Disentangling the influence of parasite genotype, host genotype and maternal environment on different stages of bacterial infection in Daphnia magna

Individuals naturally vary in the severity of infectious disease when exposed to a parasite. Dissecting this variation into genetic and environmental components can reveal whether or not this variation depends on the host genotype, parasite genotype or a range of environmental conditions. Complicating this task, however, is that the symptoms of disease result from the combined effect of a series of events, from the initial encounter between a host and parasite, through to the activation of the host immune system and the exploitation of host resources. Here, we use the crustacean Daphnia magna and its parasite Pasteuria ramosa to show how disentangling genetic and environmental factors at different stages of infection improves our understanding of the processes shaping infectious disease. Using compatible host-parasite combinations, we experimentally exclude variation in the ability of a parasite to penetrate the host, from measures of parasite clearance, the reduction in host fecundity and the proliferation of the parasite. We show how parasite resistance consists of two components that vary in environmental sensitivity, how the maternal environment influences all measured aspects of the within-host infection process and how host-parasite interactions following the penetration of the parasite into the host have a distinct temporal component.     

Parasites do not adapt to elevated temperature,as evidenced from experimental evolution of a phytoplankton–fungus system

Global warming is predicted to impact the prevalence and severity of infectious diseases. However, empirical data supporting this statement usually stem from experiments in which parasite fitness and disease outcome are measured directly after temperature increase. This might exclude the possibility of parasite adaptation. To incorporate the adaptive response of parasites into predictions of disease severity in a warmer world, we undertook an experimental evolution assay in which a fungal parasite of phytoplankton was maintained at elevated or control temperatures for six months, corresponding to 100–200 parasite generations. Host cultures were maintained at the respective temperatures and provided as substrate, but were not under parasite pressure. A reciprocal infection experiment conducted after six-month serial passages revealed no evidence of parasite adaptation. In fact, parasite fitness at elevated temperatures was inferior in parasite populations reared at elevated temperatures compared with those maintained under control temperature. However, this effect was reversed after parasites were returned to control temperatures for a few (approx. 10) generations. The absence of parasite adaptation to elevated temperatures suggests that, in phytoplankton–fungus systems, disease outcome under global warming will be largely determined by both host and parasite thermal ecology.     

Molecular characterization of the 18S rDNA gene of an avian Hepatozoon reveals that it is closely related to Lankesterella

As a part of intensive study of blood parasite infections in a population of the passerine bird blue tit (Cyanistes caeruleus, Paridae), we detected a parasite species that, based on its morphological similarity, was tentatively identified as Hepatozoon parus, the only species of this parasite genus described from birds of this family. However, morphological measurements show that H. parus is slightly larger than the parasite detected in our population. A molecular characterization of the parasite species was conducted by amplification of the 18S rDNA gene, using primers that were reported previously to amplify in Hepatozoon sp. of water pythons. Additional primers were developed based on the new sequence obtained. The 1,484-bp fragment amplified reveals that the parasite from our bird population is more closely related to Lankesterella minima than to Hepatozoon species from other vertebrates according to analysis using the BLAST comparison method (93% identity). In addition, phylogenetic analyses using parsimony and Kimura procedures unequivocally related the parasite species detected by PCR with L. minima. The bootstrap values obtained were 97% and 100%, respectively. These results imply that this parasite is a species of a lankesterellid instead of Hepatozoon.     

No evidence for specificity between host and parasite genotypes in experimental Strongyloides ratti (Nematoda) infections

A key requirement for several theories involving the evolution of sex and sexual selection is a specificity between host and parasite genotypes, i.e. the resistance of particular host genotypes to particular parasite genotypes and the infectivity of particular parasite genotypes for particular host genotypes. Determining the scope and nature of any such specificity is also of applied relevance, since any specificity for different parasite genotypes to infect particular host genotypes may affect the level of protection afforded by vaccination, the efficacy of selective breeding of livestock for parasite resistance and the long-term evolution of parasite populations in response to these control measures. Whereas we have some evidence for the role of specificity between host and pathogen genotypes in viral and bacterial infections, its role in macroparasitic infections is seldom considered. The first empirical test of this specificity for a vertebrate–nematode system is provided here using clonal lines of parasite and inbred and congenic strains of rat that differ either across the genome or only at the major histocompatibility complex. Although significant differences between the resistance of host genotypes to infection and between the fitness of different parasite genotypes are found, there is no evidence for an interaction between host and parasite genotypes. It is concluded that a specificity between host and parasite genotypes is unlikely in this system.     

An alternative model for heme biosynthesis in the malarial parasite

The malarial parasite imports an infected host's red blood cell enzymes for heme biosynthesis during the intraerythrocytic stage. This is despite all the genes of the heme-biosynthetic pathway having been identified on the parasite genome. On the basis of predictions of parasite genome-coded enzyme localization, functionality of some of these enzymes and shuttling of intermediates between different parasite compartments, a hybrid model for parasite heme biosynthesis has been proposed. However, this model does not take into account the possible role of imported host enzymes in parasite heme biosynthesis. We propose an alternative model with an extrinsic heme-biosynthetic pathway in the parasite cytosol that uses imported host enzymes, and an intrinsic pathway confined to the organellar fractions that uses the parasite-genome-encoded enzymes.     

Importance of infection of haemosporidia blood parasites during different life history stages for long‐term reproductive fitness of collared flycatchers

The interaction between birds and haemosporidia blood parasites is a well‐used system in the study of parasite biology. However, where, when and how parasites are transmitted is often unclear and defining parasite transmission dynamics is essential because of how they influence parasite‐mediated costs to the host. In this study, we used cross‐sectional and longitudinal data taken from a collared flycatcher Ficedula albicollis population to investigate the temporal dynamics of haemosporidia parasite infection and parasite‐mediated costs to host fitness. We investigated host–parasite interactions starting at the nestling stage of the bird's life‐cycle and then followed their progress over three breeding attempts to quantify their fitness – measured as the number of offspring they produced that recruited back into the breeding population. We found that the majority of haemosporidia blood parasite infections occurred within the first year of life and that the most common parasite lineages that infected the breeding population also infected juvenile birds in the natal environment. Moreover, our findings suggest that collared flycatcher nestlings in poorer condition could be at a higher risk of haemosporidia blood parasite infection. In this study, only female and not male bird fitness was adversely affected by parasite infection and the cost of infection on female fitness depended on the timing of transmission. In conclusion, our study indicates that in collared flycatchers, early‐life is potentially important for many of the interactions with haemosporidia parasite lineages, and evidence of parasite‐mediated costs to fitness suggest that these parasites may have influenced the host population dynamics.     

Molecular analysis of a haplosporidian parasite from cultured New Zealand abalone Haliotis iris

In the Austral summer and autumn of 2000 and 2001, mortalities of black-footed abalone Haliotis iris (Martyn, 1784) occurred in a commercial facility in New Zealand. Histological analyses suggested that infection by a haplosporidian parasite was responsible. To confirm identification as a haplosporidian and to help determine if this parasite represented a new, undescribed species, DNA was extracted from infected host tissues scored as positive for infection by histological examination. Small-subunit rRNA (SSU rRNA) gene sequences from both the host abalone and a parasitic organism were amplified by PCR and characterized. Although the sequence for this parasite was novel, not matching any known SSU rRNA gene sequences, phylogenetic analyses strongly supported grouping this parasite with the haplosporidians. Parsimony analyses placed the parasite at the base of the phylum Haplosporidia, ancestral to Urosporidium crescens and the Haplosporidium, Bonamia, and Minchinia species. Sequencing of multiple parasite DNA clones revealed a single polymorphic site in the haplosporidian SSU rRNA gene sequence.     

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

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

How can immunopathology shape the evolution of parasite virulence?

Immunopathology (immune-mediated pathology) is a ubiquitous cause of disease during infection, but how will parasite exploitation strategies evolve in its presence? Immunopathology can act to increase parasite fitness if it increases transmission rate, but can equally act to decrease parasite fitness if it increases host mortality. The focus here is on understanding how immunopathology, mediated through different immune mechanisms, can influence parasite fitness and how experimental manipulations of the immune system can be carried out to examine this. A better understanding of how parasite fitness scales with, or responds to, immunopathology is crucial to understanding the nature of selection acting on parasite virulence traits and will allow more informed predictions to be made regarding the trajectory of parasite virulence evolution.     

The impact of clonalty on parasite population genetic structure

In this paper, we briefly review the consequences of clonal reproduction on the apportionment of genetic diversity in parasite populations. We distinguish three kinds of parasite lifecycle where clonal reproduction occurs. The consequences of this mode of reproduction for the different kinds of parasite life-cycles are described. We here particularly focus on clonal diploids.     

Asexual and Sexual Stages of a Malaria Parasite in the Thrombocytes of Tropidurus torquatus (Iguanidae) Infected with Plasmodium tropiduri*

SYNOPSIS. Sexual and asexual stages of a parasite found in the thrombocyte-like cells of some Tropidurus torquatus infected with Plasmodium tropiduri are described. The strong ultrastructural similarities between the gametocytes of this parasite and the gametocytes of P. tropiduri, and the finding of this parasite in lizards inoculated with P. tropiduri suggest that this malaria parasite can develop both in erythrocytes and thrombocytes. Evidence in favor of this hypothesis is discussed.     

The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation

Numerous parasites with complex life cycles are able to manipulate the behaviour of their intermediate host in a way that increases their trophic transmission to the definitive host. Pomphorhynchus laevis, an acanthocephalan parasite, is known to reverse the phototactic behaviour of its amphipod intermediate host, Gammarus pulex, leading to an increased predation by fish hosts. However, levels of behavioural manipulation exhibited by naturally-infected gammarids are extremely variable, with some individuals being strongly manipulated whilst others are almost not affected by infection. To investigate parasite age and parasite intensity as potential sources of this variation, we carried out controlled experimental infections on gammarids using parasites from two different populations. We first determined that parasite intensity increased with exposure dose, but found no relationship between infection and host mortality. Repeated measures confirmed that the parasite alters host behaviour only when it reaches the cystacanth stage which is infective for the definitive host. They also revealed, we believe for the first time, that the older the cystacanth, the more it manipulates its host. The age of the parasite is therefore a major source of variation in parasite manipulation. The number of parasites within a host was also a source of variation. Manipulation was higher in hosts infected by two parasites than in singly infected ones, but above this intensity, manipulation did not increase. Since the development time of the parasite was also different according to parasite intensity (it was longer in doubly infected hosts than in singly infected ones, but did not increase more in multi-infected hosts), individual parasite fitness could depend on the compromise between development time and manipulation efficiency. Finally, the two parasite populations tested induced slightly different degrees of behavioural manipulation.     

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.     

Meta-analysis and research on host–parasite interactions: past and future

Host–parasite interactions are characterised by a lack of stable species-specific traits that limits generalisations one can make even about particular host or parasite species. For instance, the virulence, life history traits or transmission mode of a given parasite species can depend on which of its suitable hosts it infects. In the search for general rules or patterns, meta-analysis provides a possible solution to the challenges posed by the highly variable outcomes of host–parasite interactions. It allows an estimate of the overall association between any factor and its biological response that transcends the particulars of given host and parasite taxonomic combinations. In this review, we begin with a historical overview of the use of meta-analysis in research on the ecology and evolution of host–parasite interactions. We then identify several key conceptual advances that were made possible only through meta-analytical synthesis. For example, meta-analysis revealed the predominant association between rates of host and parasite gene flow and local adaptation, as well as an unexpected latitudinal gradient in parasite virulence, or parasite-induced host mortality. Finally, we propose some areas of research on host–parasite interactions that are based on a mature theoretical foundation and for which there now exist sufficient primary results to make them ripe for meta-analysis. The search for the processes causing variability in parasite species richness among host species, and the link between the expression of host resistance and the specificity of parasites, are two such research areas. The main objective of this review is to promote meta-analysis as a synthetic tool overriding the idiosyncrasies of specific host–parasite combinations and capable of uncovering the universal trends, if any, in the evolutionary ecology of parasitism.     

Diet quality determines interspecific parasite interactions in host populations

The widespread occurrence of multiple infections and the often vast range of nutritional resources for their hosts allow that interspecific parasite interactions in natural host populations might be determined by host diet quality. Nevertheless, the role of diet quality with respect to multispecies parasite interactions on host population level is not clear. We here tested the effect of host population diet quality on the parasite community in an experimental study using Daphnia populations. We studied the effect of diet quality on Daphnia population demography and the interactions in multispecies parasite infections of this freshwater crustacean host. The results of our experiment show that the fitness of a low‐virulent microsporidian parasite decreased in low, but not in high‐host‐diet quality conditions. Interestingly, infections with the microsporidium protected Daphnia populations against a more virulent bacterial parasite. The observed interspecific parasite interactions are discussed with respect to the role of diet quality‐dependent changes in host fecundity. This study reflects that exploitation competition in multispecies parasite infections is environmentally dependent, more in particular it shows that diet quality affects interspecific parasite competition within a single host and that this can be mediated by host population‐level effects.     

Functional genomics of Plasmodium falciparum through transposon-mediated mutagenesis

Plasmodium falciparum is the causative agent for the most lethal form of human malaria, killing millions annually. Genetic analyses of P. falciparum have been relatively limited due to the lack of robust techniques to manipulate this parasite. Development of transfection technologies and whole genome analyses have helped in understanding the complex biology of this parasite. Even with this wealth of information functional genomics approaches are still very limited in P. falciparum due to the cumbersome and inefficient methods of genetic manipulation. This review focuses on a recently developed, highly efficient method for transposon-based mutagenesis and transgene expression in P. falciparum that will allow functional genomics studies to be performed proficiently on this deadly malaria parasite. By using a piggyBac-based transposition system, multiple random integrations have been obtained into the genome of the parasite. This technique could hence be employed to set up several biological screens in this lethal protozoan parasite that may lead to identification of novel drug targets and vaccine candidates.     

Short-term in vitro culture of field isolates of Plasmodium vivax using umbilical cord blood

Plasmodium vivax research has been hampered by the lack of technology for culturing this parasite. Culturing P. vivax is difficult because the parasite selectively invades reticulocytes. Here we describe a modified procedure to establish and maintain short-term cultures of freshly collected P. vivax parasites using reticulocyte-enriched cord blood. Using this method, parasites could be cultured for a month. Manipulation of the culture allowed procurement of synchronized stages of the parasite. This short-term culture method can be easily adapted to study various aspects of the parasite biology.     

Partitioning average competition and extreme-genotype effects in genetically diverse infections

Competition between parasite genotypes in genetically diverse infections is widespread. However, experimental evidence on how genetic diversity influences total parasite load is variable. Here we use an additive partition equation to quantify the negative effect of inter-genotypic competition on total parasite load in diverse infections. Our approach controls for extreme-genotype effects, a process that can potentially neutralise, or even reverse, the negative effect of competition on total parasite load. A single extreme-genotype can have a disproportionate effect on total parasite load if it causes the highest parasite load in its single-infection, while increasing its performance in diverse relative to single infections. We show that in theory such disproportionate effects of extreme-genotypes can lead to a higher total parasite load in diverse infections than expected, even if competition reduces individual parasite performance on average. Controlling for the extreme-genotype effect is only possible if the competition effect on total parasite load is measured appropriately as the average difference between the realised number of each parasite genotype in mixed infections and the expected number based on single infection parasite loads. We apply this approach to sticklebacks that were experimentally infected with different trematode genotypes. On average, genetically diverse infections had lower parasite loads than expected from single-infection results. For the first time we demonstrate that competition between co-infecting genotypes per se caused the parasite load reduction, while extreme-genotype effects were not significant. We thus suggest that to correctly quantify the effect of competition alone on total parasite load in genetically diverse infections, the extreme-genotype effect has to be controlled for.     

Parasites, emerging disease and wildlife conservation

In this review some emerging issues of parasite infections in wildlife, particularly in Australia, are considered. We discuss the importance of understanding parasite biodiversity in wildlife in terms of conservation, the role of wildlife as reservoirs of parasite infection, and the role of parasites within the broader context of the ecosystem. Using a number of parasite species, the value of undertaking longitudinal surveillance in natural systems using non-invasive sampling and molecular tools to characterise infectious agents is illustrated in terms of wildlife health, parasite biodiversity and ecology.     

Temperature-dependent costs of parasitism and maintenance of polymorphism under genotype-by-environment interactions

The maintenance of genetic variation for infection-related traits is often attributed to coevolution between hosts and parasites, but it can also be maintained by environmental variation if the relative fitness of different genotypes changes with environmental variation. To gain insight into how infection-related traits are sensitive to environmental variation, we exposed a single host genotype of the freshwater crustacean Daphnia magna to four parasite isolates (which we assume to represent different genotypes) of its naturally co-occurring parasite Pasteuria ramosa at 15, 20 and 25 degrees C. We found that the cost to the host of becoming infected varied with temperature, but the magnitude of this cost did not depend on the parasite isolate. Temperature influenced parasite fitness traits; we found parasite genotype-by-environment (G x E) interactions for parasite transmission stage production, suggesting the potential for temperature variation to maintain genetic variation in this trait. Finally, we tested for temperature-dependent relationships between host and parasite fitness traits that form a key component of models of virulence evolution, and we found them to be stable across temperatures.