Capture and care of moths
Bogong moths (A. infusa) of both sexes were caught in the wild during their autumn and spring migrations (2019 and 2018) using a LepiLED insect light (www.gunnarbrehm.de), or a vertical beam search light (model GT175, Ammon Luminaire Company), placed in front of a white sheet suspended between two trees. Almost all of the animals were caught near the Mount Selwyn Snowfields (southeast New South Wales, Australia: 35.914° S, 148.444° E; elevation, 1,600 m), which is approximately 70 km north-northeast of the nearest aestivation cave in the Main Range of the New South Wales Alps. Thus, to reach these caves in spring, these moths (a tiny subset of all moths travelling to the mountains in a multitude of directions from across southeast Australia) would be expected to fly south-southwest in spring, and returning moths might be expected to travel north-northeast in autumn (which agrees with our behavioural results). A few animals were also caught near Thredbo (Dead Horse Gap, southeast New South Wales, Australia: 36.524° S, 148.260° E, elevation 1,580 m). These moths were used for electrophysiology only. Each captured moth was transferred to its own plastic container to isolate it from influence by other moths. After capture, moths were transported to the testing site Glenhare, a rural property near Adaminaby New South Wales (36.040° S, 148.864° E; elevation, 1,250 m), fed with 20% honey solution (in water) and stored in a cool and sheltered place (exposed to the natural light cycle) to recover from stress induced by capture.
Laboratory for controlled indoor experiments
A purpose-built ferromagnetic-free laboratory located at Glenhare, Adaminaby (built on a concrete slab reinforced with fibreglass and constructed entirely from non-magnetic materials) housed the indoor behavioural and electrophysiological experiments (Extended Data Fig. 1c). Each experimental apparatus (behaviour and electrophysiology) has its own dedicated earth separated from the mains earth (through a 6-mm thick, 30-mm wide and 12-m long copper strap dug into the ground below the concrete slab). Background levels of radio-frequency disturbances at this rural site are extraordinarily low4. All of the experiments were performed on dark-adapted moths in darkness at night (beginning at least 1 h after sunset). Darkness was achieved with black-out blinds (to remove residual starlight and moonlight from outside) and dark cloth around the experimental apparatus (to shield from the minimal stray light emitted from the equipment).
Non-magnetic electrophysiological apparatus
The non-magnetic electrophysiological apparatus (such as table and animal mounts; Extended Data Fig. 1f,g) was constructed from Thorlabs aluminium optomechanical components using high-grade stainless-steel fasteners. Vibration isolation between the aluminium pillar legs and the aluminium bread board table (on which the moth and manipulators were mounted) was provided by four high-grade stainless steel Stillpoints Ultra 6 (with Ultra base) isolators (Stillpoints). The moth was mounted (see below) onto a pillar attached to the bread board table, and a custom-built non-magnetic Sensapex piezo micromanipulator (Sensapex Oy, Oulu), also attached to the pillar, was used to move and advance a glass microelectrode. A removable circular UV-transmissive Perspex disc (diameter, 250 mm; thickness, 5 mm), covered in a layer of UV-transmissive diffusing paper (Lee Filters 251 1/4 white diffuser) and mounted 127 mm above the moth, was used for projection of celestial visual stimuli (see below). The electrophysiological apparatus was placed at the centre of a computer-controlled, double-wrapped41 three-axis (3D) Helmholtz coil system custom built in aluminium and copper (University of Oldenburg workshop; outer coil diameters; x, 900 mm; y, 835 mm; z, 775 mm) to create a nulled magnetic field (Extended Data Fig. 8) around the experimental moth. These coils were mounted onto the experimental table holding the moth and manipulators. The coil systems were powered by constant-current power supplies (Kepco, BOP 50-2M) and the current running through the coil systems was controlled through High-Speed USB Carriers (USB-9162, National Instruments) and custom-written codes in MATLAB (v.2019a and 2022b, MathWorks). Further details were reported previously3,4. Before each experimental session, Meda FVM-400 magnetometer measurements ensured that the magnetic field was nulled within the apparatus (Extended Data Fig. 8b).
Non-magnetic behavioural apparatus
The non-magnetic behavioural apparatus (Extended Data Fig. 1h,i) consisted of a modified Mouritsen–Frost flight simulator3,4,5,42 used to record the virtual flight path of tethered migratory Bogong moths. In brief, each flight simulator consisted of a cylindrical Perspex arena (diameter, 50 cm; height, 35 cm) placed vertically onto an aluminium table with a clear Perspex top within a 3D Helmholtz coil system (as described above, but with coil outer dimensions: x, 1,245 mm; y, 1,300 mm; z, 780 mm). Again, the nulled magnetic field conditions within the coils were carefully monitored using the Meda FVM-400 magnetometer (Extended Data Fig. 8a). The arena walls were covered with two layers of black felt. An optical encoder (E4T Miniature Optical Kit, US Digital) was mounted in the middle of a UV-transmissive Perspex disc (diameter, 50 cm; thickness, 0.5 cm), which was placed on top of the arena like a lid. A layer of UV-transmissive diffusing paper (Lee Filters 251 1/4 white diffuser) was placed on top of the disc (and served as a screen for dorsal projection of celestial stimuli; see below). A fine vertical tungsten rod (the encoder shaft: diameter, 0.5 mm; length, 153 mm), inserted into the axial centre of the optical encoder, extended downwards into the arena and allowed the attachment of tethered flying moths (see below). We used the encoder manufacturer’s software (USB1 Digital Explorer 1.07, US Digital) to continuously record the moth’s heading relative to geographical north (gN), therefore allowing us to reconstruct the moth’s virtual flight path in the presence of celestial visual cues. An LED projector (ASUS S1 Mobile), neutral density (ND) filters (optical density between 4 and 5 log units) and a mirror placed at 45° under the Perspex tabletop were used to project a dim moving (10 mm s−1) pattern of optic flow onto a screen (Lee Filters 251 1/4 white diffusing paper) placed beneath the arena and therefore below the tethered flying moth (Extended Data Fig. 1d). The direction of the optic flow was controlled by custom written software (M. York), which coupled the encoder system (USB1 or USB4 encoder data acquisition USB device, US Digital) through a feedback loop. Thus, the optic flow would always move from head to tail below the moth, instantaneously changing direction as the moth changed direction. The mean radiance of the optic flow at the location of the performing moth was 2.06 ± 0.19 × 109 photons cm−2 s−1 sr−1.
Stimulation with natural starry skies
A natural moonless starry austral night sky, in both the electrophysiological and the behavioural rigs, was projected using a downward pointing projector (behavioural rig: LED projector ASUS S1 Mobile; electrophysiological rig: Sony MP-CL1A laser projector; spectra of both projectors are shown in Extended Data Fig. 9d). Each projector was mounted sufficiently high above the moth in each rig to provide clear and correctly sized dorsal images of the night sky. To avoid unwanted stray light, each projector was housed in a custom-built 3D-printed plastic box featuring an opening in front of the lens and ventilation slits above the projector.
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