Food & You: A digital cohort on personalized nutrition.

Nutrition is a key contributor to health. Recently, several studies have identified associations between factors such as microbiota composition and health-related responses to dietary intake, raising the potential of personalized nutritional recommendations. To further our understanding of personalized nutrition, detailed individual data must be collected from participants in their day-to-day lives. However, this is challenging in conventional studies that require clinical measurements and site visits. So-called digital or remote cohorts allow in situ data collection on a daily basis through mobile applications, online services, and wearable sensors, but they raise questions about study retention and data quality. "Food & You" is a personalized nutrition study implemented as a digital cohort in which participants track food intake, physical activity, gut microbiota, glycemia, and other data for two to four weeks. Here, we describe the study protocol, report on study completion rates, and describe the collected data, focusing on assessing their quality and reliability. Overall, the study collected data from over 1000 participants, including high-resolution data of nutritional intake of more than 46 million kcal collected from 315,126 dishes over 23,335 participant days, 1,470,030 blood glucose measurements, 49,110 survey responses, and 1,024 stool samples for gut microbiota analysis. Retention was high, with over 60% of the enrolled participants completing the study. Various data quality assessment efforts suggest the captured high-resolution nutritional data accurately reflect individual diet patterns, paving the way for digital cohorts as a typical study design for personalized nutrition.

Resource limitation prevents the emergence of drug resistance by intensifying within-host competition.

Slowing the evolution of antimicrobial resistance is essential if we are to continue to successfully treat infectious diseases. Whether a drug-resistant mutant grows to high densities, and so sickens the patient and spreads to new hosts, is determined by the competitive interactions it has with drug-susceptible pathogens within the host. Competitive interactions thus represent a good target for resistance management strategies. Using an in vivo model of malaria infection, we show that limiting a resource that is disproportionately required by resistant parasites retards the evolution of drug resistance by intensifying competitive interactions between susceptible and resistant parasites. Resource limitation prevented resistance emergence regardless of whether resistant mutants arose de novo or were experimentally added before drug treatment. Our work provides proof of principle that chemotherapy paired with an "ecological" intervention can slow the evolution of resistance to antimicrobial drugs, even when resistant pathogens are present at high frequencies. It also suggests that a broad range of previously untapped compounds could be used for treating infectious diseases.

Industry-Wide Surveillance of Marek's Disease Virus on Commercial Poultry Farms.

Marek's disease virus is a herpesvirus of chickens that costs the worldwide poultry industry more than US$1 billion annually. Two generations of Marek's disease vaccines have shown reduced efficacy over the last half century due to evolution of the virus. Understanding where the virus is present may give insight into whether continued reductions in efficacy are likely. We conducted a 3-yr surveillance study to assess the prevalence of Marek's disease virus on commercial poultry farms, determine the effect of various factors on virus prevalence, and document virus dynamics in broiler chicken houses over short (weeks) and long (years) timescales. We extracted DNA from dust samples collected from commercial chicken and egg production facilities in Pennsylvania, USA. Quantitative PCR was used to assess wild-type virus detectability and concentration. Using data from 1018 dust samples with Bayesian generalized linear mixed effects models, we determined the factors that correlated with virus prevalence across farms. Maximum likelihood and autocorrelation function estimation on 3727 additional dust samples were used to document and characterize virus concentrations within houses over time. Overall, wild-type virus was detectable at least once on 36 of 104 farms at rates that varied substantially between farms. Virus was detected in one of three broiler-breeder operations (companies), four of five broiler operations, and three of five egg layer operations. Marek's disease virus detectability differed by production type, bird age, day of the year, operation (company), farm, house, flock, and sample. Operation (company) was the most important factor, accounting for between 12% and 63.4% of the variation in virus detectability. Within individual houses, virus concentration often dropped below detectable levels and reemerged later. These data characterize Marek's disease virus dynamics, which are potentially important to the evolution of the virus.

Understanding genetic variation in in vivo tolerance to artesunate: implications for treatment efficacy and resistance monitoring.

Artemisinin-based drugs are the front-line weapon in the treatment of human malaria cases, but there is concern that recent reports of slow clearing infections may signal developing resistance to treatment. In the absence of molecular markers for resistance, current efforts to monitor drug efficacy are based on the rate at which parasites are cleared from infections. However, some knowledge of the standing variation in parasite susceptibility is needed to identify a meaningful increase in infection half-life. Here, we show that five previously unexposed genotypes of the rodent malaria parasite Plasmodium chabaudi differ substantially in their in vivo response to treatment. Slower clearance rates were not linked to parasite virulence or growth rate, going against the suggestion that drug treatment will drive the evolution of virulence in this system. The level of variation observed here in a relatively small number of genotypes suggests existing 'resistant' parasites could be present in the population and therefore, increased parasite clearance rates could represent selection on pre-existing variation rather than de novo resistance events. This has implications for resistance monitoring as susceptibility may depend on evolved traits unrelated to drug exposure.

Rapid response to selection, competitive release and increased transmission potential of artesunate-selected Plasmodium chabaudi malaria parasites.

The evolution of drug resistance, a key challenge for our ability to treat and control infections, depends on two processes: de-novo resistance mutations, and the selection for and spread of resistant mutants within a population. Understanding the factors influencing the rates of these two processes is essential for maximizing the useful lifespan of drugs and, therefore, effective disease control. For malaria parasites, artemisinin-based drugs are the frontline weapons in the fight against disease, but reports from the field of slower parasite clearance rates during drug treatment are generating concern that the useful lifespan of these drugs may be limited. Whether slower clearance rates represent true resistance, and how this provides a selective advantage for parasites is uncertain. Here, we show that Plasmodium chabaudi malaria parasites selected for resistance to artesunate (an artemisinin derivative) through a step-wise increase in drug dose evolved slower clearance rates extremely rapidly. In single infections, these slower clearance rates, similar to those seen in the field, provided fitness advantages to the parasite through increased overall density, recrudescence after treatment and increased transmission potential. In mixed infections, removal of susceptible parasites by drug treatment led to substantial increases in the densities and transmission potential of resistant parasites (competitive release). Our results demonstrate the double-edged sword for resistance management: in our initial selection experiments, no parasites survived aggressive chemotherapy, but after selection, the fitness advantage for resistant parasites was greatest at high drug doses. Aggressive treatment of mixed infections resulted in resistant parasites dominating the pool of gametocytes, without providing additional health benefits to hosts. Slower clearance rates can evolve rapidly and can provide a strong fitness advantage during drug treatment in both single and mixed strain infections.

The invasion of southern South America by imported bumblebees and associated parasites.

The Palaearctic Bombus ruderatus (in 1982/1983) and Bombus terrestris (1998) have both been introduced into South America (Chile) for pollination purposes. We here report on the results of sampling campaigns in 2004, and 2010-2012 showing that both species have established and massively expanded their range. Bombus terrestris, in particular, has spread by some 200 km year(-1) and had reached the Atlantic coast in Argentina by the end of 2011. Both species, and especially B. terrestris, are infected by protozoan parasites that seem to spread along with the imported hosts and spillover to native species. Genetic analyses by polymorphic microsatellite loci suggest that the host population of B. terrestris is genetically diverse, as expected from a large invading founder population, and structured through isolation by distance. Genetically, the populations of the trypanosomatid parasite, Crithidia bombi, sampled in 2004 are less diverse, and distinct from the ones sampled later. Current C. bombi populations are highly heterozygous and also structured through isolation by distance correlating with the genetic distances of B. terrestris, suggesting the latter's expansion to be a main structuring factor for the parasite. Remarkably, wherever B. terrestris spreads, the native Bombus dahlbomii disappears although the reasons remain unclear. Our ecological and genetic data suggest a major invasion event that is currently unfolding in southern South America with disastrous consequences for the native bumblebee species.

Probing mixed-genotype infections I: extraction and cloning of infections from hosts of the trypanosomatid Crithidia bombi.

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.

Temporal variation and lack of host specificity among bacterial endosymbionts of Osedax bone worms (Polychaeta: Siboglinidae).

BACKGROUND: Osedax worms use a proliferative root system to extract nutrients from the bones of sunken vertebrate carcasses. The roots contain bacterial endosymbionts that contribute to the nutrition of these mouthless and gutless worms. The worms acquire these essential endosymbionts locally from the environment in which their larvae settle. Here we report on the temporal dynamics of endosymbiont diversity hosted by nine Osedax species sampled during a three-year investigation of an experimental whale fall at 1820-m depth in the Monterey Bay, California. The host species were identified by their unique mitochondrial COI haplotypes. The endosymbionts were identified by ribotyping with PCR primers specifically designed to target Oceanospirillales. RESULTS: Thirty-two endosymbiont ribotypes associated with these worms clustered into two distinct bacterial ribospecies that together comprise a monophyletic group, mostly restricted to deep waters (>1000 m). Statistical analyses confirmed significant changes in the relative abundances of host species and the two dominant endosymbiont ribospecies during the three-year sampling period. Bone type (whale vs. cow) also had a significant effect on host species, but not on the two dominant symbiont ribospecies. No statistically significant association existed between the host species and endosymbiont ribospecies. CONCLUSIONS: Standard PCR and direct sequencing proved to be an efficient method for ribotyping the numerically dominant endosymbiont strains infecting a large sample of host individuals; however, this method did not adequately represent the frequency of mixed infections, which appears to be the rule rather than an exception for Osedax individuals. Through cloning and the use of experimental dilution series, we determined that minority ribotypes constituting less than 30% of a mixture would not likely be detected, leading to underestimates of the frequency of multiple infections in host individuals.

The genotypic structure of a multi-host bumblebee parasite suggests a role for ecological niche overlap.

The genotypic structure of parasite populations is an important determinant of ecological and evolutionary dynamics of host-parasite interactions with consequences for pest management and disease control. Genotypic structure is especially interesting where multiple hosts co-exist and share parasites. We here analyze the natural genotypic distribution of Crithidia bombi, a trypanosomatid parasite of bumblebees (Bombus spp.), in two ecologically different habitats over a time period of three years. Using an algorithm to reconstruct genotypes in cases of multiple infections, and combining these with directly identified genotypes from single infections, we find a striking diversity of infection for both data sets, with almost all multi-locus genotypes being unique, and are inferring that around half of the total infections are resulting from multiple strains. Our analyses further suggest a mixture of clonality and sexuality in natural populations of this parasite species. Finally, we ask whether parasite genotypes are associated with host species (the phylogenetic hypothesis) or whether ecological factors (niche overlap in flower choice) shape the distribution of parasite genotypes (the ecological hypothesis). Redundancy analysis demonstrates that in the region with relatively high parasite prevalence, both host species identity and niche overlap are equally important factors shaping the distribution of parasite strains, whereas in the region with lower parasite prevalence, niche overlap more strongly contributes to the distribution observed. Overall, our study underlines the importance of ecological factors in shaping the natural dynamics of host-parasite systems.

Genetic exchange and emergence of novel strains in directly transmitted trypanosomatids.

The breeding structure of protozoan infections, i.e. whether and how frequently parasites exchange genes ("sexual reproduction"), is a crucially important parameter for many important questions; it also matters for how new virulent strains might emerge. Whether protozoan parasites are clonal or sexual is therefore a hotly debated issue. For trypanosomatids, few experimental tests of breeding structure exist to date and are limited to the vector-borne human diseases Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. We infected the natural host (Bombus terrestris) of the monoxenous parasite Crithidia bombi (Trypanosomatida) either with a single strain of the parasite or in mixed infections and tested for genetic exchange among co-infecting strains using microsatellite markers. We show that strains regularly exchange genetic material, with occasional self-crossing during mixed infections. Most offspring clones fit the expected allelic pattern from a standard Mendelian segregation. In some cases, alleles are lost or gained, leading to an entirely new genotype different from either parent. Genetic exchange in C. bombi therefore does occur and the process also leads to allelic loss or gain that could result from slippage during recombination. The majority of novel offspring types correspond to a recombination of parental alleles. The case of C. bombi demonstrates that directly transmitted, monoxenic trypanosomatids can also exchange genes. Sex therefore seems to be found in very different lineages of the trypanosomatids. Furthermore, the data allowed estimating a frequency at which C. bombi shows genetic exchange in populations.

Mutation accumulation in space and the maintenance of sexual reproduction.

The maintenance of sexual reproduction remains one of the major puzzles of evolutionary biology, since, all else being equal, an asexual mutant should have a twofold fitness advantage over the sexual wildtype. Most theories suggest that sex helps either to purge deleterious mutations, or to adapt to changing environments. Both mechanisms have their limitations if they act in isolation because they require either high genomic mutation rates or very virulent pathogens, and it is therefore often thought that they must act together to maintain sex. Typically, however, these theories have in common that they are not based on spatial processes. Here, we show that local dispersal and local competition can explain the maintenance of sexual reproduction as a means of purging deleterious mutations. Using a spatially explicit individual-based model, we find that even with reasonably low genomic mutation rates and large total population sizes, asexual clones cannot invade a sexual population. Our results demonstrate how spatial processes affect mutation accumulation such that it can fully erode the twofold benefit of asexuality faster than an asexual clone can take over a sexual population. Thus, the cost of sex is generally overestimated in models that ignore the effects of space on mutation accumulation.