Monarch butterflies memorize the spatial location of a food source.

Spatial memory helps animals to navigate familiar environments. In insects, spatial memory has extensively been studied in central place foragers such as ants and bees. However, if butterflies memorize a spatial location remains unclear. Here, we conducted behavioural experiments to test whether monarch butterflies (Danaus plexippus) can remember and retrieve the spatial location of a food source. We placed several visually identical feeders in a flight cage, with only one feeder providing sucrose solution. Across multiple days, individual butterflies predominantly visited the rewarding feeder. Next, we displaced a salient landmark close to the feeders to test which visual cue the butterflies used to relocate the rewarding feeder. While occasional landmark displacements were ignored by the butterflies and did not affect their decisions, systematic displacement of both the landmark and the rewarding feeder demonstrated that the butterflies associated the salient landmark with the feeder's position. Altogether, we show that butterflies consolidate and retrieve spatial memory in the context of foraging.

The influence of stimulus history on directional coding in the monarch butterfly brain.

The central complex is a brain region in the insect brain that houses a neural network specialized to encode directional information. Directional coding has traditionally been investigated with compass cues that revolve in full rotations and at constant angular velocities around the insect's head. However, these stimulus conditions do not fully simulate an insect's sensory perception of compass cues during navigation. In nature, an insect flight is characterized by abrupt changes in moving direction as well as constant changes in velocity. The influence of such varying cue dynamics on compass coding remains unclear. We performed long-term tetrode recordings from the brain of monarch butterflies to study how central complex neurons respond to different stimulus velocities and directions. As these butterflies derive directional information from the sun during migration, we measured the neural response to a virtual sun. The virtual sun was either presented as a spot that appeared at random angular positions or was rotated around the butterfly at different angular velocities and directions. By specifically manipulating the stimulus velocity and trajectory, we dissociated the influence of angular velocity and direction on compass coding. While the angular velocity substantially affected the tuning directedness, the stimulus trajectory influenced the shape of the angular tuning curve. Taken together, our results suggest that the central complex flexibly adjusts its directional coding to the current stimulus dynamics ensuring a precise compass even under highly demanding conditions such as during rapid flight maneuvers.

Stimulus-dependent orientation strategies in monarch butterflies.

Insects are well known for their ability to keep track of their heading direction based on a combination of skylight cues and visual landmarks. This allows them to navigate back to their nest, disperse throughout unfamiliar environments, as well as migrate over large distances between their breeding and non-breeding habitats. The monarch butterfly (Danaus plexippus), for instance, is known for its annual southward migration from North America to certain trees in Central Mexico. To maintain a constant flight route, these butterflies use a time-compensated sun compass, which is processed in a region in the brain, termed the central complex. However, to successfully complete their journey, the butterflies' brain must generate a multitude of orientation strategies, allowing them to dynamically switch from sun-compass orientation to a tactic behavior toward a certain target. To study whether monarch butterflies exhibit different orientation modes and if they can switch between them, we observed the orientation behavior of tethered flying butterflies in a flight simulator while presenting different visual cues to them. We found that the butterflies' behavior depended on the presented visual stimulus. Thus, while a dark stripe was used for flight stabilization, a bright stripe was fixated by the butterflies in their frontal visual field. If we replaced a bright stripe with a simulated sun stimulus, the butterflies switched their behavior and exhibited compass orientation. Taken together, our data show that monarch butterflies rely on and switch between different orientation modes, allowing the animal to adjust orientation to its actual behavioral demands.

Multimodal cue integration in the dung beetle compass.

South African ball-rolling dung beetles exhibit a unique orientation behavior to avoid competition for food: after forming a piece of dung into a ball, they efficiently escape with it from the dung pile along a straight-line path. To keep track of their heading, these animals use celestial cues, such as the sun, as an orientation reference. Here we show that wind can also be used as a guiding cue for the ball-rolling beetles. We demonstrate that this mechanosensory compass cue is only used when skylight cues are difficult to read, i.e., when the sun is close to the zenith. This raises the question of how the beetles combine multimodal orientation input to obtain a robust heading estimate. To study this, we performed behavioral experiments in a tightly controlled indoor arena. This revealed that the beetles register directional information provided by the sun and the wind and can use them in a weighted manner. Moreover, the directional information can be transferred between these 2 sensory modalities, suggesting that they are combined in the spatial memory network in the beetle's brain. This flexible use of compass cue preferences relative to the prevailing visual and mechanosensory scenery provides a simple, yet effective, mechanism for enabling precise compass orientation at any time of the day.

How Dung Beetles Steer Straight.

Distant and predictable features in the environment make ideal compass cues to allow movement along a straight path. Ball-rolling dung beetles use a wide range of different signals in the day or night sky to steer themselves along a fixed bearing. These include the sun, the Milky Way, and the polarization pattern generated by the moon. Almost two decades of research into these remarkable creatures have shown that the dung beetle's compass is flexible and readily adapts to the cues available in its current surroundings. In the morning and afternoon, dung beetles use the sun to orient, but at midday, they prefer to use the wind, and at night or in a forest, they rely primarily on polarized skylight to maintain straight paths. We are just starting to understand the neuronal substrate underlying the dung beetle's compass and the mystery of why these beetles start each journey with a dance.

Sun compass neurons are tuned to migratory orientation in monarch butterflies.

Every autumn, monarch butterflies migrate from North America to their overwintering sites in Central Mexico. To maintain their southward direction, these butterflies rely on celestial cues as orientation references. The position of the sun combined with additional skylight cues are integrated in the central complex, a region in the butterfly's brain that acts as an internal compass. However, the central complex does not solely guide the butterflies on their migration but also helps monarchs in their non-migratory form manoeuvre on foraging trips through their habitat. By comparing the activity of input neurons of the central complex between migratory and non-migratory butterflies, we investigated how a different lifestyle affects the coding of orientation information in the brain. During recording, we presented the animals with different simulated celestial cues and found that the encoding of the sun was narrower in migratory compared to non-migratory butterflies. This feature might reflect the need of the migratory monarchs to rely on a precise sun compass to keep their direction during their journey. Taken together, our study sheds light on the neural coding of celestial cues and provides insights into how a compass is adapted in migratory animals to successfully steer them to their destination.

A micro-CT-based standard brain atlas of the bumblebee.

In recent years, bumblebees have become a prominent insect model organism for a variety of biological disciplines, particularly to investigate learning behaviors as well as visual performance. Understanding these behaviors and their underlying neurobiological principles requires a clear understanding of brain anatomy. Furthermore, to be able to compare neuronal branching patterns across individuals, a common framework is required, which has led to the development of 3D standard brain atlases in most of the neurobiological insect model species. Yet, no bumblebee 3D standard brain atlas has been generated. Here we present a brain atlas for the buff-tailed bumblebee Bombus terrestris using micro-computed tomography (micro-CT) scans as a source for the raw data sets, rather than traditional confocal microscopy, to produce the first ever micro-CT-based insect brain atlas. We illustrate the advantages of the micro-CT technique, namely, identical native resolution in the three cardinal planes and 3D structure being better preserved. Our Bombus terrestris brain atlas consists of 30 neuropils reconstructed from ten individual worker bees, with micro-CT allowing us to segment neuropils completely intact, including the lamina, which is a tissue structure often damaged when dissecting for immunolabeling. Our brain atlas can serve as a platform to facilitate future neuroscience studies in bumblebees and illustrates the advantages of micro-CT for specific applications in insect neuroanatomy.

Spatial orientation based on multiple visual cues in non-migratory monarch butterflies.

Monarch butterflies (Danaus plexippus) are prominent for their annual long-distance migration from North America to their overwintering area in Central Mexico. To find their way on this long journey, they use a sun compass as their main orientation reference but will also adjust their migratory direction with respect to mountain ranges. This indicates that the migratory butterflies also attend to the panorama to guide their travels. Although the compass has been studied in detail in migrating butterflies, little is known about the orientation abilities of non-migrating butterflies. Here, we investigated whether non-migrating butterflies - which stay in a more restricted area to feed and breed - also use a similar compass system to guide their flights. Performing behavioral experiments on tethered flying butterflies in an indoor LED flight simulator, we found that the monarchs fly along straight tracks with respect to a simulated sun. When a panoramic skyline was presented as the only orientation cue, the butterflies maintained their flight direction only during short sequences, suggesting that they potentially use it for flight stabilization. We further found that when we presented the two cues together, the butterflies incorporate both cues in their compass. Taken together, we show here that non-migrating monarch butterflies can combine multiple visual cues for robust orientation, an ability that may also aid them during their migration.

A dung beetle that path integrates without the use of landmarks.

Unusual amongst dung beetles, Scarabaeus galenus digs a burrow that it provisions by making repeated trips to a nearby dung pile. Even more remarkable is that these beetles return home moving backwards, with a pellet of dung between their hind legs. Here, we explore the strategy that S. galenus uses to find its way home. We find that, like many other insects, they use path integration to calculate the direction and distance to their home. If they fail to locate their burrow, the beetles initiate a distinct looping search behaviour that starts with a characteristic sharp turn, we have called a 'turning point'. When homing beetles are passively displaced or transferred to an unfamiliar environment, they initiate a search at a point very close to the location of their fictive burrow-that is, a spot at the same relative distance and direction from the pick-up point as the original burrow. Unlike other insects, S. galenus do not appear to supplement estimates of the burrow location with landmark information. Thus, S. galenus represents a rare case of a consistently backward-homing animal that does not use landmarks to augment its path integration strategy.

The brain behind straight-line orientation in dung beetles.

For many insects, celestial compass cues play an important role in keeping track of their directional headings. One well-investigated group of celestial orientating insects are the African ball-rolling dung beetles. After finding a dung pile, these insects detach a piece, form it into a ball and roll it away along a straight path while facing backwards. A brain region, termed the central complex, acts as an internal compass that constantly updates the ball-rolling dung beetle about its heading. In this review, we give insights into the compass network behind straight-line orientation in dung beetles and place it in the context of the orientation mechanisms and neural networks of other insects. We find that the neuronal network behind straight-line orientation in dung beetles has strong similarities to the ones described in path-integrating and migrating insects, with the central complex being the key control point for this behavior. We conclude that, despite substantial differences in behavior and navigational challenges, dung beetles encode compass information in a similar way to other insects.

Orienting to polarized light at night - matching lunar skylight to performance in a nocturnal beetle.

For polarized light to inform behaviour, the typical range of degrees of polarization observable in the animal's natural environment must be above the threshold for detection and interpretation. Here, we present the first investigation of the degree of linear polarization threshold for orientation behaviour in a nocturnal species, with specific reference to the range of degrees of polarization measured in the night sky. An effect of lunar phase on the degree of polarization of skylight was found, with smaller illuminated fractions of the moon's surface corresponding to lower degrees of polarization in the night sky. We found that the South African dung beetle Escarabaeus satyrus can orient to polarized light for a range of degrees of polarization similar to that observed in diurnal insects, reaching a lower threshold between 0.04 and 0.32, possibly as low as 0.11. For degrees of polarization lower than 0.23, as measured on a crescent moon night, orientation performance was considerably weaker than that observed for completely linearly polarized stimuli, but was nonetheless stronger than in the absence of polarized light.

Evidence for a southward autumn migration of nocturnal noctuid moths in central Europe.

Insect migrations are spectacular natural events and resemble a remarkable relocation of biomass between two locations in space. Unlike the well-known migrations of daytime flying butterflies, such as the painted lady (Vanessa cardui) or the monarch butterfly (Danaus plexippus), much less widely known are the migrations of nocturnal moths. These migrations - typically involving billions of moths from different taxa - have recently attracted considerable scientific attention. Nocturnal moth migrations have traditionally been investigated by light trapping and by observations in the wild, but in recent times a considerable improvement in our understanding of this phenomenon has come from studying insect orientation behaviour, using vertical-looking radar. In order to establish a new model organism to study compass mechanisms in migratory moths, we tethered each of two species of central European Noctuid moths in a flight simulator to study their flight bearings: the red underwing (Catocala nupta) and the large yellow underwing (Noctua pronuba). Both species had significantly oriented flight bearings under an unobscured view of the clear night sky and in the Earth's natural magnetic field. Red underwings oriented south-southeast, while large yellow underwings oriented southwest, both suggesting a southerly autumn migration towards the Mediterranean. Interestingly, large yellow underwings became disoriented on humid (foggy) nights while red underwings remained oriented. We found no evidence in either species for a time-independent sky compass mechanism as previously suggested for the large yellow underwing.

Neural coding underlying the cue preference for celestial orientation.

Diurnal and nocturnal African dung beetles use celestial cues, such as the sun, the moon, and the polarization pattern, to roll dung balls along straight paths across the savanna. Although nocturnal beetles move in the same manner through the same environment as their diurnal relatives, they do so when light conditions are at least 1 million-fold dimmer. Here, we show, for the first time to our knowledge, that the celestial cue preference differs between nocturnal and diurnal beetles in a manner that reflects their contrasting visual ecologies. We also demonstrate how these cue preferences are reflected in the activity of compass neurons in the brain. At night, polarized skylight is the dominant orientation cue for nocturnal beetles. However, if we coerce them to roll during the day, they instead use a celestial body (the sun) as their primary orientation cue. Diurnal beetles, however, persist in using a celestial body for their compass, day or night. Compass neurons in the central complex of diurnal beetles are tuned only to the sun, whereas the same neurons in the nocturnal species switch exclusively to polarized light at lunar light intensities. Thus, these neurons encode the preferences for particular celestial cues and alter their weighting according to ambient light conditions. This flexible encoding of celestial cue preferences relative to the prevailing visual scenery provides a simple, yet effective, mechanism for enabling visual orientation at any light intensity.

Spectral information as an orientation cue in dung beetles.

During the day, a non-uniform distribution of long and short wavelength light generates a colour gradient across the sky. This gradient could be used as a compass cue, particularly by animals such as dung beetles that rely primarily on celestial cues for orientation. Here, we tested if dung beetles can use spectral cues for orientation by presenting them with monochromatic (green and UV) light spots in an indoor arena. Beetles kept their original bearing when presented with a single light cue, green or UV, or when presented with both light cues set 180° apart. When either the UV or the green light was turned off after the beetles had set their bearing in the presence of both cues, they were still able to maintain their original bearing to the remaining light. However, if the beetles were presented with two identical green light spots set 180° apart, their ability to maintain their original bearing was impaired. In summary, our data show that ball-rolling beetles could potentially use the celestial chromatic gradient as a reference for orientation.

Integration of polarization and chromatic cues in the insect sky compass.

Animals relying on a celestial compass for spatial orientation may use the position of the sun, the chromatic or intensity gradient of the sky, the polarization pattern of the sky, or a combination of these cues as compass signals. Behavioral experiments in bees and ants, indeed, showed that direct sunlight and sky polarization play a role in sky compass orientation, but the relative importance of these cues are species-specific. Intracellular recordings from polarization-sensitive interneurons in the desert locust and monarch butterfly suggest that inputs from different eye regions, including polarized-light input through the dorsal rim area of the eye and chromatic/intensity gradient input from the main eye, are combined at the level of the medulla to create a robust compass signal. Conflicting input from the polarization and chromatic/intensity channel, resulting from eccentric receptive fields, is eliminated at the level of the anterior optic tubercle and central complex through internal compensation for changing solar elevations, which requires input from a circadian clock. Across several species, the central complex likely serves as an internal sky compass, combining E-vector information with other celestial cues. Descending neurons, likewise, respond both to zenithal polarization and to unpolarized cues in an azimuth-dependent way.

Diurnal dung beetles use the intensity gradient and the polarization pattern of the sky for orientation.

To escape competition at the dung pile, a ball-rolling dung beetle forms a piece of dung into a ball and rolls it away. To ensure their efficient escape from the dung pile, beetles rely on a 'celestial compass' to move along a straight path. Here, we analyzed the reliability of different skylight cues for this compass and found that dung beetles rely not only on the sun but also on the skylight polarization pattern. Moreover, we show the first evidence of an insect using the celestial light-intensity gradient for orientation. Using a polarizer, we manipulated skylight so that the polarization pattern appeared to turn by 90 deg. The beetles then changed their bearing close to the expected 90 deg. This behavior was abolished if the sun was visible to the beetle, suggesting that polarized light is hierarchically subordinate to the sun. When the sky was depolarized and the sun was invisible, the beetles could still move along straight paths. Therefore, we analyzed the use of the celestial light-intensity gradient for orientation. Artificial rotation of the intensity pattern by 180 deg caused beetles to orient in the opposite direction. This light-intensity cue was also found to be subordinate to the sun and could play a role in disambiguating the polarization signal, especially at low sun elevations.

The neurobiology of the Monarch butterfly compass.

Monarch butterflies (Danaus plexippus) have become a superb model system to unravel how the tiny insect brain controls an impressive navigation behavior, such as long-distance migration. Moreover, the ability to compare the neural substrate between migratory and nonmigratory Monarch butterflies provides us with an attractive model to specifically study how the insect brain is adapted for migration. We here review our current progress on the neural substrate of spatial orientation in Monarch butterflies and how their spectacular annual migration might be controlled by their brain. We also discuss open research questions, the answers to which will provide important missing pieces to obtain a full picture of insect migration - from the perception of orientation cues to the neural control of migration.

Neural representation of goal direction in the monarch butterfly brain.

Neural processing of a desired moving direction requires the continuous comparison between the current heading and the goal direction. While the neural basis underlying the current heading is well-studied, the coding of the goal direction remains unclear in insects. Here, we used tetrode recordings in tethered flying monarch butterflies to unravel how a goal direction is represented in the insect brain. While recording, the butterflies maintained robust goal directions relative to a virtual sun. By resetting their goal directions, we found neurons whose spatial tuning was tightly linked to the goal directions. Importantly, their tuning was unaffected when the butterflies changed their heading after compass perturbations, showing that these neurons specifically encode the goal direction. Overall, we here discovered invertebrate goal-direction neurons that share functional similarities to goal-direction cells reported in mammals. Our results give insights into the evolutionarily conserved principles of goal-directed spatial orientation in animals.

Receptive field properties and intensity-response functions of polarization-sensitive neurons of the optic tubercle in gregarious and solitarious locusts.

Many migrating insects rely on the plane of sky polarization as a cue to detect spatial directions. Desert locusts (Schistocerca gregaria), like other insects, perceive polarized light through specialized photoreceptors in a dorsal eye region. Desert locusts occur in two phases: a gregarious swarming phase, which migrates during the day, and a solitarious nocturnal phase. Neurons in a small brain area, the anterior optic tubercle (AOTu), are critically involved in processing polarized light in the locust brain. While polarization-sensitive intertubercle cells [lobula-tubercle neuron 1 (LoTu1) and tubercle-tubercle neuron 1 (TuTu1)] interconnect the AOTu of both hemispheres, tubercle-lateral accessory lobe tract (TuLAL1) neurons transmit sky compass signals to a polarization compass in the central brain. To better understand the neural network underlying polarized light processing in the AOTu and to investigate possible adaptations of the polarization vision system to a diurnal versus nocturnal lifestyle, we analyzed receptive field properties, intensity-response relationships, and daytime dependence of responses of AOTu neurons in gregarious and solitarious locusts. Surprisingly, no differences in the physiology of these neurons were found between the two locust phases. Instead, clear differences were observed between the different types of AOTu neurons. Whereas TuTu1 and TuLAL1 neurons encoded E-vector orientation independent of light intensity and would thus be operational in bright daylight, LoTu1 neurons were inhibited by high light intensity and provided strong polarization signaling only under dim light conditions. The presence of high- and low-intensity polarization channels might, therefore, allow solitarious and gregarious locusts to use the same polarization coding system despite their different activity cycles.