppm of HCP with molecular weight >60 kDa, such as rPLBL2. Application of our method to analyze a high-purity NIST monoclonal antibody standard RM 8670 derived from a murine cell line expression system resulted in detection of 60 mouse HCPs; twice as many as previously reported with 2D-UPLC/IM/MSE method. A control monoclonal antibody used for 70 analyses over 450 days demonstrated that our method is robust.

A multivariate test of disease risk reveals conditions leading to disease amplification.

Theory predicts that increasing biodiversity will dilute the risk of infectious diseases under certain conditions and will amplify disease risk under others. Yet, few empirical studies demonstrate amplification. This contrast may occur because few studies have considered the multivariate nature of disease risk, which includes richness and abundance of parasites with different transmission modes. By combining a multivariate statistical model developed for biodiversity-ecosystem-multifunctionality with an extensive field manipulation of host (plant) richness, composition and resource supply to hosts, we reveal that (i) host richness alone could not explain most changes in disease risk, and (ii) shifting host composition allowed disease amplification, depending on parasite transmission mode. Specifically, as predicted from theory, the effect of host diversity on parasite abundance differed for microbes (more density-dependent transmission) and insects (more frequency-dependent transmission). Host diversity did not influence microbial parasite abundance, but nearly doubled insect parasite abundance, and this amplification effect was attributable to variation in host composition. Parasite richness was reduced by resource addition, but only in species-rich host communities. Overall, this study demonstrates that multiple drivers, related to both host community and parasite characteristics, can influence disease risk. Furthermore, it provides a framework for evaluating multivariate disease risk in other systems.

Effects of native diversity, soil nutrients, and natural enemies on exotic invasion in experimental plant communities.

Many factors can promote exotic plant success. Three of these factors-greater pressure from natural enemies on natives, increased soil nutrient supply, and low native species richness-may interact during invasions. To test for independent and interactive effects of these drivers, we planted herbaceous perennial communities at two levels of native richness (monocultures and five-species polycultures). We then factorially manipulated soil nutrient supply and access to these communities by aboveground foliar enemies (fungal pathogens and insect herbivores), and allowed natural colonization to proceed for four years. We predicted that nutrient addition would increase exotic success, while enemy exclusion and increasing native richness would reduce exotic success. Additionally, we expected that enemy exclusion would reduce the benefits of nutrient addition to exotic species most in species-poor communities, and that this effect would be weaker in species-rich communities. In total, we found no evidence that nutrient supply, enemy access, and native richness interacted to influence exotic success. Furthermore, native richness had no effect on exotic success. Instead, nutrient addition increased, and enemy exclusion decreased, exotic success independently. As predicted, enemy exclusion reduced exotic success, primarily by slowing the decline in abundance of planted native species. Together, these results demonstrate that multiple drivers of exotic success can act independently within a single system.

Within-Host Niche Differences and Fitness Trade-offs Promote Coexistence of Plant Viruses.

Pathogens live in diverse, competitive communities, yet the processes that maintain pathogen diversity remain elusive. Here, we use a species-rich, well-studied plant virus system, the barley yellow dwarf viruses, to examine the mechanisms that regulate pathogen diversity. We empirically parameterized models of three viruses, their two aphid vectors, and one perennial grass host. We found that high densities of both aphids maximized virus diversity and that competition limited the coexistence of two closely related viruses. Even limited ability to simultaneously infect (coinfect) host individuals strongly promoted virus coexistence; preventing coinfection led to priority effects. Coinfection generated stabilizing niche differences by allowing viruses to share hosts. However, coexistence also required trade-offs between vector generalist and specialist life-history strategies. Our predicted outcomes broadly concur with previous field observations. These results show how competition within individual hosts and vectors may lead to unexpected population-level outcomes between pathogens, including coexistence, competitive exclusion, and priority effects, and how contemporary coexistence theory can help to predict these outcomes.

Joint effects of nutrient addition and enemy exclusion on exotic plant success.

Worldwide, ecosystems are increasingly dominated by exotic plant species, a shift hypothesized to result from numerous ecological factors. Two of these, increased resource availability and enemy release, may act in concert to increase exotic success in plant communities (Resource-Enemy Release Hypothesis, R-ERH). To test this, we manipulated the availability of soil nutrients and access of vertebrate herbivores, insect herbivores, and fungal pathogens to intact grassland communities containing both native and exotic species. Our results supported both conditions necessary for R-ERH. First, exotics were less damaged than natives, experiencing less foliar damage (insect herbivory and fungal disease) than native species, particularly in communities where soil nutrients were added. Second, fertilization increased foliar damage on native species, but not exotic species. As well as fulfilling both conditions for R-ERH, these results demonstrate the importance of considering the effects of resource availability when testing for enemy release. When both conditions are fulfilled, R-ERH predicts that increasing resource availability will increase exotic abundance only in the presence of enemies. Our results fully supported this prediction for vertebrate herbivores: fertilization increased exotic cover only in communities exposed to vertebrate herbivores. Additionally, the prediction was partially supported for insect herbivores and fungal pathogens, excluding these enemies reduced exotic cover as predicted, but inconsistent with R-ERH, this effect occurred only in unfertilized communities. These results highlight the need to consider the influence of multiple enemy guilds on community processes like exotic plant invasions. Moreover, this study experimentally demonstrates that resource availability and natural enemies can jointly influence exotic success in plant communities.

Impacts of biodiversity on the emergence and transmission of infectious diseases.

Current unprecedented declines in biodiversity reduce the ability of ecological communities to provide many fundamental ecosystem services. Here we evaluate evidence that reduced biodiversity affects the transmission of infectious diseases of humans, other animals and plants. In principle, loss of biodiversity could either increase or decrease disease transmission. However, mounting evidence indicates that biodiversity loss frequently increases disease transmission. In contrast, areas of naturally high biodiversity may serve as a source pool for new pathogens. Overall, despite many remaining questions, current evidence indicates that preserving intact ecosystems and their endemic biodiversity should generally reduce the prevalence of infectious diseases.

The community ecology of pathogens: coinfection, coexistence and community composition.

Disease and community ecology share conceptual and theoretical lineages, and there has been a resurgence of interest in strengthening links between these fields. Building on recent syntheses focused on the effects of host community composition on single pathogen systems, we examine pathogen (microparasite) communities using a stochastic metacommunity model as a starting point to bridge community and disease ecology perspectives. Such models incorporate the effects of core community processes, such as ecological drift, selection and dispersal, but have not been extended to incorporate host-pathogen interactions, such as immunosuppression or synergistic mortality, that are central to disease ecology. We use a two-pathogen susceptible-infected (SI) model to fill these gaps in the metacommunity approach; however, SI models can be intractable for examining species-diverse, spatially structured systems. By placing disease into a framework developed for community ecology, our synthesis highlights areas ripe for progress, including a theoretical framework that incorporates host dynamics, spatial structuring and evolutionary processes, as well as the data needed to test the predictions of such a model. Our synthesis points the way for this framework and demonstrates that a deeper understanding of pathogen community dynamics will emerge from approaches working at the interface of disease and community ecology.

Plant diversity predicts beta but not alpha diversity of soil microbes across grasslands worldwide.

Aboveground-belowground interactions exert critical controls on the composition and function of terrestrial ecosystems, yet the fundamental relationships between plant diversity and soil microbial diversity remain elusive. Theory predicts predominantly positive associations but tests within single sites have shown variable relationships, and associations between plant and microbial diversity across broad spatial scales remain largely unexplored. We compared the diversity of plant, bacterial, archaeal and fungal communities in one hundred and forty-five 1 m(2) plots across 25 temperate grassland sites from four continents. Across sites, the plant alpha diversity patterns were poorly related to those observed for any soil microbial group. However, plant beta diversity (compositional dissimilarity between sites) was significantly correlated with the beta diversity of bacterial and fungal communities, even after controlling for environmental factors. Thus, across a global range of temperate grasslands, plant diversity can predict patterns in the composition of soil microbial communities, but not patterns in alpha diversity.

Viral pathogen production in a wild grass host driven by host growth and soil nitrogen.

Nutrient limitation is a basic ecological constraint that has received little attention in studies on virus production and disease dynamics. Nutrient availability could directly limit the production of viral nucleic acids and proteins, or alternatively limit host growth and thus indirectly limit metabolic pathways necessary for viral replication. In order to compare direct and indirect effects of nutrient limitation on virus production within hosts, we manipulated soil nitrogen (N) and phosphorus (P) availability in a glasshouse for the wild grass host Bromus hordeaceus and the viral pathogen Barley yellow dwarf virus-PAV. We found that soil N additions increased viral concentrations within host tissues, and the effect was mediated by host growth. Specifically, in statistical models evaluating the roles of host biomass production, leaf N and leaf P, viral production depended most strongly on host biomass, rather than the concentration of either nutrient. Furthermore, at low soil N, larger plants supported greater viral concentrations than smaller ones, whereas at high N, smaller plants supported greater viral concentrations. Our results suggest that enhanced viral productivity under N enrichment is an indirect consequence of nutrient stimulation to host growth rate. Heightened pathogen production in plants has important implications for a world facing increasing rates of nutrient deposition.

Graptoloid diversity and disparity became decoupled during the Ordovician mass extinction.

The morphological study of extinct taxa allows for analysis of a diverse set of macroevolutionary hypotheses, including testing for change in the magnitude of morphological divergence, extinction selectivity on form, and the ecological context of radiations. Late Ordovician graptoloids experienced a phylogenetic bottleneck at the Hirnantian mass extinction (∼445 Ma), when a major clade of graptoloids was driven to extinction while another clade simultaneously radiated. In this study, we developed a dataset of 49 ecologically relevant characters for 183 species with which we tested two main hypotheses: (i) could the biased survival of one graptoloid clade over another have resulted from morphological selectivity alone and (ii) are the temporal patterns of morphological disparity and innovation during the recovery consistent with an interpretation as an adaptive radiation resulting from ecological release? We find that a general model of morphological selectivity has a low probability of producing the observed pattern of taxonomic selectivity. Contrary to predictions from theory on adaptive radiations and ecological speciation, changes in disparity and species richness are uncoupled. We also find that the early recovery is unexpectedly characterized by relatively low morphological disparity and innovation, despite also being an interval of elevated speciation. Because it is necessary to invoke factors other than ecology to explain the graptoloid recovery, more complex models may be needed to explain recovery dynamics after mass extinctions.

Why is living fast dangerous? Disentangling the roles of resistance and tolerance of disease.

Primary axes of host developmental tempo (HDT; e.g., slow-quick return continuum) represent latent biological processes and are increasingly used to a priori identify hosts that contribute disproportionately more to pathogen transmission. The influence of HDT on host contributions to transmission depends on how HDT influences both resistance and tolerance of disease. Here, we use structural equation modeling to address known limitations of conventional measures of resistance and tolerance. We first provide a general resistance-tolerance metamodel from which system-specific models can be derived. We then develop a model specific to a group of vector-transmitted viruses that infect hundreds of grass species worldwide. We tested the model using experimental inoculations of six phylogenetically paired grass species. We found that (1) host traits covaried according to a prominent HDT axis, the slow-quick continuum; (2) infection caused a greater reduction in the performance of quick returns, with >80% of that greater impact explained by lesser resistance; (3) resistance-tolerance trade-off did not occur; and (4) phylogenetic control was necessary to measure the slow-quick continuum, resistance, and tolerance. These results support the conclusion that HDT's main influence on host contributions to transmission is via resistance. More broadly, this study provides a framework for quantifying HDT's influence on host contributions to transmission.