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Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. In this manuscript, we present a genome for a laboratory-tractable Entomophthora muscae isolate that infects fruit flies. Our E. muscae assembly is 1.03 Gb, consists of 7810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with recent datasets from entomophthoralean fungi, we show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity. In addition, we find that E. muscae and its closest allies possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae's species-specific, biotrophic lifestyle. Finally, we offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex that support the need for taxonomic revision within this group. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.

Many entomopathogenic fungi cause infections that kill their insect host. Little is understood about changes in the reproductive investment that occurs during an infection by a lethal disease over the waning life of an insect. Life-history theory suggests the host will respond by investing resources into fighting the disease or increasing reproduction. Here, we investigate how the reproductive life of adult house flies, Musca domestica, is impacted by its host-specific fungal pathogen, Entomophthora muscae. Specifically, we test how the week-long infection alters the mating behavior of virgin adult male house flies. We find that the pathogen significantly decreases male libido, an effect which grows stronger over the course of the infection. Furthermore, females were significantly less likely to choose an infected male, reducing male mating success. Additionally, we assessed sperm viability to understand the reproductive costs for monandrous females to mate with infected males. Analyses revealed that sperm quality decreases as early as 3 days post-infection. These results show that E. muscae, which can have a prevalence near 100% in wild populations, causes severe lifetime reproductive costs to male house flies. Understanding how host–pathogen interactions affect host life history is crucial for elucidating all the negative effects pathogen virulence exerts on hosts., Many entomopathogenic fungi cause infections that kill their insect host. Little is understood about changes in the reproductive investment that occurs during an infection by a lethal disease over the waning life of an insect. Life-history theory suggests the host will respond by investing resources into fighting the disease or increasing reproduction. Here, we investigate how the reproductive life of adult house flies, Musca domestica, is impacted by its host-specific fungal pathogen, Entomophthora muscae. Specifically, we test how the week-long infection alters the mating behavior of virgin adult male house flies. We find that the pathogen significantly decreases male libido, an effect which grows stronger over the course of the infection. Furthermore, females were significantly less likely to choose an infected male, reducing male mating success. Additionally, we assessed sperm viability to understand the reproductive costs for monandrous females to mate with infected males. Analyses revealed that sperm quality decreases as early as 3 days post-infection. These results show that E. muscae, which can have a prevalence near 100% in wild populations, causes severe lifetime reproductive costs to male house flies. Understanding how host–pathogen interactions affect host life history is crucial for elucidating all the negative effects pathogen virulence exerts on hosts.

Dead sporulating female fly cadavers infected by the house fly-pathogenic fungus Entomophthora muscae are attractive to healthy male flies, which by their physical inspection may mechanically trigger spore release and by their movement create whirlwind airflows that covers them in infectious conidia. The fungal artillery of E. muscae protrudes outward from the fly cadaver, and consists of a plethora of micrometric stalks that each uses a liquid-based turgor pressure build-up to eject a jet of protoplasm and the initially attached spore. The biophysical processes that regulate the release and range of spores, however, are unknown. To study the physics of ejection, we design a biomimetic 'soft cannon' that consists of a millimetric elastomeric barrel filled with fluid and plugged with a projectile. We precisely control the maximum pressure leading up to the ejection, and study the cannon efficiency as a function of its geometry and wall elasticity. In particular, we predict that ejection velocity decreases with spore size. The calculated flight trajectories under aerodynamic drag predict that the minimum spore size required to traverse a quiescent layer of a few millimetres around the fly cadaver is approximately 10 µm. This corroborates with the natural size of E. muscae conidia (approx. 27 µm) being large enough to traverse the boundary layer but small enough (less than 40 µm) to be lifted by air currents. Based on this understanding, we show how the fungal spores are able to reach a new host.

Dead sporulating female fly cadavers infected by the house fly-pathogenic fungus Entomophthora muscae are attractive to healthy male flies, which by their physical inspection may mechanically trigger spore release and by their movement create whirlwind airflows that covers them in infectious conidia. The fungal artillery of E. muscae protrudes outward from the fly cadaver, and consists of a plethora of micrometric stalks that each uses a liquid-based turgor pressure build-up to eject a jet of protoplasm and the initially attached spore. The biophysical processes that regulate the release and range of spores, however, are unknown. To study the physics of ejection, we design a biomimetic ‘soft cannon’ that consists of a millimetric elastomeric barrel filled with fluid and plugged with a projectile. We precisely control the maximum pressure leading up to the ejection, and study the cannon efficiency as a function of its geometry and wall elasticity. In particular, we predict that ejection velocity decreases with spore size. The calculated flight trajectories under aerodynamic drag predict that the minimum spore size required to traverse a quiescent layer of a few millimetres around the fly cadaver is approximately 10 µm. This corroborates with the natural size of E. muscae conidia (approx. 27 µm) being large enough to traverse the boundary layer but small enough (less than 40 µm) to be lifted by air currents. Based on this understanding, we show how the fungal spores are able to reach a new host.

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 chim

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 chim

En ny trussel imod frugt og bær producenter i Europa, fluen Drosophila suzukii (Diptera), kræver udviklingen af effektive bekæmpelsesmetoder. Dette speciale har undersøgt potentialet af den insektpatogene specialist Entomophthora muscae, som en potential naturlig fjende og derfor biologisk kontrol agent af den invaderende flue. Dette er blevet undersøgt via mortalitetsstudier i tre værter: Den primære vært Musca domestica og de alternative værter Drosophila melanogaster og D. suzukii. Ydermere er der blevet udført studier af de kompositoriske forskelle mellem sunde og inficerede fluers kutikulære kulbrinter gennem GC- MS-analyse. Endeligt er der blevet udført adfærdsforsøg i forhold til D. melanogaster hanners præference mellem raske og inficerede hunner. Der blev fundet en signifikant effekt af E. muscae i alle de eksponerede værtsarter. Entomophthora muscae udviklede sig forskelligt mellem værterne, da D. melanogaster fluerne generelt døde en dag før M. domestica fluerne. Flere stoffer blev kun fundet i inficerede fluer på tværs af arterne M. domestica og D. melanogaster. Disse stoffer inkluderede octadecyl acetate og flere syre-estere. Adfærdsforsøgene viste at hannerne havde en præference for inficerede hunner. Det konkluderes at E. muscae er en potentiel naturlig fjende af D. suzukii og at fundet af flere stoffer, unikke for de inficerede fluer, tilskynder yderligere studier af stoffernes rolle i de kemiske interaktioner og mulige værts-manipulerende egenskaber involveret i infektioner med E. muscae. Kemiske stoffer der virker tiltrækkende på fluer, vil potentielt kunne anvendes i feromonfælder til bekæmpelsen af D. suzukii. Nøgleord: Biologisk kontrol, vært-parasit interaktioner, Drosophila suzukii, Entomophthora muscae, kutikulære kulbrinter, patogen værtskifter, adfærdsmanipulation., An emerging threat against fruit and berry production systems throughout Europe, the fly Drosophila suzukii (Diptera), requires the development of effective management tools. This thesis has investigated the potential of the specialist entomopathogenic fungus Entomophthora muscae (Entomophthorales) as a natural enemy and therefore possible biological control agent of the invading fly. This has been studied via mortality experiments in three hosts: The main host Musca domestica and the alternative hosts Drosophila melanogaster and D. suzukii. Studies of the compositional differences between healthy and infected flies’ cuticular hydro carbons (CHs) have been conducted via GC-MS-analysis. Finally, behavioral studies have been conducted of the mating preference of male D. melanogaster being presented with infected vs. healthy females. A significant effect of E. muscae was found in all exposed host species. The ontogeny of the disease varied between the hosts as D. melanogaster flies generally died one day earlier than M. domestica flies. Several compounds only found in infected flies were found in both M. domestica and D. melanogaster flies. These compounds include octadecyl acetate and several acid-esters. The mating assays in D. melanogaster showed a male preference for infected females. It is concluded that E. muscae is a possible natural enemy of D. suzukii and that the findings of several compounds only found in the infected flies, encourages further studies in their role in the chemical interactions and possible host-manipulating traits of the infection by E. muscae. Chemical attractants involved in the infection would be highly relevant in the development of attract-and-kill bait systems to combat D. suzukii. Key words: Biological control, host-parasite interactions, Drosophila suzukii, Entomophthora muscae, cuticular hydro carbons, pathogenic host-shifts, behavioral manipulation.

En ny trussel imod frugt og bær producenter i Europa, fluen Drosophila suzukii (Diptera), kræver udviklingen af effektive bekæmpelsesmetoder. Dette speciale har undersøgt potentialet af den insektpatogene specialist Entomophthora muscae, som en potential naturlig fjende og derfor biologisk kontrol agent af den invaderende flue. Dette er blevet undersøgt via mortalitetsstudier i tre værter: Den primære vært Musca domestica og de alternative værter Drosophila melanogaster og D. suzukii. Ydermere er der blevet udført studier af de kompositoriske forskelle mellem sunde og inficerede fluers kutikulære kulbrinter gennem GC- MS-analyse. Endeligt er der blevet udført adfærdsforsøg i forhold til D. melanogaster hanners præference mellem raske og inficerede hunner. Der blev fundet en signifikant effekt af E. muscae i alle de eksponerede værtsarter. Entomophthora muscae udviklede sig forskelligt mellem værterne, da D. melanogaster fluerne generelt døde en dag før M. domestica fluerne. Flere stoffer blev kun fundet i inficerede fluer på tværs af arterne M. domestica og D. melanogaster. Disse stoffer inkluderede octadecyl acetate og flere syre-estere. Adfærdsforsøgene viste at hannerne havde en præference for inficerede hunner. Det konkluderes at E. muscae er en potentiel naturlig fjende af D. suzukii og at fundet af flere stoffer, unikke for de inficerede fluer, tilskynder yderligere studier af stoffernes rolle i de kemiske interaktioner og mulige værts-manipulerende egenskaber involveret i infektioner med E. muscae. Kemiske stoffer der virker tiltrækkende på fluer, vil potentielt kunne anvendes i feromonfælder til bekæmpelsen af D. suzukii. Nøgleord: Biologisk kontrol, vært-parasit interaktioner, Drosophila suzukii, Entomophthora muscae, kutikulære kulbrinter, patogen værtskifter, adfærdsmanipulation., An emerging threat against fruit and berry production systems throughout Europe, the fly Drosophila suzukii (Diptera), requires the development of effective management tools. This thesis has investigated the potential of the specialist entomopathogenic fungus Entomophthora muscae (Entomophthorales) as a natural enemy and therefore possible biological control agent of the invading fly. This has been studied via mortality experiments in three hosts: The main host Musca domestica and the alternative hosts Drosophila melanogaster and D. suzukii. Studies of the compositional differences between healthy and infected flies’ cuticular hydro carbons (CHs) have been conducted via GC-MS-analysis. Finally, behavioral studies have been conducted of the mating preference of male D. melanogaster being presented with infected vs. healthy females. A significant effect of E. muscae was found in all exposed host species. The ontogeny of the disease varied between the hosts as D. melanogaster flies generally died one day earlier than M. domestica flies. Several compounds only found in infected flies were found in both M. domestica and D. melanogaster flies. These compounds include octadecyl acetate and several acid-esters. The mating assays in D. melanogaster showed a male preference for infected females. It is concluded that E. muscae is a possible natural enemy of D. suzukii and that the findings of several compounds only found in the infected flies, encourages further studies in their role in the chemical interactions and possible host-manipulating traits of the infection by E. muscae. Chemical attractants involved in the infection would be highly relevant in the development of attract-and-kill bait systems to combat D. suzukii. Key words: Biological control, host-parasite interactions, Drosophila suzukii, Entomophthora muscae, cuticular hydro carbons, pathogenic host-shifts, behavioral manipulation.

Three species of Entomophthorales were found on adult carrot flies (Psila rosae F.) during two successive seasons:Entomophthora muscae (C.) Fres., Condiobolus apiculatus (Thax.) Remaud. & Keller and Erynia sp. E. muscae was by far the most common species and caused epizootics in one carrot fly population up to 3 times per year. Flies cuaght in the hedge showed a higher infection level than flies caught in the field close to the hedge. Flies caught 200 m away from the hedge showed the lowest infection level. Apparently the hedge was the site of infection, since carrot flies, killed by E. muscae, were found there attached to the underside of the leaves. Flies caught on yellow sticky traps developed only to a limited extend symptoms and gave little information about the fungus infection levels.

Entomophthora muscae (C.) Fres. can be grown in vitro as protoplasts. Light and electron microscopical studies of the in vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape of E. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.

Entomophthora muscae (C.) Fres. can be grown in vitro as protoplasts. Light and electron microscopical studies of the in vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape of E. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.

Three species of Entomophthorales were found on adult carrot flies (Psila rosae F.) during two successive seasons:Entomophthora muscae (C.) Fres., Condiobolus apiculatus (Thax.) Remaud. & Keller and Erynia sp. E. muscae was by far the most common species and caused epizootics in one carrot fly population up to 3 times per year. Flies cuaght in the hedge showed a higher infection level than flies caught in the field close to the hedge. Flies caught 200 m away from the hedge showed the lowest infection level. Apparently the hedge was the site of infection, since carrot flies, killed by E. muscae, were found there attached to the underside of the leaves. Flies caught on yellow sticky traps developed only to a limited extend symptoms and gave little information about the fungus infection levels.

A method for maintaining an in vivo culture of Entomophthora muscae (C) Fres. on its original host, adult carrot flies (Psila rosae F.), is described. The lethal time for adult carrot flies was greatly influenced by temperature, both for infected and for uninfected flies. In the range 8.2°C-20.2°C the LT50 for infected flies was about 5.4 times shorter than the estimated average life-span for uninfected flies. The discharge of primary spores was also strongly dependent on temperature. The total number of primary spores discharged per fly at 100% RH and in darkness ranged between 1.2×104 and 9.6×104 with a mean of 5.1×104.

Female carrot flies (Psila rosae F.) infected with Entomophthora muscae (C.) Fres. on days 1 to 4 of adult life were unable to lay their eggs near the food plant. Flies infected on day 5 of adult life showed an egg‐laying behaviour similar to that of uninfected flies. E. muscae‐infected female carrot flies caught in the field showed an egg‐laying behaviour comparable to that of the flies infected in the laboratory during the first day of their live. Eggs laid by infected females were equally as fertile as eggs laid by uninfected females, and developed into normal flies. It is concluded that female carrot flies infected with E. muscae in the field do not in general contribute to the development of the carrot fly population, due to their abnormal egg‐laying behaviour. Abnormales Eiablageverhalten echter Môhrenfliegen (Psila rosae) verursacht von dem Pilz Entomophthora muscae Wenn Weibchen der echten Môhrenfliegen (Psila rosae) zwischen den ersten und der vierten Tag in dem Laboratorium mit E. muscae infiziert worden waren, kônnten sie ihre Eier an den Futterpflanze nicht ablegen. E. muscae‐infizierte Weibchen, die ins Feld gefangen worden waren, zeigten auch ein abweichendes Eiablageverhalten. Ihr Behalten war mit dem Verhalten von Fliegen vergleichbar, die an ihrem ersten Lebenstag im Laboratorium infiziert worden waren. Von infizierten Weibchen gelegte Eier waren ebenso fruchtbar wie die von nicht infizierten Weibchen gelegten Eiern. Es wird konkludiert, dass E. muscae‐infizierten Weibchen der echten Môhrenfliegen der Entwicklung der Population keinen Beitrag leisten. 1987 The Netherlands Entomological Society

Three species of Entomophthorales were found on adult carrot flies (Psila rosae F.) during two successive seasons:Entomophthora muscae (C.) Fres., Condiobolus apiculatus (Thax.) Remaud. & Keller and Erynia sp. E. muscae was by far the most common species and caused epizootics in one carrot fly population up to 3 times per year. Flies cuaght in the hedge showed a higher infection level than flies caught in the field close to the hedge. Flies caught 200 m away from the hedge showed the lowest infection level. Apparently the hedge was the site of infection, since carrot flies, killed by E. muscae, were found there attached to the underside of the leaves. Flies caught on yellow sticky traps developed only to a limited extend symptoms and gave little information about the fungus infection levels.

Entomophthora muscae (C.) Fres. can be grown in vitro as protoplasts. Light and electron microscopical studies of the in vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape of E. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.

Female carrot flies (Psila rosae F.) infected with Entomophthora muscae (C.) Fres. on days 1 to 4 of adult life were unable to lay their eggs near the food plant. Flies infected on day 5 of adult life showed an egg‐laying behaviour similar to that of uninfected flies. E. muscae‐infected female carrot flies caught in the field showed an egg‐laying behaviour comparable to that of the flies infected in the laboratory during the first day of their live. Eggs laid by infected females were equally as fertile as eggs laid by uninfected females, and developed into normal flies. It is concluded that female carrot flies infected with E. muscae in the field do not in general contribute to the development of the carrot fly population, due to their abnormal egg‐laying behaviour. Abnormales Eiablageverhalten echter Môhrenfliegen (Psila rosae) verursacht von dem Pilz Entomophthora muscae Wenn Weibchen der echten Môhrenfliegen (Psila rosae) zwischen den ersten und der vierten Tag in dem Laboratorium mit E. muscae infiziert worden waren, kônnten sie ihre Eier an den Futterpflanze nicht ablegen. E. muscae‐infizierte Weibchen, die ins Feld gefangen worden waren, zeigten auch ein abweichendes Eiablageverhalten. Ihr Behalten war mit dem Verhalten von Fliegen vergleichbar, die an ihrem ersten Lebenstag im Laboratorium infiziert worden waren. Von infizierten Weibchen gelegte Eier waren ebenso fruchtbar wie die von nicht infizierten Weibchen gelegten Eiern. Es wird konkludiert, dass E. muscae‐infizierten Weibchen der echten Môhrenfliegen der Entwicklung der Population keinen Beitrag leisten. 1987 The Netherlands Entomological Society

A method for maintaining an in vivo culture of Entomophthora muscae (C) Fres. on its original host, adult carrot flies (Psila rosae F.), is described. The lethal time for adult carrot flies was greatly influenced by temperature, both for infected and for uninfected flies. In the range 8.2°C-20.2°C the LT50 for infected flies was about 5.4 times shorter than the estimated average life-span for uninfected flies. The discharge of primary spores was also strongly dependent on temperature. The total number of primary spores discharged per fly at 100% RH and in darkness ranged between 1.2×104 and 9.6×104 with a mean of 5.1×104.