Zygosity-based sex determination in a butterfly drives hypervariability of Masculinizer.

Nature has devised many ways of producing males and females. Here, we report on a previously undescribed mechanism for Lepidoptera that functions without a female-specific gene. The number of alleles or allele heterozygosity in a single Z-linked gene (BaMasc) is the primary sex-determining switch in Bicyclus anynana butterflies. Embryos carrying a single BaMasc allele develop into WZ (or Z0) females, those carrying two distinct alleles develop into ZZ males, while (ZZ) homozygotes initiate female development, have mismatched dosage compensation, and die as embryos. Consequently, selection against homozygotes has favored the evolution of spectacular allelic diversity: 205 different coding sequences of BaMasc were detected in a sample of 246 females. The structural similarity of a hypervariable region (HVR) in BaMasc to the HVR in Apis mellifera csd suggests molecular convergence between deeply diverged insect lineages. Our discovery of this primary switch highlights the fascinating diversity of sex-determining mechanisms and underlying evolutionary drivers.

Virus-Associated CD8+ T-Cells Are Not Activated Through Antigen-Mediated Interaction Inside Atherosclerotic Lesions.

INTRODUCTION: Viral infections have been associated with the progression of atherosclerosis and CD8+ T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8+ T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8+ T-cells in the atherosclerotic lesion. METHODS: The presence, clonality, tissue enrichment, and phenotype of virus-associated CD8+ T-cells in atherosclerotic lesions were assessed by exploiting bulk T-cell receptor-ß sequencing and single-cell T-cell receptor (α and ß) sequencing datasets on human endarterectomy samples and patient-matched blood samples. To investigate if virus-specific CD8+ T-cells can be activated through T-cell receptor stimulation in the atherosclerotic lesion, the immunopeptidome of human plaques was determined. RESULTS: Virus-associated CD8+ T-cells accumulated more in the atherosclerotic lesion (mean=2.0%), compared with patient-matched blood samples (mean=1.4%; P=0.05), and were more clonally expanded and tissue enriched in the atherosclerotic lesion in comparison with nonassociated CD8+ T-cells from the lesion. Single-cell T-cell receptor sequencing and flow cytometry revealed that these virus-associated CD8+ T-cells were phenotypically highly similar to other CD8+ T-cells in the lesion and that both exhibited a more activated phenotype compared with circulating T-cells. Interestingly, virus-associated CD8+ T-cells are unlikely to be activated through antigen-specific interactions in the atherosclerotic lesion, as no virus-derived peptides were detected on HLA-I in the lesion. CONCLUSIONS: This study suggests that virus-specific CD8+ T-cells are tissue enriched in atherosclerotic lesions; however, their potential contribution to inflammation may involve antigen-independent mechanisms.

Rapid evolution of novel biotic interactions in the UK Brown Argus butterfly uses genomic variation from across its geographical range.

Understanding the rate and extent to which populations can adapt to novel environments at their ecological margins is fundamental to predicting the persistence of biological communities during ongoing and rapid global change. Recent range expansion in response to climate change in the UK butterfly Aricia agestis is associated with the evolution of novel interactions with a larval food plant, and the loss of its ability to use an ancestral host species. Using ddRAD analysis of 61,210 variable SNPs from 261 females from throughout the UK range of this species, we identify genomic regions at multiple chromosomes that are associated with evolutionary responses, and their association with demographic history and ecological variation. Gene flow appears widespread throughout the range, despite the apparently fragmented nature of the habitats used by this species. Patterns of haplotype variation between selected and neutral genomic regions suggest that evolution associated with climate adaptation is polygenic, resulting from the independent spread of alleles throughout the established range of this species, rather than the colonization of pre-adapted genotypes from coastal populations. These data suggest that rapid responses to climate change do not depend on the availability of pre-adapted genotypes. Instead, the evolution of novel forms of biotic interaction in A. agestis has occurred during range expansion, through the assembly of novel genotypes from alleles from multiple localities.

Artificial selection reveals complex genetic architecture of shoot branching and its response to nitrate supply in Arabidopsis.

Quantitative traits may be controlled by many loci, many alleles at each locus, and subject to genotype-by-environment interactions, making them difficult to map. One example of such a complex trait is shoot branching in the model plant Arabidopsis, and its plasticity in response to nitrate. Here, we use artificial selection under contrasting nitrate supplies to dissect the genetic architecture of this complex trait, where loci identified by association mapping failed to explain heritability estimates. We found a consistent response to selection for high branching, with correlated responses in other traits such as plasticity and flowering time. Genome-wide scans for selection and simulations suggest that at least tens of loci control this trait, with a distinct genetic architecture between low and high nitrate treatments. While signals of selection could be detected in the populations selected for high branching on low nitrate, there was very little overlap in the regions selected in three independent populations. Thus the regulatory network controlling shoot branching can be tuned in different ways to give similar phenotypes.

Single-cell profiling reveals age-associated immunity in atherosclerosis.

AIMS: Aging is a dominant driver of atherosclerosis and induces a series of immunological alterations, called immunosenescence. Given the demographic shift towards elderly, elucidating the unknown impact of aging on the immunological landscape in atherosclerosis is highly relevant. While the young Western diet-fed Ldlr-deficient (Ldlr-/-) mouse is a widely used model to study atherosclerosis, it does not reflect the gradual plaque progression in the context of an aging immune system as occurs in humans. METHODS AND RESULTS: Here, we show that aging promotes advanced atherosclerosis in chow diet-fed Ldlr-/- mice, with increased incidence of calcification and cholesterol crystals. We observed systemic immunosenescence, including myeloid skewing and T-cells with more extreme effector phenotypes. Using a combination of single-cell RNA-sequencing and flow cytometry on aortic leucocytes of young vs. aged Ldlr-/- mice, we show age-related shifts in expression of genes involved in atherogenic processes, such as cellular activation and cytokine production. We identified age-associated cells with pro-inflammatory features, including GzmK+CD8+ T-cells and previously in atherosclerosis undefined CD11b+CD11c+T-bet+ age-associated B-cells (ABCs). ABCs of Ldlr-/- mice showed high expression of genes involved in plasma cell differentiation, co-stimulation, and antigen presentation. In vitro studies supported that ABCs are highly potent antigen-presenting cells. In cardiovascular disease patients, we confirmed the presence of these age-associated T- and B-cells in atherosclerotic plaques and blood. CONCLUSIONS: Collectively, we are the first to provide comprehensive profiling of aged immunity in atherosclerotic mice and reveal the emergence of age-associated T- and B-cells in the atherosclerotic aorta. Further research into age-associated immunity may contribute to novel diagnostic and therapeutic tools to combat cardiovascular disease.

Single-cell T cell receptor sequencing of paired human atherosclerotic plaques and blood reveals autoimmune-like features of expanded effector T cells.

Atherosclerosis is a lipid-driven chronic inflammatory disease; however, whether it can be classified as an autoimmune disease remains unclear. In this study, we applied single-cell T cell receptor seqencing (scTCR-seq) on human carotid artery plaques and matched peripheral blood mononuclear cell samples to assess the extent of TCR clonality and antigen-specific activation within the various T cell subsets. We observed the highest degree of plaque-specific clonal expansion in effector CD4+ T cells, and these clonally expanded T cells expressed genes such as CD69, FOS and FOSB, indicative of recent TCR engagement, suggesting antigen-specific stimulation. CellChat analysis suggested multiple potential interactions of these effector CD4+ T cells with foam cells. Finally, we integrated a published scTCR-seq dataset of the autoimmune disease psoriatic arthritis, and we report various commonalities between the two diseases. In conclusion, our data suggest that atherosclerosis has an autoimmune compondent driven by autoreactive CD4+ T cells.

Alternative splicing in seasonal plasticity and the potential for adaptation to environmental change.

Seasonal plasticity is accomplished via tightly regulated developmental cascades that translate environmental cues into trait changes. Little is known about how alternative splicing and other posttranscriptional molecular mechanisms contribute to plasticity or how these mechanisms impact how plasticity evolves. Here, we use transcriptomic and genomic data from the butterfly Bicyclus anynana, a model system for seasonal plasticity, to compare the extent of differential expression and splicing and test how these axes of transcriptional plasticity differ in their potential for evolutionary change. Between seasonal morphs, we find that differential splicing affects a smaller but functionally unique set of genes compared to differential expression. Further, we find strong support for the novel hypothesis that spliced genes are more susceptible than differentially expressed genes to erosion of genetic variation due to selection on seasonal plasticity. Our results suggest that splicing plasticity is especially likely to experience genetic constraints that could affect the potential of wild populations to respond to rapidly changing environments.

Integrated molecular and behavioural data reveal deep circadian disruption in response to artificial light at night in male Great tits (Parus major).

Globally increasing levels of artificial light at night (ALAN) are associated with shifting rhythms of behaviour in many wild species. However, it is unclear whether changes in behavioural timing are paralleled by consistent shifts in the molecular clock and its associated physiological pathways. Inconsistent shifts between behavioural and molecular rhythms, and between different tissues and physiological systems, disrupt the circadian system, which coordinates all major body functions. We therefore compared behavioural, transcriptional and metabolomic responses of captive great tits (Parus major) to three ALAN intensities or to dark nights, recording activity and sampling brain, liver, spleen and blood at mid-day and midnight. ALAN advanced wake-up time, and this shift was paralleled by advanced expression of the clock gene BMAL1 in all tissues, suggesting close links between behaviour and clock gene expression across tissues. However, further analysis of gene expression and metabolites revealed that clock shifts were inconsistent across physiological systems. Untargeted metabolomic profiling showed that only 9.7% of the 755 analysed metabolites followed the behavioural shift. This high level of desynchronization indicates that ALAN disrupted the circadian system on a deep, easily overlooked level. Thus, circadian disruption could be a key mediator of health impacts of ALAN on wild animals.

Tc17 CD8+ T cells accumulate in murine atherosclerotic lesions, but do not contribute to early atherosclerosis development.

AIMS: CD8+ T cells can differentiate into subpopulations that are characterized by a specific cytokine profile, such as the Tc17 population that produces interleukin-17. The role of this CD8+ T-cell subset in atherosclerosis remains elusive. In this study, we therefore investigated the contribution of Tc17 cells to the development of atherosclerosis. METHODS AND RESULTS: Flow cytometry analysis of atherosclerotic lesions from apolipoprotein E-deficient mice revealed a pronounced increase in RORγt+CD8+ T cells compared to the spleen, indicating a lesion-specific increase in Tc17 cells. To study whether and how the Tc17 subset affects atherosclerosis, we performed an adoptive transfer of Tc17 cells or undifferentiated Tc0 cells into CD8-/- low-density lipoprotein receptor-deficient mice fed a Western-type diet. Using flow cytometry, we showed that Tc17 cells retained a high level of interleukin-17A production in vivo. Moreover, Tc17 cells produced lower levels of interferon-γ than their Tc0 counterparts. Analysis of the aortic root revealed that the transfer of Tc17 cells did not increase atherosclerotic lesion size, in contrast to Tc0-treated mice. CONCLUSION: These findings demonstrate a lesion-localized increase in Tc17 cells in an atherosclerotic mouse model. Tc17 cells appeared to be non-atherogenic, in contrast to their Tc0 counterpart.

Recommendations for Services in a FAIR Data Ecosystem.

The development and growing adoption of the FAIR data principles and associated standards as a part of research policies and practices place novel demands on research data services. This article highlights common challenges and priorities and proposes a set of recommendations on how data infrastructures can evolve and collaborate to provide services that support the implementation of the FAIR data principles, in particular in the context of building the European Open Science Cloud (EOSC). The recommendations cover a broad area of topics, including certification, infrastructure components, stewardship, costs, rewards, collaboration, training, support, and data management. These recommendations were prioritized according to their perceived urgency by different stakeholder groups and associated with actions as well as suggested action owners. This article is the output of three workshops organized by the projects FAIRsFAIR, RDA Europe, OpenAIRE, EOSC-hub, and FREYA designed to explore, discuss, and formulate recommendations among stakeholders in the scientific community. While the results are a work-in-progress, the challenges and priorities outlined provide a detailed and unique overview of current issues seen as crucial by the community that can sharpen and improve the roadmap toward a FAIR data ecosystem.

Erratum: Recommendations for Services in a FAIR Data Ecosystem.

[This corrects the article DOI: 10.1016/j.patter.2020.100058.].

Artificial light at night, in interaction with spring temperature, modulates timing of reproduction in a passerine bird.

The ecological impact of artificial light at night (ALAN) on phenological events such as reproductive timing is increasingly recognized. In birds, previous experiments under controlled conditions showed that ALAN strongly advances gonadal growth, but effects on egg-laying date are less clear. In particular, effects of ALAN on timing of egg laying are found to be year-dependent, suggesting an interaction with climatic conditions such as spring temperature, which is known have strong effects on the phenology of avian breeding. Thus, we hypothesized that ALAN and temperature interact to regulate timing of reproduction in wild birds. Field studies have suggested that sources of ALAN rich in short wavelengths can lead to stronger advances in egg-laying date. We therefore tested this hypothesis in the Great Tit (Parus major), using a replicated experimental set-up where eight previously unlit forest transects were illuminated with either white, green, or red LED light, or left dark as controls. We measured timing of egg laying for 619 breeding events spread over six consecutive years and obtained temperature data for all sites and years. We detected overall significantly earlier egg-laying dates in the white and green light vs. the dark treatment, and similar trends for red light. However, there was a strong interannual variability in mean egg-laying dates in all treatments, which was explained by spring temperature. We did not detect any fitness consequence of the changed timing of egg laying due to ALAN, which suggests that advancing reproduction in response to ALAN might be adaptive.

Natural variation in Arabidopsis shoot branching plasticity in response to nitrate supply affects fitness.

The capacity of organisms to tune their development in response to environmental cues is pervasive in nature. This phenotypic plasticity is particularly striking in plants, enabled by their modular and continuous development. A good example is the activation of lateral shoot branches in Arabidopsis, which develop from axillary meristems at the base of leaves. The activity and elongation of lateral shoots depends on the integration of many signals both external (e.g. light, nutrient supply) and internal (e.g. the phytohormones auxin, strigolactone and cytokinin). Here, we characterise natural variation in plasticity of shoot branching in response to nitrate supply using two diverse panels of Arabidopsis lines. We find extensive variation in nitrate sensitivity across these lines, suggesting a genetic basis for variation in branching plasticity. High plasticity is associated with extreme branching phenotypes such that lines with the most branches on high nitrate have the fewest under nitrate deficient conditions. Conversely, low plasticity is associated with a constitutively moderate level of branching. Furthermore, variation in plasticity is associated with alternative life histories with the low plasticity lines flowering significantly earlier than high plasticity lines. In Arabidopsis, branching is highly correlated with fruit yield, and thus low plasticity lines produce more fruit than high plasticity lines under nitrate deficient conditions, whereas highly plastic lines produce more fruit under high nitrate conditions. Low and high plasticity, associated with early and late flowering respectively, can therefore be interpreted alternative escape vs mitigate strategies to low N environments. The genetic architecture of these traits appears to be highly complex, with only a small proportion of the estimated genetic variance detected in association mapping.

Long-term declines of European insectivorous bird populations and potential causes.

Evidence of declines in insect populations has recently received considerable scientific and societal attention. However, the lack of long-term insect monitoring makes it difficult to assess whether declines are geographically widespread. By contrast, bird populations are well monitored and often used as indicators of environmental change. We compared the population trends of European insectivorous birds with those of other birds to assess whether patterns in bird population trends were consistent with declines of insects. We further examined whether declines were evident for insectivores with different habitats, foraging strata, and other ecological preferences. Bird population trends were estimated for Europe (1990-2015) and Denmark (1990-2016). On average, insectivores declined over the study period (13% across Europe and 28% in Denmark), whereas omnivores had stable populations. Seedeaters also declined (28% across Europe; 34% in Denmark), but this assessment was based on fewer species than for other groups. The effects of insectivory were stronger for farmland species (especially grassland species), for ground feeders, and for cold-adapted species. Insectivory was associated with long-distance migration, which was also linked to population declines. However, many insectivores had stable populations, especially habitat generalists. Our findings suggest that the decline of insectivores is primarily associated with agricultural intensification and loss of grassland habitat. The loss of both seed and insect specialists indicates an overall trend toward bird communities dominated by diet generalists.

Declinaciones a Largo Plazo de Poblaciones de Aves Insectívoras en Europa y las Causas Probables Resumen La evidencia de las declinaciones poblacionales de insectos ha recibido recientemente una atención considerable por parte de la comunidad científica y la sociedad. Sin embargo, la falta de un monitoreo prolongado de los insectos complica valorar si estas declinaciones tienen una distribución extensa geográficamente. Como contraste, las poblaciones de aves tienen un monitoreo constante y con frecuencia se usan como indicadores del cambio climático. Comparamos las tendencias poblacionales de las aves insectívoras de Europa con las de otras aves para valorar si los patrones en las tendencias poblacionales de aves son consistentes con las declinaciones de insectos. Además examinamos si las declinaciones eran evidentes para aves insectívoras con diferentes hábitats, estratos de alimentación, y otras preferencias ecológicas. Las tendencias poblacionales de las aves se estimaron para Europa (1990 - 2015) y para Dinamarca (1990 - 2016). En promedio, las aves insectívoras declinaron a lo largo del periodo de estudio (13% en Europa y 28% en Dinamarca) mientras que las aves omnívoras tuvieron poblaciones estables. Las poblaciones de aves que se alimentan de semillas también declinaron (28% en Europa; 34% en Dinamarca), pero esta valoración se basó en menos especies que para los otros grupos. Los efectos de la insectivoría fueron más evidentes para las especies de tierras agrícolas (especialmente las especies de pastizales), para las especies que se alimentan sobre el suelo y para las especies adaptadas al frío. La insectivoría estuvo asociada con la migración de larga distancia, la cual también estuvo ligada a las declinaciones poblacionales. Sin embargo, muchas aves insectívoras tuvieron poblaciones estables, especialmente aquellas generalistas de hábitat. Nuestros hallazgos sugieren que la declinación de las aves insectívoras está asociada principalmente con la intensificación agrícola y la pérdida de pastizales. La pérdida de aves cuya alimentación es especialista en insectos o en semillas indica una tendencia general hacia comunidades de aves dominadas por aquellas con dietas generalistas.

Dose-response effects of light at night on the reproductive physiology of great tits (Parus major): Integrating morphological analyses with candidate gene expression.

Artificial light at night (ALAN) is increasingly recognized as a potential threat to wildlife and ecosystem health. Among the ecological effects of ALAN, changes in reproductive timing are frequently reported, but the mechanisms underlying this relationship are still poorly understood. Here, we experimentally investigated these mechanisms by assessing dose-dependent photoperiodic responses to ALAN in the great tit (Parus major). We individually exposed photosensitive male birds to one of three nocturnal light levels (0.5, 1.5, and 5 lux), or to a dark control. Subsequent histological and molecular analyses on their testes indicated a dose-dependent reproductive response to ALAN. Specifically, different stages of gonadal growth were activated after exposure to different levels of light at night. mRNA transcript levels of genes linked to the development of germ cells (stra8 and spo11) were increased under 0.5 lux compared to the dark control. The 0.5 and 1.5 lux groups showed slight increases in testis size and transcript levels associated with steroid synthesis (lhr and hsd3b1) and spermatogenesis (fshr, wt1, sox9, and cldn11), although spermatogenesis was not detected in histological analysis. In contrast, all birds under 5 lux had 10 to 30 times larger testes than birds in all other groups, with a parallel strong increase in mRNA transcript levels and clear signs of spermatogenesis. Across treatments, the volume of the testes was generally a good predictor of testicular transcript levels. Overall, our findings indicate that even small changes in nocturnal light intensity can increase, or decrease, effects on the reproductive physiology of wild organisms.

Effects of experimental light at night on extra-pair paternity in a songbird.

Light pollution is increasing worldwide and significantly affects animal behavior. In birds, these effects include advancement of morning activity and onset of dawn song, which may affect extra-pair paternity. Advanced dawn song of males may stimulate females to engage in extra-pair copulations, and the earlier activity onset may affect the males' mate guarding behavior. Earlier work showed an effect of light at night on extra-pair behavior, but this was in an area with other anthropogenic disturbances. Here, we present a two-year experimental study on effects of light at night on extra-pair paternity of great tits (Parus major). Previously dark natural areas were illuminated with white, red, and green LED lamps and compared to a dark control. In 2014, the proportion of extra-pair young in broods increased with distance to the red and white lamps (i.e., at lower light intensities), but decreased with distance to the poles in the dark control. In 2013, we found no effects on the proportion of extra-pair young. The total number of offspring sired by a male was unaffected by artificial light at night in both years, suggesting that potential changes in female fidelity in pairs breeding close to white and red light did not translate into fitness benefits for the males of these pairs. Artificial light at night might disrupt the natural patterns of extra-pair paternity, possibly negates potential benefits of extra-pair copulations and thus could alter sexual selection processes in wild birds.

Early Birds by Light at Night: Effects of Light Color and Intensity on Daily Activity Patterns in Blue Tits.

Artificial light at night disturbs the daily rhythms of many organisms. To what extent this disturbance depends on the intensity and spectral composition of light remain obscure. Here, we measured daily activity patterns of captive blue tits ( Cyanistes caeruleus) exposed to similar intensities of green, red, or white light at night. Birds advanced their onset of activity in the morning under all light colors but more under red and white light than under green light. Offset of activity was slightly delayed in all light colors. The total activity over a 24-h period did not change but birds moved a part of their daily activity into the night. Since the effect of red and white lights are comparable, we tested the influence of light intensity in a follow-up experiment, where we compared the activity of the birds under different intensities of green and white light only. While in the higher range of intensities, the effects of white and green light were comparable; at lower intensities, green light had a less disturbing effect as compared with white light on daily rhythms in blue tits. Our results show that the extent of this disturbance can be mitigated by modulating the spectral characteristics and intensity of outdoor lighting, which is now feasible through the use of LED lighting.

Restless roosts: Light pollution affects behavior, sleep, and physiology in a free-living songbird.

The natural nighttime environment is increasingly polluted by artificial light. Several studies have linked artificial light at night to negative impacts on human health. In free-living animals, light pollution is associated with changes in circadian, reproductive, and social behavior, but whether these animals also suffer from physiologic costs remains unknown. To fill this gap, we made use of a unique network of field sites which are either completely unlit (control), or are artificially illuminated with white, green, or red light. We monitored nighttime activity of adult great tits, Parus major, and related this activity to within-individual changes in physiologic indices. Because altered nighttime activity as a result of light pollution may affect health and well-being, we measured oxalic acid concentrations as a biomarker for sleep restriction, acute phase protein concentrations and malaria infection as indices of immune function, and telomere lengths as an overall measure of metabolic costs. Compared to other treatments, individuals roosting in the white light were much more active at night. In these individuals, oxalic acid decreased over the course of the study. We also found that individuals roosting in the white light treatment had a higher probability of malaria infection. Our results indicate that white light at night increases nighttime activity levels and sleep debt and affects disease dynamics in a free-living songbird. Our study offers the first evidence of detrimental effects of light pollution on the health of free-ranging wild animals.

Experimental illumination of a forest: no effects of lights of different colours on the onset of the dawn chorus in songbirds.

Light pollution is increasing exponentially, but its impact on animal behaviour is still poorly understood. For songbirds, the most repeatable finding is that artificial night lighting leads to an earlier daily onset of dawn singing. Most of these studies are, however, correlational and cannot entirely dissociate effects of light pollution from other effects of urbanization. In addition, there are no studies in which the effects of different light colours on singing have been tested. Here, we investigated whether the timing of dawn singing in wild songbirds is influenced by artificial light using an experimental set-up with conventional street lights. We illuminated eight previously dark forest edges with white, green, red or no light, and recorded daily onset of dawn singing during the breeding season. Based on earlier work, we predicted that onset of singing would be earlier in the lighted treatments, with the strongest effects in the early-singing species. However, we found no significant effect of the experimental night lighting (of any colour) in the 14 species for which we obtained sufficient data. Confounding effects of urbanization in previous studies may explain these results, but we also suggest that the experimental night lighting may not have been strong enough to have an effect on singing.