Lunar-linked biological rhythms in the immune system of freshwater three-spined stickleback.

Immune responses are widely accepted to be under circadian regulation via a molecular clock, with many practical consequences, but much less is known of how other biological rhythms could affect the immune system. In this study, we search for lunar rhythms (circalunar, circasemilunar, and circatidal cycles) in the immune expression of the recently marine-derived freshwater fish, the low-plate morph of the three-spined stickleback. We employed time series of immune expression (mRNA) measurements for 14 immune-associated genes, representing a variety of immunological pathways. Times series measurements were taken on fish populations in the wild, in seminatural outdoor mesocosms, and in the laboratory, according to sampling regimens originally designed to study circannual variation but with the additional potential to provide information about lunar variation. Our evidence best supported the existence of a very small endogenous tidal rhythm. This is consistent with previous suggestions of the existence of a primordial tidal endogenous clock, some elements of which may be conserved in animals evolving outside the marine environment.

Remotely sensed localised primary production anomalies predict the burden and community structure of infection in long-term rodent datasets.

The increasing frequency and cost of zoonotic disease emergence due to global change have led to calls for the primary surveillance of wildlife. This should be facilitated by the ready availability of remotely sensed environmental data, given the importance of the environment in determining infectious disease dynamics. However, there has been little evaluation of the temporal predictiveness of remotely sensed environmental data for infection reservoirs in vertebrate hosts due to a deficit of corresponding high-quality long-term infection datasets. Here we employ two unique decade-spanning datasets for assemblages of infectious agents, including zoonotic agents, in rodents in stable habitats. Such stable habitats are important, as they provide the baseline sets of pathogens for the interactions within degrading habitats that have been identified as hotspots for zoonotic emergence. We focus on the enhanced vegetation index (EVI), a measure of vegetation greening that equates to primary productivity, reasoning that this would modulate infectious agent populations via trophic cascades determining host population density or immunocompetence. We found that EVI, in analyses with data standardised by site, inversely predicted more than one-third of the variation in an index of infectious agent total abundance. Moreover, in bipartite host occupancy networks, weighted network statistics (connectance and modularity) were linked to total abundance and were also predicted by EVI. Infectious agent abundance and, perhaps, community structure are likely to influence infection risk and, in turn, the probability of transboundary emergence. Thus, the present results, which were consistent in disparate forest and desert systems, provide proof-of-principle that within-site fluctuations in satellite-derived greenness indices can furnish useful forecasting that could focus primary surveillance. In relation to the well-documented global greening trend of recent decades, the present results predict declining infection burden in wild vertebrates in stable habitats; but if greening trends were to be reversed, this might magnify the already upwards trend in zoonotic emergence.

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.

Effects of an IgE receptor polymorphism acting on immunity, susceptibility to infection, and reproduction in a wild rodent.

The genotype of an individual is an important predictor of their immune function, and subsequently, their ability to control or avoid infection and ultimately contribute offspring to the next generation. However, the same genotype, subjected to different intrinsic and/or extrinsic environments, can also result in different phenotypic outcomes, which can be missed in controlled laboratory studies. Natural wildlife populations, which capture both genotypic and environmental variability, provide an opportunity to more fully understand the phenotypic expression of genetic variation. We identified a synonymous polymorphism in the high-affinity Immunoglobulin E (IgE) receptor (GC and non-GC haplotypes) that has sex-dependent effects on immune gene expression, susceptibility to infection, and reproductive success of individuals in a natural population of field voles (Microtus agrestis). We found that the effect of the GC haplotype on the expression of immune genes differed between sexes. Regardless of sex, both pro-inflammatory and anti-inflammatory genes were more highly relatively expressed in individuals with the GC haplotype than individuals without the haplotype. However, males with the GC haplotype showed a stronger signal for pro-inflammatory genes, while females showed a stronger signal for anti-inflammatory genes. Furthermore, we found an effect of the GC haplotype on the probability of infection with a common microparasite, Babesia microti, in females - with females carrying the GC haplotype being more likely to be infected. Finally, we found an effect of the GC haplotype on reproductive success in males - with males carrying the GC haplotype having a lower reproductive success. This is a rare example of a polymorphism whose consequences we are able to follow across immunity, infection, and reproduction for both males and females in a natural population.

Shame at the Gates of Medicine: A Hermeneutic Exploration of Premedical Students' Experiences of Shame.

PURPOSE: Little is known about the nature of shame in students attempting to enter medical school, despite its potential to impact well-being and professional identity formation during training. In this study, the authors used hermeneutic phenomenology to ask: How do premedical students experience shame as they apply to medical school? METHOD: From September 2020 to March 2021, the authors recruited 12 students from a U.S. Master of Biomedical Sciences program who intended to apply to medical school. Data collection consisted of each participant creating a "rich picture" depicting a shame experience during their premedical training, a semistructured interview that deeply explored this and other shame experiences, and a debriefing session. Data were analyzed using Ajjawi and Higgs's 6 steps of hermeneutic analysis. RESULTS: Self-concept, composed of an individual's identities and contingencies of self-esteem, was central to participants' shame experiences. Through a confluence of past and future self-concepts and under the influence of external factors and the weight of expectations, shame often destabilized participants' present self-concepts. This destabilization occurred because of events related to application processes (repeat Medical College Admission Test attempts), interpersonal interactions (prehealth advisor meetings), and objective performance measures (grades, test scores). Participants' efforts to restabilize their self-concept catalyzed specific identity processes and self-concept formation. CONCLUSIONS: Shame provided a window into the emotional experiences, identity processes, and ideologies that shape students' attempts to enter medical school. The authors discuss the central role of contingencies of self-esteem, the potential origins of performance-based self-esteem in trainees, and the identity negotiation and identity work involved in shame reactions. They call for the adoption of contingencies of self-esteem within current conceptualizations of professional identity formation; training for faculty and prehealth advisors about the nature of shame in premedical learners; and consideration of the consequential validity of standardized tests, which may trigger damaging shame.

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.

Some observations on meaningful and objective inference in radioecological field studies.

Anthropogenic releases of radiation are of ongoing importance for environmental protection, but the radiation doses at which natural systems begin to show effects are controversial. More certainty is required in this area to achieve optimal regulation for radioactive substances. We recently carried out a large survey (268 sampled animals and 20 sites) of the association between environmental radiation exposures and small mammal gut-associated microbiomes (fungal and bacterial) in the Chornobyl Exclusion zone (CEZ). Using individual measurements of total absorbed dose rates and a study design and analyses that accounted for spatial non-independence, we found no, or only limited, association. Watts et al. have criticised our study: for not filtering candidate non-resident components prior to our fungal microbiome analyses, for our qualified speculations on the relative merits of faecal and gut samples, and for the design of our study which they felt lacked sufficient replication. The advantage of filtering non-resident-fungal taxa is not clear and it would not have changed the null (spatially adjusted) association we found between radioactive dose and mycobiome composition because the most discriminatory fungal taxa with regard to dose were non-resident taxa. We maintain that it was legitimate for us to make qualified discussion comments on the differences in results between our faecal and gut microbiome analyses and on the relative merits of these sample types. Most importantly, the criticism of our study design by Watts et al. and the designs and analysis of their recent studies in the CEZ show a misunderstanding of the true nature of independent replication in field studies. Recognising the importance of spatial non-independence is essential in the design and analysis of radioecological field surveys.

Impacts of radiation exposure on the bacterial and fungal microbiome of small mammals in the Chernobyl Exclusion Zone.

Environmental impacts of the 1986 Chernobyl Nuclear Power Plant accident are much debated, but the effects of radiation on host microbiomes have received little attention to date. We present the first analysis of small mammal gut microbiomes from the Chernobyl Exclusion Zone in relation to total absorbed dose rate, including both caecum and faeces samples. We provide novel evidence that host species determines fungal community composition, and that associations between microbiome (both bacterial and fungal) communities and radiation exposure vary between host species. Using ambient versus total weighted absorbed dose rates in analyses produced different results, with the latter more robust for interpreting microbiome changes at the individual level. We found considerable variation between results for faecal and gut samples of bank voles, suggesting faecal samples are not an accurate indicator of gut composition. Associations between radiation exposure and microbiome composition of gut samples were not robust against geographical variation, although we identified families of bacteria (Lachnospiraceae and Muribaculaceae) and fungi (Steccherinaceae and Strophariaceae) in the guts of bank voles that may serve as biomarkers of radiation exposure. Further studies considering a range of small mammal species are needed to establish the robustness of these potential biomarkers.

Long-term trends in helminth infections of wood mice (Apodemus sylvaticus) from the vicinity of Malham Tarn in North Yorkshire, England.

Helminth infections in wood mice (n = 483), trapped over a period of 26 years in the woods surrounding Malham Tarn in North Yorkshire, were analysed. Although 10 species of helminths were identified, the overall mean species richness was 1.01 species/mouse indicating that the helminth community was relatively depauperate in this wood mouse population. The dominant species was Heligmosomoides polygyrus, the prevalence (64.6%) and abundance (10.4 worms/mouse) of which declined significantly over the study period. Because of the dominance of this species, analyses of higher taxa (combined helminths and combined nematodes) also revealed significantly declining values for prevalence, although not abundance. Helminth species richness (HSR) and Brillouin's index of diversity (BID) did not show covariance with year, neither did those remaining species whose overall prevalence exceeded 5% (Syphacia stroma, Aonchotheca murissylvatici and Plagiorchis muris). Significant age effects were detected for the prevalence and abundance of all higher taxa, H. polygyrus and P. muris, and for HSR and BID, reflecting the accumulation of helminths with increasing host age. Only two cases of sex bias were found; male bias in abundance of P. muris and combined Digenea. We discuss the significance of these results and hypothesize about the underlying causes.

Partitioning the environmental drivers of immunocompetence.

By determining susceptibility to disease, environment-driven variation in immune responses can affect the health, productivity and fitness of vertebrates. Yet how the different components of the total environment control this immune variation is remarkably poorly understood. Here, through combining field observation, experimentation and modelling, we are able to quantitatively partition the key environmental drivers of constitutive immune allocation in a model wild vertebrate (three-spined stickleback, Gasterosteus aculeatus). We demonstrate that, in natural populations, thermal conditions and diet alone are sufficient (and necessary) to explain a dominant (seasonal) axis of variation in immune allocation. This dominant axis contributes to both infection resistance and tolerance and, in turn, to the vital rates of infectious agents and the progression of the disease they cause. Our results illuminate the environmental regulation of vertebrate immunity (given the evolutionary conservation of the molecular pathways involved) and they identify mechanisms through which immunocompetence and host-parasite dynamics might be impacted by changing environments. In particular, we predict a dominant sensitivity of immunocompetence and immunocompetence-driven host-pathogen dynamics to host diet shifts.

Transcriptome-wide analysis reveals different categories of response to a standardised immune challenge in a wild rodent.

Individuals vary in their immune response and, as a result, some are more susceptible to infectious disease than others. Little is known about the nature of this individual variation in natural populations, or which components of immune pathways are most responsible, but defining this underlying landscape of variation is an essential first step to understanding the drivers of this variation and, ultimately, predicting the outcome of infection. We describe transcriptome-wide variation in response to a standardised immune challenge in wild field voles. We find that genes (hereafter 'markers') can be categorised into a limited number of types. For the majority of markers, the response of an individual is dependent on its baseline expression level, with significant enrichment in this category for conventional immune pathways. Another, moderately sized, category contains markers for which the responses of different individuals are also variable but independent of their baseline expression levels. This category lacks any enrichment for conventional immune pathways. We further identify markers which display particularly high individual variability in response, and could be used as markers of immune response in larger studies. Our work shows how a standardised challenge performed on a natural population can reveal the patterns of natural variation in immune response.

Not going with the flow: Locomotor activity does not constrain immunity in a wild fish.

Immunity is a central component of fitness in wild animals, but its determinants are poorly understood. In particular, the importance of locomotory activity as a constraint on immunity is unresolved. Using a piscine model (Gasterosteus aculeatus), we combined a 25-month observational time series for a wild lotic habitat with an open flume experiment to determine the influence of locomotor activity (countercurrent swimming) on natural variation in immune function. To maximize the detectability of effects in our flume experiment, we set flow velocity and duration (10 cm/s for 48 hr) just below the point at which exhaustion would ensue. Following this treatment, we measured expression in a set of immune-associated genes and infectious disease resistance through a standard challenge with an ecologically relevant monogenean infection (Gyrodactylus gasterostei). In the wild, there was a strong association of water flow with the expression of immune-associated genes, but this association became modest and more complex when adjusted for thermal effects. Our flume experiment, although statistically well-powered and based on a scenario near the limits of swimming performance in stickleback, detected no countercurrent swimming effect on immune-associated gene expression or infection resistance. The field association between flow rate and immune expression could thus be due to an indirect effect, and we tentatively advance hypotheses to explain this. This study clarifies the drivers of immune investment in wild vertebrates; although locomotor activity, within the normal natural range, may not directly influence immunocompetence, it may still correlate with other variables that do.

Diet in the Driving Seat: Natural Diet-Immunity-Microbiome Interactions in Wild Fish.

Natural interactions between the diet, microbiome, and immunity are largely unstudied. Here we employ wild three-spined sticklebacks as a model, combining field observations with complementary experimental manipulations of diet designed to mimic seasonal variation in the wild. We clearly demonstrate that season-specific diets are a powerful causal driver of major systemic immunophenotypic variation. This effect occurred largely independently of the bulk composition of the bacterial microbiome (which was also driven by season and diet) and of host condition, demonstrating neither of these, per se, constrain immune allocation in healthy individuals. Nonetheless, through observations in multiple anatomical compartments, differentially exposed to the direct effects of food and immunity, we found evidence of immune-driven control of bacterial community composition in mucus layers. This points to the interactive nature of the host-microbiome relationship, and is the first time, to our knowledge, that this causal chain (diet → immunity → microbiome) has been demonstrated in wild vertebrates. Microbiome effects on immunity were not excluded and, importantly, we identified outgrowth of potentially pathogenic bacteria (especially mycolic-acid producing corynebacteria) as a consequence of the more animal-protein-rich summertime diet. This may provide part of the ultimate explanation (and possibly a proximal cue) for the dramatic immune re-adjustments that we saw in response to diet change.

Physical Cues Controlling Seasonal Immune Allocation in a Natural Piscine Model.

Seasonal patterns in immunity are frequently observed in vertebrates but are poorly understood. Here, we focused on a natural piscine model, the three-spined stickleback (Gasterosteus aculeatus), and asked how seasonal immune allocation is driven by physical variables (time, light, and heat). Using functionally-relevant gene expression metrics as a reporter of seasonal immune allocation, we synchronously sampled fish monthly from the wild (two habitats), and from semi-natural outdoors mesocosms (stocked from one of the wild habitats). This was repeated across two annual cycles, with continuous within-habitat monitoring of environmental temperature and implementing a manipulation of temperature in the mesocosms. We also conducted a long-term laboratory experiment, subjecting acclimated wild fish to natural and accelerated (×2) photoperiodic change at 7 and 15°C. The laboratory experiment demonstrated that immune allocation was independent of photoperiod and only a very modest effect, at most, was controlled by a tentative endogenous circannual rhythm. On the other hand, experimentally-determined thermal effects were able to quantitatively predict much of the summer-winter fluctuation observed in the field and mesocosms. Importantly, however, temperature was insufficient to fully predict, and occasionally was a poor predictor of, natural patterns. Thermal effects can thus be overridden by other (unidentified) natural environmental variation and do not take the form of an unavoidable constraint due to cold-blooded physiology. This is consistent with a context-dependent strategic control of immunity in response to temperature variation, and points to the existence of temperature-sensitive regulatory circuits that might be conserved in other vertebrates.

Physiological, but not fitness, effects of two interacting haemoparasitic infections in a wild rodent.

In contrast to the conditions in most laboratory studies, wild animals are routinely challenged by multiple infections simultaneously, and these infections can interact in complex ways. This means that the impact of a parasite on its host's physiology and fitness cannot be fully assessed in isolation, and requires consideration of the interactions with other co-infections. Here we examine the impact of two common blood parasites in the field vole (Microtus agrestis): Babesia microti and Bartonella spp., both of which have zoonotic potential. We collected longitudinal and cross-sectional data from four populations of individually tagged wild field voles. This included data on biometrics, life history, ectoparasite counts, presence/absence of microparasites, immune markers and, for a subset of voles, more detailed physiological and immunological measurements. This allowed us to monitor infections over time and to estimate components of survival and fecundity. We confirm, as reported previously, that B. microti has a preventative effect on infection with Bartonella spp., but that the reverse is not true. We observed gross splenomegaly following B. microti infection, and an increase in IL-10 production together with some weight loss following Bartonella spp. infection. However, these animals appeared otherwise healthy and we detected no impact of infection on survival or fecundity due to the two haemoparasite taxa. This is particularly remarkable in the case of B. microti which induces apparently drastic long-term changes to spleen sizes, but without major adverse effects. Our work sheds light on the ecologies of these important zoonotic agents, and more generally on the influence that interactions among multiple parasites have on their hosts in the wild.

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.

Half the story: Thermal effects on within-host infectious disease progression in a warming climate.

Immune defense is temperature dependent in cold-blooded vertebrates (CBVs) and thus directly impacted by global warming. We examined whether immunity and within-host infectious disease progression are altered in CBVs under realistic climate warming in a seasonal mid-latitude setting. Going further, we also examined how large thermal effects are in relation to the effects of other environmental variation in such a setting (critical to our ability to project infectious disease dynamics from thermal relationships alone). We employed the three-spined stickleback and three ecologically relevant parasite infections as a "wild" model. To generate a realistic climatic warming scenario we used naturalistic outdoors mesocosms with precise temperature control. We also conducted laboratory experiments to estimate thermal effects on immunity and within-host infectious disease progression under controlled conditions. As experimental readouts we measured disease progression for the parasites and expression in 14 immune-associated genes (providing insight into immunophenotypic responses). Our mesocosm experiment demonstrated significant perturbation due to modest warming (+2°C), altering the magnitude and phenology of disease. Our laboratory experiments demonstrated substantial thermal effects. Prevailing thermal effects were more important than lagged thermal effects and disease progression increased or decreased in severity with increasing temperature in an infection-specific way. Combining laboratory-determined thermal effects with our mesocosm data, we used inverse modeling to partition seasonal variation in Saprolegnia disease progression into a thermal effect and a latent immunocompetence effect (driven by nonthermal environmental variation and correlating with immune gene expression). The immunocompetence effect was large, accounting for at least as much variation in Saprolegnia disease as the thermal effect. This suggests that managers of CBV populations in variable environments may not be able to reliably project infectious disease risk from thermal data alone. Nevertheless, such projections would be improved by primarily considering prevailing thermal effects in the case of within-host disease and by incorporating validated measures of immunocompetence.

From the animal house to the field: Are there consistent individual differences in immunological profile in wild populations of field voles (Microtus agrestis)?

Inbred mouse strains, living in simple laboratory environments far removed from nature, have been shown to vary consistently in their immune response. However, wildlife populations are typically outbreeding and face a multiplicity of challenges, parasitological and otherwise. In this study we seek evidence of consistent difference in immunological profile amongst individuals in the wild. We apply a novel method in this context, using longitudinal (repeated capture) data from natural populations of field voles, Microtus agrestis, on a range of life history and infection metrics, and on gene expression levels. We focus on three immune genes, IFN-γ, Gata3, and IL-10, representing respectively the Th1, Th2 and regulatory elements of the immune response. Our results show that there was clear evidence of consistent differences between individuals in their typical level of expression of at least one immune gene, and at most all three immune genes, after other measured sources of variation had been taken into account. Furthermore, individuals that responded to changing circumstances by increasing expression levels of Gata3 had a correlated increase in expression levels of IFN-γ. Our work stresses the importance of acknowledging immunological variation amongst individuals in studies of parasitological and infectious disease risk in wildlife populations.

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.