Long-term balancing selection for pathogen resistance maintains trans-species polymorphisms in a planktonic crustacean.

Balancing selection is an evolutionary process that maintains genetic polymorphisms at selected loci and strongly reduces the likelihood of allele fixation. When allelic polymorphisms that predate speciation events are maintained independently in the resulting lineages, a pattern of trans-species polymorphisms may occur. Trans-species polymorphisms have been identified for loci related to mating systems and the MHC, but they are generally rare. Trans-species polymorphisms in disease loci are believed to be a consequence of long-term host-parasite coevolution by balancing selection, the so-called Red Queen dynamics. Here we scan the genomes of three crustaceans with a divergence of over 15 million years and identify 11 genes containing identical-by-descent trans-species polymorphisms with the same polymorphisms in all three species. Four of these genes display molecular footprints of balancing selection and have a function related to immunity. Three of them are located in or close to loci involved in resistance to a virulent bacterial pathogen, Pasteuria, with which the Daphnia host is known to coevolve. This provides rare evidence of trans-species polymorphisms for loci known to be functionally relevant in interactions with a widespread and highly specific parasite. These findings support the theory that specific antagonistic coevolution is able to maintain genetic diversity over millions of years.

The Role of Microbiome and Genotype in Daphnia magna upon Parasite Re-Exposure.

Recently, it has been shown that the community of gut microorganisms plays a crucial role in host performance with respect to parasite tolerance. Knowledge, however, is lacking on the role of the gut microbiome in mediating host tolerance after parasite re-exposure, especially considering multiple parasite infections. We here aimed to fill this knowledge gap by studying the role of the gut microbiome on tolerance in Daphnia magna upon multiple parasite species re-exposure. Additionally, we investigated the role of the host genotype in the interaction between the gut microbiome and the host phenotypic performance. A microbiome transplant experiment was performed in which three germ-free D. magna genotypes were exposed to a gut microbial inoculum and a parasite community treatment. The gut microbiome inocula were pre-exposed to the same parasite communities or a control treatment. Daphnia performance was monitored, and amplicon sequencing was performed to characterize the gut microbial community. Our experimental results showed that the gut microbiome plays no role in Daphnia tolerance upon parasite re-exposure. We did, however, find a main effect of the gut microbiome on Daphnia body size reflecting parasite specific responses. Our results also showed that it is rather the Daphnia genotype, and not the gut microbiome, that affected parasite-induced host mortality. Additionally, we found a role of the genotype in structuring the gut microbial community, both in alpha diversity as in the microbial composition.

Oligosaccharides derived from dragon fruit modulate gut microbiota, reduce oxidative stress and stimulate toll-pathway related gene expression in freshwater crustacean Daphnia magna.

Dragon fruit oligosaccharide (DFO) is an indigestible prebiotic. In this study, we aimed to investigate the effects of DFO on gut microbiota, oxidative stress and immune-related gene expression in Daphnia magna. The 10-day-old D. magna were treated with 0, 9, and 27 mg l-1 DFO for 85 h. The gut bacterial communities, superoxide dismutase (SOD) activity, lipid peroxidation and the expressions of genes in Toll signaling pathway were observed. The results showed that D. magna treated with 9 and 27 mg l-1 DFO altered gut microbiota composition by increasing Limnohabitans and Lactobacillus, and significantly increased SOD activity and reduced lipid peroxidation. Moreover, the expressions of Toll2, Toll3, Toll5, Toll7 and Pelle genes were significantly increased in D. magna treated with 9 and 27 mg l-1 DFO. Our results suggested that DFO changed the composition of the gut microbiota of D. magna by increasing the beneficial bacteria. DFO also had the ability to stimulate innate immunity in D. magna by increasing SOD activity, reducing lipid peroxidation, and increasing the expression of immune-related genes.

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.

Genetic resistance and specificity in sister taxa of Daphnia: insights from the range of host susceptibilities.

BACKGROUND: Host genetic diversity can affect various aspects of host-parasite interactions, including individual-level effects on parasite infectivity, production of transmission stages and virulence, as well as population-level effects that reduce disease spread and prevalence, and buffer against widespread epidemics. However, a key aspect of this diversity, the genetic variation in host susceptibility, has often been neglected in interpreting empirical data and in theoretical studies. Daphnia similis naturally coexists with its competitor Daphnia magna and is more resistant to the endoparasitic microsporidium Hamiltosporidium tvaerminnensis, as suggested by a previous survey of waterbodies, which detected this parasite in D. magna, but not in D. similis. However, under laboratory conditions D. similis was sometimes found to be susceptible. We therefore asked if there is genetic variation for disease trait expression, and if the genetic variation in disease traits in D. similis is different from that of D. magna. METHODS: We exposed ten clones of D. similis and ten clones of D. magna to three isolates of H. tvaerminnensis, and measured infection rates, parasite-induced host mortality and parasite spore production. RESULTS: The two Daphnia species differ in the range and variation of their susceptibilities. The parasite produced on average two-fold more spores when growing in D. magna clones than in D. similis clones. CONCLUSIONS: We confirm that D. similis is indeed much more resistant than D. magna and suggest that this could create a dilution effect in habitats where both species coexist.

Variation in Immune Defense Shapes Disease Outcomes in Laboratory and Wild Daphnia.

Host susceptibility may be critical for the spread of infectious disease, and understanding its basis is a goal of ecological immunology. Here, we employed a series of mechanistic tests to evaluate four factors commonly assumed to influence host susceptibility: parasite exposure, barriers to infection, immune responses, and body size. We tested these factors in an aquatic host-parasite system (Daphnia dentifera and the fungal parasite, Metschnikowia bicuspidata) using both laboratory-reared and field-collected hosts. We found support for each factor as a driver of infection. Elevated parasite exposure, which occurs through consumption of infectious fungal spores, increased a host's probability of infection. The host's gut epithelium functioned as a barrier to infection, but in the opposite manner from which we predicted: thinner anterior gut epithelia were more resistant to infectious spores than thick epithelia. This relationship may be mediated by structural attributes associated with epithelial cell height. Fungal spores that breached the host's gut barrier elicited an intensity-dependent hemocyte response that decreased the probability of infection for some Daphnia. Although larger body sizes were associated with increased levels of spore ingestion, larger hosts also had lower frequencies of parasite attack, less penetrable gut barriers, and stronger hemocyte responses. After investigating which mechanisms underlie host susceptibility, we asked: do these four factors contribute equally or asymmetrically to the outcome of infection? An information-theoretic approach revealed that host immune defenses (barriers and immune responses) played the strongest roles in mediating infection outcomes. These two immunological traits may be valuable metrics for linking host susceptibility to the spread of infectious disease.

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.

Phenotypic plasticity in three Daphnia genotypes in response to predator kairomone: evidence for an involvement of chitin deacetylases.

The genetic background of inducible morphological defences in Daphnia is still largely unknown. Dissolved infochemicals from the aquatic larvae of the phantom midge Chaoborus induce so-called 'neck-teeth' in the first three post-embryonic stages of Daphnia pulex This defence has become a textbook example of inducible defences. In a target gene approach, by using three Daphnia genotypes which show a gradient of neck-teeth induction in response to equal amounts of kairomone, we report a high correlation of neck-teeth induction in Daphnia pulex and relative gene expression of two chitin deacetylases. Further, previous studies suggested genes from both the juvenoid and the insulin hormone signalling pathways as well as several morphogenetic genes downstream to be responsible for neck-teeth induction in D. pulex However, these data were not supported by our study. None of the three D. pulex clones showed an upregulation of these previously proposed candidate genes as a response to predator kairomone, which is interpreted as the result of refined methods used for both RNA sampling and kairomone enrichment yielding unambiguous results compared with earlier studies. The assessment of a clonal gradient of Daphnia in the presence and absence of infochemicals provides a promising approach to identify further genes involved in the induction of morphological defences by correlating gene expression and morphology.

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna.

To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 µg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna.

The Red Queen lives: Epistasis between linked resistance loci.

A popular theory explaining the maintenance of genetic recombination (sex) is the Red Queen Theory. This theory revolves around the idea that time-lagged negative frequency-dependent selection by parasites favors rare host genotypes generated through recombination. Although the Red Queen has been studied for decades, one of its key assumptions has remained unsupported. The signature host-parasite specificity underlying the Red Queen, where infection depends on a match between host and parasite genotypes, relies on epistasis between linked resistance loci for which no empirical evidence exists. We performed 13 genetic crosses and tested over 7000 Daphnia magna genotypes for resistance to two strains of the bacterial pathogen Pasteuria ramosa. Results reveal the presence of strong epistasis between three closely linked resistance loci. One locus masks the expression of the other two, while these two interact to produce a single resistance phenotype. Changing a single allele on one of these interacting loci can reverse resistance against the tested parasites. Such a genetic mechanism is consistent with host and parasite specificity assumed by the Red Queen Theory. These results thus provide evidence for a fundamental assumption of this theory and provide a genetic basis for understanding the Red Queen dynamics in the Daphnia-Pasteuria system.

Infections by Pasteuria do not protect its natural host Daphnia magna from subsequent infections.

The existence of immunological memory in invertebrates remains a contentious topic. Exposure of Daphnia magna crustaceans to a noninfectious dose of the bacterium Pasteuria ramosa has been reported to reduce the chance of future infection upon exposure to higher doses. Using clonal hosts and parasites, we tested whether initial exposure of the host to the parasite (priming), followed by clearing of the parasite with antibiotic, protects the host from a second exposure (challenge). Our experiments included three treatments: priming and challenge with the same or with a different parasite clone, or no priming. Two independent experiments showed that both the likelihood of infection and the degree of parasite proliferation did not differ between treatments, supporting the conclusion that there is no immunological memory in this system. We discuss the possibility that previous discordant reports could result from immune or stress responses that did not fade following initial priming.

The development of pathogen resistance in Daphnia magna: implications for disease spread in age-structured populations.

Immunity in vertebrates is well established to develop with time, but the ontogeny of defence in invertebrates is markedly less studied. Yet, age-specific capacity for defence against pathogens, coupled with age structure in populations, has widespread implications for disease spread. Thus, we sought to determine the susceptibility of hosts of different ages in an experimental invertebrate host-pathogen system. In a series of experiments, we show that the ability of Daphnia magna to resist its natural bacterial pathogen Pasteuria ramosa changes with host age. Clonal differences make it difficult to draw general conclusions, but the majority of observations indicate that resistance increases early in the life of D. magna, consistent with the idea that the defence system develops with time. Immediately following this, at about the time when a daphnid would be most heavily investing in reproduction, resistance tends to decline. Because many ecological factors influence the age structure of Daphnia populations, our results highlight a broad mechanism by which ecological context can affect disease epidemiology. We also show that a previously observed protective effect of restricted maternal food persists throughout the entire juvenile period, and that the protective effect of prior treatment with a small dose of the pathogen ('priming') persists for 7 days, observations that reinforce the idea that immunity in D. magna can change over time. Together, our experiments lead us to conclude that invertebrate defence capabilities have an ontogeny that merits consideration with respect to both their immune systems and the epidemic spread of infection.

Effects of juvenile host density and food availability on adult immune response, parasite resistance and virulence in a Daphnia-parasite system.

Host density can increase infection rates and reduce host fitness as increasing population density enhances the risk of becoming infected either through increased encounter rate or because host condition may decline. Conceivably, potential hosts could take high host density as a cue to up-regulate their defence systems. However, as host density usually covaries with food availability, it is difficult to examine the importance of host density in isolation. Thus, we performed two full-factorial experiments that varied juvenile densities of Daphnia magna (a freshwater crustacean) and food availability independently. We also included a simulated high-density treatment, where juvenile experimental animals were kept in filtered media that previously maintained Daphnia at high-density. Upon reaching adulthood, we exposed the Daphnia to their sterilizing bacterial parasite, Pasteuria ramosa, and examined how the juvenile treatments influenced the likelihood and severity of infection (Experiment I) and host immune investment (Experiment II). Neither juvenile density nor food treatments affected the likelihood of infection; however, well-fed hosts that were well-fed as juveniles produced more offspring prior to sterilization than their less well-fed counterparts. By contrast, parasite growth was independent of host juvenile resources or host density. Parasite-exposed hosts had a greater number of circulating haemocytes than controls (i.e., there was a cellular immune response), but the magnitude of immune response was not mediated by food availability or host density. These results suggest that density dependent effects on disease arise primarily through correlated changes in food availability: low food could limit parasitism and potentially curtail epidemics by reducing both the host's and parasite's reproduction as both depend on the same food.

A matching-allele model explains host resistance to parasites.

The maintenance of genetic variation and sex despite its costs has long puzzled biologists. A popular idea, the Red Queen Theory, is that under rapid antagonistic coevolution between hosts and their parasites, the formation of new rare host genotypes through sex can be advantageous as it creates host genotypes to which the prevailing parasite is not adapted. For host-parasite coevolution to lead to an ongoing advantage for rare genotypes, parasites should infect specific host genotypes and hosts should resist specific parasite genotypes. The most prominent genetics capturing such specificity are matching-allele models (MAMs), which have the key feature that resistance for two parasite genotypes can reverse by switching one allele at one host locus. Despite the lack of empirical support, MAMs have played a central role in the theoretical development of antagonistic coevolution, local adaptation, speciation, and sexual selection. Using genetic crosses, we show that resistance of the crustacean Daphnia magna against the parasitic bacterium Pasteuria ramosa follows a MAM. Simulation results show that the observed genetics can explain the maintenance of genetic variation and contribute to the maintenance of sex in the facultatively sexual host as predicted by the Red Queen Theory.

The cellular immune response of Daphnia magna under host-parasite genetic variation and variation in initial dose.

In invertebrate-parasite systems, the likelihood of infection following parasite exposure is often dependent on the specific combination of host and parasite genotypes (termed genetic specificity). Genetic specificity can maintain diversity in host and parasite populations and is a major component of the Red Queen hypothesis. However, invertebrate immune systems are thought to only distinguish between broad classes of parasite. Using a natural host-parasite system with a well-established pattern of genetic specificity, the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa, we found that only hosts from susceptible host-parasite genetic combinations mounted a cellular response following exposure to the parasite. These data are compatible with the hypothesis that genetic specificity is attributable to barrier defenses at the site of infection (the gut), and that the systemic immune response is general, reporting the number of parasite spores entering the hemocoel. Further supporting this, we found that larger cellular responses occurred at higher initial parasite doses. By studying the natural infection route, where parasites must pass barrier defenses before interacting with systemic immune responses, these data shed light on which components of invertebrate defense underlie genetic specificity.

Fecundity compensation and tolerance to a sterilizing pathogen in Daphnia.

Hosts are armed with several lines of defence in the battle against parasites: they may prevent the establishment of infection, reduce parasite growth once infected or persevere through mechanisms that reduce the damage caused by infection, called tolerance. Studies on tolerance in animals have focused on mortality, and sterility tolerance has not been investigated experimentally. Here, we tested for genetic variation in the multiple steps of defence when the invertebrate Daphnia magna is infected with the sterilizing bacterial pathogen Pasteuria ramosa: anti-infection resistance, anti-growth resistance and the ability to tolerate sterilization once infected. When exposed to nine doses of a genetically diverse pathogen inoculum, six host genotypes varied in their average susceptibility to infection and in their parasite loads once infected. How host fecundity changed with increasing parasite loads did not vary between genotypes, indicating that there was no genetic variation for this measure of fecundity tolerance. However, genotypes differed in their level of fecundity compensation under infection, and we discuss how, by increasing host fitness without targeting parasite densities, fecundity compensation is consistent with the functional definition of tolerance. Such infection-induced life-history shifts are not traditionally considered to be part of the immune response, but may crucially reduce harm (in terms of fitness loss) caused by disease, and are a distinct source of selection on pathogens.

Daphnia magna shows reduced infection upon secondary exposure to a pathogen.

Previous pathogen exposure is an important predictor of the probability of becoming infected. This is deeply understood for vertebrate hosts, and increasingly so for invertebrate hosts. Here, we test if an initial pathogen exposure changes the infection outcome to a secondary pathogen exposure in the natural host-pathogen system Daphnia magna and Pasteuria ramosa. Hosts were initially exposed to an infective pathogen strain, a non-infective pathogen strain or a control. The same hosts underwent a second exposure, this time to an infective pathogen strain, either immediately after the initial encounter or 48 h later. We observed that an initial encounter with a pathogen always conferred protection against infection compared with controls.

Immune genes undergo more adaptive evolution than non-immune system genes in Daphnia pulex.

BACKGROUND: Understanding which parts of the genome have been most influenced by adaptive evolution remains an unsolved puzzle. Some evidence suggests that selection has the greatest impact on regions of the genome that interact with other evolving genomes, including loci that are involved in host-parasite co-evolutionary processes. In this study, we used a population genetic approach to test this hypothesis by comparing DNA sequences of 30 putative immune system genes in the crustacean Daphnia pulex with 24 non-immune system genes. RESULTS: In support of the hypothesis, results from a multilocus extension of the McDonald-Kreitman (MK) test indicate that immune system genes as a class have experienced more adaptive evolution than non-immune system genes. However, not all immune system genes show evidence of adaptive evolution. Additionally, we apply single locus MK tests and calculate population genetic parameters at all loci in order to characterize the mode of selection (directional versus balancing) in the genes that show the greatest deviation from neutral evolution. CONCLUSIONS: Our data are consistent with the hypothesis that immune system genes undergo more adaptive evolution than non-immune system genes, possibly as a result of host-parasite arms races. The results of these analyses highlight several candidate loci undergoing adaptive evolution that could be targeted in future studies.

Candidate innate immune system gene expression in the ecological model Daphnia.

The last ten years have witnessed increasing interest in host-pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host-pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia-pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia-Pasteuria system will need to balance a candidate gene approach with more comprehensive approaches to de novo identify immune system genes specific to the Daphnia-Pasteuria interaction.

Phenoloxidase but not lytic activity reflects resistance against Pasteuria ramosa in Daphnia magna.

The field of ecological immunology strongly relies on indicators of immunocompetence. Two major indicators in invertebrates, the activity of phenoloxidase (PO) and lytic activity have recently been questioned in studies showing that, across a natural range of baseline levels, these indicators did not predict resistance against a manipulated challenge with natural parasites. We confirmed this finding by showing that baseline levels of PO and lytic activity in the host Daphnia magna were not related to spore load of the parasite Pasteuria ramosa. Yet, PO levels in infected hosts did predict spore load, indicating PO activity can be useful as an indicator of immunocompetence in this model parasite-host system.