The migrated Australian moth finds caves hundreds of miles away
The cue comes from the heavens: Bogong moths find their way through the starry sky over a three-night period
Andrea Adden, a researcher at the Francis Crick Institute, states that the Bogong moths of Australia are not much to look at. The brown moths have arrow-like markings on their wings. They’re not very interesting.
In a paper published in the journal Nature, Adden and her colleagues show that the cue comes from the heavens. That is, the starry sky allows the Bogong moths to both orient and navigate.
Behavioural data was obtained under natural clear skies over a three night period. There was a single night where data was obtained under natural overcast skies. Data was obtained under projected skies in the form of independent studies that were run over several nights with each night’s group of insects used only once. The same results were also obtained over two separate spring and autumn seasons. Thus, all of these experiments deal with biological replicates. Sample sizes were based on the availability of moths, with at least 40 moths (and as many as 70) being used for each experiment. Experiments were done without a specific randomization protocol, apart from random selection of moths used as replicates. There were recordings and dye injections that were obtained from the single visual cells in the brain. Since each recording (and subsequent dye injection) was a unique observation, replication was not possible. This is due to the stochastic nature of intracellular recordings—single neurons were penetrated randomly from target brain regions that contain many thousands of cells.
The Killing of the Moths: Exploring the Nature of the Milky Way through a Living Dead of Night Experiment in the Australian Alps
In the summer in southeast Australia, it gets hot and they hatch in their breeding grounds. “If they had to reproduce immediately, they would not have enough food to live on.” says Adden.
Instead, the moths migrate over a number of nights in order to get to the Australian Alps where they tend to settle in cooler caves by the millions.
“Then the new moths hatch in the next year,” says Adden. They have never been to the mountains. They have no parent who can point them in the right direction.
She was hoping the stars would offer the cue they needed. “The Milky Way is such a stunning sight,” she says. It seems like an obvious thing to use when you are living in that environment.
To test her theory, Adden, who was doing her Ph.D. at Lund University in Sweden at the time, and her colleagues caught moths in the Australian Alps and ran them through one of two experiments in the dead of night.
The first was a behavioral test. It involved putting a moth inside the mini-planetarium that contained a projector of the night sky and no magnetic field.
“It’s not an experiment that works frequently,” says Adden. We rely on the moths to cooperate with us. Fortunately, enough moths did cooperate. And the result surprised the researchers.
A new ferromagnetic-free Bogong moth flight simulator on a hilltop at Glenhare: a view from the head of the Milky Way
She said they chose a stable direction even if they circle and do twists and turns. It was also their migratory direction.
Adden’s next question involved what was happening in the moth’s brain. She kept a record of the electrical activity of each neuron as she projected the Milky Way.
Two ferromagnetic-free Mouritsen–Frost flight simulators (of the same type used in the laboratory) were placed on a hilltop at Glenhare and used to record the heading directions of tethered migratory Bogong moths experiencing the full local surrounding landscape and the entire dome of the natural sky. To achieve this, each flight simulator arena consisted of a transparent UV-transmissive Perspex cylinder (again with diameter, 50 cm; height, 35 cm), placed vertically onto an aluminium table (Extended Data Fig. 1a,b. The table top was also made of transparent Perspex. The two flight simulators (and their tables) were placed around 15 m apart. A tethered moths was connected to a optical encoder in the centre of each arena with a thin horizontal UV-transmissive Perspex arm. The two arenas were controlled from computers that were operated from within a black light-tight cubical tent that was placed midway between the two arenas, approximately 7 m from each. The tent was therefore a landscape feature that the moths could potentially see during their tethered flights.
The table and animal mounts have non-magnetic components constructed 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 bread board table had a pillar attached that the moth was mounted on and a custom- built non-magnetic Sensapex Micromanipulator attached to it. 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. The experimental bug is around the 8. The tables were mounted with coils to hold the moths 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. The Meda FVM-400 magnetometer made sure the magnetic field was destroyed before the experiment began.
Upward light intensity and spectrum measurements of the starry night sky were made underneath the projection screen in each rig with the probe located at the position of the moth (Extended Data Fig. 9b–d). The light metre used was a calibrated Ocean Optics QE65PRO Spectrometer (Ocean Optics).
The data used for the analyses described below are available online48. There were no statistical differences in the results of males and females.
Intracellular stimulation of Bogong moth brain cells with artificial compass cues above the Carina nebula
The brain samples were scanned with a laser, and then viewed with a 20 oil-immersion objective. The settings for the scanning were set to 1,024 1,024 pixels, 12-bit depth, and 400 lines per second in the photon-counting mode. Neurons and relevant neuropils were then reconstructed in Amira v.5.3 (Thermo Fisher Scientific) and registered into the Bogong moth standard brain22,23.
Glass electrodes were pulled from borosilicate glass on a P-97 Flaming-Brown micropipette puller (Sutter Instrument), and had a typical resistance of 50–100 MΩ. The electrode tip was filled with Neurobiotin solution (4% Neurobiotin in 1 M KCl, Vector Laboratories), and the remainder of the electrode was filled with 1 M KCl. Electrodes were moved into position with a non-magnetic Sensapex micromanipulator. Signals were amplified using a BA-03X intracellular amplifier and headstage (NPI Electronic), and were then digitized using a CED Micro 1401-3 (Cambridge Electronic Design) and recorded with Spike2 software (v.8.03, Cambridge Electronic Design). Stimulus control signals from MATLAB (v.2019a and 2022b, MathWorks) were simultaneously recorded in Spike2. During the recording, the brain was hydrated through regular application of a solution.
To test the sensitivity of visual brain cells to areas of the Milky Way, we stimulated them with artificial compass cues that filled the projection screen above the moths. The Milky Way’s bright area around the Carina nebula was mimicked in these cues by its stripes-like shape. The main stripe of our natural Milky Way is mimicked by the bar length and width. The intensities of these artificial stimuli were also adjusted to provide a good mimic of the intensities of the stars and the dark background sky in our natural stimulus (see above) and had the same mean grey level (61 ± 1 for values between 0 to 255, darkest to brightest).
To maximize the success rate of the demanding intracellular recordings during the short migratory season, these experiments were performed both in the afternoon and during the natural nocturnal flight time of the moths. For afternoon experiments, we removed the two 1.2 log unit ND filters in front of the projector lens (as described above) to generate a starry sky projection around 250 times brighter than the one used at night (to account for the circadian-rhythm-induced light-adapted state of the moths). For night experiments, the ND filters were reinserted. No obvious differences were found in results obtained in the two situations. The insects were mounted on a holder made from 3D-printed materials. The antennae were fixed to the front of the head with a drop of wax, and a square piece of cuticle was removed from the head capsule to expose the brain. The neural sheath was washed after it was digested with Pronase. It was then removed using a pair of fine forceps. A second small hole was taken out of the cuticle and a silver wire was put into the proboscis muscle for reference.
Most behavioural procedures have been described before. Before attachment of tethering stalks, moths were chilled in a freezer for 5–10 min to immobilize them. The scales were removed using a micro-vacuum pump, built by B.F. Afterwards a thin vertical tungsten stalk (which is ferromagnetic free), fashioned at its end to create a small circular footplate, was glued to the dorsal thorax using contact cement while being restrained by a weighted-down plastic mesh. On the day of the attachment, the moths were tested.
Creating randomized starry skies for experiments in fall 2018 and spring 2019 using a multiscale Laplacian and UV LED-ring
To create randomized starry skies for experiments in autumn 2018 and spring 2018, the positions of all individual pixels of the natural night sky stimulus image were reassigned randomly to new positions, and the resulting randomized images were likewise saved as PNG files (Extended Data Fig. 9b). These featureless stellar conditions provided an identical stimulation intensity but provided no celestial spatial information. The stars were retained in the randomized skies in the spring and autumn due to the randomizing of groups of stars, but there were fewer stars in the night sky due to spatial variations. As the bright star in the image had roughly these dimensions, the picture was subdivided into squares with a size 13 13 pixels. The squares were randomly shuffled and a new image was created. The final improved randomized stimulus (used in spring 2019) was generated from the test by randomizing the positions of the individual visible stars. This was achieved by first detecting the position and size of each star in the test stimulus using a multiscale Laplacian of a Gaussian convolution of a greyscale version of the test stimulus, followed by local maxima detection. The resulting spatial information was then used to extract and save the image of each star from the natural night sky stimulus, before replacing them on a new background image, with a uniform colour and intensity equal to that of the mean of all pixels in the test stimulus that were not part of a star. The location of the stars on the randomized image can be drawn from any distribution. A uniform distribution was used for the location of all of the stars, and it was placed in the center of the image.
The projector didn’t emit UV light, so we installed a ring made by T. McIntyre that had 8 UV LEDs in front of it. The brightness of the LED-ring was controlled using custom written software using MATLAB (v.2019a and 2022b, MathWorks) and several layers of ND filters that were fixed in front of the LED-ring to bring the UV intensity into a quasi-natural range (the behavioural rig is shown in Extended Data Fig. 9d).
Spike train data were analysed using custom-written code in MATLAB (v.2019a and 2022b, MathWorks). Circular statistical analysis was performed in R (v.3.6.1; www.r-project.org) using the circular maximum-likelihood estimation package (CircMLE54). Each model was categorized into a unimodal (model M2A, M2B, or M2C) or bimodal (models M4A or M4B) based on the criteria of the Akaike information criterion. Only responses that were classified as unimodal were analysed with respect to the half-width of the rotation tuning curve.
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When they first saw them flying under the night sky, they had to hold on to the edge of the table, says study co-author Eric Warrant.
