Bogong moths under nightly movements of the stars, moon, and cloud cover: orientation measurements in a normal geomagnetic field
The moths are in danger. According to Adden, her findings can help conserve these insects and everything they rely on for food. Reducing light pollution would help these moths to continue their journey across the Australian bush.
The same behavioural methods (and criteria) that were used indoors were also used outdoors. The goal of these experiments was to understand how migratory Bogong moths deal with natural skies (in particular the nightly movements of the stars and moon, as well as cloud cover) while experiencing a normal geomagnetic field and the local surrounding landscape under natural illumination. Experiments were performed under clear starry skies during autumn 2023 (over three nights during the last week of March), as well as at two times of night, to test whether the migratory orientations of moths were affected by the nightly movements of the stars and moon (which was approximately half-full): (1) between 20:32 and 21:06 (about 1.5 h after sunset), and (2) between 23:25 and 23:59. The same moths were used for orientation measurements at both times, and moths that were flown in one arena at the earlier time (and saw the black tent on their eastern side) were flown in the other arena at the later time (and now saw the black tent on their western side) and vice versa. Moreover, there was a stand of trees close to the arena on the eastern side of the tent, and single trees close to the arena on the western side of the tent. Thus, other panoramic landmarks (apart from the tent) differed markedly in their spatial positions from within the two arenas. During earlier experiments, moths were kept isolated and in the dark in a suitcase that was warmed with hot water bottles. Experiments were also carried out on a fourth completely overcast night that totally covered the stars and moon (between 21:12 and 21:48).
It’s the first time that we’ve ever seen an insect using stars to navigate. “And also the first time that anyone had seen neurons that specifically respond to the starry sky in the insect brain.”
Bogong moths (A. infusa) fly south-southeast during their autumn and spring migrations near the mountain range of the Northern Alps
In the summer it gets really hot in southeast Australia where they hatch in the spring. “So if they were to reproduce immediately, their larvae would starve because there is not enough food,” says Adden.
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.
The new moths hatch next year, says Adden. “And they’ve never been to the mountains. They don’t have parents who know how to get there.
She thought the stars might be able to give them a cue. She says that the sky is a beautiful sight. It seemed an obvious thing to use in that environment, if you are a moth.
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. In this case it involved putting a bug in a mini-planetarium and projecting the night sky without a magnetic field.
“It’s not an experiment that always works,” says Adden. “We rely on the moths collaborating with us.” Fortunately, enough moths did cooperate. And the result surprised the researchers.
A Non-Magnetic Lab for the Study of Dark-Adapted Moths in the Burst of a Milky Way Projection
She said that they chose a stable direction but did not just circle and do twists and turns. “Not only that, it was their migratory direction.”
Adden’s next question involved what was happening in the moth’s brain. She recorded the electrical activity of individual neurons while rotating a projection of the Milky Way.
The lab was built entirely from non-magnetic materials at Adaminaby, and had two indoor experiments in it. 1c). Each experimental apparatus has its own dedicated earth separated from the mains earth through a 30mm wide and 12m long copper strap. Background levels of radio-frequency disturbances at this rural site are extraordinarily low4. The experiments were performed on dark-adapted moths in darkness at night, beginning at 1 h after sunset. To remove residual starlight and moonlight from the outdoors, black-out blinds were used and dark cloth around the experimental apparatus.
The non-magnetic apparatus was constructed using high-gradestainless-steelfasteners and Thorlabs aluminum components. The Stillpoints Ultra 6 isolators provided the isolation between the pillar legs and the bread board table. 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. The Magnetic Field was nulled within the apparatus before each experimental session because Meda FVM-400 magnetometer measurements were made.
The free software Stellarium43 was used to simulate the moonless starry night sky over Canberra (about 80 km from Adaminaby as the crow flies) at 21:30 on four respective dates: 1 October 2018 (spring 2018), 21 March 2018 (autumn 2018), 21 October 2019 (spring 2019) and 27 February 2019 (autumn 2019). Screenshots (screen resolution 7,480 × 720 pixels) of these simulated starry skies were taken, cut in a circular shape using CorelDRAW X5 and saved as PNG files (300 dpi) to create the stimulus images (Fig. 3a,b and Extended Data Fig. 9a). The 160 and 100 fields of view of the sky from each rig were provided by the projected images on the screens. Celestial images contained grey level values ranging from 4 (darkest) to 255 (brightest, on a scale of 0–255), with an average grey level of 62. The quality of the night sky provided by these images was comparable to that provided by the natural rural night sky at Glenhare (as measured with a Unihedron Sky Quality Metre; Extended Data Table 1). Before each experiment, the PNG files were opened using IrfanView64 on a PC using a screen resolution of 1,280 × 720 pixels. The projector was connected to the PC through a cable. Each rig had a circular screen and the sky was projected to be the same size.
The behavioural analysis used in this study has been previously described4,5. The instantaneous heading directions of a tethering flying moths were recorded and saved in a text file by theusb1 digital explorer v.1.07 We used custom-written MATLAB code (v.2019a and 2022b, MathWorks) to visualize the virtual flight paths of all tested moths and calculated a mean orientation vector based on each virtual flight path. Each vector for each moth in the circular plots (Fig. 3c–f) encodes the mean orientation direction of a moth’s individual recorded flight path as well as its r value (that is, length, or directedness, of the flight path vector). To take advantage of the extra information in our data arising from the fact that the flight trajectories of moths not only had a mean direction (as used for a classic Rayleigh test49) but also a mean directedness (vector length), we used the circular statistics software Oriana (v.4 (2011), KCS) and Excel (Microsoft Office 2019, Microsoft) to apply a one-sided Moore’s modified Rayleigh test4,50,51 with Bonferroni correction for multiple comparisons (Supplementary Table 1). The combined flight direction of the tested moths will likely be different from random due to the directedness of the population.
Counting, clearing, drying and incubating brains in Bogong mosaics: results of a Leica SP8 confocal brain scan
Brain samples were scanned with the 633 nm laser of a Leica SP8 confocal microscope and viewed with a ×20 oil-immersion objective (Leica Microsystems). For optimal resolution, the scan settings were set to 1,024 × 1,024 pixels, 12-bit pixel depth, 3 times line accumulation and 400 lines per s in the photon-counting mode of the hybrid detector. The brain was registered in the Amira v.5.3 and the standard brain in the Bogong moths.
After recording from a suitable cell, a positive current (range: 1–3 nA for 3 min) was applied to the electrode to inject Neurobiotin into the cell. After the brain was removed from the head capsule, the brain was removed from the electrode. Brains were fixed in 4 °C overnight in paraformaldehyde solution (4% PFA in phosphate buffer) and then washed in 0.1 M PBS (4 times for 15 min). The retinas were removed during washing. Brains were then incubated with streptavidin–Cy5 (Jackson Immuno Research, 1:1,000 in PBS with 0.3% Triton X-100) at 4 °C for 3 days and kept in the dark from this point onwards. The brains were washed multiple times in PBS-Triton X 100 and PBS, and then dehydrated in an increasing amount of alcohol. Brains were left to clear in a mixture of 100% and1:1 after 15 min, with the rest being left to clear for 75 min. The cleared brains were mounted in Permount (Thermo Fisher Scientific) mounting media between two coverslips and left to dry for at least 2 days.
We targeted an area of the central brain in which we expected to find both CX and lateral complex neurons, as well as optic lobe neurons traversing the brain in the posterior optic tract. After a sky rotation, the neuron that didn’t respond was discarded and no recording was made. 79 neurons were assessed as potentially responding to the stimulus and, of these, 28 (35%) met the inclusion criteria of a unimodal or bimodal response profile. The remaining 51 were excluded from the analysis because of their status as uniform in their response to stellar rotation.
Experiments were performed in the afternoon and night in order to maximize the success rate of the recordings. In order to make the sky look bigger, we took out the two 1.2 log unitND filters in front of the projector lens and used it to make a projection of the sky around 250 times larger than the one used at night. For night experiments, the ND filters were reinserted. No obvious differences were found in results obtained in the two situations. The moths were mounted onto the 3D-printed animal holder. The antennae and cuticle were fixed with wax to expose the brain, while a square piece of the cuticle was removed from the head capsule. The neural sheath was digested with Pronase (Sigma-Aldrich) for about 30 s and then carefully washed. It was then removed using a pair of fine forceps. A second small hole was cut into the cuticle above the proboscis muscle and a chlorinated silver wire was inserted into this muscle to serve as reference electrode.
Most behavioural procedures used in this study have been previously described4,5. For 5–10 min, moths were chilled in the freezer. The scales were removed by removing them with 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 attachment, they were tested.
Improving the randomized sky using a UV LED-ring: a circular statistical analysis using MATLAB at the CERN SPS in spring 2019
The positions of the individual points of the natural night sky image were randomly assigned to new positions, and the resulting randomized images were saved as PNG files. 9a). These featureless stellar conditions provided an identical stimulation intensity but provided no celestial spatial information. The randomized skies were improved by randomizing the group of stars, therefore retaining the stars and not removing the spatial variations in the sky that could be used for orientation. The star in the picture has roughly the same dimensions as the squares that formed the picture, a size of 13 13 pixels. The positions of the squares were randomly redrawn and the resulting picture was saved in a file. A final improved randomized stimulus (used during spring 2019) was generated from the test stimulus by randomizing the positions of the individual visible stars. This was achieved with a multiscale Laplacian of a greyscale version of the test signal, 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. This way, the location of the stars on the randomized image could be drawn from any desired distribution. A uniform distribution was used for the location of the rest of the image, and it was placed in the centre.
As the projectors did not emit UV light, we installed a custom-made LED-ring (built by T. McIntyre; diameter, 120 mm; inner diameter, 50 mm) featuring eight UV LEDs (LED370E Ultra Bright Deep Violet LED, Thorlabs) in front of the projector. 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).
The data was analysed using custom-written code in MATLAB. A circular statistical analysis was done using a package called the circular maximum-lihood estimation package. Responses were classified as unimodal (models M2A, M2B or M2C in the R-library CircMLE) or bimodal (models M4A or M4B) based on the Akaike information criterion. The responses that were classified as unimodal in the R-library circMLE were analysed further with respect to the half-width of the rotation tuning curve.
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