Neural mechanisms of parasite-induced summiting behavior in ‘zombie’ Drosophila
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eLife assessment
The phenomenon of summit disease, where complex animal behaviours are controlled by single-celled parasites, captivates biologists and non-scientists alike. In this valuable study, the authors use a laboratory model (Drosophila melanogaster infected with Entomophthora muscae) for this disease to provide compelling evidence for the neuroanatomical and physiological underpinnings of summit disease. This is an excellent example of how seemingly intractable questions in behavioural ecology can be effectively addressed in laboratory settings using decades of work in creating 'models' for biology.
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Abstract
For at least two centuries, scientists have been enthralled by the “zombie” behaviors induced by mind-controlling parasites. Despite this interest, the mechanistic bases of these uncanny processes have remained mostly a mystery. Here, we leverage the Entomophthora muscae - Drosophila melanogaster “zombie fly” system to reveal the mechanistic underpinnings of summit disease, a manipulated behavior evoked by many fungal parasites. Using a high-throughput approach to measure summiting, we discovered that summiting behavior is characterized by a burst of locomotion and requires the host circadian and neurosecretory systems, specifically DN1p circadian neurons, pars intercerebralis to corpora allata projecting (PI-CA) neurons and corpora allata (CA), the latter being solely responsible for juvenile hormone (JH) synthesis and release. Using a machine learning classifier to identify summiting animals in real time, we observed that PI-CA neurons and CA appeared intact in summiting animals, despite invasion of adjacent regions of the “zombie fly” brain by E. muscae cells and extensive host tissue damage in the body cavity. The blood-brain barrier of flies late in their infection was significantly permeabilized, suggesting that factors in the hemolymph may have greater access to the central nervous system during summiting. Metabolomic analysis of hemolymph from summiting flies revealed differential abundance of several compounds compared to non-summiting flies. Transfusing the hemolymph of summiting flies into non-summiting recipients induced a burst of locomotion, demonstrating that factor(s) in the hemolymph likely cause summiting behavior. Altogether, our work reveals a neuro-mechanistic model for summiting wherein fungal cells perturb the fly’s hemolymph, activating a neurohormonal pathway linking clock neurons to juvenile hormone production in the CA, ultimately inducing locomotor activity in their host.
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Author Response
Reviewer #1 (Public Review):
This manuscript investigates the mechanisms of 'summiting disease' using a previously characterised Drosophila model. The authors also show that E. muscae infiltrates the brain likey through a defective blood-brain barrier and populates regions of the brain in the medial protocerebrum. It likely releases metabolites into the haemolymph of summiting flies that has the ability to induce summiting in uninfected flies. They also show that a burst of locomotor activity precedes death. To understand the circuit basis of this, they perform a screen of more than a hundred neuronal lines and genes to identify an active DPN1>pars intercerebralis neurons> corpora allata>JH axis as being invovled in the summiting behaviour while not affecting death.
Thank you for your succinct summary of our paper.
R…
Author Response
Reviewer #1 (Public Review):
This manuscript investigates the mechanisms of 'summiting disease' using a previously characterised Drosophila model. The authors also show that E. muscae infiltrates the brain likey through a defective blood-brain barrier and populates regions of the brain in the medial protocerebrum. It likely releases metabolites into the haemolymph of summiting flies that has the ability to induce summiting in uninfected flies. They also show that a burst of locomotor activity precedes death. To understand the circuit basis of this, they perform a screen of more than a hundred neuronal lines and genes to identify an active DPN1>pars intercerebralis neurons> corpora allata>JH axis as being invovled in the summiting behaviour while not affecting death.
Thank you for your succinct summary of our paper.
Reviewer #2 (Public Review):
In this study, the authors aim to uncover the neuroanatomical and metabolite underpinnings of an intriguing phenomenon observed in some insects due to the infection of fungal pathogens. They very cleverly develop a high-throughput assay to examine and quantify this behaviour in a tractable model organism - Drosophila melanogaster which the authors have previously shown to also exhibit this phenomenon. They characterize the details of this behaviour and clearly show the temporal gating of this summiting-followed-by-death behavior to occur shortly before the dusk transition. They go on to examine using a candidate (over 200) screen approach potential neuronal circuits and genes based on the hypothesis that they may be related to 'arousal and gravitaxis'. They narrow down to a line that is restricted to the PI based on the fact that it has a significant effect on the summiting behaviour and that it is known to affect locomotion. They can demonstrate that flies when a subset of PI neurons (R19G10) are transiently activated, they will show summiting even without exposure to the pathogen. Based on Syt-eGFP staining they conclude that PI communicates with the carpora cardiaca (CA). They also show that CA itself is necessary for this behavior, but cannot demonstrate the role of Juvenile hormones using their pharmacological methods.
The authors then describe an automated classifier to identify an upcoming summiting behaviour. Further, they use this real-time classifier to stage different steps of the summiting and match it to the extent of pathology observed by microscopy. They also ask whether the constituents of the hemolymph differ between the summiting and not-yet summiting flies for which they conduct metabolome analysis of the hemolymphs. They are also able to show that cross-injection of uninfected or infected but not summiting flies can be induced to show summiting-like behaviour upon injection with the hemolymph. Finally, they propose the sequence by which the fungal pathogen may modulate the behaviours of the host fly so as to execute this highly gated act of increased locomotion prior to death.
This is a good summary of our findings.
Strengths
• The detailed characterization of the behaviour in D melanogaster and development of the high-throughput behavioural arena.
• Development of the automated classifier which appears to accurately predict this behaviour.
• Narrowing down to a small group of PI neurons having a strong impact on this behaviour although sufficiency is not clearly demonstrated.
Thank you for highlighting these areas of our paper. With respect to demonstrating sufficiency of the PI neurons, we believe this actually an area of comparative strength for the manuscript. With thermogenetic and complementary optogenetic experiments, we demonstrated that activating the PI-CA neurons induces a burst of locomotion consistent with that seen during summiting. A similar burst of locomotion is seen when thermogenetically activating DN1ps. These experiments demonstrate that activity in these neurons is sufficient to induce a pattern of activity like that seen during summiting. In future studies, once the molecular effectors are identified, we may be able to show that fungal alteration of the physiology of these neurons alone is sufficient to induce a burst of locomotion, but that experiment is beyond our current capabilities and beyond the scope of this study.
Weaknesses
• The evidence of temporal (circadian) gating is weak despite the proposed DN1p - PI - CA connections.
• The eventual modification of the behavior to enable enhanced locomotion and negative geotaxis to occur appears to be mediated by yet unknown factors
• The metabolite analysis did not help to narrow down to candidates that can be speculated to cause this behaviour.
With respect to evidence for temporal gating, in this study we did not aim to address the underpinnings of the timing of summiting behavior in this study and did not mean to suggest that the timing of summiting behavior is explained by DN1ps being fly clock neurons. As previously stated in response to high level comments from the editor, we interpret the data presented here as evidence that host neurons (which just happen to be clock neurons) are manipulated by the fungus to inducethe characteristic burst of pre-death locomotor activity that we believe is the key feature of summiting. We have added the following paragraph in the discussion (see Host circadian and pars intercerebralis neurons mediate summiting) to clarify this point:
“Our data indicate that the host circadian network is involved in mediating the increased locomotor activity that we now understand to define summiting. However, our data do not speak to how the timing of this behavior is determined in the zombie-fly system. That is, we have yet to address the mechanisms underlying the temporal gating of summiting and death. Our observation that E. muscae-infected fruit flies continue to die at specific times of day in the absence of proximal lighting cues (Fig 1-S1) suggests that the timing of death is under circadian control and aligns with previous work in E. muscae-infected house flies (Krasnoff et al., 1995). Given that molecular clocks are prevalent across the tree of life, it is likely that two clocks (one in the fly, one in E. muscae) are present in this system. Additional work is needed to determine if the host clock is required for the timing of death under free-running conditions and to assess if E. muscae can keep time.”
We agree that there are many unknown factors at play in this behavior. These include molecular effectors produced by the fungus that alter the physiology of host neurons, and the specific mechanisms by which JH release from the CA alters locomotion. We have endeavored to transparently present what we do and don’t know at this time and hope to be able to address these additional elements in subsequent studies.
It is true that we were unable to determine the identity of compounds driving summiting behavior. However, our analysis did serve to inform which compounds may play a role in summiting by virtue of their overabundance. While we do not yet know the structure of these compounds, their consistent detection in our samples and our new knowledge of their molecular weight with very high accuracy means that these are prime candidates to isolate, purify and functionally test moving forward.
Reviewer #3 (Public Review):
The fungus Entomophthora muscae infects flies and in turn manipulates the flies to produce a summiting behavior that is believed to enhance spore dispersal that happens upon the eventual death of the fly. In this study, the authors undertake a Herculean effort to identify the neural pathways that are manipulated by the fungus to cause summiting. In a major advance, the authors develop techniques that allow them to track behaviors of infected flies over the course of several days. This allows them to investigate summiting behaviors that occur just prior to death with unprecedented detail. In their analysis, the authors find that summiting flies show a burst of increased locomotion just prior to death. Importantly, they show that this burst of locomotion is not seen in flies that are dying from other causes (starvation or desiccation). The burst of locomotion is also found to coincide with an increase in elevation that occurs with summiting, but other results indicate that a change in elevation may be an indirect consequence of increased locomotion. With this new knowledge in hand, the authors screen for genes and neuronal pathways that either disrupt or enhance the burst of locomotion that is characteristic of summiting. These experiments clearly indicate that neurons and genes controlling circadian rhythms play a major role in summiting behaviors. The authors focus their attention on a particular subset of clock neurons (DN1p) as potentially mediating summiting behavior. It is worth noting that DN1p neurons have been implicated in a variety (and in some cases contradictory) of circadian processes and that the interpretation of manipulations of these neurons may be an oversimplification. In particular, prior studies have implicated these cells in temperature entrainment/compensation so interpreting thermogenetic manipulations of these cells might be complicated. The authors also zoom in on a specific region of the brain containing neurons of the pars intercebralis, since they find infiltration by the fungus in this region and the effects of drivers targeting the PI. Converging and convincing lines of evidence to suggest that the PI neurons output to the corpora allata and effects of summing may be mediated by the CA. The already impressive series of experiments are further clinched by the development of a machine vision-based classifier that allows the authors to automatically identify summiting flies so that they may be collected for metabolomic analyses. The authors are automatically emailed and seemingly roused themselves in the middle of the night in order to obtain the precious flies they needed. They find a bunch of compounds that appear in summiting flies and even inject hemolymph from the infected animals into naive flies to find that circulating compounds can affect behaviors. Overall, this paper is a tour de force that addresses a system of long-standing interest and brings it into the modern age. Many new questions are now raised for the future by this fascinating study.
Thank you for your gracious summary of our work and for recognizing the multifaceted approach we have taken to begin to understand the mechanistic basis of summiting. We agree that there are many new questions raised by this work and hope to address them in future publications.
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eLife assessment
The phenomenon of summit disease, where complex animal behaviours are controlled by single-celled parasites, captivates biologists and non-scientists alike. In this valuable study, the authors use a laboratory model (Drosophila melanogaster infected with Entomophthora muscae) for this disease to provide compelling evidence for the neuroanatomical and physiological underpinnings of summit disease. This is an excellent example of how seemingly intractable questions in behavioural ecology can be effectively addressed in laboratory settings using decades of work in creating 'models' for biology.
-
Reviewer #1 (Public Review):
This manuscript investigates the mechanisms of 'summiting disease' using a previously characterised Drosophila model. The authors also show that E. muscae infiltrates the brain likey through a defective blood-brain barrier and populates regions of the brain in the medial protocerebrum. It likely releases metabolites into the haemolymph of summiting flies that has the ability to induce summiting in uninfected flies. They also show that a burst of locomotor activity precedes death. To understand the circuit basis of this, they perform a screen of more than a hundred neuronal lines and genes to identify an active DPN1>pars intercerebralis neurons> corpora allata>JH axis as being invovled in the summiting behaviour while not affecting death.
-
Reviewer #2 (Public Review):
In this study, the authors aim to uncover the neuroanatomical and metabolite underpinnings of an intriguing phenomenon observed in some insects due to the infection of fungal pathogens. They very cleverly develop a high-throughput assay to examine and quantify this behaviour in a tractable model organism - Drosophila melanogaster which the authors have previously shown to also exhibit this phenomenon. They characterize the details of this behaviour and clearly show the temporal gating of this summiting-followed-by-death behavior to occur shortly before the dusk transition. They go on to examine using a candidate (over 200) screen approach potential neuronal circuits and genes based on the hypothesis that they may be related to 'arousal and gravitaxis'. They narrow down to a line that is restricted to the PI …
Reviewer #2 (Public Review):
In this study, the authors aim to uncover the neuroanatomical and metabolite underpinnings of an intriguing phenomenon observed in some insects due to the infection of fungal pathogens. They very cleverly develop a high-throughput assay to examine and quantify this behaviour in a tractable model organism - Drosophila melanogaster which the authors have previously shown to also exhibit this phenomenon. They characterize the details of this behaviour and clearly show the temporal gating of this summiting-followed-by-death behavior to occur shortly before the dusk transition. They go on to examine using a candidate (over 200) screen approach potential neuronal circuits and genes based on the hypothesis that they may be related to 'arousal and gravitaxis'. They narrow down to a line that is restricted to the PI based on the fact that it has a significant effect on the summiting behaviour and that it is known to affect locomotion. They can demonstrate that flies when a subset of PI neurons (R19G10) are transiently activated, they will show summiting even without exposure to the pathogen. Based on Syt-eGFP staining they conclude that PI communicates with the carpora cardiaca (CA). They also show that CA itself is necessary for this behavior, but cannot demonstrate the role of Juvenile hormones using their pharmacological methods.
The authors then describe an automated classifier to identify an upcoming summiting behaviour. Further, they use this real-time classifier to stage different steps of the summiting and match it to the extent of pathology observed by microscopy. They also ask whether the constituents of the hemolymph differ between the summiting and not-yet summiting flies for which they conduct metabolome analysis of the hemolymphs. They are also able to show that cross-injection of uninfected or infected but not summiting flies can be induced to show summiting-like behaviour upon injection with the hemolymph.
Finally, they propose the sequence by which the fungal pathogen may modulate the behaviours of the host fly so as to execute this highly gated act of increased locomotion prior to death.Strengths
• The detailed characterization of the behaviour in D melanogaster and development of the high-throughput behavioural arena.
• Development of the automated classifier which appears to accurately predict this behaviour.
• Narrowing down to a small group of PI neurons having a strong impact on this behaviour although sufficiency is not clearly demonstrated.Weaknesses
• The evidence of temporal (circadian) gating is weak despite the proposed DN1p - PI - CA connections.
• The eventual modification of the behavior to enable enhanced locomotion and negative geotaxis to occur appears to be mediated by yet unknown factors
• The metabolite analysis did not help to narrow down to candidates that can be speculated to cause this behaviour. -
Reviewer #3 (Public Review):
The fungus Entomophthora muscae infects flies and in turn manipulates the flies to produce a summiting behavior that is believed to enhance spore dispersal that happens upon the eventual death of the fly. In this study, the authors undertake a Herculean effort to identify the neural pathways that are manipulated by the fungus to cause summiting. In a major advance, the authors develop techniques that allow them to track behaviors of infected flies over the course of several days. This allows them to investigate summiting behaviors that occur just prior to death with unprecedented detail. In their analysis, the authors find that summiting flies show a burst of increased locomotion just prior to death. Importantly, they show that this burst of locomotion is not seen in flies that are dying from other causes …
Reviewer #3 (Public Review):
The fungus Entomophthora muscae infects flies and in turn manipulates the flies to produce a summiting behavior that is believed to enhance spore dispersal that happens upon the eventual death of the fly. In this study, the authors undertake a Herculean effort to identify the neural pathways that are manipulated by the fungus to cause summiting. In a major advance, the authors develop techniques that allow them to track behaviors of infected flies over the course of several days. This allows them to investigate summiting behaviors that occur just prior to death with unprecedented detail. In their analysis, the authors find that summiting flies show a burst of increased locomotion just prior to death. Importantly, they show that this burst of locomotion is not seen in flies that are dying from other causes (starvation or desiccation). The burst of locomotion is also found to coincide with an increase in elevation that occurs with summiting, but other results indicate that a change in elevation may be an indirect consequence of increased locomotion. With this new knowledge in hand, the authors screen for genes and neuronal pathways that either disrupt or enhance the burst of locomotion that is characteristic of summiting. These experiments clearly indicate that neurons and genes controlling circadian rhythms play a major role in summiting behaviors. The authors focus their attention on a particular subset of clock neurons (DN1p) as potentially mediating summiting behavior. It is worth noting that DN1p neurons have been implicated in a variety (and in some cases contradictory) of circadian processes and that the interpretation of manipulations of these neurons may be an oversimplification. In particular, prior studies have implicated these cells in temperature entrainment/compensation so interpreting thermogenetic manipulations of these cells might be complicated. The authors also zoom in on a specific region of the brain containing neurons of the pars intercebralis, since they find infiltration by the fungus in this region and the effects of drivers targeting the PI. Converging and convincing lines of evidence to suggest that the PI neurons output to the corpora allata and effects of summing may be mediated by the CA. The already impressive series of experiments are further clinched by the development of a machine vision-based classifier that allows the authors to automatically identify summiting flies so that they may be collected for metabolomic analyses. The authors are automatically emailed and seemingly roused themselves in the middle of the night in order to obtain the precious flies they needed. They find a bunch of compounds that appear in summiting flies and even inject hemolymph from the infected animals into naive flies to find that circulating compounds can affect behaviors. Overall, this paper is a tour de force that addresses a system of long-standing interest and brings it into the modern age. Many new questions are now raised for the future by this fascinating study.
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