...this framework should also lead to a better understanding of the evolution of virulence itself. Supporting Information Accession Numbers Information on genetic sequences for the mentioned proteins ...can be found in GenBank (http://www.ncbi.nlm.nih.gov/entrez/): Shigella flexeneri (IpaB, 1238055; IpaC, 1238054); Escherichia coli intimin (915471); Listeria monocytogenes internalins (InlA, 985151; InlB, 98692); Vibrio cholerae (O139, ctxA, ctxB:
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We here present an efficient, precise and reliable method to isolate and cultivate healthy and viable single Crithidia bombi cells from bumblebee faeces using flow cytometry. We report a precision of ...>99% in obtaining single trypanosomatid cells for further culture and analysis ("cloning"). In the study, we have investigated the use of different liquid media to cultivate C. bombi and present an optimal medium for obtaining viable clones from all tested, infected host donors. We show that this method can be applied to genotype a collection of clones from natural infections. Furthermore, we show how to cryo-preserve C. bombi cells to be revived to become infective clones after at least 4 years of storage. Considering the high prevalence of infections in natural populations, our method provides a powerful tool in studying the level and diversity of these infections, and thus enriches the current methodology for the studies of complex host-parasite interactions.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Senescence may evolve in response to externally imposed schedules of survival and reproduction. It may result from cumulative damage to several tissues and organs leading to the progressive ...malfunction of somatic defences including the immune system, which is a relevant and accessible trait to study the evolution and the mechanisms of senescence. In social insects, reproduction of workers is negligible and their fitness relies on the colony reproductive success, which depends on workers' longevity. Consequently, evolution of senescence in workers will rely on age-dependent survival rate and the residual reproductive value of the colony. Using the bumblebee, Bombus terrestris, as model system, we investigated changes in constitutive and inducible immune defences. Such changes were monitored in relation to worker age and colony age and as well under a high, persistent level of parasitism. The results show that the inducible production of antibacterial peptides was not affected by the age of individual worker or the age of their colony. In contrast, constitutive defences such as haemocyte density and phenoloxidase (PO) activity decline with individual worker age and potential accumulation of physiological defects due to high and persistent levels of parasitism did not explain this pattern of immunosenescence. Interestingly, levels of worker constitutive defences were found to increase with colony age. We found antibacterial activity was strongly traded-off against PO activity. Such a relationship was not caused by resource shortage. Overall, these data suggest that patterns of variation of immune defences with worker age and colony age are likely the result of plastic temporal adjustments of immune responses in accordance with life history predictions. Hence, control of pathogenic threat in a social system may involve optimal adjustments of immune defences at both the individual and colony levels.
Detecting functional homology between invertebrate and vertebrate immunity is of interest in terms of understanding the dynamics and evolution of immune systems. Trans-generational effects on ...immunity are well known from vertebrates, but their existence in invertebrates remains controversial. Earlier work on invertebrates has interpreted increased offspring resistance to pathogens as trans-generational immune priming. However, interpretation of these earlier studies involves some caveats and thus full evidence for a direct effect of maternal immune experience on offspring immunity is still lacking in invertebrates. Here we show that induced levels of antibacterial activity are higher in the worker offspring of the bumblebee, Bombus terrestris L., when their mother queen received a corresponding immune challenge prior to colony founding. This shows trans-generational immune priming in an insect, with ramifications for the evolution of sociality.
1. Parasites can have dramatic effects on the ecology of their hosts. Such strong host-parasite interactions are the result of either parasites with generally high virulence, or generally benign ...parasites that nevertheless express context-dependent virulence. Theoretically, one indication that an apparently benign parasite nevertheless has a large impact on its host should be the existence of strong genotypic interactions between host and parasite. 2. Crithidia bombi (Trypanosomatidae) is a highly prevalent but generally benign gut parasite of the bumble-bee Bombus terrestris. The demonstration of strong genotypic interactions between C. bombi and B. terrestris, however, suggests that context-dependent virulence may have a large impact on the host population. We thus investigated the effects of C. bombi across the entire life cycle of its host, including the stressful times of hibernation and colony-founding. Owing to the high prevalence and rates of transmission of the parasite in field populations, we used a large-scale laboratory experiment. 3. Under stressful hibernation, infected queens lost more mass. Infection also significantly reduced colony-founding success, colony size, male production and overall fitness, by up to 40%. These findings show that strong genotypic host-parasite interactions may indeed be a reliable indicator that apparently benign and highly prevalent parasites are nevertheless exerting a dramatic impact on their host populations.
Mixed-genotype infections have major consequences for many essential elements of host-parasite interactions. With genetic exchange between co-infecting parasite genotypes increased diversity among ...parasite offspring and the emergence of novel genotypes from infected hosts is possible. We here investigated mixed- genotype infections using the host, Bombus spp. and its trypanosome parasite Crithidia bombi as our study case. The natural infections of C. bombi were genotyped with a novel method for a representative sample of workers and spring queens in Switzerland. We found that around 60% of all infected hosts showed mixed-genotype infections with an average of 2.47±0.22 (S.E.) and 3.65±1.02 genotypes per worker or queen, respectively. Queens, however, harboured up to 29 different genotypes. Based on the genotypes of co-infecting strains, these could be putatively assigned to either 'primary' and 'derived' genotypes - the latter resulting from genetic exchange among the primary genotypes. High genetic relatedness among co-infecting derived but not primary genotypes supported this scenario. Co-infection in queens seems to be a major driver for the diversity of genotypes circulating in host populations.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Female multiple mating with different males (polyandry) can be advantageous because the resulting genetic heterogeneity among offspring reduces the effects of parasitism. However, the underlying ...assumption that offspring fathered by different males vary in their susceptibility to parasites is so far only supported indirectly. Here we tested this crucial assumption using data from a study on the bumblebee Bombus terrestris L. with queens inseminated with sperm of either one or several males that originated from different sire groups (i.e. groups of brothers). We found that, under field conditions, workers from different sire groups, forming a patriline within a given colony, indeed differ in their susceptibility to the common intestinal parasite, Crithidia bombi, and do so independently of queen mating frequency.
The adaptive value of facultative maternal adjustment of offspring immunity, or trans-generational immune-priming, will depend on the ecological background. In particular, where there is a mismatch ...between the immune adjustment and offspring environment, the immunological link between mothers and offspring may be disadvantageous owing to the presence of associated costs. Costs to an individual of responding to an immune challenge are extensively documented. However, in addition to parents, the relevant costs for trans-generational immune-priming also pertain to offspring, but as yet it is unknown what costs offspring will bear. In bumble-bees, higher antibacterial activity has been shown as a trans-generational effect when mothers receive a bacterial-based immune challenge prior to colony founding. Here we show that while naive offspring from immune-challenged mothers do not show evidence for a direct energy-related survival cost, they do show increased susceptibility to a parasite distinctly unrelated to the maternal challenge. The presence of costs associated with trans-generational immune-priming will shape the evolution of this trait depending on the ecological setting.
Ecological immunology relies on variation in resistance to parasites. Colonies of the bumblebee Bombus terrestris vary in their susceptibility to the trypanosome gut parasite Crithidia bombi, which ...reduces colony fitness. To understand the possible origin of this variation in resistance we assayed the expression of 28 immunologically important genes in foraging workers. We deliberately included natural variation of the host "environment" by using bees from colonies collected in two locations and sampling active foraging workers that were not age controlled. Immune gene expression patterns in response to C. bombi showed remarkable variability even among genetically similar sisters. Nevertheless, expression varied with parasite exposure, among colonies and, perhaps surprisingly, strongly among populations (collection sites). While only the antimicrobial peptide abaecin is universally up regulated upon exposure, linear discriminant analysis suggests that the overall exposure effect is driven by a combination of several immune pathways and further immune functions such as ROS regulation. Also, the differences among colonies in their immune gene expression profiles provide clues to the mechanistic basis of well-known inter-colony variation in susceptibility to this parasite. Our results show that transcriptional responses to parasite exposure can be detected in ecologically heterogeneous groups despite strong background noise.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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