A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance.

Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F. hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F. hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F. hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field.

Plasmid fitness costs are caused by specific genetic conflicts enabling resolution by compensatory mutation.

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid carriage are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or gene expression level. By combining the results of experimental evolution with genetics and transcriptomics, we show here that fitness costs of 2 divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid carriage. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by up-regulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained up-regulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer (HGT) due to their propensity for amelioration by single compensatory mutations, helping to explain why plasmids are so common in bacterial genomes.

Discrete patterns of microbiome variability across timescales in a wild rodent population.

Mammalian gastrointestinal microbiomes are highly variable, both within individuals and across populations, with changes linked to time and ageing being widely reported. Discerning patterns of change in wild mammal populations can therefore prove challenging. We used high-throughput community sequencing methods to characterise the microbiome of wild field voles (Microtus agrestis) from faecal samples collected across 12 live-trapping field sessions, and then at cull. Changes in α- and ß-diversity were modelled over three timescales. Short-term differences (following 1-2 days captivity) were analysed between capture and cull, to ascertain the degree to which the microbiome can change following a rapid change in environment. Medium-term changes were measured between successive trapping sessions (12-16 days apart), and long-term changes between the first and final capture of an individual (from 24 to 129 days). The short period between capture and cull was characterised by a marked loss of species richness, while over medium and long-term in the field, richness slightly increased. Changes across both short and long timescales indicated shifts from a Firmicutes-dominant to a Bacteroidetes-dominant microbiome. Dramatic changes following captivity indicate that changes in microbiome diversity can be rapid, following a change of environment (food sources, temperature, lighting etc.). Medium- and long-term patterns of change indicate an accrual of gut bacteria associated with ageing, with these new bacteria being predominately represented by Bacteroidetes. While the patterns of change observed are unlikely to be universal to wild mammal populations, the potential for analogous shifts across timescales should be considered whenever studying wild animal microbiomes. This is especially true if studies involve animal captivity, as there are potential ramifications both for animal health, and the validity of the data itself as a reflection of a 'natural' state of an animal.

Living with chronic infection: Persistent immunomodulation during avirulent haemoparasitic infection in a wild rodent.

Apicomplexans are a protozoan phylum of obligate parasites which may be highly virulent during acute infections, but may also persist as chronic infections which appear to have little fitness cost. Babesia microti is an apicomplexan haemoparasite that, in immunocompromised individuals, can cause severe, potentially fatal disease. However, in its natural host, wild field voles (Microtus agrestis), it exhibits chronic infections that have no detectable impact on survival or female fecundity. How is damage minimized, and what is the impact on the host's immune state and health? We examine the differences in immune state (here represented by expression of immune-related genes in multiple tissues) associated with several common chronic infections in a population of wild field voles. While some infections show little impact on immune state, we find strong associations between immune state and B. microti. These include indications of clearance of infected erythrocytes (increased macrophage activity in the spleen) and activity likely associated with minimizing damage from the infection (anti-inflammatory and antioxidant activity in the blood). By analysing gene expression from the same individuals at multiple time points, we show that the observed changes are a response to infection, rather than a risk factor. Our results point towards continual investment to minimize the damage caused by the infection. Thus, we shed light on how wild animals can tolerate some chronic infections, but emphasize that this tolerance does not come without a cost.

Early-life immune expression profiles predict later-life health and fitness in a wild rodent.

Individuals differ in the nature of the immune responses they produce, affecting disease susceptibility and ultimately health and fitness. These differences have been hypothesized to have an origin in events experienced early in life that then affect trajectories of immune development and responsiveness. Here, we investigate how early-life immune expression profiles influence life history outcomes in a natural population of field voles, Microtus agrestis, in which we are able to monitor variation between and within individuals through time by repeat sampling of individually marked animals. We analysed the co-expression of 20 immune genes in early life to create a correlation network consisting of three main clusters, one of which (containing Gata3, Il10 and Il17) was associated with later-life reproductive success and susceptibility to chronic bacterial (Bartonella) infection. More detailed analyses supported associations between early-life expression of Il17 and reproductive success later in life, and of Il10 expression early in life and later infection with Bartonella. We also found significant association between an Il17 genotype and the early-life expression of Il10. Our results demonstrate that immune expression profiles can be manifested during early life with effects that persist through adulthood and that shape the variability among individuals in susceptibility to infection and fitness widely seen in natural populations.

Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.

Rapid decline of adaptation of Pseudomonas fluorescens to soil biotic environment.

Interactions between microbes can both constrain and enhance their adaptation to the environment. However, most studies to date have employed simplified microbial communities and environmental conditions. We determined how the presence of a commercial potting compost microbial community affected adaptation of the soil bacterium Pseudomonas fluorescens SBW25 in potting compost. Pseudomonas fluorescens clones isolated from populations evolved in both the presence and absence of the community showed similar fitness increases when measured in the absence of the community. This suggests the presence of the community did not constrain adaptation. By contrast, fitness measured in the presence of the community increased for community-evolved populations, but decreased below the ancestral state for populations evolved in the absence of the community. This suggests some, but not all, mutations that were beneficial with respect to the abiotic environment were costly in the presence of the community, with the former selected against in the presence of the community. Whole-genome sequencing supports this interpretation: most mutations underpinning fitness changes were clone-specific, suggesting multiple genetic pathways to adaptation. Such extreme mutational effects have not been observed in comparable in vitro studies, suggesting that caution is needed when extrapolating results from simplified in vitro systems to natural contexts.

The diversity-generating benefits of a prokaryotic adaptive immune system.

Prokaryotic CRISPR-Cas adaptive immune systems insert spacers derived from viruses and other parasitic DNA elements into CRISPR loci to provide sequence-specific immunity. This frequently results in high within-population spacer diversity, but it is unclear if and why this is important. Here we show that, as a result of this spacer diversity, viruses can no longer evolve to overcome CRISPR-Cas by point mutation, which results in rapid virus extinction. This effect arises from synergy between spacer diversity and the high specificity of infection, which greatly increases overall population resistance. We propose that the resulting short-lived nature of CRISPR-dependent bacteria-virus coevolution has provided strong selection for the evolution of sophisticated virus-encoded anti-CRISPR mechanisms.

Compost spatial heterogeneity promotes evolutionary diversification of a bacterium.

Spatial resource heterogeneity is expected to be a key driver for the evolution of diversity. However, direct empirical support for this prediction is limited to studies carried out in simplified laboratory environments. Here, we investigate how altering spatial heterogeneity of potting compost-by the addition of water and mixing-affects the evolutionary diversification of a bacterial species, Pseudomonas fluorescens, that is naturally found in the environment. There was a greater propensity of resource specialists to evolve in the unmanipulated compost, while more generalist phenotypes dominated the compost-water mix. Genomic data were consistent with these phenotypic findings. Competition experiments strongly suggest these results are due to diversifying selection as a result of resource heterogeneity, as opposed to other covariables. Overall, our findings corroborate theoretical and in vitro findings, but in semi-natural, more realistic conditions.

Resource quality determines the evolution of resistance and its genetic basis.

Parasites impose strong selection on their hosts, but the level of any evolved resistance may be constrained by the availability of resources. However, studies identifying the genomic basis of such resource-mediated selection are rare, particularly in nonmodel organisms. Here, we investigated the role of nutrition in the evolution of resistance to a DNA virus (PiGV), and any associated trade-offs in a lepidopteran pest species (Plodia interpunctella). Through selection experiments and whole-genome resequencing, we identify genetic markers of resistance that vary between the nutritional environments during selection. We do not find consistent evolution of resistance in the presence of virus but rather see substantial variation among replicate populations. Resistance in a low-nutrition environment is negatively correlated with growth rate, consistent with an established trade-off between immunity and development, but this relationship is highly context dependent. Whole-genome resequencing of the host shows that resistance mechanisms are likely to be highly polygenic and although the underlying genetic architecture may differ between high and low-nutrition environments, similar mechanisms are commonly used. As a whole, our results emphasize the importance of the resource environment on influencing the evolution of resistance.

Transposable temperate phages promote the evolution of divergent social strategies in Pseudomonas aeruginosa populations.

Transposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments, where the cost of producing cooperative iron-chelating siderophores is high and low, respectively, we show that transposable phages promote divergence into extreme siderophore production phenotypes. Iron-limited populations with transposable phages evolved siderophore overproducing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvd genes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signalling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.

Resistance is futile: RNA-sequencing reveals differing responses to bat fungal pathogen in Nearctic Myotis lucifugus and Palearctic Myotis myotis.

Resistance and tolerance allow organisms to cope with potentially life-threatening pathogens. Recently introduced pathogens initially induce resistance responses, but natural selection favors the development of tolerance, allowing for a commensal relationship to evolve. Mycosis by Pseudogymnoascus destructans, causing white-nose syndrome (WNS) in Nearctic hibernating bats, has resulted in population declines since 2006. The pathogen, which spread from Europe, has infected species of Palearctic Myotis for a longer period. We compared ecologically relevant responses to the fungal infection in the susceptible Nearctic M. lucifugus and less susceptible Palearctic M. myotis, to uncover factors contributing to survival differences in the two species. Samples were collected from euthermic bats during arousal from hibernation, a naturally occurring phenomenon, during which transcriptional responses are activated. We compared the whole-transcriptome responses in wild bats infected with P. destructans hibernating in their natural habitat. Our results show dramatically different local transcriptional responses to the pathogen between uninfected and infected samples from the two species. Whereas we found 1526 significantly upregulated or downregulated transcripts in infected M. lucifugus, only one transcript was downregulated in M. myotis. The upregulated response pathways in M. lucifugus include immune cell activation and migration, and inflammatory pathways, indicative of an unsuccessful attempt to resist the infection. In contrast, M. myotis appears to tolerate P. destructans infection by not activating a transcriptional response. These host-microbe interactions determine pathology, contributing to WNS susceptibility, or commensalism, promoting tolerance to fungal colonization during hibernation that favors survival.

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms.

Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote within-host evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as free-floating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts.

Increased socially mediated plasticity in gene expression accompanies rapid adaptive evolution.

Recent theory predicts that increased phenotypic plasticity can facilitate adaptation as traits respond to selection. When genetic adaptation alters the social environment, socially mediated plasticity could cause co-evolutionary feedback dynamics that increase adaptive potential. We tested this by asking whether neural gene expression in a recently arisen, adaptive morph of the field cricket Teleogryllus oceanicus is more responsive to the social environment than the ancestral morph. Silent males (flatwings) rapidly spread in a Hawaiian population subject to acoustically orienting parasitoids, changing the population's acoustic environment. Experimental altering crickets' acoustic environments during rearing revealed broad, plastic changes in gene expression. However, flatwing genotypes showed increased socially mediated plasticity, whereas normal-wing genotypes exhibited negligible expression plasticity. Increased plasticity in flatwing crickets suggests a coevolutionary process coupling socially flexible gene expression with the abrupt spread of flatwing. Our results support predictions that phenotypic plasticity should rapidly evolve to be more pronounced during early phases of adaptation.

A candidate tolerance gene identified in a natural population of field voles (Microtus agrestis).

The animal immune response has hitherto been viewed primarily in the context of resistance only. However, individuals can also employ a tolerance strategy to maintain good health in the face of ongoing infection. To shed light on the genetic and physiological basis of tolerance, we use a natural population of field voles, Microtus agrestis, to search for an association between the expression of the transcription factor Gata3, previously identified as a marker of tolerance in this system, and polymorphism in 84 immune and nonimmune genes. Our results show clear evidence for an association between Gata3 expression and polymorphism in the Fcer1a gene, with the explanatory power of this polymorphism being comparable to that of other nongenetic variables previously identified as important predictors of Gata3 expression. We also uncover the possible mechanism behind this association using an existing protein-protein interaction network for the mouse model rodent, Mus musculus, which we validate using our own expression network for M. agrestis. Our results suggest that the polymorphism in question may be working at the transcriptional level, leading to changes in the expression of the Th2-related genes, Tyrosine-protein kinase BTK and Tyrosine-protein kinase TXK, and hence potentially altering the strength of the Th2 response, of which Gata3 is a mediator. We believe our work has implications for both treatment and control of infectious disease.

Endemic Hantavirus in Field Voles, Northern England.

We report a PCR survey of hantavirus infection in an extensive field vole (Microtus agrestis) population present in the Kielder Forest, northern England. A Tatenale virus-like lineage was frequently detected (≈17% prevalence) in liver tissue. Lineages genetically similar to Tatenale virus are likely to be endemic in northern England.

Adaptation to public goods cheats in Pseudomonas aeruginosa.

Cooperation in nature is ubiquitous, but is susceptible to social cheats who pay little or no cost of cooperation yet reap the benefits. The effect such cheats have on reducing population productivity suggests that there is selection for cooperators to mitigate the adverse effects of cheats. While mechanisms have been elucidated for scenarios involving a direct association between producer and cooperative product, it is less clear how cooperators may suppress cheating in an anonymous public goods scenario, where cheats cannot be directly identified. Here, we investigate the real-time evolutionary response of cooperators to cheats when cooperation is mediated by a diffusible public good: the production of iron-scavenging siderophores by Pseudomonas aeruginosa We find that siderophore producers evolved in the presence of a high frequency of non-producing cheats were fitter in the presence of cheats, at no obvious cost to population productivity. A novel morphotype independently evolved and reached higher frequencies in cheat-adapted versus control populations, exhibiting reduced siderophore production but increased production of pyocyanin-an extracellular toxin that can also increase the availability of soluble iron. This suggests that cooperators may have mitigated the negative effects of cheats by downregulating siderophore production and upregulating an alternative iron-acquisition public good. More generally, the study emphasizes that cooperating organisms can rapidly adapt to the presence of anonymous cheats without necessarily incurring fitness costs in the environment they evolve in.

High virulence sub-populations in Pseudomonas aeruginosa long-term cystic fibrosis airway infections.

BACKGROUND: Pseudomonas aeruginosa typically displays loss of virulence-associated secretions over the course of chronic cystic fibrosis infections. This has led to the suggestion that virulence is a costly attribute in chronic infections. However, previous reports suggest that overproducing (OP) virulent pathotypes can coexist with non-producing mutants in the CF lung for many years. The consequences of such within-patient phenotypic diversity for the success of this pathogen are not fully understood. Here, we provide in-depth quantification of within-host variation in the production of three virulence associated secretions in the Liverpool cystic fibrosis epidemic strain of P. aeruginosa, and investgate the effect of this phenotypic variation on virulence in acute infections of an insect host model. RESULTS: Within-patient variation was present for all three secretions (pyoverdine, pyocyanin and LasA protease). In two out of three patients sampled, OP isolates coexisted with under-producing mutants. In the third patient, all 39 isolates were under-producers of all three secretions relative to the transmissible ancestor LESB58. Finally, this phenotypic variation translated into variation in virulence in an insect host model. CONCLUSIONS: Within population variation in the production of P. aeruginosa virulence-associated secretions can lead to high virulence sub-populations persisting in patients with chronic CF infections.

Co-evolutionary dynamics between a defensive microbe and a pathogen driven by fluctuating selection.

Microbes that protect their hosts from pathogenic infection are widespread components of the microbiota of both plants and animals. It has been found that interactions between 'defensive' microbes and pathogens can be genotype-specific and even underlie the variation in host resistance to pathogenic infection. These observations suggest a dynamic co-evolutionary association between pathogens and defensive microbes, but direct evidence of co-evolution is lacking. We tested the hypothesis that defensive microbes and pathogens could co-evolve within host populations by co-passaging a microbe with host-defensive properties (Enterococcus faecalis) and a pathogen (Staphylococcus aureus) within Caenorhabditis elegans nematodes. Using both phenotypic and genomic analyses across evolutionary time, we found patterns of pathogen local adaptation and defensive microbe-pathogen co-evolution via fluctuating selection dynamics. Moreover, co-evolution with defensive microbes resulted in more rapid and divergent pathogen evolution compared to pathogens evolved independently in host populations. Taken together, our results indicate the potential for defensive microbes and pathogens to co-evolve, driving interaction specificity and pathogen evolutionary divergence in the absence of host evolution.

Conflicting selection alters the trajectory of molecular evolution in a tripartite bacteria-plasmid-phage interaction.

Bacteria engage in a complex network of ecological interactions, which includes mobile genetic elements (MGEs) such as phages and plasmids. These elements play a key role in microbial communities as vectors of horizontal gene transfer but can also be important sources of selection for their bacterial hosts. In natural communities, bacteria are likely to encounter multiple MGEs simultaneously and conflicting selection among MGEs could alter the bacterial evolutionary response to each MGE. Here, we test the effect of interactions with multiple MGEs on bacterial molecular evolution in the tripartite interaction between the bacterium, Pseudomonas fluorescens, the lytic bacteriophage, SBW25φ2, and conjugative plasmid, pQBR103, using genome sequencing of experimentally evolved bacteria. We show that individually, both plasmids and phages impose selection leading to bacterial evolutionary responses that are distinct from bacterial populations evolving without MGEs, but that together, plasmids and phages impose conflicting selection on bacteria, constraining the evolutionary responses observed in pairwise interactions. Our findings highlight the likely difficulties of predicting evolutionary responses to multiple selective pressures from the observed evolutionary responses to each selective pressure alone. Understanding evolution in complex microbial communities comprising many species and MGEs will require that we go beyond studies of pairwise interactions.