bioRxiv preprint doi: https://doi.org/10.1101/375105; this version posted December 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying 2 cicada pathogens 3 4 Greg R. Boyce1, Emile Gluck-Thaler2, Jason C. Slot2, Jason E. Stajich3, William J. Davis4, Tim 5 Y. James4, John R. Cooley5, Daniel G. Panaccione1, Jørgen Eilenberg6, Henrik H. De Fine Licht6, 6 Angie M. Macias1, Matthew C. Berger1, Kristen L. Wickert1, Cameron M. Stauder1, Ellie J. 7 Spahr1, Matthew D. Maust1, Amy M. Metheny1, Chris Simon5, Gene Kritsky7, Kathie T. Hodge8, 8 Richard A. Humber8,9, Terry Gullion10, Dylan P. G. Short11, Teiya Kijimoto1, Dan Mozgai12, 9 Nidia Arguedas13, Matt T. Kasson1,* 10 11 1Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia, 12 26506, USA. 13 2Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210, USA. 14 3Department of Microbiology and Plant Pathology and Institute for Integrative Genome Biology, 15 University of California, Riverside, California 92521, USA. 16 4Department of Ecology and Evolution, University of Michigan, Ann Arbor, Michigan 48109, 17 USA. 18 5Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, 19 Connecticut 06269, USA. 20 6Department of Plant and Environmental Science, University of Copenhagen, Denmark. 21 7Department of Biology, Mount St. Joseph University, Cincinnati, Ohio 45233, USA. 22 8Plant Pathology & Plant-Microbe Biology, School of Integrative Plant Science, Cornell 23 University, Ithaca, New York 14853, USA. bioRxiv preprint doi: https://doi.org/10.1101/375105; this version posted December 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Boyce et al. 24 9USDA-ARS-NAA-BioIPM, Ithaca, New York 14853, USA. 25 10Department of Chemistry, West Virginia University, Morgantown, West Virginia, 26506, 26 USA. 27 11Amycel Spawnmate, Royal Oaks, California, 95067, USA. 28 12Cicadamania.com, Sea Bright, New Jersey, 07760 USA. 29 13Cleveland Metroparks, Cleveland, Ohio, USA. 30 31 *Corresponding author. Email: [email protected] 32 33 Abstract 34 Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but select 35 species keep insects alive while sporulating, which enhances dispersal. Transcriptomics and 36 metabolomics studies of entomopathogens with post-mortem dissemination from their 37 parasitized hosts have unraveled infection processes and host responses, yet mechanisms 38 underlying active spore transmission by Entomophthoralean fungi in living insects remain 39 elusive. Here we report the discovery, through metabolomics, of the plant-associated 40 amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, 41 and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with 42 Massospora platypediae or Massospora levispora, which appear to represent a single fungal 43 species. The absence of some fungal enzymes necessary for cathinone and psilocybin 44 biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are 45 consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities 2 bioRxiv preprint doi: https://doi.org/10.1101/375105; this version posted December 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Boyce et al. 46 of these compounds suggest the extended phenotype of Massospora that modifies cicada 47 behavior to maximize dissemination is chemically-induced. 48 49 Introduction 50 Many animal parasites including viruses, horsehair worms, protists, and fungi modulate 51 the behavior of their hosts, and thereby increase disease transmission (Berdoy et al., 2000; 52 Gryganskyi et al., 2017; Hoover et al., 2011; Thomas et al., 2002). Each parasite possesses 53 adaptive traits that maximize dispersal, and often the modification of host behavior is interpreted 54 as a dispersal-enhancing “extended phenotype” of the parasite (Dawkins, 1982; Hughes et al., 55 2013). “Summit disease” (SD) behavior, is an extended phenotype of multiple species of insect- 56 infecting (entomopathogenic) fungi, where parasitized insects ascend and affix to elevated 57 substrates prior to death, which facilitates post-mortem dissemination of spores later emitted 58 from their mummified carcasses (Elya et al., 2018; Gryganskyi et al., 2017; Hughes et al., 59 2016; Roy et al., 2006). A more rarely seen extended phenotype among entomopathogenic fungi 60 is “active host transmission” (AHT) behavior where the fungus maintains or accelerates 61 “normal” host activity during sporulation, enabling rapid and widespread dispersal prior to host 62 death (Hughes et al., 2016; Roy et al., 2006). 63 The Entomophthorales (Zoopagomycota) are among the most important insect- and non- 64 insect arthropod-destroying fungi and include all known species with AHT behavior (Roy et al., 65 2006, Spatafora et al., 2017). Massospora is only one of two known genera where AHT is the 66 only reported form of behavior modification across all known species. Massospora contains 67 more than a dozen obligate, sexually transmissible pathogenic species that infect at least 21 68 species of cicadas (Hemiptera) worldwide (Ciferri et al., 1957; Kobayashi, 1951; Ohbayashi et 69 al., 1999; Soper, 1963; Soper, 1974; Soper, 1981). Infectious asexual spores (conidia) are 3 bioRxiv preprint doi: https://doi.org/10.1101/375105; this version posted December 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Boyce et al. 70 actively disseminated from the posterior end of the infected cicada during mating attempts and 71 flights, which are not diminished despite these conspicuous infections (Cooley et al., 2018; 72 Soper 1963; Soper, 1974; White et al., 1983; Figure 1; Movie 1). Instead, hypersexual 73 behaviors are observed in Massospora-infected cicadas, where male cicadas, in addition to 74 typical mating behavior, also attract copulation attempts from conspecific males (Cooley et al., 75 2018; Murphy and Redden, 2003). Massospora effectively hijacks cicadas, turning them into 76 efficient vectors for conidial transmission (Cooley et al., 2018). 77 Because of their ephemeral nature, obligate lifestyle, and large genome size, 78 Entomophthorales with both SD and AHT behaviors have been grossly underrepresented in in 79 vitro lab investigations as well as in phylogenetic and phylogenomic studies (Gryganskyi et al., 80 2017; Spatafora et al., 2017). However, recent genomic, transcriptomic, and metabolomic 81 approaches have further helped elucidate important aspects of SD-inducing entomopathogens 82 (Arnesen et al., 2018; De Fine Licht et al., 2017; Elya et al., 2018; Grell et al., 2011; 83 Małagocka et al., 2015; Wrońska et al., 2018). Likewise, -omics based investigations of SD- 84 inducing Hypocrealean entomopathogens have helped unravel infection processes and host 85 responses, especially for the “zombie ant fungus” Ophiocordyceps unilateralis (De Bekker et al., 86 2014; De Bekker et al., 2017; Fredericksen et al. 2017). This pathogen is thought to modify 87 host behavior via fungus-derived neurogenic metabolites and enterotoxins, thus providing a 88 chemical basis for this behavioral phenotype (De Bekker et al., 2014; De Bekker et al., 2017). 89 The mechanisms underlying AHT are completely unexplored, largely due to the inability to 90 axenically culture most Entomophthorales and rear colonies of their insect hosts (such as 91 cicadas) under laboratory conditions. Here, we applied global and targeted liquid 92 chromatography–mass spectrometry (LC-MS)-based metabolomics to freshly collected and 4 bioRxiv preprint doi: https://doi.org/10.1101/375105; this version posted December 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Boyce et al. 93 archived parasitized cicadas in order to identify candidate small molecules potentially 94 contributing to a hypersexual behavioral phenotype of three Massospora species (Cooley et al., 95 2018) or more broadly to AHT (Batko and Weiser, 1965; Hughes et al., 2016; Hutchison, 96 1962; Roy et al., 2006). 97 98 Results 99 Morphology of Massospora specimens 100 Morphological studies were conducted to permit species-level identification of collected 101 specimens based on previously published studies (Soper, 1963; Soper, 1974). Conidia (n = 37) 102 and resting spore (n = 13) measurements were acquired from freshly collected Mas. cicadina- 103 infected periodical cicadas (Magicicada spp.) and Mas. platypediae-infected wing-banger annual 104 cicadas (Platypedia putnami), as well as from archived Mas. levispora-infected Okanagana 105 rimosa and Mas. cicadina-infected periodical cicadas (Figure 1, Table 1). Morphology of 106 conidia were studied in 15, 14, and 8 specimens of Mas. cicadina (Mc), Mas. platypediae (Mp), 107 and Mas. levispora (Ml), respectively, while morphology of resting spores was examined in 11, 1 108 and 1 specimens