The objectives of the study were (1) to determine whether nocturnal Bogong moths (Agrotis infusa) possess a magnetic sense, and if so (2) to determine how information from the Earth’s magnetic field might be used with other sensory information (in particular visual information) to steer long-distance migration in the inherited migratory direction at night. To achieve these objectives, wild Bogong moths were captured during their autumn migration, tethered within a flight simulator and subjected to controlled azimuthal changes of a natural Earth-strength magnetic field and correlated visual landmarks (placed in congruent and conflicting configurations).

Before attachment of tethering stalks, moths were chilled in a freezer for 5-10 min in order to immobilize them. The scales on the moth’s dorsal thorax were removed by suction using a micro-vacuum pump (custom built by B.F.). Afterward a thin vertical tungsten stalk (which is ferromagnetic free), fashioned at its end to create a small circular footplate, was glued to their dorsal thorax using contact cement while being restrained by a weighted-down plastic mesh ( Figure S2 C). Each moth was transferred to its own clear UV-transmissive Perspex container (not airtight), given a drop of 10% honey solution on cotton wool and stored in a cool outdoor location with a natural light cycle. Moths were tested on the day of stalk attachment. Shortly before sunset, containers holding moths were placed on an elevated location (such as a rock) and provided with a clear view of the western sky and the setting sun (and the skylight polarization pattern), in case these cues were important for calibrating the magnetic compass (as found in birds []). After sunset moths could also see the stars (and the celestial rotation). On cold nights (< 10°C), the containers holding moths were placed in a warmed plastic box to maintain ambient temperature at ≈20°C and prevent them from cooling.

For the cue conflict experiments (performed at Adaminaby on migratory moths returning from the Australian Alps: Figure 2 ), the outside of each arena was covered with black cardboard, while the inside of the arena was covered with a white and very even felt glued to the arena wall. At the bottom of the arena wall, a strip (12 cm high) of black felt was glued over the white felt in order to create the visual sensation of a “dark horizon” against a “less dark” sky. In order to check for a putative role of landmarks during compass orientation, a triangular-shaped (isosceles) piece of cardboard-supported black felt (height 12 cm, base width 10.5 cm)) was connected to the top edge of the arena wall by a thin strip of diffusing paper, superimposed on the horizon to provide a dominant (“mountain-like”) landmark. To ensure that the moths were not able to use celestial cues (or the optical encoder arm) for compass orientation, the sky was occluded by a rotatable 15 cm diameter disc made (for rigidity) from a layer of translucent UV-transmissive diffusing filter paper (Lee Filters 251 1/4 white diffuser) centered above the moths such that its angular subtense at the moth diffused all visual details above the top of the arena walls. A thin (1 cm) strip of black cardboard extending from the center of the disc to its edge served as a secondary landmark. As we had no a priori knowledge of which visual field areas moths used for analyzing landmarks, the two landmarks were chosen so that they were visible in most parts of the dorsal and frontal visual fields (or even in the lateral or posterior visual fields if tethered moths turned away from the mountain-like landmark). During some experimental nights in which the moon was visible in the sky, large garden parasols made from thick fabric were positioned to shade the arenas, thus preventing direct moonlight from interfering with the experiment. All light-generating components of the equipment (such as laptops and magnetic coil power supplies) were positioned at a distance behind a tarpaulin wall in a tent that served as a visual “hide” ( Figure S2 B).

All moths that were tested to determine their general migratory direction (see Figure 1 ) at testing sites 1, 2 and 3, were tested under ambient local magnetic field conditions. To prevent unnoticed stray light generated by the electronic equipment (such as battery indicators) entering the experimental arenas, the interior walls of the testing arenas were initially covered with black cardboard. Even though all efforts were made to remove visual landmark cues from the interior of the arena, defects in the cardboard (such as slight buckles and seams) may have provided confounding visual cues during later initial experiments using magnetic field manipulation alone. For the main experiments reported in this paper, the cardboard lining was replaced with a very even felt (see below).

All experiments were conducted outdoors, after sunset, during the migratory periods in the Australian spring (test sites 1 and 2 (October): Australian Cotton Research Institute, Narrabri, NSW (Google Maps coordinates: 30.200°S, 149.612°E; magnetic declination +10°53’) and a helipad in Mount Kaputar National Park, Narrabri, NSW (Google Maps coordinates: 30.279°S, 150.174°E) and autumn (test site 3 (March): “Glenhare,” Adaminaby, NSW (Google Maps coordinates 36.042°S, 148.862°E; magnetic declination +12°29’). Background levels of radio-frequency disturbances were measured at the experimental sites using a Rohde and Schwarz FSV7 Signal and Spectrum Analyzer and a calibrated passive loop antenna (ETS Lindgren, Model 6511). The experimental sites were in very rural locations, and measured noise levels were extraordinarily low and similar to the screened conditions under which the magnetic compass of night-migratory songbirds is not disturbed [] ( Figure S1 A).

The modification to the simulator consisted of incorporating an optic-flow system below the moths to produce sustained stable flight and to provide them with the appearance of forward progress over terrain. For each flight simulator, a projector (Philips PicoPix PPX3610 or BENQ GP2/3), a mirror placed at 45° and neutral density filters (optical density between 4 and 5 log units), were used to produce a very dim bitmap image of the Earth’s surface near Narrabri (at approximately 800 m altitude) that was back-projected onto a tracing paper screen placed below the arena ( Figure S2 A). Custom written software (Martin York, Queens University, Canada), that controlled the direction of movement of the image (but not its orientation), was coupled to the encoder system (USB1, USB4: US Digital, Vancouver, WA, USA) via a feedback loop. This feedback ensured that the resultant ventral flow-field image always moved 180° relative to the moth’s heading (i.e., backward from nose to tail), creating a visual sensation of forward movement irrespective of which direction the moth flew, instantaneously turning with the moth as it changed flight direction. The average light intensities of the optic flow at the moth were 1.31 × 10cd m(Flight Simulator 1) and 6.70 × 10cd m(Flight Simulator 2) and in both simulators it moved continuously at 10 mm s. From their position in the center of an arena, the moths could see a 2D 120° sector of sky (equivalent to a 3D solid angle of 3.5 steradians). Great care was taken to ensure that landmarks external to the arena, such as trees or other structures, could not be seen by moths while they were performing navigational flight behavior in the simulator.

Two ferromagnetic-free modified Mouritsen-Frost flight simulators [] (built from aluminum, plastic and UV-transmissive Perspex) were used to continuously record the heading directions of tethered migratory Bogong moths. Briefly, each flight simulator consisted of a cylindrical Perspex arena (diameter 50 cm, height 35 cm), placed vertically on an aluminum table ( Figure S2 A). The table top was made of clear Perspex. The two flight simulators (and their tables) were placed around 8 m apart. For tethering, the tungsten stalk of the moth was attached to the bottom end of a second long, fine vertical tungsten rod (the encoder shaft) using a short length of thin rubber intravenous medical tubing (see below). The encoder shaft was connected to an optical encoder suspended at the center of the open top end of the arena by a thin horizontal Perspex arm. The encoder instantaneously measured the moth’s heading and allowed us to reconstruct its virtual flight path, thus tracking the heading of the flying moth relative to magnetic north (mN) in the presence or absence of induced sensory cues.

At test sites 1 and 2 (Narrabri), the electrical power required to run the coil systems (and all other devices) was provided by a portable petrol-powered generator (Honda, EU2000i) displaced from the experimental area by at least 30 m using extension cables. Access to the local power grid was provided at test site 3 (Adaminaby).

During cue conflict experiments, the azimuthal direction of mN in a natural Earth-strength magnetic field was turned within each flight simulator arena by a computer controlled, double-wrapped [] Helmholtz two-coil system (custom built by the workshops of the University of Oldenburg) placed around each arena with its long axes 60° relative to mN ( Figure S2 A). The azimuthal direction was turned clockwise by ∼120° (120.4°) without significantly altering the field’s strength or inclination ( Figures S1 B–S1F). A switchbox (also custom built by the workshops of the University of Oldenburg) placed between the power-supplies and the coils, enabled the current to be directed in the same (or parallel) direction through the double wound coils, or in opposite (or antiparallel) directions. In the antiparallel configuration the magnetic field produced by the coils cancels out so that only the Earth’s natural magnetic field remains (Natural Magnetic Field, NMF). In the parallel mode, the current sent through the coils was carefully adjusted to create a resultant magnetic field vector similar to the natural local magnetic field vector but with mN deflected to a clockwise azimuth of ∼120° (30° south of the actual (real) magnetic East: Changed Magnetic Field, CMF). Control of the magnetic field shifts in CMN was enabled by a High-Speed USB Carrier (USB-9162, National Instruments) connected between a laptop computer (see below) and the constant-current power supplies feeding the coil-systems (Kepco, BOP 50-2M). Fine adjustment of the magnetic fields was controlled by a custom-written code in MATLAB (Mathworks, Natick, MA). Before each experimental session, the local magnetic field parameters at the experimental site and at the centers of the two flight simulator arenas were measured using a Meda FVM-400 magnetometer.

Experimental procedures

An individual moth was taken out of its container by using a pair of forceps (a haemostat) to grasp the stalk attached to its dorsal thorax. The stalk was then inserted into a small section of intraveneous tubing attached to the encoder shaft, thus enabling the moth to rotate freely around its yaw axis and choose a direction that its conspecifics would choose in the wild.

Each moth was first aligned by hand to mN and the encoder reset to zero to calibrate the system. The instantaneous heading directions (relative to mN) for each individual moth were recorded as angles by the encoder system at a sampling rate of 1 kHz (with a horizontal resolution of 3°) and saved as text files on the hard drive of a notebook (Dell Latitude E6430ATG). Using the recording software (USB1, USB4: US Digital), we sampled 5 heading directions per second. The encoder software featured a graphical rotary dial interface, enabling the experimenter to continuously monitor the current heading direction of the moth in the arena.

Moths chosen for analysis were required to fulfil three ante hoc and, in the case of the cue conflict experiments ( Figure 2 ), one post hoc criterion (see below). The three ante hoc criteria were: (1) the tethering stalk was perfectly vertical, (2) wing flapping was vigorous and its amplitude was large and equal for both wings (indicating that the contact cement had not interfered with the wings), and (3) that the moth flew continuously for 10 min ( Figure 1 ) or 25 min ( Figure 2 ). In the case of the third ante hoc criterion, an individual tethered moth would generally start vigorously flapping its wings (creating a faint noise), “flying” for many minutes. If a moth stopped flying, the arena was gently tapped in order to stimulate the moth to continue flight behavior. A moth that stopped flying 4 times was rejected and the recording aborted. Likewise, moths that flew in continuous spirals (usually an indication of a non-vertical stalk) were also rejected. If a moth was suspected of having stopped flying, the arena was approached from below to check acoustically if the animals were truly flying – the sound of powered flight is easily distinguished from gliding. Headlamps with dim red LEDs were used while the moths were handled to keep them in a dark-adapted state.

1 and C 2 , the magnetic field direction was shifted back to NMN (0°), while the landmarks remained at 180°, misaligning visual and magnetic cues and creating a cue conflict. Phases C 1 and C 2 were together 10 min long in order to see the effect of the cue conflict in detail over time. In phase D, partly to ensure that moths performed robustly, the magnetic field was left at NMN (0°), while the landmarks were manually returned to 60°. Thus, phase D is a repeat of phase A. In cue-conflict experiments, moths were subjected to two five-phase experiments, each phase requiring the moth to fly for 5 min (25 min total). In experiment 1 ( Figure 2 A), the two landmarks in phase A were displaced from the magnetic field vector by 60° toward the East (with the magnetic field vector at natural mN (NMN) = 0°). At the transition to phase B, the triangle at 60° was manually flipped from the inside to the outside of the arena wall, while as quickly as possible a second triangle (located at 180° at mS) was flipped from the outside to the inside. The stripe on the diffusing landmark disc was likewise rotated 120° toward mS. At the same time, the magnetic coil system was remotely switched from anti-parallel to parallel, which caused a shift of the magnetic field vector from NMN (0°) to CMN (120°). Thus, in phase B the magnetic field azimuth and the two landmarks remained correlated in position (and unchanged from the moth’s perspective). In phase Cand C, the magnetic field direction was shifted back to NMN (0°), while the landmarks remained at 180°, misaligning visual and magnetic cues and creating a cue conflict. Phases Cand Cwere together 10 min long in order to see the effect of the cue conflict in detail over time. In phase D, partly to ensure that moths performed robustly, the magnetic field was left at NMN (0°), while the landmarks were manually returned to 60°. Thus, phase D is a repeat of phase A.

Experiment 2 ( Figure 2 C) was the same as experiment 1, apart from the initial starting conditions for correlated visual and magnetic cues in phase A. Here landmarks were again at 60°, but the magnetic coil system was switched to parallel, meaning that the magnetic field direction started at CMN (120°). At the transition to phase B, the magnetic coil system was switched to anti-parallel, causing an anti-clockwise shift of the field by 120° to mN (NMN), and landmarks were removed from the 60° position, and shifted anti-clockwise by 120° by insertion at the 320° (−60°) position – from the moth’s point of view, the relative positions of the magnetic field and visual landmarks remained correlated. The rest of experiment 2 proceeded according to the same logic as experiment 1.

During the course of an evening, experiments 1 and 2 were alternated randomly on the two flight simulators (i.e., any systematic bias on either experiment due to the apparatus itself could thus be excluded).

1 and C 2 in the two experiments was simply due to fatigue. This control was identical to experiment 1 except that the cue conflict of phases C 1 and C 2 was not included in the control. Instead, in the control, phase B was repeated a further two times (phases B 1 and B 2 ). The only difference between the control and experiment 1 was the magnetic field direction in phases C 1 and C 2 , which was turned by 120° (from 0° (mN) in phase B) to break its correlation with the visual landmarks – in phases B 1 and B 2 the field direction remained unchanged at 0°. A control experiment ( Figure 2 B) was performed to check whether the disorientation seen for the cue conflict during phases Cand Cin the two experiments was simply due to fatigue. This control was identical to experiment 1 except that the cue conflict of phases Cand Cwas not included in the control. Instead, in the control, phase B was repeated a further two times (phases Band B). The only difference between the control and experiment 1 was the magnetic field direction in phases Cand C, which was turned by 120° (from 0° (mN) in phase B) to break its correlation with the visual landmarks – in phases Band Bthe field direction remained unchanged at 0°.