Behavioral responses of free-flying Drosophila melanogaster to shiny, reflecting surfaces.

Active locomotion plays an important role in the life of many animals, permitting them to explore the environment, find vital resources, and escape predators. Most insect species rely on a combination of visual cues such as celestial bodies, landmarks, or linearly polarized light to navigate or orient themselves in their surroundings. In nature, linearly polarized light can arise either from atmospheric scattering or from reflections off shiny non-metallic surfaces like water. Multiple reports have described different behavioral responses of various insects to such shiny surfaces. Our goal was to test whether free-flying Drosophila melanogaster, a molecular genetic model organism and behavioral generalist, also manifests specific behavioral responses when confronted with such polarized reflections. Fruit flies were placed in a custom-built arena with controlled environmental parameters (temperature, humidity, and light intensity). Flight detections and landings were quantified for three different stimuli: a diffusely reflecting matt plate, a small patch of shiny acetate film, and real water. We compared hydrated and dehydrated fly populations, since the state of hydration may change the motivation of flies to seek or avoid water. Our analysis reveals for the first time that flying fruit flies indeed use vision to avoid flying over shiny surfaces.

Systematic functional analysis of rab GTPases reveals limits of neuronal robustness to environmental challenges in flies.

Rab GTPases are molecular switches that regulate membrane trafficking in all cells. Neurons have particular demands on membrane trafficking and express numerous Rab GTPases of unknown function. Here, we report the generation and characterization of molecularly defined null mutants for all 26 rab genes in Drosophila. In flies, all rab genes are expressed in the nervous system where at least half exhibit particularly high levels compared to other tissues. Surprisingly, loss of any of these 13 nervous system-enriched Rabs yielded viable and fertile flies without obvious morphological defects. However, all 13 mutants differentially affected development when challenged with different temperatures, or neuronal function when challenged with continuous stimulation. We identified a synaptic maintenance defect following continuous stimulation for six mutants, including an autophagy-independent role of rab26. The complete mutant collection generated in this study provides a basis for further comprehensive studies of Rab GTPases during development and function in vivo.

Modular assays for the quantitative study of visually guided navigation in both flying and walking flies.

BACKGROUND: The quantitative study of behavioral responses to visual stimuli provides crucial information about the computations executed by neural circuits. Insects have long served as powerful model systems, either when walking on air suspended balls (spherical treadmill), or flying while glued to a needle (virtual flight arena). NEW METHOD: Here we present detailed instructions for 3D-printing and assembly of arenas optimized for visually guided navigation, including codes for presenting both celestial and panorama cues. These modular arenas can be used either as virtual flight arenas, or as spherical treadmills and consist entirely of commercial and 3D-printed components placed in a temperature and humidity controlled environment. COMPARISON TO EXISTING METHOD(S): Previous assays often include a combination of rather cost-intensive and technically complex, custom-built mechanical, electronic, and software components. Implementation amounts to a major challenge when working in an academic environment without the support of a professional machine shop. RESULTS: Robust optomotor responses are induced in flyingDrosophila by displaying moving stripes in a cylinder surrounding the magnetically tethered fly. Similarly, changes in flight heading are induced by presenting changes in the orientation of linearly polarized UV light presented from above. Finally, responses to moving patterns are induced when individual flies are walking on an air-suspended ball. CONCLUSION: These modular assays allow for the investigation of a diverse combination navigational cues (sky and panorama) in both flying and walking flies. They can be used for the molecular dissection of neural circuitry in Drosophila and can easily be rescaled for accommodating other insects.

Heading choices of flying Drosophila under changing angles of polarized light.

Many navigating insects include the celestial polarization pattern as an additional visual cue to orient their travels. Spontaneous orientation responses of both walking and flying fruit flies (Drosophila melanogaster) to linearly polarized light have previously been demonstrated. Using newly designed modular flight arenas consisting entirely of off-the-shelf parts and 3D-printed components we present individual flying flies with a slow and continuous rotational change in the incident angle of linear polarization. Under such open-loop conditions, single flies choose arbitrary headings with respect to the angle of polarized light and show a clear tendency to maintain those chosen headings for several minutes, thereby adjusting their course to the slow rotation of the incident stimulus. Importantly, flies show the tendency to maintain a chosen heading even when two individual test periods under a linearly polarized stimulus are interrupted by an epoch of unpolarized light lasting several minutes. Finally, we show that these behavioral responses are wavelength-specific, existing under polarized UV stimulus while being absent under polarized green light. Taken together, these findings provide further evidence supporting Drosophila's abilities to use celestial cues for visually guided navigation and course correction.

Modality-Specific Circuits for Skylight Orientation in the Fly Visual System.

In the fly optic lobe, ∼800 highly stereotypical columnar microcircuits are arranged retinotopically to process visual information. Differences in cellular composition and synaptic connectivity within functionally specialized columns remain largely unknown. Here, we describe the cellular and synaptic architecture in medulla columns located downstream of photoreceptors in the dorsal rim area (DRA), where linearly polarized skylight is detected for guiding orientation responses. We show that only in DRA medulla columns both R7 and R8 photoreceptors target to the bona fide R7 target layer where they form connections with previously uncharacterized, modality-specific Dm neurons: two morphologically distinct DRA-specific cell types (termed Dm-DRA1 and Dm-DRA2) stratify in separate sublayers and exclusively contact polarization-sensitive DRA inputs, while avoiding overlaps with color-sensitive Dm8 cells. Using the activity-dependent GRASP and trans-Tango techniques, we confirm that DRA R7 cells are synaptically connected to Dm-DRA1, whereas DRA R8 form synapses with Dm-DRA2. Finally, using live imaging of ingrowing pupal photoreceptor axons, we show that DRA R7 and R8 termini reach layer M6 sequentially, thus separating the establishment of different synaptic connectivity in time. We propose that a duplication of R7→Dm circuitry in DRA ommatidia serves as an ideal adaptation for detecting linearly polarized skylight using orthogonal e-vector analyzers.