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Abstract Background Magnetic orientation is a taxonomically widespread phenomenon in the animal kingdom, but has been little studied in anuran amphibians. We collected Common Toads (Bufo bufo) during their migration towards their spawning pond and tested them shortly after displacement for possible magnetic orientation in arena experiments. Animals were tested in two different set-ups, in the geomagnetic field and in a reversed magnetic field. To the best of our knowledge, this is the first study testing orientation of adult anurans with a controlled magnetic field of a known strength and alignment. Results After displacement, toads oriented themselves unimodally under the geomagnetic field, following their former migration direction (d-axis). When the magnetic field was reversed, the distribution of bearings changed from a unimodal to a bimodal pattern, but still along the d-axis. The clustering of bearings was only significant after the toads reached the outer circle, 60.5 cm from their starting point. At a virtual inner circle (diameter 39 cm) and at the start of the experiment, orientation of toads did not show any significant pattern. Conclusions The experimental set-up used in our study is suitable to test orientation behaviour of the Common Toad. We speculate that toads had not enough time to relocate their position on an internal map. Hence, they followed their former migration direction. Bimodality in orientation when exposed to the reversed magnetic field could be the result of a cue conflict, between magnetic and possibly celestial cues. For maintaining their migration direction toads use, at least partly, the geomagnetic field as a reference system.

Biologging has proven to be a powerful approach to investigate diverse questions related to movement ecology across a range of spatiotemporal scales and increasingly relies on multidisciplinary expertise. However, the variety of animal-borne equipment, coupled with little consensus regarding analytical approaches to interpret large, complex data sets presents challenges and makes comparison between studies and study species difficult. Here, we present a combined hardware and analytical approach for standardizing the collection, analysis, and interpretation of multisensor biologging data. Here, we present (i) a custom-designed integrated multisensor collar (IMSC), which was field tested on 71 free-ranging wild boar ( Sus scrofa ) over 2 years; (ii) a machine learning behavioral classifier capable of identifying six behaviors in free-roaming boar, validated across individuals equipped with differing collar designs; and (iii) laboratory and field-based calibration and accuracy assessments of animal magnetic heading measurements derived from raw magnetometer data. The IMSC capacity and durability exceeded expectations, with a 94% collar recovery rate and a 75% cumulative data recording success rate, with a maximum logging duration of 421 days. The behavioral classifier had an overall accuracy of 85% in identifying the six behavioral classes when tested on multiple collar designs and improved to 90% when tested on data exclusively from the IMSC. Both laboratory and field tests of magnetic compass headings were in precise agreement with expectations, with overall median magnetic headings deviating from ground truth observations by 1.7° and 0°, respectively. Although multisensor equipment and sophisticated analyses are now commonplace in biologging studies, the IMSC hardware and analytical framework presented here provide a valuable tool for biologging researchers and will facilitate standardization of biologging data across studies. In addition, we highlight the potential of additional analyses available using this framework that can be adapted for use in future studies on terrestrial mammals., Competing Interests: The authors have no conflicts of interest to declare., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

Many animals return to their home areas (i.e., 'homing') after translocation to sites further away. Such translocations have traditionally been used in behavioral ecology to understand the orientation and migration behavior of animals. The movement itself can then be followed by marking and recapturing animals or by tracking, for example, using GPS systems. Most detailed studies investigating this behavior have been conducted in smaller vertebrates (e.g., birds, amphibians, and mice), whereas information on larger mammals, such as red deer, is sparse. We conducted GPS-assisted translocation experiments with red deer at two sites in the Czech Republic. Individuals were translocated over a distance of approximately 11 km and their home journey was tracked. Circular statistics were used to test for significant homeward orientation at distances of 100, 500, 1000, and 5000 m from the release site. In addition, we applied Lavielle trajectory segmentation to identify the different phases of homing behavior. Thirty-one out of 35 translocations resulted in successful homing, with a median time of 4.75 days (range 1.23-100 days). Animals were significantly oriented towards home immediately after release and again when they came closer to home; however, they did not show a significant orientation at the distances in between. We were able to identify three homing phases, an initial 'exploratory phase', followed by a 'homing phase' which sometimes was again followed by an 'arrival phase'. The 'homing phase' was characterized by the straightest paths and fastest movements. However, the variation between translocation events was considerable. We showed good homing abilities of red deer after translocation. Our results demonstrate the feasibility of conducting experiments with environmental manipulations (e.g., to impede the use of sensory cues) close to the release site. The homing behavior of red deer is comparable to that of other species, and might represent general homing behavior patterns in animals. Follow-up studies should further dissect and investigate the drivers of the individual variations observed and try to identify the sensory cues used during homing., (© 2024. The Author(s).)

Locomotion in benthic invertebrates can strongly affect habitat selection and ecosystem nutrient cycling. In the case of freshwater mussels, the drivers of locomotion are largely unresolved. Our aim was to assess the influence of light presence and intensity on the locomotory behaviour of freshwater mussels in controlled laboratory experiments. The species investigated in our study were Anodonta anatina and Unio pictorum, two widely distributed mussels in European lentic and lotic inland waters. At low algal concentrations, known to be associated with more frequent locomotory activities, we found that both species moved primarily in the absence of light (72.7% of all movements across experiments). However, the movements of both species were directed towards the light source, resembling a net-positive 'phototactic' response but in the absence of light. The distance to the light source, which was negatively correlated to light intensity, had a positive effect on the distance covered in locomotory activities by A. anatina but not by U. pictorum. Intraspecific variation in shell size had no impact on movement distance, indicating that the energetic costs of movement were not a limiting factor. We suggest that the observed movement towards brighter locations helps to enhance food quantity and quality, whilst movement in darkness mitigates predation risks., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Some data are collected on circular (rather than linear) scales. Often researchers are interested in comparing two samples of such circular data to test the hypothesis that they came from the same underlying population. Recently, we compared 18 statistical approaches to testing such a hypothesis, and recommended two as particularly effective. A very recent publication introduced a novel statistical approach that was claimed to outperform the methods that we had indicated were highest performing. However, the evidence base for this claim was limited. Here we perform simulation studies to offer a more detailed comparison of the new "Angular Randomisation Test" (ART) with existing tests. We expand previous evaluations in two ways: exploring small and medium sized samples, and exploring a range of different shapes for the underlying distribution(s). We find that the ART controls type I error rates at the nominal level. The ART had greater power than established methods in detecting a difference in underlying distribution caused by a shift around the circle. Its performance advantage in this case was strongest when samples where small and unbalanced in size. When the difference between underlying unimodal distributions was in shape rather than central tendency, then the ART was at least as good (and sometimes considerably more powerful) than the established methods, except when distributions samples were small and uneven in size, and the smaller sample came from a more concentrated underlying distribution. In such cases its power could be markedly inferior to established alternatives. The ART was also inferior to alternatives in dealing with axially distributed data. We conclude that under widely-encountered circumstances the ART test can be recommended for its simplicity of implementation, but researchers should be aware of situations where it cannot be recommended., (© 2023. The Author(s).)

Background: Sex ratios of animal populations are important factors of population demographics. In pond-breeding amphibians, the operational sex ratio (OSR) among the breeding population is usually male-biased. Also, in European green toads (Bufotes viridis), males usually outnumber females at breeding sites, while the sex ratio of the total adult population (ASR) is assumed to be balanced. It has been suggested that sex-specific breeding behavior causes male-predominance at the breeding sites. We used a dataset of 5 years of street patrols to test this hypothesis. For this we analyzed local sex ratios of green toads in terrestrial habitats and at two artificial breeding ponds. We expected temporal and/or spatial changes of local sex ratios which would indicate sex dependent differences in breeding behavior., Results: Overall observed ASR among 2111 green toads, counted in the course of street patrols from 2016 to 2020, was slightly male-biased (ASR = 0.56, annual ASRs = 0.49-0.63). Based on the data of 1631 toads (920 males, 711 females) captured within a radius of 300 m around nine main breeding sites, temporal and spatial variations in local ASRs were evaluated. Resulting values were compared to the calculated OSR at two artificial breeding ponds in 2021 (645 adult: 553 males, 92 females). Estimates predict more equally distributed females and males prior to the main breeding season. During breeding season, males predominated at both breeding sites (B1: 0.83, B2: 0.89), whereas females are estimated to outnumber males in terrestrial habitats. Proportions of females highly significantly increased with advancing time of the year and increasing distance to the breeding sites. While males tended to accumulate in proximity to water bodies, females dispersed soon after breeding to more distant areas., Conclusions: Observed sex ratios in the studied green toad population changed with time and sampling site, deviating from the population-wide sex ratio. Expanding sampling effort in amphibian conservation assessments in time and space, i.e., outside the main breeding season and away from the breeding sites, would be important to encompass such variations., (© 2023. The Author(s).)

To better understand the influence of land use and meteorological parameters on air pollutants, we deployed passive air samplers in 15 regions with different land use in eastern Austria. The samplers consisted of polyurethane PUF and polyester PEF filter matrices, which were analyzed for 566 substances by gas-chromatography/mass-spectrometry. In a previous article, we highlighted a widespread contamination of ambient air with pesticides that depends on the surrounding land use and meteorological parameters. Here we report that, in addition to agricultural pesticides, eight other substances were frequently detected in ambient air: Nitrapyrin, a nitrification inhibitor used to increase nitrogen use efficiency of fertilizers and banned in Austria since 1993; biocides against insects (DEET and transfluthrin) used mainly outside agriculture; piperonyl butoxide (PBO), a synergist mixed into pesticide formulations; and four industrially used polychlorinated biphenyls (PCBs), long banned worldwide. Concentrations of the detected substances were positively related to air temperature, but only slightly related to agricultural land use in the sampler's vicinity. The city center showed the highest concentrations of biocides, PCBs and PBO, but also medium concentrations of nitrapyrin. Four sites had no air contamination with these substances; including two national parks dominated by grassland or forest, but also two sites with mixed land use. The potential human toxicity of the detected substances based on globally harmonized hazard classifications was high: seven substances had specific organ toxicity, six were cancerogenic, and two were acutely toxic; however, several substances had incomplete information of hazard profiles. Moreover, all substances were acutely and chronically toxic to aquatic life. We recommend that substances of different origins be included in the air pollution monitoring portfolio to comprehensively assess the potential hazards to humans and the environment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kruse, Schlechtriemen, Gruber reports financial support was provided by Verein zur Förderung einer enkeltauglichen Umwelt in Österreich., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Advancements in tracking technologies provide an increasingly important tool in animal monitoring and conservation that can describe animal spatial behavior in native habitats and uncover migratory routes that otherwise may be difficult or impossible to map. In addition, high-resolution accelerometer sensors provide powerful insights into animal activity patterns and can help to identify specific behaviors from accelerometer profiles alone. Previously, such accelerometers were restricted to larger animals due to size and mass constraints. However, recent advances make it possible to use such devices on smaller animals such as the European green toad ( Bufotes viridis ), the focus of our current study. We deploy custom made tracking devices, that consist of very-high-frequency transmitters and tri-axial accelerometers, to track toads in their native urban environment in Vienna (Austria). A total of nine toads were tracked, ranging from three to nine tracking days per individual during the post-breeding season period. We demonstrate that our devices could reliably monitor toad movement and activity during the observation period. Hence, we confirmed the predominantly nocturnal activity patterns and recorded low overall movement at this urban site. Accelerometer data revealed that toads exhibited brief but intense activity bursts between 10 pm and midnight, resting periods during the night and intermittent activity during the day. Positional tracking alone would have missed the major activity events as they rarely resulted in large positional displacements. This underscores the importance of and value in integrating multiple tracking sensors for studies of movement ecology. Our approach could be adapted for other amphibians or other animals with mass constraints and may become standard monitoring equipment in the near future., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Urban areas are increasing worldwide, which poses threats to animal wildlife. However, in certain cases cities can provide refuges for endangered animals. The European green toad (Bufotes viridis) is one of such examples, which is known from cities throughout their distribution. In contrast, considerable areas of their former (primary) habitats have been degraded. The primary habitats of this species include steppes and wild river floodplains, both characterized by dynamic changes and the presence of open areas. We used available green toad observation data (2007-2020) to model the effects of land-use types on occurrence probability in the city of Vienna. Forest and densely populated areas were highly significantly negatively associated with green toad presence, while transformation/construction site areas showed a strong positive effect. Such occurrence pattern might be characteristic for early succession species, which depend on stochastic environmental disturbances (e.g., droughts and floods) in their primary habitats. We argue that urban landscape planning should appreciate the potential ecological value of open land in cities which is either in a transition phase or a permanent 'wasteland'. Ecological managing of such landscape could vastly increase urban biodiversity., (© 2023. The Author(s).)

Body condition is increasingly used to assess the status of populations and as a proxy for individual fitness. A common, quick and non-invasive approach is to estimate condition from the relation between body length and mass. Among the methods developed for this purpose, the Scaled Mass Index (SMI) appears best suited for comparisons among populations. We assembled data from 17 populations of European green toads (Bufotes viridis) with the aim of devising a standard formula applicable for monitoring this species. The mean value of the exponents describing length-mass allometry in these samples was 3.0047. Hence, we propose using 3 as a scaling coefficient for calculating the SMI in green toads. From the contrast of SMI values for both sexes within populations, estimated with either the population-specific or the standard coefficient, we conclude that applying the standard formula not only facilitates comparisons among populations but may also help to avoid misinterpretation of variation within populations., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Little is known about (i) how numbers and concentrations of airborne pesticide residues are influenced by land use, interactions with meteorological parameters, or by substance-specific chemo-physical properties, and (ii) what potential toxicological hazards this could pose to non-target organisms including humans. We installed passive air samplers (polyurethane PUF and polyester PEF filter matrices) in 15 regions with different land uses in eastern Austria for up to 8 months. Samples were analyzed for 566 substances by gas-chromatography/mass-spectrometry. We analyzed relationships between frequency and concentrations of pesticides, land use, meteorological parameters, substance properties, and season. We found totally 67 pesticide active ingredients (24 herbicides, 30 fungicides, 13 insecticides) with 10-53 pesticides per site. Herbicides metolachlor, pendimethalin, prosulfocarb, terbuthylazine, and the fungicide HCB were found in all PUF samplers, and glyphosate in all PEF samplers; chlorpyrifos-ethyl was the most abundant insecticide found in 93% of the samplers. Highest concentrations showed the herbicide prosulfocarb (725 ± 1218 ng sample -1 ), the fungicide folpet (412 ± 465 ng sample -1 ), and the insecticide chlorpyrifos-ethyl (110 ± 98 ng sample -1 ). Pesticide numbers and concentrations increased with increasing proportions of arable land in the surroundings. However, pesticides were also found in two National Parks (10 and 33 pesticides) or a city center (17 pesticides). Pesticide numbers and concentrations changed between seasons and correlated with land use, temperature, radiation, and wind, but were unaffected by substance volatility. Potential ecotoxicological exposure of mammals, birds, earthworms, fish, and honeybees increased with increasing pesticide numbers and concentrations. Human toxicity potential of detected pesticides was high, with averaged 54% being acutely toxic, 39% reproduction toxic, 24% cancerogenic, and 10% endocrine disrupting. This widespread pesticide air pollution indicates that current environmental risk assessments, field application techniques, protective measures, and regulations are inadequate to protect the environment and humans from potentially harmful exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Animals that create structures often display non-random patterns in the direction of their constructions. This tendency of oriented construction is widely presumed to be an adaptive trait of the constructor's extended phenotype, but there is little empirical support for this hypothesis. Particularly, for cavity nesting-birds there is a lack of studies examining this issue. In this study of a primary cavity excavator, the endangered red-cockaded woodpecker (Dryobates borealis), we show that cavity entrances exhibited a strong westward bias in all 11 of the populations examined throughout the geographic range of the species in the southeastern United States. This species requires cavities in living pine trees for roosting and nesting that often take many years to complete, resulting in many incomplete excavations on the landscape. We used population monitoring data to show that orientation was stronger among completed cavities than incomplete cavities. There was a significant correlation between latitude and average cavity direction among populations, turning northward with increasing latitude, suggesting adaptation to local conditions. Long-term monitoring data showed that cavity orientation and breeding group size are correlated with egg hatching rates, fledging rates, and the total number of fledglings produced per nest. Our results provide empirical evidence from extensive long-term data that directional orientation in animal constructions is an important feature of the extended animal phenotype and have immediate implications for animal ecology and the conservation of endangered species., (© 2022. The Author(s).)

Background: A broad range of scientific studies involve taking measurements on a circular, rather than linear, scale (often variables related to times or orientations). For linear measures there is a well-established statistical toolkit based on linear modelling to explore the associations between this focal variable and potentially several explanatory factors and covariates. In contrast, statistical testing of circular data is much simpler, often involving either testing whether variation in the focal measurements departs from circular uniformity, or whether a single explanatory factor with two levels is supported., Methods: We use simulations and example data sets to investigate the usefulness of a MANOVA approach for circular data in comparison to commonly used statistical tests., Results: Here we demonstrate that a MANOVA approach based on the sines and cosines of the circular data is as powerful as the most-commonly used tests when testing deviation from a uniform distribution, while additionally offering extension to multi-factorial modelling that these conventional circular statistical tests do not., Conclusions: The herein presented MANOVA approach offers a substantial broadening of the scientific questions that can be addressed statistically using circular data., (© 2022. The Author(s).)

Most amphibians have a complex life cycle with an aquatic larval and an adult (semi-) terrestrial stage. However, studies concerning spatial behaviour and orientation mainly focus on either the aquatic larvae or the adult animals on land. Consequently, behavioural changes that happen during metamorphosis and the consequences for emigration and population distribution are less understood. This paper aims to summarize the knowledge concerning specific topics of early amphibian life history stages and proposes several testable hypotheses within the following fields of research: larval and juvenile orientation, influences of environmental and genetic factors on juvenile emigration, their habitat choice later in life as well as population biology. I argue that studying larval and juvenile amphibian spatial behaviour is an understudied field of research, however, could considerably improve our understanding of amphibian ecology.

A magnetic compass sense has been demonstrated in all major classes of vertebrates, as well as in many invertebrates. In mammals, controlled laboratory studies of mice have provided evidence for a robust magnetic compass that is comparable to, or exceeds, the performance of that in other animals. Nevertheless, the vast majority of laboratory studies of spatial behavior and cognition in murine rodents have failed to produce evidence of sensitivity to magnetic cues. Given the central role that a magnetic compass sense plays in the spatial ecology and cognition of non-mammalian vertebrates, and the potential utility that a global/universal reference frame derived from the magnetic field would have in mammals, the question of why responses to magnetic cues have been so difficult to demonstrate reliably is of considerable importance. In this paper, we review evidence that the magnetic compass of murine rodents shares a number of properties with light-dependent compasses in a wide variety of other animals generally believed to be mediated by a radical pair mechanism (RPM) or related quantum process. Consistent with the RPM, we summarize both published and previously unpublished findings suggesting that the murine rodent compass is sensitive to low-level radio frequency (RF) fields. Finally, we argue that the presence of anthropogenic RF fields in laboratory settings, may be an important source of variability in responses of murine rodents to magnetic cues., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The European green toad, Bufotes viridis (Laurenti, 1768), is a rare and protected species in Vienna. In spring and summer 2020, we conducted a survey to assess size and status of its population in Donaufeld, an agricultural area designated for real estate development. Recaptures of photographically registered toads allowed to estimate the population size with 137 individuals (confidence interval: 104-181). Comparatively large body size indicates the presence of a well-established population. Reproductive success was high in the study year. A mismatch mating of a male B . viridis with a female Bufo bufo was observed. Mitigation measures are needed to support this population facing imminent habitat deterioration.

Many biological variables are recorded on a circular scale and therefore need different statistical treatment. A common question that is asked of such circular data involves comparison between two groups: Are the populations from which the two samples are drawn differently distributed around the circle? We compared 18 tests for such situations (by simulation) in terms of both abilities to control Type-I error rate near the nominal value, and statistical power. We found that only eight tests offered good control of Type-I error in all our simulated situations. Of these eight, we were able to identify the Watson's U 2 test and a MANOVA approach, based on trigonometric functions of the data, as offering the best power in the overwhelming majority of our test circumstances. There was often little to choose between these tests in terms of power, and no situation where either of the remaining six tests offered substantially better power than either of these. Hence, we recommend the routine use of either Watson's U 2 test or MANOVA approach when comparing two samples of circular data., (© 2021. The Author(s).)

Background: One third of the worldwide amphibian species are threatened, therefore, efficient monitoring efforts are needed. Amphibians which adopt a hidden lifestyle, such as the common spadefoot toad, are often missed with standard surveying efforts. Spadefoot toads can be identified in regurgitated pellets of the barn owl, which provides an effective way to estimate toad activity. In our study we analyzed frequency of spadefoot toad remains from 2004 to 2016 in a steppe landscape in eastern Austria., Methods: We used an automated model selection procedure together with a GLM analysis using a zero inflated error Poisson distribution, to analyze the presence of Pelobates fuscus in barn owl pellets. All analyses were done in the statistical software R, and the scripts to reproduce our results are available within this publication. Our approach may provide a template for other researchers to use for their own pellet data., Conclusions: Our analysis suggested that activity of the common spadefoot toad is mainly influenced by rainfalls, while time of the year and temperature had small but significant effects. Interestingly, our data confirmed the possibility of a second breeding period in summer, triggered by heavy rainfalls. There were no indications for a population decrease in the observed years and locations. Our study shows that barn owl pellets can be used effectivley to assess pelobatid activity in an area. This might constitute a useful monitoring tool for conservation management for amphibians.

Microtubules play a critical role in multiple aspects of neurodevelopment, including the generation, migration and differentiation of neurons. A recurrent mutation (R402H) in the α-tubulin gene TUBA1A is known to cause lissencephaly with cerebellar and striatal phenotypes. Previous work has shown that this mutation does not perturb the chaperone-mediated folding of tubulin heterodimers, which are able to assemble and incorporate into the microtubule lattice. To explore the molecular mechanisms that cause the disease state we generated a new conditional mouse line that recapitulates the R402H variant. We show that heterozygous mutants present with laminar phenotypes in the cortex and hippocampus, as well as a reduction in striatal size and cerebellar abnormalities. We demonstrate that homozygous expression of the R402H allele causes neuronal death and exacerbates a cell intrinsic defect in cortical neuronal migration. Microtubule sedimentation assays coupled with quantitative mass spectrometry demonstrated that the binding and/or levels of multiple microtubule associated proteins (MAPs) are perturbed by the R402H mutation including VAPB, REEP1, EZRIN, PRNP and DYNC1l1/2. Consistent with these data we show that the R402H mutation impairs dynein-mediated transport which is associated with a decoupling of the nucleus to the microtubule organising center. Our data support a model whereby the R402H variant is able to fold and incorporate into microtubules, but acts as a gain of function by perturbing the binding of MAPs., Competing Interests: The authors have declared that no competing interests exist.

Research on navigation in animals is hampered by conflicting results and failed replications. In order to assess the generality of previous results, male Bufo bufo were collected during their breeding migration and translocated to two testing sites, 2.4 and 2.9 km away, respectively, from their breeding pond in the north of Vienna (Austria). There each toad was tested twice for orientation responses in a circular arena, on the night of collection and four days later. On the first test day, the toads showed significant axial orientation along their individual former migration direction. On the second test day, no significant homeward orientation was detected. Both results accord with findings of previous experiments with toads from another population. We analysed the potential influence of environmental factors (temperature, cloud cover and lunar cycle) on toad orientations using a MANOVA approach. Although cloud cover and lunar cycle had small effects on the second test day, they could not explain the absence of homeward orientation. The absence of homing responses in these tests may be either caused by the absence of navigational capabilities of toads beyond their home ranges, or by inadequacies of the applied method. To resolve this question, tracking of freely moving toads should have greater potential than the use of arena experiments.

The biophysical and molecular mechanisms that enable animals to detect magnetic fields are unknown. It has been proposed that birds have a light-dependent magnetic compass that relies on the formation of radical pairs within cryptochrome molecules. Using spectroscopic methods, we show that pigeon cryptochrome clCRY4 is photoreduced efficiently and forms long-lived spin-correlated radical pairs via a tetrad of tryptophan residues. We report that clCRY4 is broadly and stably expressed within the retina but enriched at synapses in the outer plexiform layer in a repetitive manner. A proteomic survey for retinal-specific clCRY4 interactors identified molecules that are involved in receptor signaling, including glutamate receptor-interacting protein 2, which colocalizes with clCRY4. Our data support a model whereby clCRY4 acts as an ultraviolet-blue photoreceptor and/or a light-dependent magnetosensor by modulating glutamatergic synapses between horizontal cells and cones., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Many studies in biology involve data measured on a circular scale. Such data require different statistical treatment from those measured on linear scales. The most common statistical exploration of circular data involves testing the null hypothesis that the data show no aggregation and are instead uniformly distributed over the whole circle. The most common means of performing this type of investigation is with a Rayleigh test. An alternative might be to compare the fit of the uniform distribution model to alternative models. Such model-fitting approaches have become a standard technique with linear data, and their greater application to circular data has been recently advocated. Here we present simulation data that demonstrate that such model-based inference can offer very similar performance to the best traditional tests, but only if adjustment is made in order to control type I error rate., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The transcription factor ZENK is an immediate early gene that has been employed as a surrogate marker to map neuronal activity in the brain. It has been used in a wide variety of species, however, commercially available antibodies have limited immunoreactivity in birds. To address this issue we generated a new mouse monoclonal antibody, 7B7-A3, raised against ZENK from the rock pigeon (Columba livia). We show that 7B7-A3 labels clZENK in both immunoblots and histological stainings with high sensitivity and selectivity for its target. Using a sound stimulation paradigm we demonstrate that 7B7-A3 can detect activity-dependent ZENK expression at key stations of the central auditory pathway of the pigeon. Finally, we compare staining efficiency across three avian species and confirm that 7B7-A3 is compatible with immunohistochemical detection of ZENK in the rock pigeon, zebra finch, and domestic chicken. Taken together, 7B7-A3 represents a useful tool for the avian neuroscience community to map functional activity in the brain.

Abstract: The most common statistical procedure with a sample of circular data is to test the null hypothesis that points are spread uniformly around the circle without a preferred direction. An array of tests for this has been developed. However, these tests were designed for continuously distributed data, whereas often (e.g. due to limited precision of measurement techniques) collected data is aggregated into a set of discrete values (e.g. rounded to the nearest degree). This disparity can cause an uncontrolled increase in type I error rate, an effect that is particularly problematic for tests that are based on the distribution of arc lengths between adjacent points (such as the Rao spacing test). Here, we demonstrate that an easy-to-apply modification can correct this problem, and we recommend this modification when using any test, other than the Rayleigh test, of circular uniformity on aggregated data. We provide R functions for this modification for several commonly used tests. In addition, we tested the power of a recently proposed test, the Gini test. However, we concluded that it lacks sufficient increase in power to replace any of the tests already in common use. In conclusion, using any of the standard circular tests (except the Rayleigh test) without modifications on rounded/aggregated data, especially with larger sample sizes, will increase the proportion of false-positive results-but we demonstrate that a simple and general modification avoids this problem., Significance Statement: Circular data are widespread across biological disciplines, e.g. in orientation studies or circadian rhythms. Often these data are rounded to the nearest 1-10 degrees. We have shown previously that this leads to an inflation of false-positive results when testing whether the data is significantly different from a random distribution using the Rao test. Here we present a modification that avoids this increase in false-positives for rounded data while retaining statistical power for a variety of tests. In sum, we provide comprehensive guidance on how best to test for departure from uniformity in non-continuous data., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

A diverse array of vertebrate species employs the Earth's magnetic field to assist navigation. Despite compelling behavioral evidence that a magnetic sense exists, the location of the primary sensory cells and the underlying molecular mechanisms remain unknown [1]. To date, most research has focused on a light-dependent radical-pair-based concept and a system that is proposed to rely on biogenic magnetite (Fe 3 O 4 ) [2, 3]. Here, we explore an overlooked hypothesis that predicts that animals detect magnetic fields by electromagnetic induction within the semicircular canals of the inner ear [4]. Employing an assay that relies on the neuronal activity marker C-FOS, we confirm that magnetic exposure results in activation of the caudal vestibular nuclei in pigeons that is independent of light [5]. We show experimentally and by physical calculations that magnetic stimulation can induce electric fields in the pigeon semicircular canals that are within the physiological range of known electroreceptive systems. Drawing on this finding, we report the presence of a splice isoform of a voltage-gated calcium channel (Ca V 1.3) in the pigeon inner ear that has been shown to mediate electroreception in skates and sharks [6]. We propose that pigeons detect magnetic fields by electromagnetic induction within the semicircular canals that is dependent on the presence of apically located voltage-gated cation channels in a population of electrosensory hair cells., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Circular data are gathered in diverse fields of science where measured traits are cyclical in nature: such as compass directions or times of day. The most common statistical question asked of a sample of circular data is whether the data seems to be drawn from a uniform distribution or one that is concentrated around one or more preferred directions. The overwhelmingly most-popular test of the null hypothesis of uniformity is the Rayleigh test, even though this test is known to have very low power in some circumstances. Here we present simulation studies evaluating the performance of tests developed as alternatives to the Rayleigh test., Results: The results of our simulations demonstrate that a single test, the Hermans and Rasson test is almost as powerful as the Rayleigh test in unimodal situations (when the Rayleigh test does well) but substantially outperforms the Rayleigh test in multimodal situations., Conclusion: We recommend researchers switch to routine use of the new Hermans and Rasson test. We also demonstrate that all available tests have low power to detect departures from uniformity involving more than two concentrated regions: we recommend that where researchers suspect such complex departures that they collect substantially-sized samples and apply another recent test due to Pycke that was designed specifically for such complex cases. We provide clear textual descriptions of how to implement each of these recommended tests and encode them in R functions that we provide.

Background: For data collected on a circular rather than linear scale, a very common procedure is to test whether the underlying distribution appears to deviate from circular uniformity. Rao's spacing test is often used to evaluate the support the data offers for the null hypothesis of uniformity. Here we demonstrate that the traditional version of this test fails to adequately control type I error rate when the data is non-continuous (i.e. is rounded/grouped to a finite number of discrete values, e.g. to the nearest degree, a common situation). To overcome this issue, we provide a numerically-intensive simulation version of the test., Methods: We use a simulation study to explore the performance of the traditional and our novel variant on Rao's spacing test, both in terms of control of type I error rate and statistical power., Results: When data is measured on a continuous circular scale then both methods offer good control of type I error and similar statistical power. If the data is rounded (even to a relatively fine scale such as to the nearest degree - giving 360 possible values), however, the traditional method produces highly inflated type I error rates, particularly with high sample sizes, that make it inappropriate for application to such data. In contrast, our simulation method retains good control of type I error while offering levels of statistical power similar to the traditional Rao test., Conclusions: The traditional method of applying Rao's spacing test should be replaced by the simulation-based variant introduced here. The two methods offer similar performance but only the simulation method retains good control of the type I error rate when circular data is rounded to a finite set of values (likely due to limited precision of measuring equipment). Adoption of the simulation variant will substantially improve the reliability of this regularly-used test in the commonplace situation where data values are rounded., Competing Interests: No ethical approvals were required.No consents were required.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Magnetic sensing is used to structure every-day, non-migratory behaviours in many animals. We show that crayfish exhibit robust spontaneous magnetic alignment responses. These magnetic behaviours are altered by interactions with Branchiobdellidan worms, which are obligate ectosymbionts. Branchiobdellidan worms have previously been shown to have positive effects on host growth when present at moderate densities, and negative effects at relatively high densities. Here we show that crayfish with moderate densities of symbionts aligned bimodally along the magnetic northeast-southwest axis, similar to passive magnetic alignment responses observed across a range of stationary vertebrates. In contrast, crayfish with high symbiont densities failed to exhibit consistent alignment relative to the magnetic field. Crayfish without symbionts shifted exhibited quadramodal magnetic alignment and were more active. These behavioural changes suggest a change in the organization of spatial behaviour with increasing ectosymbiont densities. We propose that the increased activity and a switch to quadramodal magnetic alignment may be associated with the use of systematic search strategies. Such a strategy could increase contact-rates with conspecifics in order to replenish the beneficial ectosymbionts that only disperse between hosts during direct contact. Our results demonstrate that crayfish perceive and respond to magnetic fields, and that symbionts influence magnetically structured spatial behaviour of their hosts.

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Over the last three decades, evidence has emerged that low-intensity magnetic fields can influence biological systems. It is now well established that migratory birds have the capacity to detect the Earth's magnetic field; it has been reported that power lines are associated with childhood leukemia and that pulsed magnetic fields increase the production of reactive oxidative species (ROS) in cellular systems. Justifiably, studies in this field have been viewed with skepticism, as the underlying molecular mechanisms are unknown. In the accompanying paper, Sherrard and colleagues report that low-flux pulsed electromagnetic fields (PEMFs) result in aversive behavior in Drosophila larvae and ROS production in cell culture. They further report that these responses require the presence of cryptochrome, a putative magnetoreceptor. If correct, it is conceivable that carcinogenesis associated with power lines, PEMF-induced ROS generation, and animal magnetoreception share a common mechanistic basis., Competing Interests: The authors have declared that no competing interests exist.

The formation of the vertebrate brain requires the generation, migration, differentiation and survival of neurons. Genetic mutations that perturb these critical cellular events can result in malformations of the telencephalon, providing a molecular window into brain development. Here we report the identification of an N-ethyl-N-nitrosourea-induced mouse mutant characterized by a fractured hippocampal pyramidal cell layer, attributable to defects in neuronal migration. We show that this is caused by a hypomorphic mutation in Vps15 that perturbs endosomal-lysosomal trafficking and autophagy, resulting in an upregulation of Nischarin, which inhibits Pak1 signaling. The complete ablation of Vps15 results in the accumulation of autophagic substrates, the induction of apoptosis and severe cortical atrophy. Finally, we report that mutations in VPS15 are associated with cortical atrophy and epilepsy in humans. These data highlight the importance of the Vps15-Vps34 complex and the Nischarin-Pak1 signaling hub in the development of the telencephalon.

Circular data are common in biological studies. The most fundamental question that can be asked of a sample of circular data is whether it suggests that the underlying population is uniformly distributed around the circle, or whether it is concentrated around at least one preferred direction (e.g. a migratory goal or activity phase). We compared the statistical power of five commonly used tests (the Rayleigh test, the V-test, Watson's test, Kuiper's test and Rao's spacing test) across a range of different unimodal scenarios. The V-test showed higher power for symmetrical distributions, Rao's spacing performed worst for all explored unimodal distributions tested and the remaining three tests showed very similar performance. However, the V-test only applies if the hypothesis is restricted to one (pre-specified) direction of interest. In all other unimodal cases, we recommend using the Rayleigh test. Much less explored is the multimodal case with data concentrated around several directions. We performed power simulations for a variety of multimodal situations, testing the performance of the widely used Rayleigh, Rao's, Watson, and Kuiper's tests as well as the more recent Bogdan and Hermans-Rasson tests. Our analyses of alternative statistical methods show that the commonly used tests lack statistical power in many of multimodal cases. Transformation of the raw data (e.g. doubling the angles) can overcome some of the issues, but only in the case of perfect f- fold symmetry. However, the Hermans-Rasson method, which is not yet implemented in any software package, outcompetes the alternative tests (often by substantial margins) in most of the multimodal situations explored. We recommend the wider uptake of the powerful but hitherto neglected Hermans-Rasson method. In summary, we provide guidance for biologists helping them to make decisions when testing circular data for single or multiple departures from uniformity., Competing Interests: Compliance with ethical standardsThe authors declare that they have no competing interests.

The Earth's magnetic field is involved in spatial behaviours ranging from long-distance migration to non-goal directed behaviours, such as spontaneous magnetic alignment (SMA). Mercury is a harmful pollutant most often generated from anthropogenic sources that can bio-accumulate in animal tissue over a lifetime. We compared SMA of hatchling snapping turtles from mothers captured at reference (i.e., low mercury) and mercury contaminated sites. Reference turtles showed radio frequency-dependent SMA along the north-south axis, consistent with previous studies of SMA, while turtles with high levels of maternally inherited mercury failed to show consistent magnetic alignment. In contrast, there was no difference between reference and mercury exposed turtles on standard performance measures. The magnetic field plays an important role in animal orientation behaviour and may also help to integrate spatial information from a variety of sensory modalities. As a consequence, mercury may compromise the performance of turtles in a wide variety of spatial tasks. Future research is needed to determine the threshold for mercury effects on snapping turtles, whether mercury exposure compromises spatial behaviour of adult turtles, and whether mercury has a direct effect on the magnetoreception mechanism(s) that mediate SMA or a more general effect on the nervous system., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The generation, migration, and differentiation of neurons requires the functional integrity of the microtubule cytoskeleton. Mutations in the tubulin gene family are known to cause various neurological diseases including lissencephaly, ocular motor disorders, polymicrogyria and amyotrophic lateral sclerosis. We have previously reported that mutations in TUBB5 cause microcephaly that is accompanied by severe intellectual impairment and motor delay. Here we present the characterization of a Tubb5 mouse model that allows for the conditional expression of the pathogenic E401K mutation. Homozygous knockin animals exhibit a severe reduction in brain size and in body weight. These animals do not show any significant impairment in general activity, anxiety, or in the acoustic startle response, however, present with notable defects in motor coordination. When assessed on the static rod apparatus mice took longer to orient and often lost their balance completely. Interestingly, mutant animals also showed defects in prepulse inhibition, a phenotype associated with sensorimotor gating and considered an endophenotype for schizophrenia. This study provides insight into the behavioral consequences of tubulin gene mutations., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

A wealth of evidence provides support for magnetic alignment (MA) behavior in a variety of disparate species within the animal kingdom, in which an animal, or a group of animals, show a tendency to align the body axis in a consistent orientation relative to the geomagnetic field lines. Interestingly, among vertebrates, MA typically coincides with the north-south magnetic axis, however, the mean directional preferences of an individual or group of organisms is often rotated clockwise from the north-south axis. We hypothesize that this shift is not a coincidence, and future studies of this subtle, yet consistent phenomenon may help to reveal some properties of the underlying sensory or processing mechanisms, that, to date, are not well understood. Furthermore, characterizing the fine structure exhibited in MA behaviors may provide key insights to the biophysical substrates mediating magnetoreception in vertebrates. Therefore, in order to determine if a consistent shift is exhibited in taxonomically diverse vertebrates, we performed a meta-analysis on published MA datasets from 23 vertebrate species that exhibited an axial north-south preference. This analysis revealed a significant clockwise shift from the north-south magnetic axis. We summarize and discuss possible competing hypotheses regarding the proximate mechanisms underlying the clockwise shifted MA and conclude that the most likely cause of such a shift would be a lateralization in central processing of magnetic information., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We investigated spontaneous magnetic alignment (SMA) by juvenile snapping turtles using exposure to low-level radio frequency (RF) fields at the Larmor frequency to help characterize the underlying sensory mechanism. Turtles, first introduced to the testing environment without the presence of RF aligned consistently towards magnetic north when subsequent magnetic testing conditions were also free of RF ('RF off → RF off'), but were disoriented when subsequently exposed to RF ('RF off → RF on'). In contrast, animals initially introduced to the testing environment with RF present were disoriented when tested without RF ('RF on → RF off'), but aligned towards magnetic south when tested with RF ('RF on → RF on'). Sensitivity of the SMA response of yearling turtles to RF is consistent with the involvement of a radical pair mechanism. Furthermore, the effect of RF appears to result from a change in the pattern of magnetic input, rather than elimination of magnetic input altogether, as proposed to explain similar effects in other systems/organisms. The findings show that turtles first exposed to a novel environment form a lasting association between the pattern of magnetic input and their surroundings. However, under natural conditions turtles would never experience a change in the pattern of magnetic input. Therefore, if turtles form a similar association of magnetic cues with the surroundings each time they encounter unfamiliar habitat, as seems likely, the same pattern of magnetic input would be associated with multiple sites/localities. This would be expected from a sensory input that functions as a global reference frame, helping to place multiple locales (i.e., multiple local landmark arrays) into register to form a global map of familiar space.

Introduction: The ability to relocate home or breeding sites after experimental removal has been observed in several amphibians and the sensory basis of this behavior has been studied in some temperate-region species. However, the actual return trajectories have rarely been quantified in these studies and it remains unknown how different cues guide the homing behavior. Dendrobatidae (dart-poison frogs) exhibit some of the most complex spatial behaviors among amphibians, such as territoriality and tadpole transport. Recent data showed that Allobates femoralis, a frog with paternal tadpole transport, successfully returns to the home territories after experimental translocations of up to 400 m. In the present study, we used harmonic direction finding to obtain homing trajectories. Additionally, we quantified the initial orientation of individuals, translocated 10 m to 105 m, in an arena assay., Results: Tracking experiments revealed that homing trajectories are characterized by long periods of immobility (up to several days) and short periods (several hours) of rapid movement, closely fitting a straight line towards the home territory. In the arena assay, the frogs showed significant homeward orientation for translocation distances of 35 m to 70 m but not for longer and shorter distances., Conclusions: Our results describe a very accurate homing behavior in male A. femoralis. The straightness of trajectories and initial homeward orientation suggest integration of learned landmarks providing a map position for translocated individuals. Future research should focus on the role of learning in homing behavior and the exact nature of cues being used.

Magnetoreception has been demonstrated in all five vertebrate classes. In rodents, nest building experiments have shown the use of magnetic cues by two families of molerats, Siberian hamsters and C57BL/6 mice. However, assays widely used to study rodent spatial cognition (e.g. water maze, radial arm maze) have failed to provide evidence for the use of magnetic cues. Here we show that C57BL/6 mice can learn the magnetic direction of a submerged platform in a 4-armed (plus) water maze. Naïve mice were given two brief training trials. In each trial, a mouse was confined to one arm of the maze with the submerged platform at the outer end in a predetermined alignment relative to magnetic north. Between trials, the training arm and magnetic field were rotated by 180(°) so that the mouse had to swim in the same magnetic direction to reach the submerged platform. The directional preference of each mouse was tested once in one of four magnetic field alignments by releasing it at the center of the maze with access to all four arms. Equal numbers of responses were obtained from mice tested in the four symmetrical magnetic field alignments. Findings show that two training trials are sufficient for mice to learn the magnetic direction of the submerged platform in a plus water maze. The success of these experiments may be explained by: (1) absence of alternative directional cues (2), rotation of magnetic field alignment, and (3) electromagnetic shielding to minimize radio frequency interference that has been shown to interfere with magnetic compass orientation of birds. These findings confirm that mice have a well-developed magnetic compass, and give further impetus to the question of whether epigeic rodents (e.g., mice and rats) have a photoreceptor-based magnetic compass similar to that found in amphibians and migratory birds.