Mechanisms behind the Madness: How Do Zombie-Making Fungal Entomopathogens Affect Host Behavior To Increase Transmission?

Transmission is a crucial step in all pathogen life cycles. As such, certain species have evolved complex traits that increase their chances to find and invade new hosts. Fungal species that hijack insect behaviors are evident examples. Many of these "zombie-making" entomopathogens cause their hosts to exhibit heightened activity, seek out elevated positions, and display body postures that promote spore dispersal, all with specific circadian timing. Answering how fungal entomopathogens manipulate their hosts will increase our understanding of molecular aspects underlying fungus-insect interactions, pathogen-host coevolution, and the regulation of animal behavior. It may also lead to the discovery of novel bioactive compounds, given that the fungi involved have traditionally been understudied. This minireview summarizes and discusses recent work on zombie-making fungi of the orders Hypocreales and Entomophthorales that has resulted in hypotheses regarding the mechanisms that drive fungal manipulation of insect behavior. We discuss mechanical processes, host chemical signaling pathways, and fungal secreted effectors proposed to be involved in establishing pathogen-adaptive behaviors. Additionally, we touch on effectors' possible modes of action and how the convergent evolution of host manipulation could have given rise to the many parallels in observed behaviors across fungus-insect systems and beyond. However, the hypothesized mechanisms of behavior manipulation have yet to be proven. We, therefore, also suggest avenues of research that would move the field toward a more quantitative future

Patterns and potential mechanisms of thermal preference in E. muscae-infected Drosophila melanogaster

Animals use various strategies to defend against pathogens. Behavioral fever, or fighting infection by moving to warm locations, is seen in many ectotherms. The behavior-manipulating fungal pathogen Entomophthora muscae infects numerous dipterans, including fruit flies and house flies, Musca domestica. House flies have been shown to exhibit robust behavioral fever early after exposure to E. muscae, then switch to prefer cool temperatures in the later stages of infection. Interestingly, no evidence of behavioral fever in response to any investigated pathogen has been found in the fruit fly, Drosophila melanogaster. However, they have been found to prefer cool temperatures during infections. To determine if fruit flies utilize behavioral fever, cold-seeking, or both during E. muscae infection, we used a two-choice behavioral assay to measure individual temperature preferences of E. muscae-exposed and unexposed flies at early (24-72 hour) and late (72-120 hour) infection time points. In contrast with our expectation from house flies, fruit flies did not exhibit behavioral fever. However, we found significant cold temperature-seeking in flies that died from infection on the day of the assay. To investigate whether this cold-seeking behavior was being caused by the fly or the fungus, we tested the effects of temperature on the fitness of the host, D. melanogaster, and the pathogen, E. muscae, during infection. We found that flies held at low and high temperatures for 24 hours before death from infection laid no eggs at the lower temperature. This could suggest that the fly is not causing the cold-seeking behavior because there is no apparent fitness benefit at low temperatures. Conversely, cadavers sporulating at the low temperature tended to cause more flies to eventually die from infection, indicating that E. muscae infects flies more effectively at lower temperatures. Preliminarily, our results support fungal control of temperature preference before death, though further testing is needed. The idea that E. muscae benefits from colder temperatures, and therefore drives cold-seeking behavior in D. melanogaster at the end of the host’s life, expands our current knowledge about host behavior manipulation by E. muscae and provides a fascinating avenue for investigating the mechanisms by which this fungus manipulates complex behaviors in its animal host

Discovery of Novel, Orally Bioavailable, Antileishmanial Compounds Using Phenotypic Screening

Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 μM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for in vitro absorption, distribution, metabolism, excretion, and in vivo pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC50) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug

Using Drosophila melanogaster to understand how microbes affect host behavior

Animals live in a world teeming with microbial life. Co-existing over evolutionary time, both microbes and animals have evolved methods to influence the other to maximize their respective fitness. It has been the focus of my doctoral work to study how microbes affect animal behavior using the model species Drosophila melanogaster. Like all other animals so-far encountered, the fruit fly digestive system is occupied by microbes consisting of mostly bacteria. Contrary to my expectations, I did not encounter fly behaviors that changed by varying their associated gut bacteria. In order to understand what changes were taking place in flies with manipulated gut flora, I assayed gene expression in dissected guts or in whole flies. I discovered that associating flies from the embryonic stage with zero, one or three bacterial taxa resulted in the same transcriptional profile in the adult gut, suggesting that adult guts are buffered against the bacteria that passage through them. However, associating flies with the yeast Saccharomyces cerevisiae, whether alone or in combination with these bacteria, was sufficient to recapitulate the transcriptional profile observed in guts of conventionally-reared flies. This suggests that yeast, not bacteria, are key in mediating the gut transcriptional response of adult flies. In contrast, transcription within the entire adult animal varied with exposure to different microbial populations, suggesting that different bacterial taxa can influence fly hosts prior to adulthood. Yeast, Saccharomyces cerevisiae, is a key food source for D. melanogaster both in the wild and in the laboratory. Previous work by former Eisen laboratory member Kelly Schiabor had demonstrated a correlation between attraction to yeast grown under natural (sugar-replete, nitrogen-limiting) conditions (YVN) and chimeric variant in the gene encoding a component of fly olfaction, odorant receptor 22 (Or22). In conjunction with Alli Quan, I tested the hypothesis that the chimeric Or22 allele in D. melanogaster mediates sensitivity of flies to YVN and therefore makes these flies better suited to detect yeast in the environment. Through sequence analysis, bidirectional crosses between chimeric and non-chimeric lines and ultimately, replacement of a non-chimeric Or22 allele with a chimeric allele within a non-chimeric background, we found that Or22 alone does not mediate sensitivity to YVN. Still, the signs of selection at the Or22 locus suggest that this receptor confers some adaptive function in wild flies.Entomophthora muscae is a fungal pathogen that infects, alters the behavior of, and then kills dipterans. Predominantly reported in house flies and other large Muscoidea, critically-ill flies summit, extend their proboscis, and raise their wings up and away from their dorsal abdomen in the moments prior to death, dying in an elevated position that appears to benefit fungal dispersal and therefore fitness. Despite being described over 160 years ago, we know little of E. muscae biology, especially the molecular means through which it alters host behavior. Serendipitously, I discovered a strain of E. muscae (CNE1) that infects wild Drosophila species, including D. melanogaster. I have isolated this strain in the laboratory both in vivo, through active propagation between healthy fruit flies, and in vitro, in liquid culture. By observing the isolated fungal culture with modern technology, I have been able to corroborate and add to the series of observations describing how this bizarre organism infects, grows and abandons spent hosts. Crucially, by having isolated a strain that naturally infects a model organism, I have been able to begin testing a variety of molecular hypothesis as to which host machinery is necessary for observed phenotypes, demonstrating that specific neurons and genes are not involved in mediating end-of-life behaviors. With stable E. muscae CNE1 culture, I have been able to show that the fungus first travels to the brain and central nervous system in infected flies before proliferating uncontrollably in the body cavity. I have also, with the aid of Michaels Bronski and Eisen, sequenced the genome of the isolate, and, as a consequence, have been able to assay gene expression of both host and fungus over the course of infection. Ultimately, this work represents only the beginning of what is possible in the E. muscae CNE1-D. melanogaster system, not just for understanding the molecular basis of host behavioral manipulation, but also for studying varied aspects of host-microbe interactions

Using Drosophila melanogaster to understand how microbes affect host behavior

Animals live in a world teeming with microbial life. Co-existing over evolutionary time, both microbes and animals have evolved methods to influence the other to maximize their respective fitness. It has been the focus of my doctoral work to study how microbes affect animal behavior using the model species Drosophila melanogaster. Like all other animals so-far encountered, the fruit fly digestive system is occupied by microbes consisting of mostly bacteria. Contrary to my expectations, I did not encounter fly behaviors that changed by varying their associated gut bacteria. In order to understand what changes were taking place in flies with manipulated gut flora, I assayed gene expression in dissected guts or in whole flies. I discovered that associating flies from the embryonic stage with zero, one or three bacterial taxa resulted in the same transcriptional profile in the adult gut, suggesting that adult guts are buffered against the bacteria that passage through them. However, associating flies with the yeast Saccharomyces cerevisiae, whether alone or in combination with these bacteria, was sufficient to recapitulate the transcriptional profile observed in guts of conventionally-reared flies. This suggests that yeast, not bacteria, are key in mediating the gut transcriptional response of adult flies. In contrast, transcription within the entire adult animal varied with exposure to different microbial populations, suggesting that different bacterial taxa can influence fly hosts prior to adulthood. Yeast, Saccharomyces cerevisiae, is a key food source for D. melanogaster both in the wild and in the laboratory. Previous work by former Eisen laboratory member Kelly Schiabor had demonstrated a correlation between attraction to yeast grown under natural (sugar-replete, nitrogen-limiting) conditions (YVN) and chimeric variant in the gene encoding a component of fly olfaction, odorant receptor 22 (Or22). In conjunction with Alli Quan, I tested the hypothesis that the chimeric Or22 allele in D. melanogaster mediates sensitivity of flies to YVN and therefore makes these flies better suited to detect yeast in the environment. Through sequence analysis, bidirectional crosses between chimeric and non-chimeric lines and ultimately, replacement of a non-chimeric Or22 allele with a chimeric allele within a non-chimeric background, we found that Or22 alone does not mediate sensitivity to YVN. Still, the signs of selection at the Or22 locus suggest that this receptor confers some adaptive function in wild flies.Entomophthora muscae is a fungal pathogen that infects, alters the behavior of, and then kills dipterans. Predominantly reported in house flies and other large Muscoidea, critically-ill flies summit, extend their proboscis, and raise their wings up and away from their dorsal abdomen in the moments prior to death, dying in an elevated position that appears to benefit fungal dispersal and therefore fitness. Despite being described over 160 years ago, we know little of E. muscae biology, especially the molecular means through which it alters host behavior. Serendipitously, I discovered a strain of E. muscae (CNE1) that infects wild Drosophila species, including D. melanogaster. I have isolated this strain in the laboratory both in vivo, through active propagation between healthy fruit flies, and in vitro, in liquid culture. By observing the isolated fungal culture with modern technology, I have been able to corroborate and add to the series of observations describing how this bizarre organism infects, grows and abandons spent hosts. Crucially, by having isolated a strain that naturally infects a model organism, I have been able to begin testing a variety of molecular hypothesis as to which host machinery is necessary for observed phenotypes, demonstrating that specific neurons and genes are not involved in mediating end-of-life behaviors. With stable E. muscae CNE1 culture, I have been able to show that the fungus first travels to the brain and central nervous system in infected flies before proliferating uncontrollably in the body cavity. I have also, with the aid of Michaels Bronski and Eisen, sequenced the genome of the isolate, and, as a consequence, have been able to assay gene expression of both host and fungus over the course of infection. Ultimately, this work represents only the beginning of what is possible in the E. muscae CNE1-D. melanogaster system, not just for understanding the molecular basis of host behavioral manipulation, but also for studying varied aspects of host-microbe interactions

The genus <i>Entomophthora</i>:bringing the insect destroyers into the twenty-first century

Abstract The fungal genus Entomophthora consists of highly host-specific pathogens that cause deadly epizootics in their various insect hosts. The most well-known among these is the “zombie fly” fungus E. muscae, which, like other Entomophthora species, elicits a series of dramatic behaviors in infected hosts to promote optimal spore dispersal. Despite having been first described more than 160 years ago, there are still many open questions about Entomophthora biology, including the molecular underpinnings of host behavior manipulation and host specificity. This review provides a comprehensive overview of our current understanding of the biology of Entomophthora fungi and enumerates the most pressing outstanding questions that should be addressed in the field. We briefly review the discovery of Entomophthora and provide a summary of the 21 recognized Entomophthora species, including their type hosts, methods of transmission (ejection of spores after or before host death), and for which molecular data are available. Further, we argue that this genus is globally distributed, based on a compilation of Entomophthora records in the literature and in online naturalist databases, and likely to contain additional species. Evidence for strain-level specificity of hosts is summarized and directly compared to phylogenies of Entomophthora and the class Insecta. A detailed description of Entomophthora’s life-cycle and observed manipulated behaviors is provided and used to summarize a consensus for ideal growth conditions. We discuss evidence for Entomophthora’s adaptation to growth exclusively inside insects, such as producing wall-less hyphal bodies and a unique set of subtilisin-like proteases to penetrate the insect cuticle. However, we are only starting to understand the functions of unusual molecular and genomic characteristics, such as having large > 1 Gb genomes full of repetitive elements and potential functional diploidy. We argue that the high host-specificity and obligate life-style of most Entomophthora species provides ample scope for having been shaped by close coevolution with insects despite the current general lack of such evidence. Finally, we propose six major directions for future Entomophthora research and in doing so hope to provide a foundation for future studies of these fungi and their interaction with insects

Yeast drives genome-wide difference in gut gene expression.

<p>A) Average linkage hierarchical clustering was performed in Gene Cluster 3.0 across all genes that are expressed at least at two FPKM in at least two out of 11 samples. Bacteria mono-association data has been averaged across each treatment to collapse down into a single column. FPKM values for each gene are normalized to range from -1 to 1 before plotting. Abbreviations: Ap avg = average for <i>A</i>. <i>pasteurianus</i>-mono-associated samples; Lbrev avg = average for <i>L</i>. <i>brevis</i>-mono-associated samples, Lp avg = average for <i>L</i>. <i>plantarum</i>-mono-associated samples, 3bac = poly-associated (without yeast), Ax = axenic, Conv = conventional, Yeast = <i>S</i>. <i>cerevisiae</i>-mono-associated, 4mic = poly-associated (with yeast). Scale bar is shown at bottom right. B) Top) heatmap of 579 genes that are overexpressed in axenic, bacteria-mono-associated and poly-associated (without yeast) guts compared to other gut samples (Bonferroni p-value>0.05, ANOVA). Bottom) Results from Panther GO-SLIM biological function enrichment test [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167357#pone.0167357.ref026" target="_blank">26</a>] for gene set above compared to reference set of all genes observed across all gut datasets (556 were identified by Panther and used for analysis out of 579) C) Top) Heatmap of 1728 genes that are overexpressed in conventional, yeast mono-associated and poly-associated (with yeast) compared to other gut samples (Bonferroni p-value>0.05, ANOVA). Results from Panther GO-Slim biological processes enrichment test with 1728 (1663 identified) genes compared to reference set of all genes observed across all gut datasets. Note for B) and C): all individual sample values were used for ANOVA analysis, not the average value as plotted in A).</p

Stable Host Gene Expression in the Gut of Adult <i>Drosophila melanogaster</i> with Different Bacterial Mono-Associations

<div><p>There is growing evidence that the microbes found in the digestive tracts of animals influence host biology, but we still do not understand how they accomplish this. Here, we evaluated how different microbial species commonly associated with laboratory-reared <i>Drosophila melanogaster</i> impact host biology at the level of gene expression in the dissected adult gut and in the entire adult organism. We observed that guts from animals associated from the embryonic stage with either zero, one or three bacterial species demonstrated indistinguishable transcriptional profiles. Additionally, we found that the gut transcriptional profiles of animals reared in the presence of the yeast <i>Saccharomyces cerevisiae</i> alone or in combination with bacteria could recapitulate those of conventionally-reared animals. In contrast, we found whole body transcriptional profiles of conventionally-reared animals were distinct from all of the treatments tested. Our data suggest that adult flies are insensitive to the ingestion of the bacteria found in their gut, but that prior to adulthood, different microbes impact the host in ways that lead to global transcriptional differences observable across the whole adult body.</p></div

Analysis of gene expression trends from gnotobiotic whole flies.

<p>A) Transcriptome-wide heatmap from axenic, conventional, yeast-mono-associated, bacteria-mono-associated and poly-associated whole flies clustered by gene expression. Average linkage hierarchical clustering using an uncentered correlation similarity metric was performed in Gene Cluster 3.0 across all genes that are expressed at least at two FPKM across two out of eleven samples. Abbreviations: Ap = <i>A</i>. <i>pasteurianus</i>-mono-associated; Lbrev = <i>L</i>. <i>brevis</i>-mono-associated, Lp = <i>L</i>. <i>plantarum</i>-mono-associated, 3bac = poly-associated without yeast, Ax = axenic, Conv = conventional, Yeast = <i>S</i>. <i>cerevisiae</i>-mono-associated, 4mic = poly-associated with yeast. Scale bar is shown at bottom right. B) Top) heatmap of 1159 of 1385 genes that are overexpressed in conventional whole flies compared to other whole fly samples (Bonferroni p-value>0.05, ANOVA). Genes absent in heatmap did not pass filtering criteria. Bottom) Results from Panther GO-SLIM biological function enrichment test [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167357#pone.0167357.ref026" target="_blank">26</a>] for gene set above (1278 genes were identified of 1385) compared to reference set of all genes observed across all whole fly datasets. C) Top) Heatmap 351 that are overexpressed in all non-conventional whole-fly samples compared to conventional whole flies (Bonferroni p-value>0.05, ANOVA). Results from Panther GO-Slim biological processes enrichment test with gene set above (348 genes were identified out of 351) compared to reference set of all genes observed across all whole fly datasets.</p