The starry night sky at Canberra: A simulated night sky analysis and visual cortex activity of a tethered flying moth
In southeastern Australia and parts of New Zealand, boas come out of their cells and begin the long journey to caves they have never been to before. The moths return in autumn to reproduce and die. “How on earth do they know how to find their way there?” Warrant says.
The magnetic field was manipulated by researchers in order to see if moths could navigate without this cue. They recorded the visual cortex activity of the moths’ brains to see if they have an internal representation of the stars.
The moonless starry night sky can be seen over the capital city on four separate dates: 1 October, March, andautumn, and March and October. 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. 9b). Subtended at the moth, celestial images projected onto the circular screens in each rig (see above) provided 160° (behavioural rig) and 100° (electrophysiological rig) fields of view of the starry night sky centred on the zenith. An average grey level of 62 is found in the images which ranged in values from 4 to 255. 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 PC was connected to a projector through an HDMI cable. The diameter of the circular screen in each rig matched the projection size of the circular sky.
The behavioural analysis used in this study was previously described. As mentioned above, the encoder software (USB1 Digital Explorer v.1.07, US Digital) recorded the instantaneous heading directions of a tethered flying moth every 200 ms (5 Hz) and saved these values in a text file. We used a mean orientation and flight path visualization method to see what the virtual flight path would look like. 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). We wanted to take advantage of the extra information in the data that came from the fact that the trajectory of the moths could be seen in terms of a mean direction and a mean directedness. The likelihood of a combined flight direction changing from random has been shown by the R* value.
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.
Intracellular Electrophysiological Experimental Experiments to Probe the Milky Way Stimulus and the Light-Adapted State of Moths
Brain samples were scanned with a laser and viewed with an oil-immersion objective. The settings were set to a resolution of 1,024 1,024 pixels with 12-bit and 400 lines per s in the photon-counting mode. The brain was reconstructed in Amira v.5.3 and put into the standard pattern of the insect’s brain.
The glass had a resistance of 50–100 M and was pulled from the P-97 Flaming-Brown micropipe. 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. The electrodes were put in position using a non-magnetic Sensapex micromanipulator. The signals were amplified by using a NPI electronic amplifier and headstage, and then recorded with Spike2 software. Stimulus control signals from MATLAB (v.2019a and 2022b, MathWorks) were simultaneously recorded in Spike2. The brain was hydrated through regular application of a solution.
To test the selectivity of central brain visual neurons to various parts of the Milky Way during electrophysiological experiments, we stimulated cells with artificial compass cues that, like the starry sky stimulus, filled the projection screen above the moth (Fig. 4d). The summer Milky Way had a bright region around the Carina-nucleo and a stripe-like shape. The bar length and width were made to look like the main stripe of the Milky Way. The mean grey level, which was adjusted to give a good mimic of the intensities of the stars and the dark background sky in our natural stimuli, was the same as before.
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. They were mounted onto a custom-made 3D-printed animal holder. 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 digested with Pronase (Sigma-Aldrich) for about 30 s and then carefully washed. It was then removed using a pair of fine forceps. The proboscis muscle was cut with a second small hole and a wire was placed into it to serve as reference.
Most behavioural procedures used in this study have been previously described4,5. Before attachment of tethering stalks, moths were chilled in a freezer for 5–10 min to immobilize them. The scales on the hind thorax of the moth were removed using a micro-vacuum pump. 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, pests were put through their paces.
Source: Bogong moths use a stellar compass for long-distance navigation at night
Improved randomized night sky stimulus images using UV LEDs and PNG-files for the Spike Train experiment in autumn 2018 and spring 2019
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). They gave the identical stimulation intensity, but they didn’t give the spatial information. The randomized starry skies used during spring and autumn 2019 were improved by randomizing groups of pixels containing individual stars, therefore retaining the stars but removing spatial variations in the night sky (such as the Milky Way) that could be used for orientation (again maintaining identical intensity). The star in the picture had approximately the same dimensions as the squares in the image, so the squares were split into them. The positions of these squares were now randomly reassigned and the resulting image was saved as a PNG 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 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 stars location on the randomized image can 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.
The projectors didn’t emituv light so we built a ring made by T. McIntyre featuring eight UV LEDs 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).
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). The models M2A, M2B or M2C were considered to be unimodal because they were classified based on the information criterion. Only responses which were classified as unimodal in the R library circmLE were analysed further with respect to the half-ridge of the tuning curve.
The study co-author says that the first time they saw them, they flew in the right direction, with no other cue.
The moths were unable to navigate when they didn’t have access to the sky and the field of vision. They were able to navigate in the right direction by using only the visual cue of the stars. They were able to fly in the correct direction even though they didn’t have visible stars.
