A quantitative in vitro cultivation technique to determine cell number and growth rates in strains of Crithidia bombi (Trypanosomatidae), a parasite of bumblebees

The protozoan parasite Crithidia bombi and its host, the bumblebee Bombus terrestris, are used as a model system for the study of the evolutionary ecology of host-parasite interactions. In order to study these interactions we established a method for in vitro cultivation of single parasite strains. Additionally, a high-throughput method is developed for the determination of cell numbers in cultures by means of optical density (OD) measurements. The protocol for in vitro cultivation allowed for growing different strains on agar plates as well as in culture medium. A calibration curve for the relationship between cell number and OD has been developed. Subsequently, growth rates for different genotypes of C. bombi have been recorded. Significant differences in the growth rates and generation times between these genotypes were demonstrated. As this might be related to the virulence of the parasite, this relationship may be confirmed by in vivo growth rate determination. In comparison with conventional cell counting, the application of OD measurements allows for high-throughput experiments as the time taken to record each sample is reduced by a factor of 30. The in vitro cultivation method allows for controlled infection experiments in order to study host-parasite interactions.     

Robustness of the outcome of adult bumblebee infection with a trypanosome parasite after varied parasite exposures during larval development

The outcome of defence by the invertebrate immunity has recently been shown to be more complex than previously thought. In particular, the outcome is affected by biotic and abiotic environmental variation, host genotype, parasite genotype and their interaction. Knowledge of conditions under which environmental variation affects the outcome of an infection is one important question that relates to this complexity. We here use the model system of the bumblebee, Bombus terrestris, infected by the trypanosome, Crithidia bombi, combined with a split-colony design to test the influence of the parasite environment during larval rearing on adult resistance. We find that genotype-specific interactions are maintained and adult resistance is not influenced. This demonstrates that environmental dependence of bumblebee-trypanosome interactions is not ubiquitous, and yet unknown constraints will maintain standard coevolutionary dynamics under such environmental deviations.     

Patterns of parasite infection in bumble bees (Bombus spp.) of Northern Virginia

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Food-environment mediates the outcome of specific interactions between a bumblebee and its trypanosome parasite

Specific host-parasite interactions, where the outcome of exposure to a parasite depends upon the genotypic identity of both parties, have implications for understanding host-parasite coevolution and patterns of genetic diversity. Thus, grasping the extent to which these interactions are mediated by environmental changes in a spatially and temporally heterogeneous world is vital. In this study, it is shown that the environment can influence specific host-parasite interactions in the well-studied system of the bumblebee Bombus terrestris and its trypanosome parasite Crithidia bombi. Naturally relevant variation in the quality of the food environment formed a three-way interaction with both host and parasite identity in determining the outcome of infection, with regard to the resistance of the host and the transmission of the parasite. The demonstration of such a host-genotype by parasite-genotype by environment interaction (G(H) x G(P) x E) shows the importance of considering environmental variation when investigating host-parasite interactions. Moreover, such interactions may to some extent explain levels of genetic diversity in natural host-parasite systems owing to the fact that they will create selection mosaics when environments are heterogeneous.     

Relative number of generations of hosts and parasites does not influence parasite local adaptation in coevolving populations of bacteria and phages

A potential consequence of host-parasite coevolution in spatially structured populations is parasite local adaptation: local parasites perform better than foreign parasites on their local host populations. It has been suggested that the generally shorter generation times of parasites compared with their hosts contributes to parasites, rather than hosts, being locally adapted. We tested the hypothesis that relative generation times of hosts and parasites affect local adaptation of hosts and parasites, using the bacterium Pseudomonas fluorescens and a lytic phage as host and parasite, respectively. Generation times were not directly manipulated, but instead one of the coevolving partners was regularly removed and replaced with a population from an earlier time point. Thus, one partner underwent more generations than the other. Manipulations were carried out at both early and later periods of coevolutionary interactions. At early stages of coevolution, host and parasites that underwent relatively more generations displayed higher levels of resistance and infectivity, respectively. However, the relative number of generations that bacteria and phages underwent did not change the level of local adaptation relative to control populations. This is likely because generalist hosts and parasites are favoured during early stages of coevolution, preventing local adaptation. By contrast, at later stages manipulations had no effect on either average levels of resistance or infectivity, or alter the level of local adaptation relative to the controls, possibly because traits other than resistance and infectivity were under strong selection. Taken together, these data suggest that the relative generation times of hosts and parasites may not be an important determinant of local adaptation in this system.     

Dispersal in a parasitic worm and its two hosts: consequence for local adaptation

Characterizing host and parasite population genetic structure and estimating gene flow among populations is essential for understanding coevolutionary interactions between hosts and parasites. We examined the population genetic structure of the trematode Schistosoma mansoni and its two host species (the definitive host Rattus rattus and the intermediate host Biomphalaria glabrata) using microsatellite markers. Parasites were sampled from rats. The study was conducted in five sites of the Guadeloupe Island, Lesser Antilles. Mollusks display a pattern of isolation by distance whereas such a pattern is not found neither in schistosomes nor in rats. The comparison of the distribution of genetic variability in S. mansoni and its two host species strongly suggests that migration of parasites is principally determined by that of the vertebrate host in the marshy focus of Guadeloupe. However, the comparison between genetic differentiation values in schistosomes and rats suggests that the efficacy of the schistosome rat-mediated dispersal between transmission sites is lower than expected given the prevalence, parasitic load and migration rate of rats among sites. This could notably suggest that rat migration rate could be negatively correlated to the age or the infection status of individuals. Models made about the evolution of local adaptation in function of the dispersal rates of hosts and parasites suggest that rats and mollusks should be locally adapted to their parasites.     

Complete rRNA gene sequences reveal that the microsporidium Nosema bombi infects diverse bumblebee (Bombus spp.) hosts and contains multiple polymorphic sites

Characterisation of microsporidian species and differentiation among genetic variants of the same species has typically relied on ribosomal RNA (rRNA) gene sequences. We characterised the entire rRNA gene of a microsporidium from 11 isolates representing eight different European bumblebee (Bombus) species. We demonstrate that the microsporidium Nosema bombi infected all hosts that originated from a wide geographic area. A total of 16 variable sites (all single nucleotid polymorphisms (SNPs)) was detected in the small subunit (SSU) rRNA gene and 42 (39 SNPs and 3 indels) in the large subunit (LSU) rRNA sequence. Direct sequencing of PCR-amplified DNA products of the internal transcribed spacer (ITS) region revealed identical sequences in all isolates. In contrast, ITS fragment length determined by PAGE and sequencing of cloned amplicons gave better resolution of sequences and revealed multiple SNPs across isolates and two fragment sizes in each isolate (six short and seven long amplicon variants). Genetic variants were not unique to individual host species. Moreover, two or more sequence variants were obtained from individual bumblebee hosts, suggesting the existence of multiple, variable copies of rRNA in the same microsporidium, and contrary to that expected for a class of multi-gene family under concerted evolution theory. Our data on within-genome rRNA variability call into question the usefulness of rRNA sequences to characterise intraspecific genetic variants in the Microsporidia and other groups of unicellular organisms.     

Genetic and morphometric variation in the Holarctic helminth parasite Andrya arctica (Cestoda, Anoplocephalidae) in relation to the divergence of its lemming hosts (Dicrostonyx spp.)

Andrya arctica is a cestode parasite of the family Anoplocephalidae (Cyclophyllidea), parasitizing lemmings of the genus Dicrostonyx throughout the Holarctic region. The population structure of this intestinal parasite was studied from eight different regions, six of which represented different genetic entities of lemming hosts. Molecular sequence tagged site markers and minisatellite fingerprints as well as morphology and morphometries were used to reveal the population structure of A. arctica in the Holarctic region. The results suggest that the evolutionary history of this cestode species has included different processes acting on different geographical regions. On the Siberian mainland (host D. torquatus), the division of the parasites into different genetic entities agreed perfectly with the chromosomal races of the lemming hosts that points towards a shared evolutionary history between the host and the parasite ('cospeciation'). The main phylogenetic split of Dicrostonyx between Eurasia and North America was not, however, observed in A. arctica. This suggests that in the Nearctic (host D. groenlandicus) the parasite has remained relatively unmodified because of the large cohesive populations ('coadaptation'). The uniqueness of the Greenland population, and possibly also that of the Wrangel Island, can be explained by peripheral isolation, refugial effects or founder effects.     

Divergence of ovipositor length and egg shape in a brood parasitic bitterling fish through the use of different mussel hosts

Bitterling fishes deposit their eggs on the gills of living mussels using a long ovipositor. We examined whether ovipositor length (OL) and egg shape correlated with differences in host mussel species in the family Unionidae among populations of the tabira bitterling (Acheilognathus tabira) in Japan. Bitterling populations that use mussels in the sub-family Anodontinae possessed longer ovipositors and more elongated eggs than those using mussels of Unioninae, as expected from the difference in host size between the sub-families (anodontine mussels are larger than unionine mussels). Based on a robust phylogeny of A. tabira populations, we demonstrated that the evolution of both OL and egg shape were correlated with host differences, but not with each other, suggesting that these traits have been selected for independently. Our study demonstrates how adaptive traits for brood parasitism may diverge with host shift due to different host availability and/or interspecific competition for hosts.     

Experimental evolution with a multicellular host causes diversification within and between microbial parasite populations—Differences in emerging phenotypes of two different parasite strains

Host‐parasite coevolution is predicted to have complex evolutionary consequences, potentially leading to the emergence of genetic and phenotypic diversity for both antagonists. However, little is known about variation in phenotypic responses to coevolution between different parasite strains exposed to the same experimental conditions. We infected Caenorhabditis elegans with one of two strains of Bacillus thuringiensis and either allowed the host and the parasite to experimentally coevolve (coevolution treatment) or allowed only the parasite to adapt to the host (one‐sided parasite adaptation). By isolating single parasite clones from evolved populations, we found phenotypic diversification of the ancestral strain into distinct clones, which varied in virulence toward ancestral hosts and competitive ability against other parasite genotypes. Parasite phenotypes differed remarkably not only between the two strains, but also between and within different replicate populations, indicating diversification of the clonal population caused by selection. This study highlights that the evolutionary selection pressure mediated by a multicellular host causes phenotypic diversification, but not necessarily with the same phenotypic outcome for different parasite strains.     

Genotypic and phenotypic variation in transmission traits of a complex life cycle parasite

Characterizing genetic variation in parasite transmission traits and its contribution to parasite vigor is essential for understanding the evolution of parasite life‐history traits. We measured genetic variation in output, activity, survival, and infection success of clonal transmission stages (cercaria larvae) of a complex life cycle parasite (Diplostomum pseudospathaceum). We further tested if variation in host nutritional stage had an effect on these traits by keeping hosts on limited or ad libitum diet. The traits we measured were highly variable among parasite genotypes indicating significant genetic variation in these life‐history traits. Traits were also phenotypically variable, for example, there was significant variation in the measured traits over time within each genotype. However, host nutritional stage had no effect on the parasite traits suggesting that a short‐term reduction in host resources was not limiting the cercarial output or performance. Overall, these results suggest significant interclonal and phenotypic variation in parasite transmission traits that are not affected by host nutritional status.     

Spatial population genetic structure of a bacterial parasite in close coevolution with its host

Knowledge of a species’ population genetic structure can provide insight into fundamental ecological and evolutionary processes including gene flow, genetic drift and adaptive evolution. Such inference is of particular importance for parasites, as an understanding of their population structure can illuminate epidemiological and coevolutionary dynamics. Here, we describe the population genetic structure of the bacterium Pasteuria ramosa, a parasite that infects planktonic crustaceans of the genus Daphnia. This system has become a model for investigations of host–parasite interactions and represents an example of coevolution via negative frequency‐dependent selection (aka “Red Queen” dynamics). To sample P. ramosa, we experimentally infected a panel of Daphnia hosts with natural spore banks from the sediments of 25 ponds throughout much of the species range in Europe and western Asia. Using 12 polymorphic variable number tandem repeat loci (VNTR loci), we identified substantial genetic diversity, both within and among localities, that was structured geographically among ponds. Genetic diversity was also structured among host genotypes within ponds, although this pattern varied by locality, with P. ramosa at some localities partitioned into distinct host‐specific lineages, and other localities where recombination had shuffled genetic variation among different infection phenotypes. Across the sample range, there was a pattern of isolation by distance, and principal components analysis coupled with Procrustes rotation identified congruence between patterns of genetic variation and geography. Our findings support the hypothesis that Pasteuria is an endemic parasite coevolving closely with its host. These results provide important context for previous studies of this model system and inform hypotheses for future research.     

Effects of host condition on susceptibility to infection, parasite developmental rate, and parasite transmission in a snail-trematode interaction

Whether or not organisms become infected by parasites is likely to be a complex interplay between host and parasite genotypes, as well as the physiological condition of both species. Details of this interplay are very important because physiology‐driven susceptibility has the potential to confound genetic coevolutionary responses. Here we concentrate on how physiological aspects of infection may interfere with genetic‐based infectivity in a snail–trematode (Potamopyrgus antipodarum/Microphallus sp.) interaction by asking: (1) how does host condition affect susceptibility to infection? and (2) how does host condition affect the survival of infected individuals? We manipulated host condition by experimentally varying resources. Contrary to our expectation, host condition did not affect susceptibility to infection, suggesting that genetics are more important than physiology in this regard. However, hosts in poor condition had higher parasite‐induced mortality than hosts in good condition. Taken together, these results suggest that coevolutionary interactions with parasites may depend on host condition, not by altering susceptibility, but rather by affecting the likelihood of parasite transmission.     

Host use of a hemiparasitic plant: no trade-offs in performance on different hosts

To examine putative specialization of a hemiparasitic plant to the most beneficial host species, we studied genetic variation in performance and trade-offs between performance on different host species in the generalist hemiparasite, Rhinanthus serotinus. We grew 25 maternal half-sib families of the parasite on Agrostis capillaris and Trifolium pratense and without a host in a greenhouse. Biomass and number of flowers of the parasite were the highest when grown on T. pratense. There were significant interactions between host species and R. serotinus seed-family indicating that the differences in performance on the two hosts and without a host varied among the families. However, we found no significant negative correlations between performance of R. serotinus on the host species or between performance on the two hosts and autotrophic performance. Thus, the genetic factors studied here are not likely to affect the evolution of specialization of R. serotinus to the most beneficial host.     

Contrasting definitive hosts as determinants of the genetic structure in a parasite with complex life cycle along the south‐eastern Pacific

The spatial genetic structure (and gene flow) of parasites with complex life cycles, such as digeneans, has been attributed mainly to the dispersion ability of the most mobile host, which most often corresponds to the definitive host (DH). In this study, we compared the genetic structure and diversity of adult Neolebouria georgenascimentoi in two fish species (DHs) that are extensively distributed along the south‐eastern Pacific (SEP). The analysis was based on the cytochrome oxidase subunit I gene sequences of parasites collected between 23°S and 45°S. In total, 202 sequences of N. georgenascimentoi in Pinguipes chilensis isolated from nine sites and 136 sequences of Prolatilus jugularis from five sites were analysed. Our results showed that N. georgenascimentoi is a species complex that includes three different parasite species; however, in this study, only lineage 1 and 2 found in P. chilensis and P. jugularis, respectively, were studied because they are widely distributed along the coastline. Lineage 1 parasites had two common haplotypes with wide distribution and unique haplotypes in northern sites. Lineage 2 had only one common haplotype with wide distribution and a large number of unique haplotypes with greater genetic diversity. Both lineages have experienced recent population expansion. Only lineage 1 exhibited a genetic structure that was mainly associated with a biogeographical break at approximately 30°S along the SEP. Our finding suggests that host access to different prey (=intermediate hosts) could affect the genetic structure of the parasite complex discovered here. Consequently, difference between these patterns suggests that factors other than DH dispersal are involved in the genetic structure of autogenic parasites.     

How a haemosporidian parasite of bats gets around: the genetic structure of a parasite,vector and host compared

Parasite population structure is often thought to be largely shaped by that of its host. In the case of a parasite with a complex life cycle, two host species, each with their own patterns of demography and migration, spread the parasite. However, the population structure of the parasite is predicted to resemble only that of the most vagile host species. In this study, we tested this prediction in the context of a vector‐transmitted parasite. We sampled the haemosporidian parasite Polychromophilus melanipherus across its European range, together with its bat fly vector Nycteribia schmidlii and its host, the bent‐winged bat Miniopterus schreibersii. Based on microsatellite analyses, the wingless vector, and not the bat host, was identified as the least structured population and should therefore be considered the most vagile host. Genetic distance matrices were compared for all three species based on a mitochondrial DNA fragment. Both host and vector populations followed an isolation‐by‐distance pattern across the Mediterranean, but not the parasite. Mantel tests found no correlation between the parasite and either the host or vector populations. We therefore found no support for our hypothesis; the parasite population structure matched neither vector nor host. Instead, we propose a model where the parasite's gene flow is represented by the added effects of host and vector dispersal patterns.     

Genetic diversity,population structure and sex‐biased dispersal in three co‐evolving species

Genetic diversity and spatial structure of populations are important for antagonistic coevolution. We investigated genetic variation and population structure of three closely related European ant species: the social parasite Harpagoxenus sublaevis and its two host species Leptothorax acervorum and Leptothorax muscorum. We sampled populations in 12 countries and analysed eight microsatellite loci and an mtDNA sequence. We found high levels of genetic variation in all three species, only slightly less variation in the host L. muscorum. Using a newly introduced measure of differentiation (Jost’s D_est ), we detected strong population structuring in all species and less male‐biased dispersal than previously thought. We found no phylogeographic patterns that could give information on post‐glacial colonization routes – northern populations are as variable as more southern populations. We conclude that conditions for Thompson’s geographic mosaic of coevolution are ideal in this system: all three species show ample genetic variation and strong population structure.     

Asymmetric local adaptation to host plants (Cirsium spp.) by the phytophagous ladybird beetle Henosepilachna niponica and its implications

Divergent host specialization by phytophagous insects is often detected as local adaptation and is thought to have played an important role in their diversification even within an ecological specialist. The phytophagous ladybird beetle Henosepilachna niponica Lewis (Coleoptera: Coccinellidae) predominantly depends on thistles (Cirsium spp., Asteraceae). The distribution of H. niponica occupies multiple areas dominated by different thistle species. This implies the possibility of the occurrence of host-associated divergent specialization of H. niponica. In this study, we investigated the pattern of host-use ability of three allopatric H. niponica populations (Aomori, Iwate, and Yamagata) on three thistle species – Cirsium alpicola Nakai, Cirsium nipponicum (Maxim.) Makino, and Cirsium tonense Nakai – under laboratory conditions. The results displayed asymmetric local adaptation by the beetles. The adults and larvae of the Aomori population showed sufficient acceptance and performance on C. nipponicum and C. tonense, the hosts of the Iwate and Yamagata populations, respectively. On the other hand, the Iwate and Yamagata populations fed small amounts of and performed poorly on C. alpicola, the host of the Aomori population. In contrast, the adults from all the populations clearly preferred feeding on C. nipponicum or C. tonense to C. alpicola. We concluded that the small but significant population differentiation and asymmetric local adaptation by beetles to congeneric host plant species could be a sign of the earliest stage of population divergence by divergent natural selection, given that these divergences will act as ‘immigrant inviability’ and ‘habitat isolation’ at least asymmetrically when these populations come into contact.     

Seasonal transmission ofRaphidascaris acus (Nematoda), a parasite of freshwater fishes,in definitive and intermediate hosts

Synopsis The seasonal transmission ofRaphidascaris acus was studied in two small lakes on Manitoulin Island, Ontario. Dragonfly nymphs and caddisfly larvae, acting as paratenic hosts, contained second-stage larvae. Several fishes, including percids and cyprinids, were intermediate hosts with second, third, and fourth-stage larvae in the liver. Yellow perch,Perca flavescens, was the most important of these. Intensities were up to 928 and increased with length and age of the perch; prevalence was 100%. Abundance ofR. acus tended to be higher in females but was not related to condition of the perch. Second-stage larvae were acquired from invertebrates in summer and developed to the fourth stage by November. They became surrounded by fibrous capsules during the next summer but remained alive for at least another year. The longevity of larvae in the intermediate host may ensure survival of the parasite through periods of low host abundance after winterkill. Northern pike,Esox lucius, was the definitive host. Abundance ofR. acus tended to be greater in larger pike but was not related to sex or condition of the fish. The parasite was acquired in late fall. Prevalence was 100% and mean intensities were over 200 in winter and spring, declining to 64–100% and less than 15, respectively, in summer. Mature worms were present from early spring through summer. Seasonality of infection in the definitive host is not attributable to seasonal availability of larvae in perch. Instead it may be controlled by timing of predation on perch and rate of development and longevity of the parasite. Transmission to pike apparently continues in summer. Low intensity may result from low recruitment rate and rapid turnover of the parasite population.