Biology subjects

Helm, B.

Publications and source records attributed to Helm, B..

16 recordsLinked to original sources

bioRxiv2026

Spatial atlas highlights contribution of C. difficile in early-stage colorectal cancer

Background and AimsIntratumoral heterogeneity with respect to both host and microbial components has emerged as an important contributor to colorectal cancer (CRC) biology. Although the overall taxonomy and abundance of the CRC tumor microbiome have become well characterized, much less is known about bacterial niches within the tumor microenvironment (TME). Escherichia coli and Fusobacterium nucleatum are highly prevalent and abundant species linked to CRC. Less abundant organisms, like Clostridioides difficile and Enterocloster aldenensis, are emerging as potentially important CRC-associated bacteria. Additionally, many studies are confounded by preoperative oral antibiotics or the inclusion of late-stage cancers, both of which may alter the gut microbiome. To better understand the spatial relationships between bacteria and CRC, we generated a molecular atlas based on surgically resected tissue specimens collected from a unique cohort of early-stage CRC patients in whom oral antibiotics were not administered preoperatively. MethodsFrom 20 CRC specimens, we performed matched histopathological analysis, fluorescence in situ hybridization (FISH), whole exome sequencing (WES), 16S rRNA amplicon bacterial DNA sequencing, codetection by indexing (CODEX) multiplex immunofluorescence, and spatial transcriptomics of 756 regions among the specimens. We used 16S rRNA amplicon sequencing data to design and experimentally validate custom bacterial probes that were applied to the spatial transcriptomics. Human colonic organoids were used to validate the relationship between Clostridioides difficile toxin B (TcdB) and an E-twenty-six family transcription factor, ELF3. ResultsWe identified eight intratumoral bacterial niches consisting of a variety of bacterial species, and each niche was associated with unique tumor features. We further defined tumor gene expression patterns correlating with individual bacterial species and show that C. difficile has a disproportionate impact on the tumor transcriptome given its relatively low abundance. Specifically, TcdB induces nuclear localization of ELF3, increased cytosolic {beta}-catenin protein, and upregulated WNT signaling. ConclusionOur study integrates multi-omics to identify bacterial species with biological and spatial relevance in CRC regardless of abundance. These findings will enable further studies to define diagnostic and therapeutic targets for bacteria-associated CRC.

Cancer Biology↗

bioRxiv2025

Migration patterns and hybridization within the Asian stonechat complex in response to a major geographical barrier

Long-distance avian migration is thought to be under strong natural selection. Facing geographical barriers, migrants display various patterns considered to be adaptive. For example, they may detour along either side around the barrier or cross it, requiring specialized behavioral adaptations. Variations within closely related taxa are excellent sources for understanding the evolutionary background of migration and how barriers are shaping migration routes. In Asia, some species are assumed to have a migratory divide in response to the major geographical barrier, the Qinghai-Tibet Plateau (QTP), including the stonechat taxa (Siberian Stonechat Saxicola maurus maurus and Amur Stonechat S. stejnegeri). As they detour along either side of the QTP, these taxa are believed to disfavor a crossing over the highland. However, the more southernly distributed Tibetan Stonechat (S. m. przewalskii) breeds on the QTP, suggesting adaptation to high elevation. To investigate migration patterns and the potentially associated genetic differences, we studied migration routes and population genetics of four populations around the assumed migratory divide in Russia and Mongolia, and of one from the QTP in China. Our results confirmed the existence of a migratory divide between maurus and stejnegeri, albeit with extensive hybridization. We observed both the hypothesized western and eastern routes, but also found individuals employing intermediate routes crossing the QTP, of which two-thirds were clear hybrids. Meanwhile, przewalskii followed a highland-crossing route and was genetically differentiated from maurus and stejnegeri. The diverse migration routes among Asian stonechats show differential responses towards the geographical barrier. The intermediate route may be associated with hybridization, and its conditional viability may facilitate gene flow between maurus and stejnegeri. The Asian stonechat complex thus offers great opportunities for novel research of the genetics and evolution of migration. The specific evolutionary background associated with inhabiting and crossing the QTP can offer new perspectives in this field. Teaser textMigratory divides can arise in birds because alternative routes around migratory barriers would select for behaviors to restrict hybridization. Hybrids of parental types that employ alternative routes are hypothesized to embark on intermediate routes that would expose them to suboptimal conditions, resulting in post-zygotic reproductive isolation. However, this hypothesis is challenged when a sister taxon actually breeds on the geographical barrier. This is the case in the Asian stonechat complex that breeds near or on the Qinghai-Tibet Plateau (QTP), the roof of the world. We demonstrated a migratory divide in central Siberia to Mongolia for race maurus and stejnegeri, yet showed also evidence for extensive hybridization. Hybrids migrated along a newly discovered intermediate route, seemingly viable and overlaps with the migration trajectory of race przewalskii over the eastern part of the QTP. The Asian migratory divide relative to the QTP thus provided new insights to the evolution of landbird migration.

evolutionary biology↗

bioRxiv2025

msBayesImpute: A Versatile Framework for Addressing Missing Values in Biomedical Mass Spectrometry Proteomics Data

Advancements in mass spectrometry (MS) technologies have significantly improved the ability to quantify proteins and analyse their modifications. However, MS-based proteomics datasets frequently encounter missing values due to a complex interplay of missing at random (MAR) and missing not at random (MNAR) mechanisms. If unaddressed, such missing data can result in information loss and biased outcomes in data pre-processing, such as normalisation, as well as subsequent analyses and interpretations. Few approaches effectively address both MAR and MNAR, and those that do often necessitate manual tuning of mixture percentages between them or rely on two-group experimental designs. To enhance the handling of missing values, we developed msBayesImpute, an innovative computational method that integrates Bayesian factorization with probabilistic dropout models. We evaluated msBayesImpute against several popular imputation methods using both simulated missing values and those generated through a dilution series experiment on samples from lung cancer patients. Our comprehensive benchmark demonstrated superior performance in reconstructing missing values, estimating normalization factors, and identifying differentially expressed proteins across varying levels of missingness. Notably, msBayesImpute does not require predefined experimental designs and is scalable to large-scale studies. This versatility positions msBayesImpute as an effective and robust tool for enhancing the utility of MS datasets in biological research.

bioinformatics↗

bioRxiv2025

Phenology underlies apparent urbanisation effects on avian malaria in juvenile songbirds

Urbanisation can modify species interactions, including those between parasites and their hosts. In birds, urbanisation can either increase or decrease avian malaria infection, depending on host species, parasite or study location. However, temporal coordination between parasites and hosts, which may impact infection outcomes, has not been studied in urban ecology. To fill this gap, we collected blood samples from wild blue tit nestlings (Cyanistes caeruleus) in urban and forest habitats to examine how their hatch dates affected the prevalence and intensity of malaria infection. To separate parasites and quantify parasite load, we newly developed a species-specific qPCR assay. We found that Leucocytozoon prevalence was strongly affected by urbanisation, but effects depended on study year. This was driven by hatch date: nestlings that hatched earlier in the spring had a lower probability of being infected, independent of habitat type. In the few heavily infected nestlings, intensity of infection was associated with low body weight, suggesting fitness effects of infection. These results highlight the importance of breeding early to avoid early-life infection with malaria parasites, and that apparent urbanisation effects on infection arose from phenological differences between urban and forest habitats. Underappreciated phenological changes may underline other ecological effects of urbanisation.

ecology↗

bioRxiv2024

Urban effects on timing and variability of diel activity differ across passerine species and seasons

1. Life on Earth is adapted to rhythmic cycles in environmental conditions throughout the day and year via diel patterns of behavioural activity. 2. Urban conditions can disrupt such behavioural rhythms of activity. However, most studies so far have investigated urban effects on patterns of activity of single species in a single season. Additionally, we know little about the level of between and within-individual variation in urban and non-urban populations, and whether they differ. 3. Here, we use automated radio telemetry to record patterns of daily activity in six passerine species (blackbird, robin, great tit, blue tit, dunnock and chaffinch) across two urban and two forest populations during the pre-breeding and post-breeding season. We investigate urban effects on five activity-related traits: time of activity onset, time of activity end, duration of diurnal activity, level of diurnal activity and level of nocturnal activity. We employ statistical tools that allow us to estimate urban effects on mean phenotypic values, but also quantify urban versus forest differences in between-individual and within-individual phenotypic variation. 4. We found the strongest urban effects on time of activity onset in blackbirds and robins during the pre-breeding season: urban populations of blackbird and robin started their daily activity earlier than their forest counterparts. We did not find this effect in the other species. Urban populations of all species showed higher levels of nocturnal activity than forest populations, but this effect was not offset by lower diurnal activity, suggesting that urban birds may incur higher daily energetic demands. 5. Lastly, our analysis revealed large and consistent differences in variation in the investigated timing traits. Urban birds were more variable between individuals, implying lower population synchronization, and more variable within individuals, implying less consistent behaviour than their forest counterparts. Conversely, activity levels were more variable in forest birds. 6. We conclude that, for birds, urban life is associated with less rest, less consistency, and lower synchronicity, but that effect sizes depend on species and time of the year. Our results warn against generalising the effects of urbanisation on daily rhythms of birds, and call for future studies to understand the mechanisms behind species and seasonal differences.

animal behavior and cognition↗

bioRxiv2024

Variation in diel timing in great tits is affected by the timing of their social mate

Diel rhythms are driven by genetic and environmental components. These rhythms are mediated by the circadian clock, and entail rhythmicity of various physiological and behavioural traits. Although individuals show to some extent repeatable timing (i.e., chronotype), there is ample variation of diel timing observed within and between individuals of the same species. Here, we investigated various environmental factors, including timing of the social partner, that could explain day-to-day variation within individuals. Synchronisation with the social partner during provisioning timing could increase breeding success, and decrease extrapair paternity opportunities for females during the fertile period in case of consistent timing across the breeding stages. Therefore, we also investigated fitness consequences of between-individual variation. We first assessed the magnitude of between- and within-individual variation in the timing of nest visits by great tits (Parus major). We monitored nest visits of males and females in 37 broods during chick provisioning in 2020 and 2021. Next, we explored the responsiveness of the diel timing to environmental variables, specifically comparing abiotic and social factors. The onset of nest visits varied significantly with day within the breeding season, rainfall and the diel timing of the breeding partner but not with night temperature. In response to the partners onset, females responded stronger compared to males. By contrast, offset was generally more variable within individuals and less variation was explained by the environmental variables. Both males and females delayed their activity offset with the progressing season and females also had a later onset with more daytime rainfall. Further, the reproductive output and extrapair paternity were independent of parental chronotype and their synchronisation within pairs. It is possible that consistency of chronotypes is less important for reproductive success than the ability to plastically respond to changing environmental conditions. Thus, the next step could be to investigate potential individual differences in plasticity which could be even linked to specific chronotypes. This information might be crucial to predict how species can cope with unpredictable environmental conditions.

animal behavior and cognition↗

bioRxiv2024

Lysis buffer selection guidance for mass spectrometry-based global proteomics including studies on the intersection of signal transduction and metabolism

Prerequisite for a successful proteomics experiment is a high-quality lysis of the sample of interest, resulting in a large number of identified proteins as well as a high coverage of protein sequences. Therefore, the choice of suitable lysis conditions is crucial. Many buffers were previously employed in proteomics studies, yet a comprehensive comparison of lysate preparation conditions was so far missing. In this study, we compared the efficiency of four commonly used lysis buffers, containing the agents NP40, SDS, urea or GdnHCl, in four different types of biological samples (suspension and adherent cell lines, primary mouse cells and mouse liver tissue). After liquid chromatography-mass spectrometry (LC-MS) measurement and MaxQuant analysis, we compared chromatograms, intensities, number of identified proteins and the localization of the identified proteins. Overall, SDS emerged as the most reliable reagent, ensuring stable performance and reproducibility across diverse samples. Furthermore, our data advocated for a dual-sample lysis approach, including that the resulting pellet is lysed again after the initial lysis with a urea lysis buffer and subsequently both lysates are combined for a single LC-MS run to maximize the proteome coverage. However, none of the investigated lysis buffers proved to be superior in every category, indicating that the lysis buffer of choice depends on the proteins of interest and on the biological question. Further, we demonstrated with our systematic studies the establishment of conditions that allows to perform global proteomics and affinity purification-based interactome characterization from the same lysate. In sum our results provide guidance for the best-suited lysis buffer for mass spectrometry-based proteomics depending on the question of interest.

systems biology↗

bioRxiv2023

Basal MET Phosphorylation is an Indicator of Hepatocyte Dysregulation in Liver Disease

Chronic liver diseases are worldwide on the rise. Due to the rapidly increasing incidence, in particular in Western countries, Non-alcoholic fatty liver disease (NAFLD) is gaining importance as the disease can develop into hepatocellular carcinoma. Lipid accumulation in hepatocytes has been identified as the characteristic structural change in NAFLD development, but molecular mechanisms responsible for disease progression remained unresolved. Here, we uncover in primary hepatocytes from a preclinical model fed with a Western diet (WD) a strong downregulation of the PI3K-AKT pathway and an upregulation of the MAPK pathway. Dynamic pathway modeling of hepatocyte growth factor (HGF) signal transduction combined with global proteomics identifies that an elevated basal MET phosphorylation rate is the main driver of altered signaling leading to increased proliferation of WD-hepatocytes. Model-adaptation to patient-derived hepatocytes reveal patient-specific variability in basal MET phosphorylation, which correlates with patient outcome after liver surgery. Thus, dysregulated basal MET phosphorylation could be an indicator for the health status of the liver and thereby inform on the risk of a patient to suffer from liver failure after surgery.

systems biology↗

bioRxiv2022

Reproductive fitness is associated with female chronotype in a songbird

Research on biological rhythms has revealed widespread variation in diel timing within populations. Repeatable individual chronotypes have been linked to performance in humans but, in free-living species, benefits of chronotype are poorly understood. To address this gap, we investigated fitness correlates of incubation patterns in female songbirds (great tit, Parus major) at urban and forest sites. We confirm repeatable chronotypes (r [≥] 0.31) and show novel links between chronotype and reproductive success. In both habitats, females that started activity earlier in the day raised more fledglings. We also observed that forest females started their day at a similar time throughout the breeding season, whereas urban females tied their onset of activity closely to sunrise. Our study points to possible mechanisms that underlie chronotype variation and provides sought-after evidence for its relevance to fitness.

animal behavior and cognition↗

bioRxiv2022

Pre-analytical processing of plasma and serum samples for combined proteome and metabolome analysis

Metabolomic and proteomic analyses of human plasma and serum samples harbour the power to advance our understanding of disease biology. Pre-analytical factors may contribute to variability and bias in the detection of analytes, especially when multiple labs are involved, caused by sample handling, processing time, and differing operating procedures. To better understand the impact of pre-analytical factors that are relevant to implement a unified proteomic and metabolomic approach in a clinical setting, we assessed the influence of temperature, sitting times, and centrifugation speed on the plasma and serum metabolomes and proteomes from six healthy volunteers. We used targeted metabolic profiling (497 metabolites) and data-independent acquisition (DIA) proteomics (572 proteins) on the same samples generated with well-defined pre-analytical conditions to evaluate criteria for pre-analytical SOPs for plasma and serum samples. Time and temperature showed the strongest influence on the integrity of plasma and serum proteome and metabolome. While rapid handling and low temperatures (4{degrees}C) are imperative for metabolic profiling, the analysed proteome showed variability when exposed to temperatures of 4{degrees}C for more than 2 hours, highlighting the need for compromises in a combined analysis. We formalised a quality control scoring system to objectively rate sample stability and tested this score using external data sets from other pre-analytical studies. Stringent and harmonised standard operating procedures (SOPs) are required for pre-analytical sample handling when combining proteomics and metabolomics of clinical samples to yield robust and interpretable data on a longitudinal scale and across different clinics. To ensure an adequate level of practicability in a clinical routine for metabolomics and proteomics studies we suggest to keep blood samples up to 2 hours on ice (4{degrees}C) prior to snap-freezing as a compromise between stability and operability. Finally, we provide the methodology as an open source R package allowing the systematic scoring of proteomics and metabolomics datasets to assess the stability of plasma and serum samples.

biochemistry↗

bioRxiv2022

Population-specific association of Clock gene polymorphism with annual cycle timing in stonechats

Timing is essential for survival and reproduction of organisms across the tree of life. The core circadian clock gene Clk has been implicated in annual timing and shows highly conserved sequence homology across vertebrates except for one variable region of poly Glutamine repeats. Clk genotype varies in some species with latitude, seasonal timing and migration. However, findings are inconsistent, difficult to disentangle from environmental responses, and biased towards high latitudes. Here we combine field data with a common-garden set up to study associations of Clk with latitude, migration and annual-cycle timing within the stonechat species complex with trans-equatorial distribution range. Including 950 individuals from nine populations with diverse migratory strategies. Gene diversity was lowest in resident African and Canary Island populations and increased with latitude, independently of migration distance. Repeat length and annual-cycle timing was linked in a population-specific way. Specifically, equatorial African stonechats showed delayed timing with longer repeat length for all annual-cycle stages. Our data suggest that at low latitudes with constant photoperiod, Clk genotype might orchestrate a range of consistent, individual chronotypes. In contrast, the influence of Clk on annual-cycle timing at higher latitudes might be mediated by its interactions with genes involved in (circadian) photoperiodic pathways.

genetics↗

bioRxiv2022

Repeated toxic injuries of murine liver are tolerated through microsteatosis and mild inflammation

The liver has a remarkable capacity to regenerate and thus compensates for repeated injuries through toxic chemicals, drugs, alcohol or malnutrition for decades. However, largely unknown is how and when alterations in the liver occur due to tolerable damaging insults. To that end, we induced repeated liver injuries over ten weeks in a mouse model injecting carbon tetrachloride (CCl4) twice a week. We lost 10% of the study animals within the first six weeks, which was accompanied by a steady deposition of extracellular matrix (ECM) regardless of metabolic activity of the liver. From week six onwards, all mice survived, and in these mice ECM deposition was rather reduced, suggesting ECM remodeling as a liver response contributing to better coping with repeated injuries. The data of time-resolved paired transcriptome and proteome profiling of 18 mice was subjected to multi-level network inference, using Knowledge guided Multi-Omics Network inference (KiMONo), identified multi-level key markers exclusively associated with the injury-tolerant liver response. Interestingly, pathways of cancer and inflammation were lighting up and were validated using independent data sets compiled of 1034 samples from publicly available human cohorts. A yet undescribed link to lipid metabolism in this damage-tolerant phase was identified. Immunostaining revealed an unexpected accumulation of small lipid droplets (microvesicular steatosis) in parallel to a recovery of catabolic processes of the liver to pre-injury levels. Further, mild inflammation was experimentally validated. Taken together, we identified week six as a critical time point to switch the liver response program from an acute response that fosters ECM accumulation to a tolerant "survival" phase with pronounced deposition of small lipid droplets in hepatocytes potentially protecting against the repetitive injury with toxic chemicals. Our data suggest that microsteatosis formation plus a mild inflammatory state represent biomarkers and probably functional liver requirements to resist chronic damage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/476054v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@11ecc7aorg.highwire.dtl.DTLVardef@1027df5org.highwire.dtl.DTLVardef@9ba0f7org.highwire.dtl.DTLVardef@164f80a_HPS_FORMAT_FIGEXP M_FIG C_FIG The datasets generated via transcriptomics, proteomics as well as blood, histopathological and biochemical analysis were analyzed in an independent and integrative manner. The independent analysis was performed via state-of-the-art statistical approaches i.e. differential and consistently regulated genes and proteins. Combining the results identified three fibrosis phases. Using the KiMONo algorithm, a fibrosis specific multi-omic network was inferred. Within this network we identified several nodes connecting phase III specific features forming 13 distinct multi-omic modules suggesting a tolerance scheme. Some of these modules were experimentally validated and compared to 11 independent human studies of various liver diseases.

systems biology↗

bioRxiv2020

Artificial light at night shifts the circadian system but still leads to physiological disruption in a wild bird

Globally increasing levels of artificial light at night (ALAN) are associated with shifts in circadian rhythms of behaviour in many wild species. However, it is still unclear whether changes in behavioural timing are underlined by parallel shifts in the molecular clock, and whether such internal shifts may differ between different tissues and physiological pathways, which could highlight circadian disruption. We tackled these questions in a comprehensive study that integrated behavioural, gene expression and metabolomic analyses. We exposed captive male great tits (Parus major) to three ALAN intensities or to dark nights, recorded their activity rhythms and obtained mid-day and midnight samples of brain, liver, spleen and blood. ALAN advanced wake-up time, and this shift was paralleled by an advance in the expression of the clock gene BMAL1 in all tissues, suggesting close links of brain and peripheral clock gene expression with activity rhythms. However, several metabolic and immune genes were desynchronised the shifted BMAL1 expression, suggesting circadian disruption of behaviour and physiology. This result was reinforced by untargeted metabolomic profiling, which showed that only 9.7% of the 755 analysed metabolites followed the behavioural shift. We suggest circadian as a key mediator of the health impacts of ALAN on wild animals.

zoology↗

bioRxiv2020

Repeated genomic signatures of adaptation to urbanisation in a songbird across Europe

Urbanisation is currently increasing worldwide, and there is now ample evidence of phenotypic changes in wild organisms in response to this novel environment, but the extent to which this adaptation is due to genetic changes is poorly understood. Current evidence for evolution is based on localised studies, and thus lacking replicability. Here, we genotyped great tits (Parus major) from nine cities across Europe, each paired with a rural site, and provide evidence of repeated polygenic responses to urban habitats. In addition, we show that selective sweeps occurred in response to urbanisation within the same genes across multiple cities. These genetic responses were mostly associated with genes related to neural function and development, demonstrating that genetic adaptation to urbanisation occurred around the same pathways in wildlife populations across a large geographical scale.

evolutionary biology↗

bioRxiv2017

Correlated patterns of genetic diversity and differentiation across an avian family

Comparative studies of genomic differentiation among independent lineages can provide insights into aspects of the speciation process, such as the relative importance of selection and drift in shaping genomic landscapes, the role of genomic regions of high differentiation, and the prevalence of convergent molecular evolution. We investigated patterns of genetic diversity and divergence in stonechats (genus Saxicola), a widely distributed avian species complex with phenotypic variation in plumage, morphology, and migratory behavior, to ask whether similar genomic regions are important in the evolution of independent, but closely related, taxa. We used whole-genome pooled sequencing of 262 individuals from 5 taxa and found that patterns of genetic diversity and divergence are highly similar among different stonechat taxa. We then asked if these patterns remain correlated at deeper evolutionary scales and found that homologous genomic regions have become differentiated in stonechats and the closely related Ficedula flycatchers. Such correlation across a range of evolutionary divergence and among phylogenetically independent comparisons suggests that similar processes may be driving the differentiation of these independently evolving lineages, which in turn may be the result of intrinsic properties of particular genomic regions (e.g., areas of low recombination). Consequently, studies employing genome scans to search for areas important in reproductive isolation should account for corresponding regions of differentiation, as these regions may not necessarily represent speciation islands or facilitate local adaptation.

genomics↗

bioRxiv2016

Unexpected diversity in socially synchronized rhythms of shorebirds

The behavioural rhythms of organisms are thought to be under strong selection, influenced by the rhythmicity of the environment1-4. Such behavioural rhythms are well studied in isolated individuals under laboratory conditions1,5, but free-living individuals have to temporally synchronize their activities with those of others, including potential mates, competitors, prey and predators6-10. Individuals can temporally segregate their daily activities (e.g. prey avoiding predators, subordinates avoiding dominants) or synchronize their activities (e.g. group foraging, communal defence, pairs reproducing or caring for offspring)6-9,11. The behavioural rhythms that emerge from such social synchronization and the underlying evolutionary and ecological drivers that shape them remain poorly understood5-7,9. Here, we address this in the context of biparental care, a particularly sensitive phase of social synchronization12 where pair members potentially compromise their individual rhythms. Using data from 729 nests of 91 populations of 32 biparentally-incubating shorebird species, where parents synchronize to achieve continuous coverage of developing eggs, we report remarkable within- and between-species diversity in incubation rhythms. Between species, the median length of one parents incubation bout varied from 1 - 19 hours, while period length-the time in which a parents probability to incubate cycles once between its highest and lowest value - varied from 6 - 43 hours. The length of incubation bouts was unrelated to variables reflecting energetic demands, but species relying on crypsis (the ability to avoid detection by other animals) had longer incubation bouts than those that are readily visible or actively protect their nest against predators. Rhythms entrainable to the 24-h light-dark cycle were less prevalent at high latitudes and absent in 18 species. Our results indicate that even under similar environmental conditions and despite 24-h environmental cues, social synchronization can generate far more diverse behavioural rhythms than expected from studies of individuals in captivity5-7,9. The risk of predation, not the risk of starvation, may be a key factor underlying the diversity in these rhythms.

animal behavior and cognition↗