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A Science Blog & Podcast focused on utilizing publicly available research to expand our understanding of entheogens.

  • Writer's pictureDustin Theibert

Psychoactive alkaloids from cicada-infecting parasitic fungi

Updated: Sep 9, 2021

Boyce et al. report the discovery of three alkaloids (cathinone, psilocybin & psilocin) from a family of parasitic insect fungi (entomopathogenic) of the Massospora genus in cicadas. They confirmed the presence, of traditionally plant-associated amphetamine, cathinone, in four periodical cicada populations infected with Massospora cicadina. In addition to finding the mushroom-associated tryptamines, psilocybin & psilocin, in annual cicadas infected with Massospora platypediae or Massospora levispora. The absence of biological intermediates and enzymes necessary for biosynthesis of these alkaloids likely confirms previously unknown pathways. Entomopathogenic fungi typically kill their hosts before releasing spores, but these Massospora keep cicadas alive forming a fungal “plug” as third of its body is converted into spores. This has undoubtedly lead to some interesting nomenclatures such as zombie cicadas or “flying saltshakers of death.”[1]


Targets, Takeaways, and Data Production

Among periodical cicadas (Magicicada), there are 15 geographic broods, based on the calendar year when they emerge, containing different combinations of 13 year and 17 year distinct species.[2] This study included representatives from six of seven species, spanning 12 broods. A similar number of males and females were sampled from healthy and fungus-infected cicadas. Given the scarcity of available Mas. genetic sequences to compare, morphology comparison was necessary to determine species. This was based on previously reported measurements of spores and spore growth; furthermore can be seen in Figure 1. Interestingly, all the Mas. platypediae measurements fell within the reported range for Mas. levispora.

Fungal DNA was extracted from the fungal plug for the purpose of PCR amplification and sequencing. Three targets genes were validatied against a positive control: PsiD (L-tryptophan decarboxylase), PsiK (4-hydroxytryptamine kinase), and PsiM (norbaeocystin methyltransferase). All of which can be found in the hallucinogenic mushrooms, Psilocybe cubensis and Psilocybe cyanescens [3&4]. Already developed primers were used in an attempt to amplify Psilocybe psilocybin genes.

Phylogenetic trees of the three Mas. species were constructed for further analysis of natural evolutionary relationships was completed on specimens too degraded for other approaches. A similar approach was completed on other entomopathogenic fungi that exhibit “active host transmission” by not killing the host prior to spore dispersion.

Fungal metabolites were extracted from fungi and insects and analyzed through liquid chromatography–mass spectrometry (LC–MS) for metabolite analysis. The data was processed, metabolites identified, and statistical data analysis was completed to check the quality of the samples.


Collections and Compounds

This group investigated 79 unique collections of various species and was able to perform quantification of any present cathinone, psilocin and psilocybin . Cathinone was expressed in three collections of Mas. cicadina: from Brood V in West Virginia, VI in North Carolina, and a third from archived collection spanning four broods (VIII, IX, XXII, and XXIII)[5]. They also looked for cathinone Coenzyme A (CoA) metabolites, yet none were detected. Examples of LC-MS data for cathinone in both sample and reference material can be seen in Figure 2.

The targeted screening also included psilocybin and psilocin. With psilocybin expression in Mas. levispora from annual Okanagana rimosa cicadas in Michigan and psilocin in Platypedia putnami cicadas in New Mexico. One intermediate in the psilocybin biosynthetic pathway in mushrooms, 4-hydroxytryptamine, was identified but fragmentation patterns could not be compared due to the lack of an available analytical standard. Examples of LC-MS data for psilocybin in both sample and reference material can be seen in Figure 3.


Insight into Compound Biosynthesis

After the quantification of alkaloids previously unknown to entomopathogenic fungi and cicadas in general, there was a search for the genes known to play a role in the biosynthesis of psilocybin and cathinone. Initial attempts find these similar genes from known pathways failed. Scarce sequence data for cathinone biosynthesis limited further genetic analysis; however, common psilocybin gene specific sequences have been investigated as previously mentioned. Multiple biosynthesis enzyme sequences for each alkaloid pathway were found in Mas. samples with minor exceptions. There are two novel and notable characteristics: 1) lack of fungal kinase, an enzyme which is normally found in psilocybin biosynthesis; and 2) one enzyme that catalyzes a reaction converting L-phenylalanine early in cathinone biosynthesis. In addition the lack of many pathway metabolites seems to support novel or altered pathways for both compounds; this is depicted in Figures 4 and 5.


Conclusion and Caveats

This was a landmark research moment, as psilocybin has only natively been found in basidiomycota mushrooms, which evolved separately from Massospora for approximately 900 million years. The discovery of these alkaloids with limited genetic evidence of a common biosynthetic pathway raises many questions about enzyme biosynthetic routes. There are several novel characteristics previously mentioned, in addition there is a lack of many pathway metabolites which seems to support novel or altered pathways. However the authors caveat with other evolutionary origins cannot be ruled out until the exact genes underlying this pathway are confirmed.

The activities of these psychoactive compounds and unusual host behaviors suggest cicada motor function is altered to possibly maximize Massospora dissemination. While metabolites, namely cathinone, may improve endurance required to engage in reproductive activities despite infections, it cannot easily explain the display of female courtship behaviors by infected male cicadas.[5] This may be better explained by mediated hormonal changes post infection.

Mas. platypediae and Mas. levispora likely represent a single fungal species. Based on morphological and genetic data but it’s possible they are two related species capable of infecting wing-banger cicadas.

Not all infected populations yielded alkaloids and nor were they detected from every individual. This is not unexpected given the natural variation in alkaloid production. Previously this was noted with ergot alkaloids in individual morning glory seeds across populations.[6]

The mechanisms in active host transmission are largely unexplored, due to the inability to culture entomopathogenic fungi without contamination as well as raise colonies of host insects (such as cicadas) under laboratory conditions. Theoretical lifecycle of infection by Mas. is explored in Figure 6. However the authors anticipate discoveries such as these will bring a renewed interest in fungi and their pharmacologically important secondary metabolites. One which may serve as the next frontier for novel drug discovery.



  1. Sota, T., Yamamoto, S., Cooley, J.R., Hill, K.B., Simon, C., Yoshimura, J., 2013. Independent divergence of 13-and 17-y life cycles among three periodical cicada lineages. Proc. Natl. Acad. Sci. Unit. States Am. 110 (17), 6919e6924.

  2. Fricke, J., Blei, F., Hoffmeister, D., 2017. Enzymatic synthesis of psilocybin. Angew.Chem. Int. Ed. 56 (40), 12352e12355.

  3. Reynolds, H.T., Vijayakumar, V., Gluck-Thaler, E., Korotkin, H.B., Matheny, P.B.,Slot, J.C., 2018. Horizontal gene cluster transfer increased hallucinogenic mushroom diversity. Evol. Lett. 2 (2), 88e101.

  4. Cooley, J.R., Marshall, D.C., Hill, K.B., 2018. A specialized fungal parasite (Massosporacicadina) hijacks the sexual signals of periodical cicadas (Hemiptera: Cicadidae:Magicicada). Sci. Rep. 8 (1), 1432.

  5. Nowak, J., Wozniakiewicz, M., Klepacki, P., Sowa, A., Koscielniak, P., 2016. Identification and determination of ergot alkaloids in Morning Glory cultivars. Anal. Bioanal. Chem. 408 (12), 093e3102.

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