A GPS assisted translocation experiment to study the homing behavior of red deer.

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.

C. elegans is not a robust model organism for the magnetic sense.

Magnetoreception is defined as the ability to sense and use the Earth's magnetic field, for example to orient and direct movements. The receptors and sensory mechanisms underlying behavioral responses to magnetic fields remain unclear. A previous study described magnetoreception in the nematode Caenorhabditis elegans, which requires the activity of a single pair of sensory neurons. These results suggest C. elegans as a tractable model organism for facilitating the search for magnetoreceptors and signaling pathways. The finding is controversial, however, as an attempt to replicate the experiment in a different laboratory was unsuccessful. We here independently test the magnetic sense of C. elegans, closely replicating the assays developed in the original publication. We find that C. elegans show no directional preference in magnetic fields of both natural and higher intensity, suggesting that magnetotactic behavior in the worm is not robustly evoked in a laboratory setting. Given the lack of a robust magnetic response under controlled conditions, we conclude that C. elegans is not a suitable model organism to study the mechanism of the magnetic sense.

Author Correction: Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Functional anatomy of the middle and inner ears of the red fox, in comparison to domestic dogs and cats.

Anatomical middle and inner ear parameters are often used to predict hearing sensitivities of mammalian species. Given that ear morphology is substantially affected both by phylogeny and body size, it is interesting to consider whether the relatively small anatomical differences expected in related species of similar size have a noticeable impact on hearing. We present a detailed anatomical description of the middle and inner ears of the red fox Vulpes vulpes, a widespread, wild carnivore for which a behavioural audiogram is available. We compare fox ears to those of the well-studied and similarly sized domestic dog and cat, taking data for dogs and cats from the literature as well as providing new measurements of basilar membrane (BM) length and hair cell numbers and densities in these animals. Our results show that the middle ear of the red fox is very similar to that of dogs. The most obvious difference from that of the cat is the lack of a fully formed bony septum in the bulla tympanica of the fox. The cochlear structures of the fox, however, are very like those of the cat, whereas dogs have a broader BM in the basal cochlea. We further report that the mass of the middle ear ossicles and the bulla volume increase with age in foxes. Overall, the ear structures of foxes, dogs and cats are anatomically very similar, and their behavioural audiograms overlap. However, the results of several published models and correlations that use middle and inner ear measurements to predict aspects of hearing were not always found to match well with audiogram data, especially when it came to the sharper tuning in the fox audiogram. This highlights that, although there is evidently a broad correspondence between structure and function, it is not always possible to draw direct links when considering more subtle differences between related species.

A high sensitivity ZENK monoclonal antibody to map neuronal activity in Aves.

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.

A Putative Mechanism for Magnetoreception by Electromagnetic Induction in the Pigeon Inner Ear.

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 (Fe3O4) [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 (CaV1.3) 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.

Brain atlas of the African mole-rat Fukomys anselli.

African mole-rats are subterranean rodents that spend their whole life in underground burrow systems. They show a range of morphological and physiological adaptations to their ecotope, for instance severely reduced eyes and specialized somatosensory, olfactory, and auditory systems. These adaptations are also reflected in the accessory sensory pathways in the brain that process the input coming from the sensory organs. So far, a brain atlas was available only for the naked mole-rat (Heterocephalus glaber). The Ansell's mole-rat (Fukomys anselli) has been the subject of many investigations in various disciplines (ethology, sensory physiology, and anatomy) including magnetic orientation. It is therefore surprising that an atlas of the brain of this species was not available so far. Here, we present a comprehensive atlas of the Ansell's mole-rat brain based on Nissl and Klüver-Barrera stained sections. We identify and label 375 brain regions and discuss selected differences from the brain of the closely related naked mole-rat as well as from epigeic mammals (rat), with a particular focus on the auditory brainstem. This atlas can serve as a reference for future neuroanatomical investigations of subterranean mammals.

EyeCi: Optical clearing and imaging of immunolabeled mouse eyes using light-sheet fluorescence microscopy.

Immunofluorescent imaging is an indispensable technique to study morphology and molecular aspects in tissues. Classical approaches make it necessary to cut physical sections of tissue samples to overcome the limited penetration depth of light, restricting the available information to two dimensions. Recent advances in tissue-clearing techniques enable imaging of fluorescently labeled organs and entire organisms on a cellular level in three dimensions without the need of sectioning. Volume imaging of immunolabeled and cleared tissues started a new era of systems biology, because these techniques provide information on connectivity and circuits, especially in structures with projections in three dimensions such as vascular or nervous systems. The variety of published clearing protocols allows the imaging of every organ with a single exception: the eye. Whole-eye clearing approaches were unsuccessful so far due to the strong pigmentation of the retinal pigment epithelium. Here, we present a new protocol that combines a highly effective melanin bleaching step with solvent-based clearing, termed EyeCi. The protocol is compatible with immunolabeling as demonstrated by the visualization of ocular and retinal vasculature in the intact mouse eye by means of light-sheet fluorescence microscopy. This novel protocol is rapid (1 week) and inexpensive, hence allowing high-throughput, high resolution analysis of vascular architecture of healthy and diseased eyes, in its native, three-dimensional organization within intact eyeballs. Volume imaging of whole cleared eyeballs further enables three-dimensional surface reconstruction and automated quantification of choroidal and retinal vasculature extending ocular imaging to a global level. Thus, EyeCi represents an extension to state-of-the-art light microscopy techniques and is potentially suitable for the investigation of vascular leakage or neovascularization processes.

Retinal photoreceptor and ganglion cell types and topographies in the red fox (Vulpes vulpes) and Arctic fox (Vulpes lagopus).

The red fox (Vulpes vulpes) is the carnivore with the widest distribution in the world. Not much is known about the visual system of these predominantly forest-dwelling animals. The closely related Arctic fox (Vulpes lagopus) lives in more open tundra habitats. In search for corresponding adaptations, we examined the photoreceptors and retinal ganglion cells (RGCs), using opsin immunohistochemistry, lucifer yellow injections and Nissl staining. Both species possess a majority of middle-to-longwave-sensitive (M/L) and a minority of shortwave-sensitive (S) cones, indicating dichromatic color vision. Area centralis peak cone densities are 22,600/mm2 in the red fox and 44,800/mm2 in the Arctic fox. Both have a centro-peripheral density decrease of M/L cones, and a dorsoventrally increasing density of S cones. Rod densities and rod/cone ratios are higher in the red fox than the Arctic fox. Both species possess the carnivore-typical alpha and beta RGCs. The RGC topography shows a centro-peripheral density gradient with a distinct area centralis (mean peak density 7,900 RGCs/mm2 in the red fox and 10,000 RGCs/mm2 in the Arctic fox), a prominent visual streak of higher RGC densities in the Arctic fox, and a moderate visual streak in the red fox. Visual acuity and estimated sound localization ability were nearly identical between both species. In summary, the red fox retina shows adaptations to nocturnal activity in a forest habitat, while the Arctic fox retina is better adapted to higher light levels in the open tundra.

Subcellular analysis of pigeon hair cells implicates vesicular trafficking in cuticulosome formation and maintenance.

Hair cells are specialized sensors located in the inner ear that enable the transduction of sound, motion, and gravity into neuronal impulses. In birds some hair cells contain an iron-rich organelle, the cuticulosome, that has been implicated in the magnetic sense. Here, we exploit histological, transcriptomic, and tomographic methods to investigate the development of cuticulosomes, as well as the molecular and subcellular architecture of cuticulosome positive hair cells. We show that this organelle forms rapidly after hatching in a process that involves vesicle fusion and nucleation of ferritin nanoparticles. We further report that transcripts involved in endocytosis, extracellular exosomes, and metal ion binding are differentially expressed in cuticulosome positive hair cells. These data suggest that the cuticulosome and the associated molecular machinery regulate the concentration of iron within the labyrinth of the inner ear, which might indirectly tune a magnetic sensor that relies on electromagnetic induction.

Non-Breeding Eusocial Mole-Rats Produce Viable Sperm--Spermiogram and Functional Testicular Morphology of Fukomys anselli.

Ansell's mole-rats (Fukomys anselli) are subterranean rodents living in families composed of about 20 members with a single breeding pair and their non-breeding offspring. Most of them remain with their parents for their lifetime and help to maintain and defend the natal burrow system, forage, and care for younger siblings. Since incest avoidance is based on individual recognition (and not on social suppression) we expect that non-breeders produce viable sperm spontaneously. We compared the sperm of breeding and non-breeding males, obtained by electroejaculation and found no significant differences in sperm parameters between both groups. Here, we used electroejaculation to obtain semen for the first time in a subterranean mammal. Spermiogram analysis revealed no significant differences in sperm parameters between breeders and non-breeders. We found significantly larger testes (measured on autopsies and on living animals per ultrasonography) of breeders compared to non-breeders (with body mass having a significant effect). There were no marked histological differences between breeding and non-breeding males, and the relative area occupied by Leydig cells and seminiferous tubules on histological sections, respectively, was not significantly different between both groups. The seminiferous epithelium and to a lesser degree the interstitial testicular tissue are characterized by lesions (vacuolar degenerations), however, this feature does not hinder fertilization even in advanced stages of life. The continuous production of viable sperm also in sexually abstinent non-breeders might be best understood in light of the mating and social system of Fukomys anselli, and the potential to found a new family following an unpredictable and rare encounter with an unfamiliar female ("provoked or induced dispersal"). Apparently, the non-breeders do not reproduce because they do not copulate but not because they would be physiologically infertile. The significantly increased testes volume of breeding males (compared to non-breeders) is in agreement with previously found higher testosterone levels of breeders.

Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals.

Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown, and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae, and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein.

Dogs are sensitive to small variations of the Earth's magnetic field.

INTRODUCTION: Several mammalian species spontaneously align their body axis with respect to the Earth's magnetic field (MF) lines in diverse behavioral contexts. Magnetic alignment is a suitable paradigm to scan for the occurrence of magnetosensitivity across animal taxa with the heuristic potential to contribute to the understanding of the mechanism of magnetoreception and identify further functions of magnetosensation apart from navigation. With this in mind we searched for signs of magnetic alignment in dogs. We measured the direction of the body axis in 70 dogs of 37 breeds during defecation (1,893 observations) and urination (5,582 observations) over a two-year period. After complete sampling, we sorted the data according to the geomagnetic conditions prevailing during the respective sampling periods. Relative declination and intensity changes of the MF during the respective dog walks were calculated from daily magnetograms. Directional preferences of dogs under different MF conditions were analyzed and tested by means of circular statistics. RESULTS: Dogs preferred to excrete with the body being aligned along the North-South axis under calm MF conditions. This directional behavior was abolished under unstable MF. The best predictor of the behavioral switch was the rate of change in declination, i.e., polar orientation of the MF. CONCLUSIONS: It is for the first time that (a) magnetic sensitivity was proved in dogs, (b) a measurable, predictable behavioral reaction upon natural MF fluctuations could be unambiguously proven in a mammal, and (c) high sensitivity to small changes in polarity, rather than in intensity, of MF was identified as biologically meaningful. Our findings open new horizons in magnetoreception research. Since the MF is calm in only about 20% of the daylight period, our findings might provide an explanation why many magnetoreception experiments were hardly replicable and why directional values of records in diverse observations are frequently compromised by scatter.

Directional compass preference for landing in water birds.

INTRODUCTION: Landing flight in birds is demanding on visual control of velocity, distance to target, and slope of descent. Birds flying in flocks must also keep a common course of landing in order to avoid collisions. Whereas the wind direction may provide a cue for landing, the nature of the landing direction indicator under windless conditions has been unknown. We recorded and analysed landing directions of 3,338 flocks in 14 species of water birds in eight countries. RESULTS: We show that the preferred landing direction, independently of the direction from which the birds have arrived, is along the north-south axis. We analysed the effect of the time of the year, time of the day (and thus sun position), weather (sunny versus overcast), light breeze, locality, latitude, and magnetic declination in 2,431 flocks of mallards (Anas platyrhynchos) and found no systematic effect of these factors upon the preferred direction of landing. We found that magnetic North was a better predictor for landing direction than geographic North. CONCLUSIONS: In absence of any other common denominator determining the landing direction, the alignment with the magnetic field lines seems to be the most plausible if not the only explanation for the directional landing preference under windless and overcast conditions and we suggest that the magnetic field thus provides a landing direction indicator.

Magnetic alignment in carps: evidence from the Czech christmas fish market.

While magnetoreception in birds has been studied intensively, the literature on magnetoreception in bony fish, and particularly in non-migratory fish, is quite scarce. We examined alignment of common carps (Cyprinus carpio) at traditional Christmas sale in the Czech Republic. The sample comprised measurements of the directional bearings in 14,537 individual fish, distributed among 80 large circular plastic tubs, at 25 localities in the Czech Republic, during 817 sampling sessions, on seven subsequent days in December 2011. We found that carps displayed a statistically highly significant spontaneous preference to align their bodies along the North-South axis. In the absence of any other common orientation cues which could explain this directional preference, we attribute the alignment of the fish to the geomagnetic field lines. It is apparent that the display of magnetic alignment is a simple experimental paradigm of great heuristic potential.