A distinct layer of the medulla integrates sky compass signals in the brain of an insect

Mass migration of desert locusts is a common phenomenon in North Africa and the Middle East but how these insects navigate is still poorly understood. Laboratory studies suggest that locusts are able to exploit the sky polarization pattern as a navigational cue. Like other insects locusts detect polarized light through a specialized dorsal rim area (DRA) of the eye. Polarization signals are transmitted through the optic lobe to the anterior optic tubercle (AOTu) and, finally, to the central complex in the brain. Whereas neurons of the AOTu integrate sky polarization and chromatic cues in a daytime dependent manner, the central complex holds a topographic representation of azimuthal directions suggesting a role as an internal sky compass. To understand further the integration of sky compass cues we studied polarization-sensitive (POL) neurons in the medulla that may be intercalated between DRA photoreceptors and AOTu neurons. Five types of POL-neuron were characterized and four of these in multiple recordings. All neurons had wide arborizations in medulla layer 4 and most, additionally, in the dorsal rim area of the medulla and in the accessory medulla, the presumed circadian clock. The neurons showed type-specific orientational tuning to zenithal polarized light and azimuth tuning to unpolarized green and UV light spots. In contrast to neurons of the AOTu, we found no evidence for color opponency and daytime dependent adjustment of sky compass signals. Therefore, medulla layer 4 is a distinct stage in the integration of sky compass signals that precedes the time-compensated integration of celestial cues in the AOTu.     

Central neural coding of sky polarization in insects

Many animals rely on a sun compass for spatial orientation and long-range navigation. In addition to the Sun, insects also exploit the polarization pattern and chromatic gradient of the sky for estimating navigational directions. Analysis of polarization-vision pathways in locusts and crickets has shed first light on brain areas involved in sky compass orientation. Detection of sky polarization relies on specialized photoreceptor cells in a small dorsal rim area of the compound eye. Brain areas involved in polarization processing include parts of the lamina, medulla and lobula of the optic lobe and, in the central brain, the anterior optic tubercle, the lateral accessory lobe and the central complex. In the optic lobe, polarization sensitivity and contrast are enhanced through convergence and opponency. In the anterior optic tubercle, polarized-light signals are integrated with information on the chromatic contrast of the sky. Tubercle neurons combine responses to the UV/green contrast and e-vector orientation of the sky and compensate for diurnal changes of the celestial polarization pattern associated with changes in solar elevation. In the central complex, a topographic representation of e-vector tunings underlies the columnar organization and suggests that this brain area serves as an internal compass coding for spatial directions.     

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.     

Neurobiology of polarization vision in the locust Schistocerca gregaria

The polarization pattern of the blue sky serves as an important reference for spatial orientation in insects. To understand the neural mechanisms involved in sky compass orientation we have analyzed the polarization vision system in the locust Schistocerca gregaria. As in other insects, photoreceptors adapted for the detection of sky polarization are concentrated in a dorsal rim area (DRA) of the compound eye. Stationary flying locusts show polarotactic yaw-torque responses when illuminated through a rotating polarizer from above. This response is abolished after painting the DRAs. Central stages of the polarization vision system, revealed through tracing studies, include dorsal areas in the lamina and medulla, the anterior lobe of the lobula, the anterior optic tubercle, the lateral accessory lobe and the central complex. Physiological analysis of polarization-sensitive (POL) neurons has focussed on the optic tubercle and on the central complex. Each POL neuron was maximally excited at a certain e-vector (phimax) and was maximally inhibited at an e-vector perpendicular to phimax. The neurons had large visual fields, and many neurons received input from both eyes. The neuronal organization of the central complex suggests a role as a spatial compass within the locust brain.     

Transmedulla Neurons in the Sky Compass Network of the Honeybee (Apis mellifera) Are a Possible Site of Circadian Input

Honeybees are known for their ability to use the sun’s azimuth and the sky’s polarization pattern for spatial orientation. Sky compass orientation in bees has been extensively studied at the behavioral level but our knowledge about the underlying neuronal systems and mechanisms is very limited. Electrophysiological studies in other insect species suggest that neurons of the sky compass system integrate information about the polarization pattern of the sky, its chromatic gradient, and the azimuth of the sun. In order to obtain a stable directional signal throughout the day, circadian changes between the sky polarization pattern and the solar azimuth must be compensated. Likewise, the system must be modulated in a context specific way to compensate for changes in intensity, polarization and chromatic properties of light caused by clouds, vegetation and landscape. The goal of this study was to identify neurons of the sky compass pathway in the honeybee brain and to find potential sites of circadian and neuromodulatory input into this pathway. To this end we first traced the sky compass pathway from the polarization-sensitive dorsal rim area of the compound eye via the medulla and the anterior optic tubercle to the lateral complex using dye injections. Neurons forming this pathway strongly resembled neurons of the sky compass pathway in other insect species. Next we combined tracer injections with immunocytochemistry against the circadian neuropeptide pigment dispersing factor and the neuromodulators serotonin, and γ-aminobutyric acid. We identified neurons, connecting the dorsal rim area of the medulla to the anterior optic tubercle, as a possible site of neuromodulation and interaction with the circadian system. These neurons have conspicuous spines in close proximity to pigment dispersing factor-, serotonin-, and GABA-immunoreactive neurons. Our data therefore show for the first time a potential interaction site between the sky compass pathway and the circadian clock.     

Surgical lesion of the anterior optic tract abolishes polarotaxis in tethered flying locusts, <Emphasis Type="Italic">Schistocerca gregaria</Emphasis>

Many insects can detect the polarization pattern of the blue sky and rely on polarization vision for sky compass orientation. In laboratory experiments, tethered flying locusts perform periodic changes in flight behavior under a slowly rotating polarizer even if one eye is painted black. Anatomical tracing studies and intracellular recordings have suggested that the polarization vision pathway in the locust brain involves the anterior optic tract and tubercle, the lateral accessory lobe, and the central complex of the brain. To investigate whether visual pathways through the anterior optic tract mediate polarotaxis in the desert locust, we transected the tract on one side and tested polarotaxis (1) with both eyes unoccluded and (2) with the eye of the intact hemisphere painted black. In the second group of animals, but not in the first group, polarotaxis was abolished. Sham operations did not impair polarotaxis. The experiments show that the anterior optic tract is an indispensable part of visual pathways mediating polarotaxis in the desert locust.     

Experience‐related reorganization of giant synapses in the lateral complex: Potential role in plasticity of the sky‐compass pathway in the desert ant Cataglyphis fortis

Cataglyphis desert ants undergo an age‐related polyethism from interior workers to relatively short‐lived foragers with remarkable visual navigation capabilities, predominantly achieved by path integration using a polarized skylight‐based sun compass and a stride‐integrating odometer. Behavioral and physiological experiments revealed that the polarization (POL) pattern is processed via specialized UV‐photoreceptors in the dorsal rim area of the compound eye and POL sensitive optic lobe neurons. Further information about the neuronal substrate for processing of POL information in the ant brain has remained elusive. This work focuses on the lateral complex (LX), known as an important relay station in the insect sky‐compass pathway. Neuroanatomical results in Cataglyphis fortis show that LX giant synapses (GS) connect large presynaptic terminals from anterior optic tubercle neurons with postsynaptic GABAergic profiles of tangential neurons innervating the ellipsoid body of the central complex. At the ultrastructural level, the cup‐shaped presynaptic structures comprise many active zones contacting numerous small postsynaptic profiles. Three‐dimensional quantification demonstrated a significantly higher number of GS (～13%) in foragers compared with interior workers. Light exposure, as opposed to age, was necessary and sufficient to trigger a similar increase in GS numbers. Furthermore, the increase in GS numbers was sensitive to the exclusion of UV light. As previous experiments have demonstrated the importance of the UV spectrum for sky‐compass navigation in Cataglyphis, we conclude that plasticity in LX GS may reflect processes involved in the initial calibration of sky‐compass neuronal circuits during orientation walks preceding active foraging. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 390–404, 2016     

Behavioral analysis of polarization vision in tethered flying locusts

For spatial navigation many insects rely on compass information derived from the polarization pattern of the sky. We demonstrate that tethered flying desert locusts (Schistocerca gregaria) show e-vector-dependent yaw-torque responses to polarized light presented from above. A slowly rotating polarizer (5.3° s^–1) induced periodic changes in yaw torque corresponding to the 180° periodicity of the stimulus. Control experiments with a rotating diffuser, a weak intensity pattern, and a stationary polarizer showed that the response is not induced by intensity gradients in the stimulus. Polarotaxis was abolished after painting the dorsal rim areas of the compound eyes black, but remained unchanged after painting the eyes except the dorsal rim areas. During rotation of the polarizer, two e-vectors (preferred and avoided e-vector) induced no turning responses: they were broadly distributed from 0 to 180° but, for a given animal, were perpendicular to each other. The data demonstrate polarization vision in the desert locust, as shown previously for bees, flies, crickets, and ants. Polarized light is perceived through the dorsal rim area of the compound eye, suggesting that polarization vision plays a role in compass navigation of the locust.     

Ultrastructure and orientation of ommatidia in the dorsal rim area of the locust compound eye

In many insect species, a dorsal rim area (DRA) in the compound eye is adapted to analyze the sky polarization pattern for compass orientation. In the desert locust Schistocerca gregaria, these specializations are particularly striking. The DRA of the locust consists of about 400 ommatidia. The facets have an irregular shape, and pore canals are often present in the corneae. Screening pigment is missing in the region of the dioptric apparatus suggesting large receptive fields. The rhabdoms are shorter, but about four times larger in cross-section than the rhabdoms of ordinary ommatida. Eight retinula cells contribute to the rhabdom. The microvilli of retinula cell 7 and of cells 1, 2, 5, 6, 8 are highly aligned throughout the rhabdom and form two blocks of orthogonal orientation. The microvilli in the minute rhabdomeres of retinula cells 3 and 4, in contrast, show no particular alignment. As in other insect species, microvillar orientations are arranged in a fan-like pattern across the DRA. Photoreceptor axons project to distinct areas in the dorsal lamina and medulla. The morphological specializations in the DRA of the locust eye most likely maximize the polarization sensitivity and suggest that the locust uses this eye region for analysis of the sky polarization pattern.     

Polarized light helps monarch butterflies navigate

During their spectacular migratory journey in the fall, North American monarch butterflies (Danaus plexippus) use a time-compensated sun compass to help them navigate to their overwintering sites in central Mexico. One feature of the sun compass mechanism not fully explored in monarchs is the sunlight-dependent parameters used to navigate. We now provide data suggesting that the angle of polarized skylight (the e-vector) is a relevant orientation parameter. By placing butterflies in a flight simulator outdoors and using a linear polarizing filter, we show that manipulating the e-vector alters predictably the direction of oriented flight. Butterflies studied in either the morning or afternoon showed similar responses to filter rotation. Monarch butterflies possess the anatomical structure needed for polarized skylight detection, as rhabdoms in the dorsalmost row of photoreceptor cells in monarch eye show the organization characteristic of polarized-light receptors. The existence of polarized-light detection could allow migrants to accurately navigate under a variety of atmospheric conditions and reveals a critical input pathway into the sun compass mechanism.     

昆虫对偏振光的响应及感受机理研究进展

偏振光是不同于普通光源的一种光, 常指光矢量在某一个方向振动的光波, 可分为线性偏振光、 圆偏振光和椭圆偏振光等。目前已经发现自然界的偏振光影响许多昆虫的行为, 如西方蜜蜂Apis mellifera的飞行导航、 蛱蝶Heliconius cydno chioneus的觅偶、 凤蝶Papilio aegeus产卵场所的选择等。金龟子对圆偏振光的反射可以作为一种分类的性状。昆虫复眼背部边缘区域（dorsal rim area, DRA）小眼是感受偏振光的主要器官, 电生理学研究表明前视神经节是蝗虫偏振视觉通路的一部分。在匈牙利, 已经开始利用偏振光研制步甲等昆虫的诱捕器。     

A specialized dorsal rim area for polarized light detection in the compound eye of the scarab beetle Pachysoma striatum

Many animals have been shown to use the pattern of polarized light in the sky as an optical compass. Specialised photoreceptors are used to analyse this pattern. We here present evidence for an eye design suitable for polarized skylight navigation in the flightless desert scarab Pachysoma striatum. Morphological and electrophysiological studies show that an extensive part of the dorsal eye is equivalent to the dorsal rim area used for polarized light navigation in other insects. A polarization-sensitivity of 12.8 (average) can be recorded from cells sensitive to the ultraviolet spectrum of light. Features commonly known to increase the visual fields of polarization-sensitive photoreceptors, or to decrease their spatial resolution, are not found in the eye of this beetle. We argue that in this insect an optically unspecialised area for polarized light detection allows it not be used exclusively for polarized light navigation.     

Connecting the navigational clock to sun compass input in monarch butterfly brain

Migratory monarch butterflies (Danaus plexippus) use a time-compensated sun compass to navigate to their overwintering grounds in Mexico. Although polarized light is one of the celestial cues used for orientation, the spectral content (color) of that light has not been fully explored. We cloned the cDNAs of three visual pigment-encoding opsins (ultraviolet [UV], blue, and long wavelength) and found that all three are expressed uniformly in main retina. The photoreceptors of the polarization-specialized dorsal rim area, on the other hand, are monochromatic for the UV opsin. Behavioral studies support the importance of polarized UV light for flight orientation. Next, we used clock protein expression patterns to identify the location of a circadian clock in the dorsolateral protocerebrum of butterfly brain. To provide a link between the clock and the sun compass, we identified a CRYPTOCHROME-staining neural pathway that likely connects the circadian clock to polarized light input entering brain.     

Orientation in a desert lizard (Uma notata): time-compensated compass movement and polarotaxis

Summary The diurnal escape response of fringetoed lizards (Uma notata) startled by predators demonstrates clear directional orientation not likely to depend on local landmarks in the shifting sands of their desert environment. Evidence that celestial orientation is involved in this behavior has been sought in the present experiments by testing the effects of (1) phase shifting the animal's internal clock by 6 h and (2) by training the lizards to seek shelter while exposed to natural polarization patterns. In the first case, 90° shifts in escape direction were demonstrated in outdoor tests, as expected if a time-compensated sun or sky polarized light compass is involved. In the second instance, significant bimodale-vector dependent orientation was found under an overhead polarizing light filter but this was only evident when the response data were transposed to match the zenithe-vector rotation dependent on the sun's apparent movement through the sky. This extends to reptiles the capacity to utilize overheade-vector directions as a time-compensated sky compass. The sensory site of this discrimination and the relative roles of sun and sky polarization in nature remain to be discovered.     

Sun compass integration of skylight cues in migratory monarch butterflies

Migrating monarch butterflies (Danaus plexippus) use a time-compensated sun compass to navigate from eastern North America to their overwintering grounds in central Mexico. Here we describe the neuronal layout of those aspects of the butterfly's central complex likely to establish part of the internal sun compass and find them highly homologous to those of the desert locust. Intracellular recordings from neurons in the monarch sun compass network reveal responses tuned to specific E-vector angles of polarized light, as well as azimuth-dependent responses to unpolarized light, independent of spectral composition. The neural responses to these two stimuli in individual neurons are mediated through different regions of the compound eye. Moreover, these dual responses are integrated to create a consistent representation of skylight cues in the sun compass throughout the day. The results advance our understanding of how ambiguous sensory signals are processed by the brain to elicit a robust behavioral response.     

Evidence for instantaneous e-vector detection in the honeybee using an associative learning paradigm

Many insects use the polarization pattern of the sky for obtaining compass information during orientation or navigation. E-vector information is collected by a specialized area in the dorsal-most part of the compound eye, the dorsal rim area (DRA). We tested honeybees' capability of learning certain e-vector orientations by using a classical conditioning paradigm with the proboscis extension reflex. When one e-vector orientation (CS+) was associated with sugar water, while another orientation (CS-) was not rewarded, the honeybees could discriminate CS+ from CS-. Bees whose DRA was inactivated by painting did not learn CS+. When ultraviolet (UV) polarized light (350 nm) was used for CS, the bees discriminated CS+ from CS-, but no discrimination was observed in blue (442 nm) or green light (546 nm). Our data indicate that honeybees can learn and discriminate between different e-vector orientations, sensed by the UV receptors of the DRA, suggesting that bees can determine their flight direction from polarized UV skylight during foraging. Fixing the bees' heads during the experiments did not prevent learning, indicating that they use an 'instantaneous' algorithm of e-vector detection; that is, the bees do not need to actively scan the sky with their DRAs ('sequential' method) to determine e-vector orientation.     

Honeybee navigation: critically examining the role of the polarization compass

Although it is widely accepted that honeybees use the polarized-light pattern of the sky as a compass for navigation, there is little direct evidence that this information is actually sensed during flight. Here, we ask whether flying bees can obtain compass cues derived purely from polarized light, and communicate this information to their nest-mates through the ‘waggle dance’. Bees, from an observation hive with vertically oriented honeycombs, were trained to fly to a food source at the end of a tunnel, which provided overhead illumination that was polarized either parallel to the axis of the tunnel, or perpendicular to it. When the illumination was transversely polarized, bees danced in a predominantly vertical direction with waggles occurring equally frequently in the upward or the downward direction. They were thus using the polarized-light information to signal the two possible directions in which they could have flown in natural outdoor flight: either directly towards the sun, or directly away from it. When the illumination was axially polarized, the bees danced in a predominantly horizontal direction with waggles directed either to the left or the right, indicating that they could have flown in an azimuthal direction that was 90° to the right or to the left of the sun, respectively. When the first half of the tunnel provided axial illumination and the second half transverse illumination, bees danced along all of the four principal diagonal directions, which represent four equally likely locations of the food source based on the polarized-light information that they had acquired during their journey. We conclude that flying bees are capable of obtaining and signalling compass information that is derived purely from polarized light. Furthermore, they deal with the directional ambiguity that is inherent in polarized light by signalling all of the possible locations of the food source in their dances, thus maximizing the chances of recruitment to it.     

Neural mechanisms in insect navigation: polarization compass and odometer

Insect navigation relies on path integration, a procedure by which information about compass bearings pursued and distances travelled are combined to calculate position. Three neural levels of the polarization compass, which uses the polarization of skylight as a reference, have been analyzed in orthopteran insects. A group of dorsally directed, highly specialized ommatidia serve as polarization sensors. Polarization-opponent neurons in the optic lobe condition the polarization signal by removing unreliable and irrelevant components of the celestial stimulus. Neurons found in the central complex of the brain possibly represent elements of the compass output. The odometer for measuring travelling distances in honeybees relies on optic flow experienced during flight, whereas desert ants most probably use proprioreceptive cues.     

Immunocytochemistry of histamine in the brain of the locust<Emphasis Type="Italic"> Schistocerca gregaria</Emphasis>

Histamine serves a neurotransmitter role in arthropod photoreceptor neurons, but is also present in a small number of interneurons throughout the nervous system. In search of a suitable model system for the analysis of histaminergic neurotransmission in insects, we mapped the distribution of histamine in the brain of the desert locust Schistocerca gregaria by immunocytochemistry. In the optic lobe, apparently all photoreceptor cells of the compound eye with projections to the lamina and medulla showed intense immunostaining. Photoreceptors of the dorsal rim area of the eye had particularly large fiber diameters and gave rise to uniform varicose immunostaining throughout dorsal rim areas of the lamina and medulla. In the locust midbrain 21 bilateral pairs of histamine-immunoreactive interneurons were found, and 13 of these were reconstructed in detail. While most neuropil areas contained a dense meshwork of immunoreactive processes, immunostaining in the antennal lobe and in the calyces of the mushroom body was sparse and no staining occurred in the pedunculus and lobes of the mushroom body, in the protocerebral bridge, and in the lower division of the central body. A prominent group of four immunostained neurons had large cell bodies near the median ocellar nerve root and descending axonal fibers. These neurons are probably identical to previously identified primary commissure pioneer neurons of the locust brain. The apparent lack in the desert locust of certain histamine-immunoreactive neurons which were reported in the migratory locust may be responsible for differences in the physiological role of histamine between both species.The study was supported by the Deutsche Forschungsgemeinschaft, grants Ho 950/13 and 950/14     

Honeybee navigation: following routes using polarized-light cues

While it is generally accepted that honeybees (Apis mellifera) are capable of using the pattern of polarized light in the sky to navigate to a food source, there is little or no direct behavioural evidence that they actually do so. We have examined whether bees can be trained to find their way through a maze composed of four interconnected tunnels, by using directional information provided by polarized light illumination from the ceilings of the tunnels. The results show that bees can learn this task, thus demonstrating directly, and for the first time, that bees are indeed capable of using the polarized-light information in the sky as a compass to steer their way to a food source.