A fly simulator for finding the Way of Bogong Moth (Agrotis infusa) using celestial navigation: a case study using a behavioural arena
Each spring, billions of Bogong moths (Agrotis infusa) migrate hundreds of kilometres south to the Australian Alps, guided by the austral night sky. The discovery that they can find their way using only the stars — reported in an 18 July Nature paper1 — makes this moth the first invertebrate to be observed using celestial navigation for long-distance journeys.
The lid on the behavioural arena was lifted to allow for tethering, which happened when the encoder shaft was attached through a 1.5. The moths are able to choose any flight direction if the arena lid is Returned to the arena. There was a way to point the moth towards the geographical north after it was mounted. After the heading direction count was reset, the optical encoder was set to record the flight direction of the moths under a given sky condition at a sampling rate of five frames per second. Each moth was tested for exactly 5 min in each stimulus condition.
Warrant and his team believed that the insects were guided by looking at the stars. To test this, they captured wild moths and placed them in a ‘flight simulator’ — a clear cylindrical box in which the insects were tethered and their movements recorded while being shown projections of the starry sky.
The behavioural analysis used in this study has been previously described4,5. The instantaneous heading directions of a flying bug were recorded and saved in a text file using the US Digital Encoder software. 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. The mean orientation direction of the flight path of a single moth can be summarized in its r value, as shown in the figure. 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 R* value encodes the directedness of a population of tested moths and reveals the likelihood that the combined flight direction of these moths—each with its own direction and directedness—differs significantly from random.
The data used for the analyses is online. As there were no statistical differences in results obtained from male and female moths, the results were pooled.
MRI of the Bogong moth standard brain22,23 using the Leica SP8 confocal microscopy and photon counting with a hybrid detector
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. Neurons and relevant neuropils were then reconstructed in Amira v.5.3 (Thermo Fisher Scientific) and registered into the Bogong moth standard brain22,23.
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. The brain was taken out of the head capsule after the electrodes were removed. In the morning the brains were fixed in 4 C and then washed in 0.1 MPBS for 15 minutes. 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. After incubation, the brains were washed in PBS-Triton X-100 (6 times for 20 min) and PBS (2 times for 20 min) and then dehydrated in an increasing ethanol series (50%, 70%, 90%, 95% and twice at 100%). Brains were then transferred to a fresh mixture of methylsalicylate and ethanol (1:1) and, after 15 min, were left to clear in 100% methylsalicylate for 75 min. The brains were mounted on Permount and left to dry for at least 2 days.
In the section of the central brain that was targeted, we expected to find both the choras and the gliomas in the brain. Neurons that clearly did not respond to an initial sky rotation were immediately discarded and no recording was saved. 28 of the 78 neurons that were assessed were included in the inclusion of the Unimodal or bimodal response profile. The 51 neurons that were classified as uniform for their response to stellar rotation were excluded from the analysis.
To maximize the success of the recordings, the experiments were done in the afternoon and in the night time. 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 moths were mounted on a custom-made animal holder that was 3D-printed. The head was exposed to the brain through the removal of a square piece of cuticle and the fixing of the antennae with wax. 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. There was a second hole cut in the cuticle and a chlorinated silver wire inserted in the proboscis muscle to serve as reference.
The majority of the behavioural procedures used in this study have been described before. Before attaching the stalks, they were chilled for 5 minutes in the freezer. The scales on the moth’s dorsal thorax were removed by suction using a micro-vacuum pump (custom built by B.F.). A thin vertically forged, ferromagnetic free, small circular footplate was created by attaching a contact cement bandage to the thorax with a weighted down plastic mesh, and then being restrained by a weighted down plastic mesh. Moths were tested on the day of stalk attachment.
Source: Bogong moths use a stellar compass for long-distance navigation at night
Improved Randomized Starry Sky Environment Design for Spike Train Experiments using a Custom-Designed LED-Ring and High-Luminosity UV LEDs
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. 9a). These stellar conditions weren’t featureless, but gave the same stimulation intensity. When using randomized starry skies during the spring and autumn of this year, there were groups of individual stars that were randomly chosen to maintain the same intensity, but they were able to retain the stars since there were no spatial variations in the night sky. Here, the stimulus image (of autumn 2019) was subdivided into squares with a size of 13 × 13 pixels, as the brightest star in the image had roughly these dimensions. The positions of these squares were now randomly reassigned and the resulting image was saved as a PNG file. The final improved randomizedStimulus was created by randomizing the positions of the individual visible stars. This was accomplished by detecting positions and sizes of each star in the test suck using a multiscale Laplacian of a greyscale version of the test. After the resulting spatial information was used to extract and save the image of the stars from the natural night sky environment, a new background image was created which had a uniform color and intensity equal to that of the test image. The location of the stars on the randomized image could be drawn from any desired distribution. In this case, a uniform distribution was used for the location of all but the brightest star, which was placed in the centre of the image.
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 adjusted using custom written software and a set of filters that were placed in front of the ring to bring the UV intensity into a quasi-natural range.
Some Spike train data was analysed using custom-written code in MathWorks. Circular statistical analysis was performed using the circular maximum-likelihood estimation package. The data was categorized as unimodal or bimodal based on the criteria of the Akaike information criterion. Only responses that were classified as unimodal (models M2A, M2B or M2C in the R-library CircMLE, based on the Akaike information criterion) were analysed further with respect to the half-width of the rotation tuning curve, the SNR and the variability of the response.
“The first time we saw them flying under the night sky with no other cue and flying in the right direction we had to hold on to the edge of the table,” says study co-author Eric Warrant, an entomologist at Lund University in Sweden.
When the moths did not have access to the projection of the sky or to the electromagnetic field, they were unable to navigate at all. They were able to navigate with the help of visual cue from the stars. They were also able to fly in the correct direction in the presence of an electromagnetic field, but no visible stars.