Periodic Reporting for period 3 - MagneticMoth (Hunting for the elusive “sixth” sense: navigation and magnetic sensation in a nocturnal migratory moth)
Berichtszeitraum: 2020-09-01 bis 2022-02-28
What is the problem/issue being addressed?
Our ability to sense and react to the world around us is largely restricted to five main senses – vision, hearing, smell, taste and touch. However, some aspects of our world are beyond our sensory grasp, such as the ability to detect the weak electric fields of other animals (as so exquisitely achieved in several species of fish). Another is the ability to detect the Earth’s magnetic field. Remarkably though, many other animals – as varied as birds, reptiles, fish and arthropods – can detect this field, and use it as a compass during long-distance navigation. But how they detect the Earth’s magnetic field still remains a mystery. Even though plausible mechanisms have been hypothesised to explain this ability, the receptor cell responsible has never been found. Part of the reason for this lies in the nervous system complexity of the vertebrate animal groups in which magnetoreception has been studied most. However, we have uncovered a much less complex animal that may hold the key to finding the elusive magnetoreceptor, the overarching objective of this project – the nocturnal Australian Bogong moth Agrotis infusa (Fig. 1), a long-distance migratory insect that we have discovered relies on the Earth’s magnetic field as a navigational compass.
Bogong moths make a yearly migration over enormous distances, from southern Queensland, western and northwest New South Wales (NSW) and western Victoria, to the alpine regions of NSW and Victoria (Fig. 1B; Warrant et al. 2016). After emerging from their pupae early in the austral spring, adult Bogong moths embark on a long nocturnal journey towards the Australian Alps, a journey that can take many days or even weeks and cover over 1000 km. Once in the Alps (from the end of September), Bogong moths seek out the shelter of a few selected high ridge-top caves and rock crevices (typically at elevations above 1800 m). In hundreds of thousands, moths line the interior walls of these cool alpine caves (Fig. 1C) where they “hibernate” over the summer months (referred to as “aestivation”). Towards the end of the summer (February and March), the same individuals that arrived months earlier leave the caves and begin their long return trip to their breeding grounds. Once there, moths mate, lay eggs and die. The moths that hatch in the following spring then repeat the migratory cycle afresh. Despite having had no previous experience of the migratory route, these moths find their way to the Alps and locate their aestivation caves that are dotted along the high alpine ridges of southeast Australia, a feat (normalised to body length) that would be equivalent to a person leaving Europe with nothing apart from their senses and travelling to a specific destination in Antarctica.
Why is it important for society?
Our desire to understand how animals sense and use the magnetic field is admittedly driven by sheer curiosity of how animals, even insects with tiny nervous systems, can solve such a difficult problem. Nonetheless, such robust long-distance navigational performance at night is immensely beneficial, not only for animals, but also for any kind of technological application that requires similar levels of performance. The robust high-performance solutions for control and guidance that have evolved in the animal kingdom over hundreds of millions of years hold clues for how similar systems can be implemented in technological applications. Many animals, such as sea turtles and many species of migratory birds, have evolved a map sense used to determine their exact geographic location on the Earth’s surface. This map sense is based on a combination of different sensory cues, prominent among them being the properties of the Earth’s magnetic field. A magnetic map based on the predictable (and often orthogonal) gradients of magnetic intensity and field line inclination across the surface of the Earth – has been likened to a modern GPS navigation system. Animals with a map sense are thus said to be capable of “true navigation”. And unlike a man-made GPS system based on satellites that can fail, or which can be interfered with by undesirable agents, a magnetic map based on the constant and predictable magnetic field of the Earth is always reliable.
What are the overall objectives?
Apart from the overarching objective mentioned above, namely to identify and describe the elusive magnetoreceptor, the project has three main objectives:
A. To identify the principles underpinning the highly directed return migration of Bogong moths. We are using state-of-the-art vertical radar and LIDAR methods to analyse the fine structure of Bogong moth swarms during their forward and return migrations to unravel the principles they use to maintain a precise migratory trajectory independent of the prevailing wind direction. Second, to understand the population dynamics of what is undoubtedly the most accurate and directed migration known in any nocturnal insect, we are using genetic methods and isotope analyses to determine whether moths from different geographic origins aestivate in different alpine caves (as suggested by our preliminary data). Our aim is to obtain the most comprehensive picture ever obtained of migratory strategies in a nocturnal insect, and allow us to understand how a magnetic compass sense is used to hold a precise migratory course despite an unfavourable wind direction.
B. To determine how magnetic and visual cues are used for nocturnal navigation in migratory moths. We are dissecting how visual cues (terrestrial landmarks, the moon, stars) are used in combination with the Earth’s magnetic field to steer migration. Secondly, we are attempting to determine which of the two current hypotheses for biological magnetoreception are used by the Bogong moth. Our aim is to obtain the first detailed description of how magnetic and visual cues are integrated to create a robust compass for long-distance navigation in insects.
C. To identify and physiologically characterise the peripheral receptor cells and central brain circuits responsible for the detection and analysis of the Earth’s magnetic field. We plan to target specific regions in the nervous system with intracellular electrodes, with the aim of recording from, and physiologically characterising, candidate magnetoreceptor cells during magnetic stimulation. The aim is to discover the enigmatic magnetoreceptor, which would be a major advance. We are also determining the morphological layout of Bogong moth compass neuropils in the navigational centre of the brain (the so-called central complex), and targeting candidate compass neurons with intracellular electrodes, with the aim of recording from, and physiologically characterising, these neurons during magnetic stimulation. The main objective of these studies is to determine how magnetic fields are used to encode body orientation in the brain of the moth, and how behavioural alterations in orientation in response to magnetic fields are initiated in the brain. This is currently not understood in any animal.
Our ability to sense and react to the world around us is largely restricted to five main senses – vision, hearing, smell, taste and touch. However, some aspects of our world are beyond our sensory grasp, such as the ability to detect the weak electric fields of other animals (as so exquisitely achieved in several species of fish). Another is the ability to detect the Earth’s magnetic field. Remarkably though, many other animals – as varied as birds, reptiles, fish and arthropods – can detect this field, and use it as a compass during long-distance navigation. But how they detect the Earth’s magnetic field still remains a mystery. Even though plausible mechanisms have been hypothesised to explain this ability, the receptor cell responsible has never been found. Part of the reason for this lies in the nervous system complexity of the vertebrate animal groups in which magnetoreception has been studied most. However, we have uncovered a much less complex animal that may hold the key to finding the elusive magnetoreceptor, the overarching objective of this project – the nocturnal Australian Bogong moth Agrotis infusa (Fig. 1), a long-distance migratory insect that we have discovered relies on the Earth’s magnetic field as a navigational compass.
Bogong moths make a yearly migration over enormous distances, from southern Queensland, western and northwest New South Wales (NSW) and western Victoria, to the alpine regions of NSW and Victoria (Fig. 1B; Warrant et al. 2016). After emerging from their pupae early in the austral spring, adult Bogong moths embark on a long nocturnal journey towards the Australian Alps, a journey that can take many days or even weeks and cover over 1000 km. Once in the Alps (from the end of September), Bogong moths seek out the shelter of a few selected high ridge-top caves and rock crevices (typically at elevations above 1800 m). In hundreds of thousands, moths line the interior walls of these cool alpine caves (Fig. 1C) where they “hibernate” over the summer months (referred to as “aestivation”). Towards the end of the summer (February and March), the same individuals that arrived months earlier leave the caves and begin their long return trip to their breeding grounds. Once there, moths mate, lay eggs and die. The moths that hatch in the following spring then repeat the migratory cycle afresh. Despite having had no previous experience of the migratory route, these moths find their way to the Alps and locate their aestivation caves that are dotted along the high alpine ridges of southeast Australia, a feat (normalised to body length) that would be equivalent to a person leaving Europe with nothing apart from their senses and travelling to a specific destination in Antarctica.
Why is it important for society?
Our desire to understand how animals sense and use the magnetic field is admittedly driven by sheer curiosity of how animals, even insects with tiny nervous systems, can solve such a difficult problem. Nonetheless, such robust long-distance navigational performance at night is immensely beneficial, not only for animals, but also for any kind of technological application that requires similar levels of performance. The robust high-performance solutions for control and guidance that have evolved in the animal kingdom over hundreds of millions of years hold clues for how similar systems can be implemented in technological applications. Many animals, such as sea turtles and many species of migratory birds, have evolved a map sense used to determine their exact geographic location on the Earth’s surface. This map sense is based on a combination of different sensory cues, prominent among them being the properties of the Earth’s magnetic field. A magnetic map based on the predictable (and often orthogonal) gradients of magnetic intensity and field line inclination across the surface of the Earth – has been likened to a modern GPS navigation system. Animals with a map sense are thus said to be capable of “true navigation”. And unlike a man-made GPS system based on satellites that can fail, or which can be interfered with by undesirable agents, a magnetic map based on the constant and predictable magnetic field of the Earth is always reliable.
What are the overall objectives?
Apart from the overarching objective mentioned above, namely to identify and describe the elusive magnetoreceptor, the project has three main objectives:
A. To identify the principles underpinning the highly directed return migration of Bogong moths. We are using state-of-the-art vertical radar and LIDAR methods to analyse the fine structure of Bogong moth swarms during their forward and return migrations to unravel the principles they use to maintain a precise migratory trajectory independent of the prevailing wind direction. Second, to understand the population dynamics of what is undoubtedly the most accurate and directed migration known in any nocturnal insect, we are using genetic methods and isotope analyses to determine whether moths from different geographic origins aestivate in different alpine caves (as suggested by our preliminary data). Our aim is to obtain the most comprehensive picture ever obtained of migratory strategies in a nocturnal insect, and allow us to understand how a magnetic compass sense is used to hold a precise migratory course despite an unfavourable wind direction.
B. To determine how magnetic and visual cues are used for nocturnal navigation in migratory moths. We are dissecting how visual cues (terrestrial landmarks, the moon, stars) are used in combination with the Earth’s magnetic field to steer migration. Secondly, we are attempting to determine which of the two current hypotheses for biological magnetoreception are used by the Bogong moth. Our aim is to obtain the first detailed description of how magnetic and visual cues are integrated to create a robust compass for long-distance navigation in insects.
C. To identify and physiologically characterise the peripheral receptor cells and central brain circuits responsible for the detection and analysis of the Earth’s magnetic field. We plan to target specific regions in the nervous system with intracellular electrodes, with the aim of recording from, and physiologically characterising, candidate magnetoreceptor cells during magnetic stimulation. The aim is to discover the enigmatic magnetoreceptor, which would be a major advance. We are also determining the morphological layout of Bogong moth compass neuropils in the navigational centre of the brain (the so-called central complex), and targeting candidate compass neurons with intracellular electrodes, with the aim of recording from, and physiologically characterising, these neurons during magnetic stimulation. The main objective of these studies is to determine how magnetic fields are used to encode body orientation in the brain of the moth, and how behavioural alterations in orientation in response to magnetic fields are initiated in the brain. This is currently not understood in any animal.
A major task completed during the first half of the project period – and which was essential for progress in the projects detailed below – was the construction of a new ferromagnetic-free laboratory building at our field site in Australia (Fig. 2). This is the only lab building of its kind in the southern hemisphere and only the second ferromagnetic-free lab building to have been built anywhere in the world (the other being in Germany). This new lab has greatly increased the types, reliability and quality of experiments we can perform. The building of this lab was competed in 2018, with a new non-magnetic electrophysiology rig (with Helmholtz coils to provide precise Earth-strength magnetic stimulation – Fig. 2C-D) and a behavioural arena (also with Helmholtz coils – Fig. 2F,G). The behavioural arena and the electrophysiology rig each allow the projection of natural starry skies on screens placed above the moth (using the free planetarium software Stellarium). Thus, both rigs are capable of precise visual and magnetic stimulation. The magnetic disturbances in all parts of the lab, and in both rigs, are almost negligible - the Earth’s magnetic field is entirely unperturbed within the interior of the lab, and the levels of electrical noise in the electrophysiology rig are the lowest we have ever measured.
I will now detail our progress so far in the project, according to the objectives stated above.
A. To identify the principles underpinning the highly directed return migration of Bogong moths
1. What is the fine structure of Bogong moth swarms during their forward and return migrations?
We are using state-of-the-art vertical radar methods (VLR) to analyse the fine structure of Bogong moth swarms during their forward and return migrations to unravel the principles they use to maintain a precise migratory trajectory independent of the prevailing wind direction. This work has started with an in-depth dissection of 20 years of historical vertical radar data collected at Bourke in northwest New South Wales in an attempt to discover signatures of Bogong moths in this data (work by postdoc Dr. Zhenhua Hao). The data collected from VLR is incredibly rich – night after night the radar collects information about the directions, speeds, body orientations and altitudes of vast numbers of migratory insects that cross the beam, which can then be directly correlated with local wind and weather conditions. We are now very close to being able to single out Bogong moths in these radar traces, and thus to study their navigational patterns in detail – this studied has recently been published (Hao et al. 2020). To complement the studies, we have also started collaboration with physicists Dr. Mikkel Dr. Brydegaard and Samuel Jansson at the Department of Physics in Lund to create a vertical LIDAR system (Fig. 3), a technique that has the potential to pick up morphological details from insects that can be used to identify them to species level (which is almost impossible with VLR).
2. Is there is genetic structure in the Bogong moth population?
In this section of the project (performed by PhD student Jesse Wallace), we are aiming to determine whether or not there is genetic structure in the Bogong moth population, and therefore whether it is possible to estimate the origin of a particular moth based on its genome.
We sequenced the Bogong moth’s genome de novo with SciLifeLab (National Genomics Infrastructure Uppsala and Stockholm) in Sweden, whose annotation is now nearing completion. This will establish a powerful tool to not only assess gene variants correlated with the Bogong moth’s migratory ability but also enable us to more efficiently analyse differential gene expression data between the migratory and aestivating stages resulting from transcriptome analysis performed in the current project (see Objective C.1). The genome is also allowing us to use natural genetic markers to find differences in the population structure across the breeding areas of southeast Australia, and to determine whether each population of Bogong moths travels to a specific cave for aestivation.
3. Is the migratory direction of each Bogong moth population encoded genetically and/or epigenetically, and if so, how it is made heritable?
The breeding grounds of the Bogong moth are widely dispersed across south-eastern Australia, and their aestivation sites are tightly conned to a handful of mountains. This means that the direction in which the Bogong moths must migrate to reach their aestivation sites varies across their breeding grounds. The annual migration is performed by a new generation of moths each year, so there is no opportunity to learn this direction in one year, and remember it in subsequent years. Our hypothesis is that the migratory direction is inherited by the Bogong moth. We are currently addressing the following questions: How is the migration goal direction encoded in the Bogong moth, is it heritable and if so how? It is quite possible that our population data (question 2 above), when analysed, will provide clues to these questions, particularly if the direction is encoded genetically. It is however possible that instead of being genetically encoded, the direction is encoded epigenetically, for example through differential DNA methylation. This could provide a mechanism to make the migration direction heritable without necessarily requiring a well dened population structure.
B. To determine how magnetic and visual cues are used for nocturnal navigation in migratory moths
1. How are visual landmarks and celestial cues combined with cues from the Earth’s magnetic field?
During this project, led by Lund postdoc Dr. David Dreyer, we have established that Bogong moths use the Earth’s magnetic field – in conjunction with visual landmarks – as a compass for guiding their southerly migration during the Australian Spring and their northerly return migration during the Autumn (Fig. 4). Using the flight simulator in our new ferromagnetic-free lab in Australia (see Fig. 2), we have now found compelling new evidence that suggests that the Australian night sky could function as one of the most important visual cues in this magnetovisual compass system. This remarkable result suggests that Bogong moths can use the starry night sky as a true compass to discern the geographic cardinal points (N, S, E and W), an ability previously only known from humans and a few species of nocturnally migrating birds. Via the work of our PhD student Andrea Adden, in our Australian electrophysiology rig, we have now discovered cells in the central brain of the Bogong moth that respond exclusively to the rotation of a dorsally-projected starry night sky (in a nulled magnetic field – Fig. 5). We are thus also starting to unravel the neural basis of the stellar compass
C. To identify and physiologically characterise the peripheral receptor cells and central brain circuits responsible for the detection and analysis of the Earth’s magnetic field.
1. Anatomically identifying potential magnetoreceptors
The aim of this part of the project – led by Lund postdoc Kristina Brauburger - is to shed light on the molecular basis of magnetic sensing in the Bogong moth. We have therefore sought to identify the locations, and the temporal and spatial expression patterns, of the putative molecular magnetosensors, the cryptochromes. Cryptochromes are blue-light receptors that were shown to possess the required characteristics needed for magnetic sensing at the molecular level. One theory of light-dependent magnetoreception suggests that cryptochromes are located within the photoreceptors of the eyes and enable animals to “see” the Earth’s magnetic field (Fig. 6).
To shed more light on these putative actors involved in the magnetosensory process, we first identified the different types of opsins and cryptochromes present in the visual system of Bogong moths. In the first year of the project we cloned full-length cDNAs encoding four Bogong moth opsins based on pooled tissue from two moths. By phylogenetic analyses we identified these as two long wavelength opsins (green and putatively red sensitive opsin), one blue opsin and one UV opsin and two cryptochromes (cry1 and cry2), homologous to two cryptochromes also described in the monarch butterfly, Danaus plexippus. Our sequence data suggest that cry1 of the Bogong moth is similar in sequence to cry1 of Drosophila melanogaster (Fig. 7)
In a next step, we investigated the location of these light sensors in the moth’s visual system. The visual system consists of the main compound eyes, as well as two ocelli located on the dorsal part of the Bogong moth’s head. Either type of eye might be involved in magnetoreception. We used the sequence information obtained from the cloning to design specific antisense riboprobes targeting opsin and cryptochrome mRNA. By in situ hybridization we then identified the expression patterns of the different opsin mRNAs (Fig. 8). In contrast, the detection of cryptochrome mRNA turned out to be more challenging and we have so far not found conclusive evidence for the expression of these genes in the visual system of Bogong moths, possibly because our current probes are not sensitive enough to detect low-level expression. However, by performing PCR with cryptochrome-specific primers, we could show that cry1 and cry2 is indeed expressed in both the eyes and ocelli at all (Fig. 9).
2. Responses of compass cells in the central complex (CX) to visual and magnetic stimulation
To illuminate the neural basis for the Bogong moth’s remarkable ability to migrate across a thousand kilometres of unfamiliar terrain, we first had to understand the organization of their brains. PhD student Andrea Adden has generated a standardized average-shape brain based on 10 individual male whole-mount preparations that serves as reference atlas for embedding single neuron morphologies from intracellular dye injections (see Fig. 5A). This standard brain, together with a detailed anatomical description of all major neuropils in the Bogong moth brain, was recently published in the Journal of Comparative Neurology (Adden et al. 2020).
Using the non-magnetic electrophysiological rig in the Australian lab, Andrea Adden was able to obtain intracellular sharp-electrode recordings from many CX neurons that responded to rotations of the starry night sky (see Fig.5B). The cells were tuned to specific azimuth orientations of the night sky, but did not respond to control motion stimuli, or to moving light spots with similar contrast as the starry sky. The neurons found thus appear to be driven selectively by a naturalistic starry sky stimulus. Our results demonstrate, for the first time in any insect, that the starry sky is an appropriate stimulus for activating neurons in the Bogong moth brain, and that the CX likely provides the neural substrate for starry sky-based navigation in the Bogong moth.
Adden, A., Wibrand, S., Pfeiffer, K., Warrant, E.J. and Heinze, S. The Bogong moth brain: anatomical basis of nocturnal migration. Journal of Comparative Neurology. doi: 10.1002/cne.24866 (2020).
Hao, Z., Drake, V.A. Taylor, J.R. and Warrant, E.J. (2020). Insect target classes discerned from entomological radar data. Remote Sensing 2: 673. doi:10.3390/rs12040673.
I will now detail our progress so far in the project, according to the objectives stated above.
A. To identify the principles underpinning the highly directed return migration of Bogong moths
1. What is the fine structure of Bogong moth swarms during their forward and return migrations?
We are using state-of-the-art vertical radar methods (VLR) to analyse the fine structure of Bogong moth swarms during their forward and return migrations to unravel the principles they use to maintain a precise migratory trajectory independent of the prevailing wind direction. This work has started with an in-depth dissection of 20 years of historical vertical radar data collected at Bourke in northwest New South Wales in an attempt to discover signatures of Bogong moths in this data (work by postdoc Dr. Zhenhua Hao). The data collected from VLR is incredibly rich – night after night the radar collects information about the directions, speeds, body orientations and altitudes of vast numbers of migratory insects that cross the beam, which can then be directly correlated with local wind and weather conditions. We are now very close to being able to single out Bogong moths in these radar traces, and thus to study their navigational patterns in detail – this studied has recently been published (Hao et al. 2020). To complement the studies, we have also started collaboration with physicists Dr. Mikkel Dr. Brydegaard and Samuel Jansson at the Department of Physics in Lund to create a vertical LIDAR system (Fig. 3), a technique that has the potential to pick up morphological details from insects that can be used to identify them to species level (which is almost impossible with VLR).
2. Is there is genetic structure in the Bogong moth population?
In this section of the project (performed by PhD student Jesse Wallace), we are aiming to determine whether or not there is genetic structure in the Bogong moth population, and therefore whether it is possible to estimate the origin of a particular moth based on its genome.
We sequenced the Bogong moth’s genome de novo with SciLifeLab (National Genomics Infrastructure Uppsala and Stockholm) in Sweden, whose annotation is now nearing completion. This will establish a powerful tool to not only assess gene variants correlated with the Bogong moth’s migratory ability but also enable us to more efficiently analyse differential gene expression data between the migratory and aestivating stages resulting from transcriptome analysis performed in the current project (see Objective C.1). The genome is also allowing us to use natural genetic markers to find differences in the population structure across the breeding areas of southeast Australia, and to determine whether each population of Bogong moths travels to a specific cave for aestivation.
3. Is the migratory direction of each Bogong moth population encoded genetically and/or epigenetically, and if so, how it is made heritable?
The breeding grounds of the Bogong moth are widely dispersed across south-eastern Australia, and their aestivation sites are tightly conned to a handful of mountains. This means that the direction in which the Bogong moths must migrate to reach their aestivation sites varies across their breeding grounds. The annual migration is performed by a new generation of moths each year, so there is no opportunity to learn this direction in one year, and remember it in subsequent years. Our hypothesis is that the migratory direction is inherited by the Bogong moth. We are currently addressing the following questions: How is the migration goal direction encoded in the Bogong moth, is it heritable and if so how? It is quite possible that our population data (question 2 above), when analysed, will provide clues to these questions, particularly if the direction is encoded genetically. It is however possible that instead of being genetically encoded, the direction is encoded epigenetically, for example through differential DNA methylation. This could provide a mechanism to make the migration direction heritable without necessarily requiring a well dened population structure.
B. To determine how magnetic and visual cues are used for nocturnal navigation in migratory moths
1. How are visual landmarks and celestial cues combined with cues from the Earth’s magnetic field?
During this project, led by Lund postdoc Dr. David Dreyer, we have established that Bogong moths use the Earth’s magnetic field – in conjunction with visual landmarks – as a compass for guiding their southerly migration during the Australian Spring and their northerly return migration during the Autumn (Fig. 4). Using the flight simulator in our new ferromagnetic-free lab in Australia (see Fig. 2), we have now found compelling new evidence that suggests that the Australian night sky could function as one of the most important visual cues in this magnetovisual compass system. This remarkable result suggests that Bogong moths can use the starry night sky as a true compass to discern the geographic cardinal points (N, S, E and W), an ability previously only known from humans and a few species of nocturnally migrating birds. Via the work of our PhD student Andrea Adden, in our Australian electrophysiology rig, we have now discovered cells in the central brain of the Bogong moth that respond exclusively to the rotation of a dorsally-projected starry night sky (in a nulled magnetic field – Fig. 5). We are thus also starting to unravel the neural basis of the stellar compass
C. To identify and physiologically characterise the peripheral receptor cells and central brain circuits responsible for the detection and analysis of the Earth’s magnetic field.
1. Anatomically identifying potential magnetoreceptors
The aim of this part of the project – led by Lund postdoc Kristina Brauburger - is to shed light on the molecular basis of magnetic sensing in the Bogong moth. We have therefore sought to identify the locations, and the temporal and spatial expression patterns, of the putative molecular magnetosensors, the cryptochromes. Cryptochromes are blue-light receptors that were shown to possess the required characteristics needed for magnetic sensing at the molecular level. One theory of light-dependent magnetoreception suggests that cryptochromes are located within the photoreceptors of the eyes and enable animals to “see” the Earth’s magnetic field (Fig. 6).
To shed more light on these putative actors involved in the magnetosensory process, we first identified the different types of opsins and cryptochromes present in the visual system of Bogong moths. In the first year of the project we cloned full-length cDNAs encoding four Bogong moth opsins based on pooled tissue from two moths. By phylogenetic analyses we identified these as two long wavelength opsins (green and putatively red sensitive opsin), one blue opsin and one UV opsin and two cryptochromes (cry1 and cry2), homologous to two cryptochromes also described in the monarch butterfly, Danaus plexippus. Our sequence data suggest that cry1 of the Bogong moth is similar in sequence to cry1 of Drosophila melanogaster (Fig. 7)
In a next step, we investigated the location of these light sensors in the moth’s visual system. The visual system consists of the main compound eyes, as well as two ocelli located on the dorsal part of the Bogong moth’s head. Either type of eye might be involved in magnetoreception. We used the sequence information obtained from the cloning to design specific antisense riboprobes targeting opsin and cryptochrome mRNA. By in situ hybridization we then identified the expression patterns of the different opsin mRNAs (Fig. 8). In contrast, the detection of cryptochrome mRNA turned out to be more challenging and we have so far not found conclusive evidence for the expression of these genes in the visual system of Bogong moths, possibly because our current probes are not sensitive enough to detect low-level expression. However, by performing PCR with cryptochrome-specific primers, we could show that cry1 and cry2 is indeed expressed in both the eyes and ocelli at all (Fig. 9).
2. Responses of compass cells in the central complex (CX) to visual and magnetic stimulation
To illuminate the neural basis for the Bogong moth’s remarkable ability to migrate across a thousand kilometres of unfamiliar terrain, we first had to understand the organization of their brains. PhD student Andrea Adden has generated a standardized average-shape brain based on 10 individual male whole-mount preparations that serves as reference atlas for embedding single neuron morphologies from intracellular dye injections (see Fig. 5A). This standard brain, together with a detailed anatomical description of all major neuropils in the Bogong moth brain, was recently published in the Journal of Comparative Neurology (Adden et al. 2020).
Using the non-magnetic electrophysiological rig in the Australian lab, Andrea Adden was able to obtain intracellular sharp-electrode recordings from many CX neurons that responded to rotations of the starry night sky (see Fig.5B). The cells were tuned to specific azimuth orientations of the night sky, but did not respond to control motion stimuli, or to moving light spots with similar contrast as the starry sky. The neurons found thus appear to be driven selectively by a naturalistic starry sky stimulus. Our results demonstrate, for the first time in any insect, that the starry sky is an appropriate stimulus for activating neurons in the Bogong moth brain, and that the CX likely provides the neural substrate for starry sky-based navigation in the Bogong moth.
Adden, A., Wibrand, S., Pfeiffer, K., Warrant, E.J. and Heinze, S. The Bogong moth brain: anatomical basis of nocturnal migration. Journal of Comparative Neurology. doi: 10.1002/cne.24866 (2020).
Hao, Z., Drake, V.A. Taylor, J.R. and Warrant, E.J. (2020). Insect target classes discerned from entomological radar data. Remote Sensing 2: 673. doi:10.3390/rs12040673.
1. Progress beyond the state of the art
So far in the project, we have progressed beyond the state-of-the-art in several areas. Firstly, we have shown that nocturnal migratory insects - like nocturnal migratory birds - rely on the Earth’s magnetic field to steer migration, which is a major discovery. Moreover, not only are Bogong moths capable of sensing the Earth’s magnetic field, but they use it together with visual landmarks to steer migratory flight.
Secondly, we have also shown that one of the major visual cues used by moths – in conjunction with the Earth’s magnetic field - is the Milky Way, which in the southern hemisphere is a bright stripe of light that is brighter in the southern sky than it is in the northern sky. We have discovered, using behavioural experiments, that moths are able to determine compass directions (N, S, E and W) using the starry sky alone, the first invertebrate known to do so (the only other animals known to possess this ability are humans and some species of night-migratory birds). We now suspect, and hope in the remaining part of the project to show, that by combining information from the Earth’s magnetic field and the starry night sky, Bogong moths possess a highly robust navigational compass. In addition, within this study we also discovered and identified a number of visual cells in the moth’s central brain that respond exclusively to rotations of the starry night sky, and which are therefore likely to be involved in the stellar compass network that analyses stellar information to set and correct the migratory course. These discoveries show that natural sensory cues - in this case visual and magnetic cues - are used to solve a complex navigational problem in an animal whose brain occupies a volume less than that of a grain of rice. This impressive ability adds significant new evidence supporting the idea that the insect nervous system, despite its small size, is both sophisticated and remarkable.
Thirdly, we have now completed sequencing, and have almost completed annotating, the genome of the Bogong moth. This represents a major milestone in genomics, not only because we now have the genome of an iconic Australian insect known to all Australians, but also because the genome will allow us (and others) to ask a number of very important fundamental questions regarding the biology, ecology, conservation, migration and sensory biology of this keystone species (whose mass migration to the Australian Alps is critically important for the health of the alpine ecosystem).
Fourthly, we have pioneered new methods for detecting and tracking migrating insects. Our new vertical LIDAR system, coupled with traditional vertical radar, will allow us and others to identify and track insects during their migration with unprecedented clarity. These methods promise not only to shine significant light on the migration of Bogong moths, but also herald new possibilities for monitoring the health of insect populations in regions of the world where their decline is extremely worrying.
2. Expected results until the end of the project
We have detailed many of these in the sections titled “Plans for ongoing work” (in the part of the report detailing the work performed), but in brief one can list our expected results as follows:
Objective A
We expect to complete our analysis on the historical radar data, and together with accurate weather modelling, to derive realistic trajectories and timelines for Bogong moths travelling from Bourke to the Snowy Mountains, thereby ascertaining their large-scale navigational abilities. We also expect to have a fully operational LIDAR system delivering accurate identifications of migrating Bogong moths in the field, and that we will be able to deploy this system together with radar at any location in southeast Australia we wish. We’ll also complete the population genetics of Bogong moths and determine whether individual populations are linked to individual aestivation caves. Moreover, we also plan to determine whether the migratory direction is inherited genetically or epigenetically.
Objective B
We expect to determine exactly how the Earth’s magnetic field and the starry night sky are used together as a compass during navigation, which hierarchy exists between these cues, and under what circumstances. We also aim to determine whether the magnetic sense of Bogong moths is based on cryptochrome molecules or magnetite.
Objective C
We expect to complete our investigations on the locations of cryptochrome-expressing cells in the nervous system of Bogong moths. We also expect to discover which (if either) of the two resident cryptochrome molecules possessed by Bogong moths (cry 1 and cry2) are likely to function as a magnetic sensor. With all of this information we should be able to identify the location in the nervous system that has the highest likelihood of housing a magnetoreceptor. A major (and very risky) goal of Objective C is to electrophysiologically record from cells identified as likely magnetoreceptors using the methods above. This is still our goal during the remainder of the project. Within Objective C we will also construct a new electrophysiological lab for extracellular tetrode brain recordings for the express purpose of trying to find neurons and/or regions of the brain that are responsive to changes in an externally applied earth-strength magnetic field. These recordings will be instrumental in allowing us to understand how magnetic information is processed in the Bogong moth brain.
So far in the project, we have progressed beyond the state-of-the-art in several areas. Firstly, we have shown that nocturnal migratory insects - like nocturnal migratory birds - rely on the Earth’s magnetic field to steer migration, which is a major discovery. Moreover, not only are Bogong moths capable of sensing the Earth’s magnetic field, but they use it together with visual landmarks to steer migratory flight.
Secondly, we have also shown that one of the major visual cues used by moths – in conjunction with the Earth’s magnetic field - is the Milky Way, which in the southern hemisphere is a bright stripe of light that is brighter in the southern sky than it is in the northern sky. We have discovered, using behavioural experiments, that moths are able to determine compass directions (N, S, E and W) using the starry sky alone, the first invertebrate known to do so (the only other animals known to possess this ability are humans and some species of night-migratory birds). We now suspect, and hope in the remaining part of the project to show, that by combining information from the Earth’s magnetic field and the starry night sky, Bogong moths possess a highly robust navigational compass. In addition, within this study we also discovered and identified a number of visual cells in the moth’s central brain that respond exclusively to rotations of the starry night sky, and which are therefore likely to be involved in the stellar compass network that analyses stellar information to set and correct the migratory course. These discoveries show that natural sensory cues - in this case visual and magnetic cues - are used to solve a complex navigational problem in an animal whose brain occupies a volume less than that of a grain of rice. This impressive ability adds significant new evidence supporting the idea that the insect nervous system, despite its small size, is both sophisticated and remarkable.
Thirdly, we have now completed sequencing, and have almost completed annotating, the genome of the Bogong moth. This represents a major milestone in genomics, not only because we now have the genome of an iconic Australian insect known to all Australians, but also because the genome will allow us (and others) to ask a number of very important fundamental questions regarding the biology, ecology, conservation, migration and sensory biology of this keystone species (whose mass migration to the Australian Alps is critically important for the health of the alpine ecosystem).
Fourthly, we have pioneered new methods for detecting and tracking migrating insects. Our new vertical LIDAR system, coupled with traditional vertical radar, will allow us and others to identify and track insects during their migration with unprecedented clarity. These methods promise not only to shine significant light on the migration of Bogong moths, but also herald new possibilities for monitoring the health of insect populations in regions of the world where their decline is extremely worrying.
2. Expected results until the end of the project
We have detailed many of these in the sections titled “Plans for ongoing work” (in the part of the report detailing the work performed), but in brief one can list our expected results as follows:
Objective A
We expect to complete our analysis on the historical radar data, and together with accurate weather modelling, to derive realistic trajectories and timelines for Bogong moths travelling from Bourke to the Snowy Mountains, thereby ascertaining their large-scale navigational abilities. We also expect to have a fully operational LIDAR system delivering accurate identifications of migrating Bogong moths in the field, and that we will be able to deploy this system together with radar at any location in southeast Australia we wish. We’ll also complete the population genetics of Bogong moths and determine whether individual populations are linked to individual aestivation caves. Moreover, we also plan to determine whether the migratory direction is inherited genetically or epigenetically.
Objective B
We expect to determine exactly how the Earth’s magnetic field and the starry night sky are used together as a compass during navigation, which hierarchy exists between these cues, and under what circumstances. We also aim to determine whether the magnetic sense of Bogong moths is based on cryptochrome molecules or magnetite.
Objective C
We expect to complete our investigations on the locations of cryptochrome-expressing cells in the nervous system of Bogong moths. We also expect to discover which (if either) of the two resident cryptochrome molecules possessed by Bogong moths (cry 1 and cry2) are likely to function as a magnetic sensor. With all of this information we should be able to identify the location in the nervous system that has the highest likelihood of housing a magnetoreceptor. A major (and very risky) goal of Objective C is to electrophysiologically record from cells identified as likely magnetoreceptors using the methods above. This is still our goal during the remainder of the project. Within Objective C we will also construct a new electrophysiological lab for extracellular tetrode brain recordings for the express purpose of trying to find neurons and/or regions of the brain that are responsive to changes in an externally applied earth-strength magnetic field. These recordings will be instrumental in allowing us to understand how magnetic information is processed in the Bogong moth brain.