Newswise — In an eLife publication, Carolyn Elya, a postdoc in Harvard's Department of Organismic and Evolutionary Biology, uncovers the molecular and cellular foundations of how the parasitic fungus Entomophthora muscae (E. muscae) controls fruit flies' behavior.
In her 2018 eLife study, Elya initially reported the altered behavior, known as summiting, in fruit flies. As a UC Berkeley graduate student researching microbes carried by these flies, Elya placed decaying fruit to trap wild specimens. Upon revisiting her collection, she unexpectedly discovered deceased flies in a captivating stance, exhibiting a distinct banding pattern on their abdomen. By extracting and sequencing DNA, Elya confirmed the presumed culprit behind this phenomenon as E. muscae.
At twilight, summiting takes place as infected flies ascend to higher ground and extend their proboscises towards the surface. A viscous droplet emerges from the proboscis, causing the fly to stick firmly to the surface just before their wings lift away from their body, resulting in the flies' demise.
"The climbing aspect is crucial as it strategically situates the fly in a favorable position for the fungus to infect a maximum number of potential hosts," explains Elya. "The fungus employs highly specialized and transient structures that forcefully rupture the fly's skin, releasing spores into the surroundings. These spores remain viable for just a few hours. Given the fleeting nature of this process, securing an advantageous position becomes paramount for survival."
During her time at UC Berkeley, Elya established a laboratory model known as the Entomophthora muscae-Drosophila melanogaster 'zombie fly' system, utilizing the naturally occurring fungal isolate she discovered in her own backyard. This system enabled Elya to consistently infect fruit flies, a commonly used organism in laboratories, and cultivate the fungus separately from the fly host. She employed specialized media designed to simulate the internal conditions of the fly, allowing for independent fungal culture.
Summiting had been mentioned in scientific literature on numerous occasions; however, prior studies were limited to observations of deceased house flies. The actual behavior exhibited by flies in their final hours of life remained unexplored. Elya sought to bridge this knowledge gap by devising a high-throughput behavioral assay capable of automatically tracking hundreds of infected flies. While employing this platform to monitor the behavioral changes as flies transformed into zombies, Elya made an unexpected discovery. "Contrary to our initial assumption, summiting is not solely about climbing," revealed Elya. "Instead, it entails a sudden surge of locomotor activity that commences approximately two and a half hours before the flies ultimately perish."
Upon making this breakthrough, Elya collaborated with co-authors to integrate her system with the laboratory's robust fruit fly genetic toolkit, allowing for the controlled generation of zombie flies as needed. By combining this approach with her novel behavior assay, the research team was able to pinpoint the specific genes and neurons essential for flies to engage in summiting behavior. This innovative combination of techniques provided invaluable insights into the underlying mechanisms governing summiting in fruit flies.
"In our research, we observed that the hormonal axes of the flies played a significant role in mediating summiting behavior," explains Elya. "When we silenced these specific neurons, the flies exhibited a significant decline in their ability to engage in summiting behavior." These neurons project to a neurohemal organ responsible for the production of juvenile hormone, a hormone that is widely conserved among insects. "Our hypothesis is that the fungus manipulates the activity of these neurons to stimulate the release of juvenile hormone, thereby triggering the sudden burst of locomotor activity observed in the flies," Elya adds.
Subsequently, Elya and her co-authors gathered a vast behavioral dataset comprising hundreds of infected flies. Utilizing this dataset, they trained a computer algorithm to recognize and identify flies during the summiting process. This classifier tool proved invaluable, allowing the team to make a groundbreaking observation: fungal cells infiltrate the brains of the flies in an organized manner, targeting specific regions of the brain during the summiting behavior. This discovery shed light on the intricate dynamics of the fungal invasion within the fly's brain during this peculiar behavioral manipulation.
Intriguingly, the research team made an additional finding: when the flies were exposed to the fungus, their blood-brain barrier became compromised. Ordinarily, this barrier safeguards the neurons from the circulating blood within the fly's body. However, the breakdown of the blood-brain barrier has significant implications, as it allows substances present in the bloodstream to potentially interact with the neurons in the brain. This newfound route of interaction opens up possibilities for the modulation of neural activity through the influence of circulating factors. The compromised blood-brain barrier highlights the intricate interplay between the fungus, the fly's physiology, and the manipulation of neural processes.
Elya stated, "We believe that this phenomenon could play a crucial role in the mechanism by which the fungus induces behavioral changes." Remarkably, the team discovered that by extracting blood from flies engaged in summiting behavior and introducing it to unaffected flies, they were able to induce increased locomotion in the recipient flies. This suggests that certain factors present in the blood contribute to the initiation of summiting behavior. However, the exact identity of these factors and whether they are produced by the fungus or the fly itself remains uncertain. These experiments demonstrate that blood-borne factors can influence summiting behavior, highlighting the intricate interplay between the fungus, the fly, and the systemic factors driving behavioral manipulation. Further research is required to unravel the specific nature of these factors and their roles in the summiting phenomenon.
Moving forward, Elya aims to advance her research by developing transgenic techniques that would enable the modulation of factors originating from the fungus itself, complementing the existing perturbations that can be made in the flies. She acknowledges that numerous unanswered questions persist in this field of study, emphasizing that the precise mechanisms employed by the fungus remain a mystery. Elya's future endeavors will focus on unraveling the intricate workings of the fungus and gaining a deeper understanding of its manipulative strategies. Through continued investigation, she hopes to shed light on the enigmatic nature of this fascinating phenomenon.
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