The timings of host diapause and epidemic progression mediate host genetic diversity and future epidemic size in Daphnia-parasite populations.

Epidemics commonly exert parasite-mediated selection and cause declines in host population genetic diversity. This can lead to evolution of resistance in the long term and smaller subsequent epidemics. Alternatively, the loss of genetic diversity can increase host vulnerability to future disease spread and larger future epidemics. Matters are made more complex by the fact that a great many host organisms produce diapausing life stages in response to environmental change (often as a result of sexual reproduction; e.g. plant seeds and invertebrate resting eggs). These diapausing stages can disrupt the relationship between past epidemics, host genetic diversity and future epidemics because they allow host dispersal through time. Specifically, temporally dispersing hosts avoid infection and thus selection from contemporary parasites, and also archive genetic variation for the future. We studied 80 epidemics in 20 semi-natural populations of the temporally dispersing crustacean Daphnia magna and its sterilizing bacterial parasite Pasteuria ramosa, and half of these populations experienced a simulated environmental disturbance treatment. We found that early initiation of diapause relative to the timing of the epidemic led to greater host genetic diversity and reduced epidemic size in the subsequent year, but this was unaffected by environmental disturbance.

Pathogen Dynamics across the Diversity of Aging.

AbstractReproduction, mortality, and immune function often change with age but do not invariably deteriorate. Across the tree of life, there is extensive variation in age-specific performance and changes to key life-history traits. These changes occur on a spectrum from classic senescence, where performance declines with age, to juvenescence, where performance improves with age. Reproduction, mortality, and immune function are also important factors influencing the spread of infectious disease, yet there exists no comprehensive investigation into how the aging spectrum of these traits impacts epidemics. We used a model laboratory infection system to compile an aging profile of a single organism, including traits directly linked to pathogen susceptibility and those that should indirectly alter pathogen transmission by influencing demography. We then developed generalizable epidemiological models demonstrating that different patterns of aging produce dramatically different transmission landscapes: in many cases, aging can reduce the probability of epidemics, but it can also promote severity. This work provides context and tools for use across taxa by empiricists, demographers, and epidemiologists, advancing our ability to accurately predict factors contributing to epidemics or the potential repercussions of senescence manipulation.

A Two-Locus System with Strong Epistasis Underlies Rapid Parasite-Mediated Evolution of Host Resistance.

Parasites are a major evolutionary force, driving adaptive responses in host populations. Although the link between phenotypic response to parasite-mediated natural selection and the underlying genetic architecture often remains obscure, this link is crucial for understanding the evolution of resistance and predicting associated allele frequency changes in the population. To close this gap, we monitored the response to selection during epidemics of a virulent bacterial pathogen, Pasteuria ramosa, in a natural host population of Daphnia magna. Across two epidemics, we observed a strong increase in the proportion of resistant phenotypes as the epidemics progressed. Field and laboratory experiments confirmed that this increase in resistance was caused by selection from the local parasite. Using a genome-wide association study, we built a genetic model in which two genomic regions with dominance and epistasis control resistance polymorphism in the host. We verified this model by selfing host genotypes with different resistance phenotypes and scoring their F1 for segregation of resistance and associated genetic markers. Such epistatic effects with strong fitness consequences in host-parasite coevolution are believed to be crucial in the Red Queen model for the evolution of genetic recombination.

Disentangling non-specific and specific transgenerational immune priming components in host-parasite interactions.

Exposure to a pathogen primes many organisms to respond faster or more efficiently to subsequent exposures. Such priming can be non-specific or specific, and has been found to extend across generations. Disentangling and quantifying specific and non-specific effects is essential for understanding the genetic epidemiology of a system. By combining a large infection experiment and mathematical modelling, we disentangle different transgenerational effects in the crustacean model Daphnia magna exposed to different strains of the bacterial parasite Pasteuria ramosa. In the experiment, we exposed hosts to a high dose of one of three parasite strains, and subsequently challenged their offspring with multiple doses of the same (homologous) or a different (heterologous) strain. We find that exposure of Daphnia to Pasteuria decreases the susceptibility of their offspring by approximately 50%. This transgenerational protection is not larger for homologous than for heterologous parasite challenges. Methodologically, our work represents an important contribution not only to the analysis of immune priming in ecological systems but also to the experimental assessment of vaccines. We present, for the first time, an inference framework to investigate specific and non-specific effects of immune priming on the susceptibility distribution of hosts-effects that are central to understanding immunity and the effect of vaccines.

Local adaptation of a parasite to solar radiation impacts disease transmission potential, spore yield, and host fecundity.

Environmentally transmitted parasites spend time in the abiotic environment, where they are subjected to a variety of stressors. Learning how they face this challenge is essential if we are to understand how host-parasite interactions may vary across environmental gradients. We used a zooplankton-bacteria host-parasite system where availability of sunlight (solar radiation) influences disease dynamics to look for evidence of parasite local adaptation to sunlight exposure. We also examined how variation in sunlight tolerance among parasite strains impacted host reproduction. Parasite strains collected from clearer lakes (with greater sunlight penetration) were most tolerant of the negative impacts of sunlight exposure, suggesting local adaptation to sunlight conditions. This adaptation came with both a cost and a benefit for parasites: parasite strains from clearer lakes produced relatively fewer transmission stages (spores) but these strains were more infective. After experimental sunlight exposure, the most sunlight-tolerant parasite strains reduced host fecundity just as much as spores that were never exposed to sunlight. Sunlight availability varies greatly among lakes around the world. Our results suggest that the selective pressure sunlight exposure exerts on parasites may impact both parasite and host fitness, potentially driving variation in disease epidemics and host population dynamics across sunlight availability gradients.

Host sexual dimorphism affects the outcome of within-host pathogen competition.

Natural infections often consist of multiple pathogens of the same or different species. When coinfections occur, pathogens compete for access to host resources and fitness is determined by how well a pathogen can reproduce compared to its competitors. Yet not all hosts provide the same resource pool. Males and females, in particular, commonly vary in both their acquisition of resources and investment in immunity, but their ability to modify any competition between different pathogens remains unknown. Using the Daphnia magna-Pasteuria ramosa model system, we exposed male and female hosts to either a single genotype infection or coinfections consisting of two pathogen genotypes of varying levels of virulence. We found that coinfections within females favored the transmission of the more virulent pathogen genotype, whereas coinfections within male hosts resulted in equal transmission of competing pathogen genotypes. This contrast became less pronounced when the least virulent pathogen was able to establish an infection first, suggesting that the influence of host sex is shaped by priority effects. We suggest that sex is a form of host heterogeneity that may influence the evolution of virulence within coinfection contexts and that one sex may be a reservoir for pathogen genetic diversity in nature.

Assessment of parasite virulence in a natural population of a planktonic crustacean.

BACKGROUND: Understanding the impact of disease in natural populations requires an understanding of infection risk and the damage that parasites cause to their hosts (= virulence). However, because these disease traits are often studied and quantified under controlled laboratory conditions and with reference to healthy control hosts, we have little knowledge about how they play out in natural conditions. In the Daphnia-Pasteuria host-parasite system, field assessments often show very low estimates of virulence, while controlled laboratory experiments indicate extremely high virulence. RESULTS: To examine this discrepancy, we sampled Daphnia magna hosts from the field during a parasite epidemic and recorded disease traits over a subsequent 3-week period in the laboratory. As predicted for chronic disease where infections in older (larger) hosts are also, on average, older, we found that larger D. magna females were infected more often, had fewer offspring prior to the onset of castration and showed signs of infection sooner than smaller hosts. Also consistent with laboratory experiments, infected animals were found in both sexes and in all sizes of hosts. Infected females were castrated at capture or became castrated soon after. As most females in the field carried no eggs in their brood pouch at the time of sampling, virulence estimates of infected females relative to uninfected females were low. However, with improved feeding conditions in the laboratory, only uninfected females resumed reproduction, resulting in very high relative virulence estimates. CONCLUSIONS: Overall, our study shows that the disease manifestation of P. ramosa, as expressed under natural conditions, is consistent with what we know from laboratory experiments. However, parasite induced fecundity reduction of infected, relative to uninfected hosts depended strongly on the environmental conditions. We argue that this effect is particularly strong for castrating parasites, because infected hosts have low fecundity under all conditions.

Within-Host Priority Effects Systematically Alter Pathogen Coexistence.

Coinfection of host populations alters pathogen prevalence, host mortality, and pathogen evolution. Because pathogens compete for limiting resources, whether multiple pathogens can coexist in a host population can depend on their within-host interactions, which, in turn, can depend on the order in which pathogens infect hosts (within-host priority effects). However, the consequences of within-host priority effects for pathogen coexistence have not been tested. Using laboratory studies with a coinfected zooplankton system, we found that pathogens had increased fitness in coinfected hosts when they were the second pathogen to infect a host, compared to when they were the first pathogen to infect a host. With these results, we parameterized a pathogen coexistence model with priority effects, finding that pathogen coexistence (1) decreased when priority effects increased the fitness of the first pathogen to arrive in coinfected hosts and (2) increased when priority effects increased the fitness of the second pathogen to arrive in coinfected hosts. We also identified the natural conditions under which we expect within-host priority effects to foster coexistence in our system. These outcomes were the result of positive or negative frequency dependence created by feedback loops between pathogen prevalence and infection order in coinfected hosts. This suggests that priority effects can systematically alter conditions for pathogen coexistence in host populations, thereby changing pathogen community structure and potentially altering host mortality and pathogen evolution via emergent processes.

Impact of an immunosuppressive human pharmaceutical on the interaction of a bacterial parasite and its invertebrate host.

The interaction of pollutants and pathogens may result in altered and often enhanced effects of the chemical, the biotic stressor or both. These interaction effects cannot be reliably predicted from the toxicity of the chemical or the virulence of the pathogen alone. While standardized detection methods for immunotoxic effects of chemicals exist with regard to human health, employing host-resistance assays with vertebrates, such standardized test systems are completely lacking for invertebrate species and no guidance is available on how immunotoxic effects of a chemical in invertebrates could be definitively identified. In the present study, we investigated the impact of the immunosuppressive pharmaceutical cyclosporine A (CsA) on the invertebrate host-pathogen system Daphnia magna - Pasteuria ramosa. CsA is a calcineurin-inhibitor in vertebrates and also known to have antibiotic as well as antifungal properties. Juvenile D. magna were exposed to CsA for 21 days with or without additional pathogen challenge during the first 72 h of exposure. Long-term survival of the host D. magna was synergistically impacted by co-exposure to the chemical and the pathogen, expressed e.g. in significantly enhanced hazard ratios. Additionally, enhanced virulence of the pathogen upon chemical co-exposure was expressed in an increased proportion of infected hosts and an increased speed of Pasteuria-induced host sterilization. In contrast, effects on reproduction were additive in Pasteuria-challenged, but finally non-infected D. magna. The enhancing effects of CsA occurred at and below 3 µg/L, which was in the absence of the pathogen the lowest concentration significantly impacting the standard toxicity endpoint 'reproduction' in D. magna. Hence, the present study provides evidence that a pharmaceutical intended to suppress the human immune system can also suppress disease resistance of an aquatic invertebrate organism at otherwise non-toxic concentrations. Plausible ways of direct interactions of CsA with the host's immune system are discussed, e.g. interference with phagocytosis or Toll-like receptors. Experimental verification of such a direct interference would be warranted to support the strong evidence for immunotoxic activity of CsA in invertebrates. While it remains open whether CsA concentrations in the environment are high enough to trigger adverse effects in environmental organisms, our findings highlight the need to consider immunotoxicity in an environmental risk assessment, and to develop suitable standardized methods for this purpose.

Knockdown of a mucin-like gene in Meloidogyne incognita (Nematoda) decreases attachment of endospores of Pasteuria penetrans to the infective juveniles and reduces nematode fecundity.

Mucins are highly glycosylated polypeptides involved in many host-parasite interactions, but their function in plant-parasitic nematodes is still unknown. In this study, a mucin-like gene was cloned from Meloidogyne incognita (Mi-muc-1, 1125 bp) and characterized. The protein was found to be rich in serine and threonine with numerous O-glycosylation sites in the sequence. Quantitative real-time polymerase chain reaction (qRT-PCR) showed the highest expression in the adult female and in situ hybridization revealed the localization of Mi-muc-1 mRNA expression in the tail area in the region of the phasmid. Knockdown of Mi-muc-1 revealed a dual role: (1) immunologically, there was a significant decrease in attachment of Pasteuria penetrans endospores and a reduction in binding assays with human red blood cells (RBCs), suggesting that Mi-MUC-1 is a glycoprotein present on the surface coat of infective second-stage juveniles (J2s) and is involved in cellular adhesion to the cuticle of infective J2s; pretreatment of J2s with different carbohydrates indicated that the RBCs bind to J2 cuticle receptors different from those involved in the interaction of Pasteuria endospores with Mi-MUC-1; (2) the long-term effect of RNA interference (RNAi)-mediated knockdown of Mi-muc-1 led to a significant reduction in nematode fecundity, suggesting a possible function for this mucin as a mediator in the interaction between the nematode and the host plant.

Simulated climate change, epidemic size, and host evolution across host-parasite populations.

Climate change is causing warmer and more variable temperatures as well as physical flux in natural populations, which will affect the ecology and evolution of infectious disease epidemics. Using replicate seminatural populations of a coevolving freshwater invertebrate-parasite system (host: Daphnia magna, parasite: Pasteuria ramosa), we quantified the effects of ambient temperature and population mixing (physical flux within populations) on epidemic size and population health. Each population was seeded with an identical suite of host genotypes and dose of parasite transmission spores. Biologically reasonable increases in environmental temperature caused larger epidemics, and population mixing reduced overall epidemic size. Mixing also had a detrimental effect on host populations independent of disease. Epidemics drove parasite-mediated selection, leading to a loss of host genetic diversity, and mixed populations experienced greater evolution due to genetic drift over the season. These findings further our understanding of how diversity loss will reduce the host populations' capacity to respond to changes in selection, therefore stymying adaptation to further environmental change.

Disease spread in age structured populations with maternal age effects.

Fundamental ecological processes, such as extrinsic mortality, determine population age structure. This influences disease spread when individuals of different ages differ in susceptibility or when maternal age determines offspring susceptibility. We show that Daphnia magna offspring born to young mothers are more susceptible than those born to older mothers, and consider this alongside previous observations that susceptibility declines with age in this system. We used a susceptible-infected compartmental model to investigate how age-specific susceptibility and maternal age effects on offspring susceptibility interact with demographic factors affecting disease spread. Our results show a scenario where an increase in extrinsic mortality drives an increase in transmission potential. Thus, we identify a realistic context in which age effects and maternal effects produce conditions favouring disease transmission.

Parasite transmission in a natural multihost-multiparasite community.

Understanding the transmission and dynamics of infectious diseases in natural communities requires understanding the extent to which the ecology, evolution and epidemiology of those diseases are shaped by alternative hosts. We performed laboratory experiments to test how parasite spillover affected traits associated with transmission in two co-occurring parasites: the bacterium Pasteuria ramosa and the fungus Metschnikowia bicuspidata Both parasites were capable of transmission from the reservoir host (Daphnia dentifera) to the spillover host (Ceriodaphnia dubia), but this occurred at a much higher rate for the fungus than the bacterium. We quantified transmission potential by combining information on parasite transmission and growth rate, and used this to compare parasite fitness in the two host species. For both parasites, transmission potential was lower in the spillover host. For the bacterium, virulence was higher in the spillover host. Transmission back to the original host was high for both parasites, with spillover influencing transmission rate of the fungus but not the bacterium. Thus, while inferior, the spillover host is not a dead-end for either parasite. Overall, our results demonstrate that the presence of multiple hosts in a community can have important consequences for disease transmission, and host and parasite fitness.This article is part of the themed issue 'Opening the black box: re-examining the ecology and evolution of parasite transmission'.

Survival, reproduction, growth, and parasite resistance of aquatic organisms exposed on-site to wastewater treated by advanced treatment processes.

Advanced wastewater treatment technologies are generally known to be an effective tool for reducing micropollutant discharge into the aquatic environment. Nevertheless, some processes such as ozonation result in stable transformation products with often unknown toxicity. In the present study, whole effluents originating from nine different steps of advanced treatment combinations were compared for their aquatic toxicity. Assessed endpoints were survival, growth and reproduction of Lumbriculus variegatus, Daphnia magna and Lemna minor chronically exposed in on-site flow-through tests based on standard guidelines. The treatment combinations were activated sludge treatment followed by ozonation with subsequent filtration by granular activated carbon or biofilters and membrane bioreactor treatment of raw wastewater followed by ozonation. Additionally, the impact of treated wastewater on the immune response of invertebrates was investigated by challenging D. magna with a bacterial endoparasite. Conventionally treated wastewater reduced reproduction of L. variegatus by up to 46%, but did not affect D. magna and L. minor with regard to survival, growth, reproduction and parasite resistance. Instead, parasite susceptibility was significantly reduced in D. magna exposed to conventionally treated as well as ozonated wastewater in comparison to D. magna exposed to the medium control. None of the three test organisms provided clear evidence that wastewater ozonation leads to increased aquatic toxicity. Rather than to the presence of toxic transformation products, the affected performance of L. variegatus could be linked to elevated concentrations of ammonium and nitrite that likely resulted from treatment failures.

Parasitism drives host genome evolution: Insights from the Pasteuria ramosa-Daphnia magna system.

Because parasitism is thought to play a major role in shaping host genomes, it has been predicted that genomic regions associated with resistance to parasites should stand out in genome scans, revealing signals of selection above the genomic background. To test whether parasitism is indeed such a major factor in host evolution and to better understand host-parasite interaction at the molecular level, we studied genome-wide polymorphisms in 97 genotypes of the planktonic crustacean Daphnia magna originating from three localities across Europe. Daphnia magna is known to coevolve with the bacterial pathogen Pasteuria ramosa for which host genotypes (clonal lines) are either resistant or susceptible. Using association mapping, we identified two genomic regions involved in resistance to P. ramosa, one of which was already known from a previous QTL analysis. We then performed a naïve genome scan to test for signatures of positive selection and found that the two regions identified with the association mapping further stood out as outliers. Several other regions with evidence for selection were also found, but no link between these regions and phenotypic variation could be established. Our results are consistent with the hypothesis that parasitism is driving host genome evolution.

The trans-generational impact of population density signals on host-parasite interactions.

BACKGROUND: The density of a host population is a key parameter underlying disease transmission, but it also has implications for the expression of disease through its effect on host physiology. In response to higher densities, individuals are predicted to either increase their immune investment in response to the elevated risk of parasitism, or conversely to decrease their immune capacity as a consequence of the stress of a crowded environment. However, an individual's health is shaped by many different factors, including their genetic background, current environmental conditions, and maternal effects. Indeed, population density is often sensed through the presence of info-chemicals in the environment, which may influence a host's interaction with parasites, and also those of its offspring. All of which may alter the expression of disease, and potentially uncouple the presumed link between changes in host density and disease outcomes. RESULTS: In this study, we used the water flea Daphnia magna and its obligate bacterial parasite Pasteuria ramosa, to investigate how signals of high host density impact on host-parasite interactions over two consecutive generations. We found that the chemical signals from crowded treatments induced phenotypic changes in both the parental and offspring generations. In the absence of a pathogen, life-history changes were genotype-specific, but consistent across generations, even when the signal of density was removed. In contrast, the influence of density on infected animals depended on the trait and generation of exposure. When directly exposed to signals of high-density, host genotypes responded differently in how they minimised the severity of disease. Yet, in the subsequent generation, the influence of density was rarely genotype-specific and instead related to ability of the host to minimise the onset of infection. CONCLUSION: Our findings reveal that population level correlations between host density and infection capture only part of the complex relationship between crowding and the severity of disease. We suggest that besides its role in horizontal transmission, signals of density can influence parasite epidemiology by modifying mechanisms of resistance across multiple generations, and elevating variability via genotype-by-environment interactions. Our results help resolve why some studies are able to find a positive correlation between high density and resistance, while others uncover a negative correlation, or even no direct relationship at all.

A Population Biology Perspective on the Stepwise Infection Process of the Bacterial Pathogen Pasteuria ramosa in Daphnia.

The infection process of many diseases can be divided into series of steps, each one required to successfully complete the parasite's life and transmission cycle. This approach often reveals that the complex phenomenon of infection is composed of a series of more simple mechanisms. Here we demonstrate that a population biology approach, which takes into consideration the natural genetic and environmental variation at each step, can greatly aid our understanding of the evolutionary processes shaping disease traits. We focus in this review on the biology of the bacterial parasite Pasteuria ramosa and its aquatic crustacean host Daphnia, a model system for the evolutionary ecology of infectious disease. Our analysis reveals tremendous differences in the degree to which the environment, host genetics, parasite genetics and their interactions contribute to the expression of disease traits at each of seven different steps. This allows us to predict which steps may respond most readily to selection and which steps are evolutionarily constrained by an absence of variation. We show that the ability of Pasteuria to attach to the host's cuticle (attachment step) stands out as being strongly influenced by the interaction of host and parasite genotypes, but not by environmental factors, making it the prime candidate for coevolutionary interactions. Furthermore, the stepwise approach helps us understanding the evolution of resistance, virulence and host ranges. The population biological approach introduced here is a versatile tool that can be easily transferred to other systems of infectious disease.

Does infection tilt the scales? Disease effects on the mass balance of an invertebrate nutrient recycler.

While parasites are increasingly recognized as important components of ecosystems, we currently know little about how they alter ecosystem nutrient availability via host-mediated nutrient cycling. We examined whether infection alters the flow of nutrients through hosts and whether such effects depend upon host diet quality. To do so, we compared the mass specific nutrient (i.e., nitrogen and phosphorus) release rates, ingestion rates, and elemental composition of uninfected Daphnia to those infected with a bacterial parasite, P. ramosa. N and P release rates were increased by infection when Daphnia were fed P-poor diets, but we found no effect of infection on the nutrient release of individuals fed P-rich diets. Calculations based on the first law of thermodynamics indicated that infection should increase the nutrient release rates of Daphnia by decreasing nutrient accumulation rates in host tissues. Although we found reduced nutrient accumulation rates in infected Daphnia fed all diets, this reduction did not increase the nutrient release rates of Daphnia fed the P-rich diet because infected Daphnia fed this diet ingested nutrients more slowly than uninfected hosts. Our results thus indicate that parasites can significantly alter the nutrient use of animal consumers, which could affect the availability of nutrients in heavily parasitized environments.

Host age modulates within-host parasite competition.

In many host populations, one of the most striking differences among hosts is their age. While parasite prevalence differences in relation to host age are well known, little is known on how host age impacts ecological and evolutionary dynamics of diseases. Using two clones of the water flea Daphnia magna and two clones of its bacterial parasite Pasteuria ramosa, we examined how host age at exposure influences within-host parasite competition and virulence. We found that multiply-exposed hosts were more susceptible to infection and suffered higher mortality than singly-exposed hosts. Hosts oldest at exposure were least often infected and vice versa. Furthermore, we found that in young multiply-exposed hosts competition was weak, allowing coexistence and transmission of both parasite clones, whereas in older multiply-exposed hosts competitive exclusion was observed. Thus, age-dependent parasite exposure and host demography (age structure) could together play an important role in mediating parasite evolution. At the individual level, our results demonstrate a previously unnoticed interaction of the host's immune system with host age, suggesting that the specificity of immune function changes as hosts mature. Therefore, evolutionary models of parasite virulence might benefit from incorporating age-dependent epidemiological parameters.

Expression of parasite genetic variation changes over the course of infection: implications of within-host dynamics for the evolution of virulence.

How infectious disease agents interact with their host changes during the course of infection and can alter the expression of disease-related traits. Yet by measuring parasite life-history traits at one or few moments during infection, studies have overlooked the impact of variable parasite growth trajectories on disease evolution. Here we show that infection-age-specific estimates of host and parasite fitness components can reveal new insight into the evolution of parasites. We do so by characterizing the within-host dynamics over an entire infection period for five genotypes of the castrating bacterial parasite Pasteuria ramosa infecting the crustacean Daphnia magna. Our results reveal that genetic variation for parasite-induced gigantism, host castration and parasite spore loads increases with the age of infection. Driving these patterns appears to be variation in how well the parasite maintains control of host reproduction late in the infection process. We discuss the evolutionary consequences of this finding with regard to natural selection acting on different ages of infection and the mechanism underlying the maintenance of castration efficiency. Our results highlight how elucidating within-host dynamics can shed light on the selective forces that shape infection strategies and the evolution of virulence.