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.

QTL study reveals candidate genes underlying host resistance in a Red Queen model system.

Specific interactions of host and parasite genotypes can lead to balancing selection, maintaining genetic diversity within populations. In order to understand the drivers of such specific coevolution, it is necessary to identify the molecular underpinnings of these genotypic interactions. Here, we investigate the genetic basis of resistance in the crustacean host, Daphnia magna, to attachment and subsequent infection by the bacterial parasite, Pasteuria ramosa. We discover a single locus with Mendelian segregation (3:1 ratio) with resistance being dominant, which we call the F locus. We use QTL analysis and fine mapping to localize the F locus to a 28.8-kb region in the host genome, adjacent to a known resistance supergene. We compare the 28.8-kb region in the two QTL parents to identify differences between host genotypes that are resistant versus susceptible to attachment and infection by the parasite. We identify 13 genes in the region, from which we highlight eight biological candidates for the F locus, based on presence/absence polymorphisms and differential gene expression. The top candidates include a fucosyltransferase gene that is only present in one of the two QTL parents, as well as several Cladoceran-specific genes belonging to a large family that is represented in multiple locations of the host genome. Fucosyltransferases have been linked to resistance in previous studies of Daphnia-Pasteuria and other host-parasite systems, suggesting that P. ramosa spore attachment could be mediated by changes in glycan structures on D. magna cuticle proteins. The Cladoceran-specific candidate genes suggest a resistance strategy that relies on gene duplication. Our results add a new locus to a growing genetic model of resistance in the D. magna-P. ramosa system. The identified candidate genes will be used in future functional genetic studies, with the ultimate aim to test for cycles of allele frequencies in natural populations.

Exploring the mechanisms of host-specificity of a hyperparasitic bacterium (Pasteuria spp.) with potential to control tropical root-knot nematodes (Meloidogyne spp.): insights from Caenorhabditis elegans.

Plant-parasitic nematodes are important economic pests of a range of tropical crops. Strategies for managing these pests have relied on a range of approaches, including crop rotation, the utilization of genetic resistance, cultural techniques, and since the 1950's the use of nematicides. Although nematicides have been hugely successful in controlling nematodes, their toxicity to humans, domestic animals, beneficial organisms, and the environment has raised concerns regarding their use. Alternatives are therefore being sought. The Pasteuria group of bacteria that form endospores has generated much interest among companies wanting to develop microbial biocontrol products. A major challenge in developing these bacteria as biocontrol agents is their host-specificity; one population of the bacterium can attach to and infect one population of plant-parasitic nematode but not another of the same species. Here we will review the mechanism by which infection is initiated with the adhesion of endospores to the nematode cuticle. To understand the genetics of the molecular processes between Pasteuria endospores and the nematode cuticle, the review focuses on the nature of the bacterial adhesins and how they interact with the nematode cuticle receptors by exploiting new insights gained from studies of bacterial infections of Carnorhabditis elegans. A new Velcro-like multiple adhesin model is proposed in which the cuticle surface coat, which has an important role in endospore adhesion, is a complex extracellular matrix containing glycans originating in seam cells. The genes associated with these seam cells appear to have a dual role by retaining some characteristics of stem cells.

Host Sex Modulates the Energetics of Pathogen Proliferation and Its Dependence on Environmental Resources.

AbstractSex differences in immunity are predicted to underlie much of the frequently observed sex differences in the prevalence or severity of infection. We propose the additional hypothesis that differences in the ability of males and females to acquire and use resources will also affect how readily a pathogen can convert host energy into transmission stages, thereby contributing to sex differences in infection dynamics. To test this we manipulated the resource environment of male and female Daphnia magna by altering the availability of food and then exposed hosts to a bacterial pathogen. We measured the production of transmission spores and virulence via the reduction in life span, together with feeding rates and changes in mass-independent metabolic rate, as a measure of the intake and expenditure of energy during infection. When raised in the presence of high resource levels, females more readily allowed for resources in the environment to be translated to pathogen exploitation, as represented by increased spore production, greater virulence, and higher energy use. In contrast, the traits of infected males were robust to changes in resource availability. High food availability thus exaggerated the degree of sexual dimorphism observed between the sexes. It also modified the relationship between host energy use, virulence, and pathogen spore production for each sex. These results suggest that a host's resource environment can affect how a male or female is exploited by a pathogen and may thus be an additional factor driving sex-specific patterns of disease susceptibility or severity.

Exploring Bacillus thuringiensis as a model for endospore adhesion and its potential to investigate adhesins in Pasteuria penetrans.

AIMS: Phytonematodes are a constraint on crop production and have been controlled using nematicides; these are highly toxic and legislation in Europe and elsewhere is prohibiting their use and alternatives are being sought. Pasteuria penetrans is a hyperparasitic bacterium that form endospores and have potential to control root-knot nematodes (Meloidogyne spp.), but their attachment to the nematode cuticle is host-specific. Understanding host specificity has relied upon endospore inhibition bioassays using immunological and biochemical approaches. Phylogenetic analysis of survey sequences has shown P. penetrans to be closely related to Bacillus and to have a diverse range of collagen-like fibres which we hypothesise to be involved in the endospore adhesion. However, due to the obligately hyperparasitic nature of Pasteuria species, identifying and characterizing these collagenous-like proteins through gain of function has proved difficult and new approaches are required. METHODS AND RESULTS: Using antibodies raised to synthetic peptides based on Pasteuria collagen-like genes we show similarities between P. penetrans and the more easily cultured bacterium Bacillus thuringiensis and suggest it be used as a gain of function platform/model. Using immunological approaches similar proteins between P. penetrans and B. thuringiensis are identified and characterized, one >250 kDa and another ~72 kDa are glycosylated with N-acetylglucosamine and both of which are digested if treated with collagenase. These treatments also affected endospore attachment and suggest these proteins are involved in adhesion of endospores to nematode cuticle. CONCLUSION: There are conserved similarities in the collagen-like proteins present on the surface of endospores of both P. penetrans and B. thuringiensis. SIGNIFICANCE AND IMPACT OF STUDY: As B. thuringiensis is relatively easy to culture and can be transformed, it could be developed as a platform for studying the role of the collagen-like adhesins from Pasteuria in endospore adhesion.

Temperature and pathogen exposure act independently to drive host phenotypic trajectories.

Natural populations are experiencing an increase in the occurrence of both thermal stress and disease outbreaks. How these two common stressors interact to determine host phenotypic shifts will be important for population persistence, yet a myriad of different traits and pathways are a target of both stressors, making generalizable predictions difficult to obtain. Here, using the host Daphnia magna and its bacterial pathogen Pasteuria ramosa, we tested how temperature and pathogen exposure interact to drive shifts in multivariate host phenotypes. We found that these two stressors acted mostly independently to shape host phenotypic trajectories, with temperature driving a faster pace of life by favouring early development and increased intrinsic population growth rates, while pathogen exposure impacted reproductive potential through reductions in lifetime fecundity. Studies focussed on extreme thermal stress are increasingly showing how pathogen exposure can severely hamper the thermal tolerance of a host. However, our results suggest that under milder thermal stress, and in terms of life-history traits, increases in temperature might not exacerbate the impact of pathogen exposure on host performance, and vice versa.

Compatibility of Predatory Nematode, Fictor composticola and Bacterial Parasite, Pasteuria penetrans for the Management of Root-Knot Nematode.

Compatibility of Fictor composticola and Pasteuria penetrans was evaluated through two experiments against Meloidogyne incognita. In vitro assay conducted in 5 cm diameter Petri plates containing 1% water agar. Two males and two females of F. composticola were released alongwith 200 P. penetrans encumbered or healthy J2 of M. incognita. Observations on prey consumed were taken after 24 and 48 h of release. P. penetrans-encumbered J2 were consumed more by the predator than healthy J2. In a pot assay, cucumber plants grown in one kg pots were inoculated with 2000 P. penetrans encumbered or healthy J2 of M. incognita and 400 individuals of F. composticola. After 45 days; numbers of galls, egg masses and final J2 population in soil were significantly less in the treatment with F. composticola + P. penetrans-encumbered J2 than F. composticola with healthy J2. The root galls and the number of Pasteuria-infected females were more in the absence of predatory nematode.

Ecology directs host-parasite coevolutionary trajectories across Daphnia-microparasite populations.

Host-parasite interactions often fuel coevolutionary change. However, parasitism is one of a myriad of possible ecological interactions in nature. Biotic (for example, predation) and abiotic (for example, temperature) variation can amplify or dilute parasitism as a selective force on hosts and parasites, driving population variation in (co)evolutionary trajectories. We dissected the relationships between wider ecology and coevolutionary trajectory using 16 ecologically complex Daphnia magna-Pasteuria ramosa ponds seeded with an identical starting host (Daphnia) and parasite (Pasteuria) population. We show, using a time-shift experiment and outdoor population data, how multivariate biotic and abiotic ecological differences between ponds caused coevolutionary divergence. Wider ecology drove variation in host evolution of resistance, but not parasite infectivity; parasites subsequently coevolved in response to the changing complement of host genotypes, such that parasites adapted to historically resistant host genotypes. Parasitism was a stronger interaction for the parasite than for its host, probably because the host is the principal environment and selective force, whereas for hosts, parasite-mediated selection is one of many sources of selection. Our findings reveal the mechanisms through which wider ecology creates coevolutionary hotspots and coldspots in biologically realistic arenas of host-parasite interaction, and sheds light on how the ecological theatre can affect the (co)evolutionary play.

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.

Propidium iodide enabled live imaging of Pasteuria sp.-Pratylenchus zeae infection studies under fluorescence microscopy.

Live imaging allows observations of cell structures and processes in real time, to monitor dynamic changes within living organisms compared to fixed organisms. Fluorescence microscopy was used to monitor the dynamic infection process of the nematode parasitic bacterium Pasteuria sp. and the sugarcane root-lesion nematode, Pratylenchus zeae. Under fluorescence microscopy, green-autofluorescent globules were observed in live control and Pasteuria sp.-infected nematodes. Only nematodes killed by Pasteuria sp. or heat treated displayed a diffuse pattern of autofluorescence. Propidium iodide (PI), used as a cell membrane integrity indicator, confirmed that the nematode's cuticle acts as an impermeable barrier. PI stained cells/DNA of heat-treated control and Pasteuria sp.-infected P. zeae. PI as a counterstain facilitated the location of Pasteuria endospores on the cuticle surface of P. zeae. No PI staining was observed in sporangia and in endospores within the nematode body. However, PI specifically stained endospores on the cuticle surface and within the cuticle carcass showing, in mature propagules, a ring-like pattern. Live imaging, combined with fluorescence microscopy and fluorescent dyes such as PI, appears useful in live studies on plant nematode interactions with nematophagous bacteria.

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.

Digest: Parasite adaptation to light alters transmission and fecundity†.

Natural variation as well as human impacts can alter the light environment in lakes in ways that affect aquatic host-parasite interactions. In laboratory infection assays, Rogalski and Duffy (2020) determine that the bacterial parasite Pasteuria ramosa adapts to solar radiation by increasing its transmission potential to its zooplankton host, Daphnia dentifera. Local adaptation to light can allow P. ramosa spores to retain their infectivity following light exposure. Future work should determine the underlying drivers of P. ramosa light adaptation and how adaptation might alter ecosystem dynamics.

Genome-Wide Association Analysis Identifies a Genetic Basis of Infectivity in a Model Bacterial Pathogen.

Knowledge of the genetic architecture of pathogen infectivity and host resistance is essential for a mechanistic understanding of coevolutionary processes, yet the genetic basis of these interacting traits remains unknown for most host-pathogen systems. We used a comparative genomic approach to explore the genetic basis of infectivity in Pasteuria ramosa, a Gram-positive bacterial pathogen of planktonic crustaceans that has been established as a model for studies of Red Queen host-pathogen coevolution. We sequenced the genomes of a geographically, phenotypically, and genetically diverse collection of P. ramosa strains and performed a genome-wide association study to identify genetic correlates of infection phenotype. We found multiple polymorphisms within a single gene, Pcl7, that correlate perfectly with one common and widespread infection phenotype. We then confirmed this perfect association via Sanger sequencing in a large and diverse sample set of P. ramosa clones. Pcl7 codes for a collagen-like protein, a class of adhesion proteins known or suspected to be involved in the infection mechanisms of a number of important bacterial pathogens. Consistent with expectations under Red Queen coevolution, sequence variation of Pcl7 shows evidence of balancing selection, including extraordinarily high diversity and absence of geographic structure. Based on structural homology with a collagen-like protein of Bacillus anthracis, we propose a hypothesis for the structure of Pcl7 and the physical location of the phenotype-associated polymorphisms. Our results offer strong evidence for a gene governing infectivity and provide a molecular basis for further study of Red Queen dynamics in this model host-pathogen system.

An alternative route of bacterial infection associated with a novel resistance locus in the Daphnia-Pasteuria host-parasite system.

To understand the mechanisms of antagonistic coevolution, it is crucial to identify the genetics of parasite resistance. In the Daphnia magna-Pasteuria ramosa host-parasite system, the most important step of the infection process is the one in which P. ramosa spores attach to the host's foregut. A matching-allele model (MAM) describes the host-parasite genetic interactions underlying attachment success. Here we describe a new P. ramosa genotype, P15, which, unlike previously studied genotypes, attaches to the host's hindgut, not to its foregut. Host resistance to P15 attachment shows great diversity across natural populations. In contrast to P. ramosa genotypes that use foregut attachment, P15 shows some quantitative variation in attachment success and does not always lead to successful infections, suggesting that hindgut attachment represents a less-efficient infection mechanism than foregut attachment. Using a Quantitative Trait Locus (QTL) approach, we detect two significant QTLs in the host genome: one that co-localizes with the previously described D. magna PR locus of resistance to foregut attachment, and a second, major QTL located in an unlinked genomic region. We find no evidence of epistasis. Fine mapping reveals a genomic region, the D locus, of ~13 kb. The discovery of a second P. ramosa attachment site and of a novel host-resistance locus increases the complexity of this system, with implications for both for the coevolutionary dynamics (e.g., Red Queen and the role of recombination), and for the evolution and epidemiology of the infection process.

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.

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.

Dissecting the genetic architecture of a stepwise infection process.

How a host fights infection depends on an ordered sequence of steps, beginning with attempts to prevent a pathogen from establishing an infection, through to steps that mitigate a pathogen's control of host resources or minimize the damage caused during infection. Yet empirically characterizing the genetic basis of these steps remains challenging. Although each step is likely to have a unique genetic and environmental signature, and may therefore respond to selection in different ways, events that occur earlier in the infection process can mask or overwhelm the contributions of subsequent steps. In this study, we dissect the genetic architecture of a stepwise infection process using a quantitative trait locus (QTL) mapping approach. We control for variation at the first line of defence against a bacterial pathogen and expose downstream genetic variability related to the host's ability to mitigate the damage pathogens cause. In our model, the water-flea Daphnia magna, we found a single major effect QTL, explaining 64% of the variance, that is linked to the host's ability to completely block pathogen entry by preventing their attachment to the host oesophagus; this is consistent with the detection of this locus in previous studies. In susceptible hosts allowing attachment, however, a further 23 QTLs, explaining between 5% and 16% of the variance, were mapped to traits related to the expression of disease. The general lack of pleiotropy and epistasis for traits related to the different stages of the infection process, together with the wide distribution of QTLs across the genome, highlights the modular nature of a host's defence portfolio, and the potential for each different step to evolve independently. We discuss how isolating the genetic basis of individual steps can help to resolve discussion over the genetic architecture of host resistance.

Pathogen exposure disrupts an organism's ability to cope with thermal stress.

As a result of global climate change, species are experiencing an escalation in the severity and regularity of extreme thermal events. With patterns of disease distribution and transmission predicted to undergo considerable shifts in the coming years, the interplay between temperature and pathogen exposure will likely determine the capacity of a population to persist under the dual threat of global change and infectious disease. In this study, we investigated how exposure to a pathogen affects an individual's ability to cope with extreme temperatures. Using experimental infections of Daphnia magna with its obligate bacterial pathogen Pasteuria ramosa, we measured upper thermal limits of multiple host and pathogen genotype combinations across the dynamic process of infection and under various forms (static and ramping) of thermal stress. We find that pathogens substantially limit the thermal tolerance of their host, with the reduction in upper thermal limits on par with the breadth of variation seen across similar species entire geographical ranges. The precise magnitude of any reduction, however, was specific to the host and pathogen genotype combination. In addition, as thermal ramping rate slowed, upper thermal limits of both healthy and infected individuals were reduced. Our results suggest that the capacity of a population to evolve new thermal limits, when also faced with the threat of infection, will depend not only on a host's genetic variability in warmer environments, but also on the frequency of host and pathogen genotypes. We suggest that pathogen-induced alterations of host thermal performance should be taken into account when assessing the resilience of any population and its potential for adaptation to global change.

Can pathogens optimize both transmission and dispersal by exploiting sexual dimorphism in their hosts?

Pathogens often rely on their host for dispersal. Yet, maximizing fitness via replication can cause damage to the host and an associated reduction in host movement, incurring a trade-off between transmission and dispersal. Here, we test the idea that pathogens might mitigate this trade-off between reproductive fitness and dispersal by taking advantage of sexual dimorphism in their host, tailoring responses separately to males and females. Using experimental populations of Daphnia magna and its bacterial pathogen Pasteuria ramosa as a test-case, we find evidence that this pathogen can use male hosts as a dispersal vector, and the larger females as high-quality resource patches for optimized production of transmission spores. As sexual dimorphism in dispersal and body size is widespread across the animal kingdom, this differential exploitation of the sexes by a pathogen might be an unappreciated phenomenon, possibly evolved in various systems.

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.