The ecological role of most fungal non-homeostatic secondary-metabolites is still unknown. Unlike their neighboring kingdom, Metazoa, fungi are unable to use movement as a way to avoid threats. Fungi are subject to attack from fungivores and pathogens, including animals and microorganisms (reviewed by Spiteller and Spiteller 2008). They can mediate these interactions with antagonistic compounds, which tend to be more concentrated in their sexual bodies than in their vegetative body. Fungi can produce a wide variety of chemicals that exhibit potent antimicrobial, antifungal, anthelmintic, and/or antifeedant properties. In selected tested animals, these chemicals can be highly poisonous, produce a pungent and displeasing taste, and/or produce hallucinogenic effects. Among these fungal secondary-metabolites is psilocybin and its closely related compounds (e.g. psilocin, baeocystin, norbaeocystin).
Psilocybin and its chemical cousins are alkaloids that closely resemble the structure of serotonin (5-hydroxytryptamine, 5-HT) (Figure 1), which is an important neurotransmitter in metazoa. The serotonin-binding receptor, 5-HT, evolved over 750 million years ago (Peroutka & Howell 1994) in single-celled Eukaryotes and diversified into 5-HT1-7 approximately 650 to 700 million years ago, prior to the split of protostomes and deuterostomes (Ayala et al. 1998). The invertebrate and vertebrate serotonergic receptor classes are shared due to the occurrence of this diversification early in metazoan history (Peroutka and Howell 1994; Hauser et al. 2006). Serotonin can act as a classical neurotransmitter, as a neuromodulator, or as a neurohormone and is responsible for a wide variety of physiological and behavioral processes (Weiger 1997). Examples of serotonin’s regulatory effects in animals are reviewed by __ and include rhythmic contractions in cnidarians (Anctil 1989), reproductive behavior in platyhelminthes (Maule et al. 1990), feeding and learning in molluscs (Frost et al. 1988), swimming and feeding in annelids, aggression in crustaceans, locomotion in jawless fish, and sleep, appetite, and mood in mammals.
Figure 1. Comparison of chemical structures of serotonin with those of serotonin analogs produced by psychedelic mushrooms (ref_). Psilocin is most similar to serotonin, the differences being that psilocin’s amino group is dimethylated and its hydroxyl is on the 4 position. Psilocybin, baeocystin, and norbaeocystin all have phosphate groups; they differ in the level of methylation on their amino groups.
Psilocybin and its relatives are agonists or partial-agonists of several serotonin receptors (Halberstadt and Geyer 2011). Psilocybin, the primary serotonergic compound in psychedelic mushrooms (ref_), is quickly dephosphorylated into psilocin when metabolized (Passie et al 2002). Psilocin has a high affinity for the receptors 5-HT2B and 5-HT2C, a moderately lower affinity for 5-HT2A, and a low affinity for 5-HT1.
Psilocybin and its relatives are found in many distantly related families within the fungal order Agaricales, such as Bolbitiaceae, Inocybaceae, Hymenogastraceae, and Pluteaceae (Stamets 1997; Wurst et al. 2002; Allen 2010; Dinis-Oliveira 2017). Because the phylogenetically distant psilocybin producing fungi have overlapping ecological niches (dung and late wood-decay) and contain the same gene cluster responsible for its production, it has been hypothesized that the ability to produce psilocybin was spread via horizontal gene transfer (Reynolds et al. 2017). Psilocybin’s multiple origins and continued persistence suggest that it is unlikely to simply be a byproduct and likely confers some ecological advantage. Like many other fungal secondary compounds, the original purpose of psilocybin production may have been for defense. Psilocybin and its relatives closely resemble the structures of defense compounds produced by some toads (Spiteller and Spiteller 2008). Assuming the purpose of these compounds is defense, the targets are still unknown.
The physiological effects of psilocybin and its relatives have been studied best in mammalian models, producing strong psychedelic effects (Spiteller and Spiteller 2008). In humans, psilocybin ingestion most commonly causes changes in sensory perception and mood but can less commonly cause hypertension, tachycardia, visual problems, nausea, anxiety, asthenia, vertigo, mydriasis, motor incoordination, disorientation, and hallucinations (Jo et al. 2014). The other serotonergic compounds produced by psychedelic mushrooms (e.g. psilocin, baeocystin, norbaeocystin) exhibit similar properties. Although psilocybin is the alkaloid that typically occurs in the highest quantities within the fungal sexual bodies, physiological effects of mushroom extracts on mammals have been found to be much stronger (even at ⅒th of the concentration) as compared to those of pure synthetic psilocybin, demonstrating that there are strong synergistic interactions amongst psychotropic compounds contained within the sexual bodies (Zhuk et al. 2015).
Fungi with an ability to produce psilocybin typically occupy dung and late wood-decay ecological niches, which overlap with niches of mycophagous and wood-eating insects (Reynolds et al. 2017), making these insects plausible targets. In support of this possible association, the emergence of lignified-tissues approximately 380MYA (Floudas et al. 2012) and large quantities of dung produced by herbivorous megafauna 50-40MYA (MacFadden 2000; Retallack 2001) coincide with the diversifications of fungal lineages known to produce psilocybin (Ramírez-Cruz et al. 2013; Tóth et al. 2013). Psilocybin may have significant impacts on insects due to serotonin’s importance in insect physiology. Serotonin has been shown to influence sleep (Yuan et al. 2005, 2006), memory (Sitaraman et al. 2008, 2012), visual information processing (Thamm et al. 2010), swarming behavior (Anstey et al. 2009), aggression (Dierick and Greenspan 2007), muscular contractions (Evans and Myers 1986), a variety of appetite and digestion processes (Dacks et al. 2003; Neckameyer 2010; French et al. 2014). Psilocybin may act as a neurotransmitter analog and interfere with fungivorous and competitor insects, especially social insects which require coordinated tasks of multiple individuals to function as a colony (Genise 2017). Neurotransmitter analogs have been used by other fungi against insects in different scenarios, such as Cordyceps species modifying insect behavior (de Bekker et al. 2014). Although psilocybin has not been tested on insects, one study found that spiders responded to increasing psilocybin doses by exhibiting a decrease in web thread-length and web-building frequency, with a dose of 6g/kg stopping web-building behavior altogether (Christiansen et al. 1962).
Another plausible target of the serotonergic compounds may be nematodes. A helminth’s muscular system, carbohydrate metabolism, and adenylate cyclase regulation are all controlled by the neurotransmitters acetylcholine and serotonin (Mansour 1979). Ayahuasca, a plant-composed psychedelic brew used by First Peoples of Central and South America, contains many serotonergic psychedelic alkaloids (e.g. N,N-DMT) which exhibit antimicrobial and anthelmintic properties (Levin and York 1978). The serotonergic alkaloids are effective antagonists of the neurotransmitters of the neuromuscular system of helminths and inhibit protozoan parasites. Within helminths, they can kill or paralyze the worms by interfering with crucial biochemical and physiological processes (Rodriguez et al. 1982). The drug d-LSD, another psychedelic serotonergic compound, has been shown to antagonize the stimulant action of serotonin and depress the activity of adenylate cyclase (Mansour 1979). The authors suggest that other hallucinogenic serotonin analogs, may exhibit anthelmintic properties as well. Because nematodes (a roundworm helminth) are known to consume half of the fungal biomass in soil (Spiteller and Spiteller 2008) and to be vulnerable to compounds that interfere with serotonin receptors, it is plausible that psilocybin-producing species contain their unique alkaloids to combat the fungivorous nematodes.
Mushrooms of the genus Psilocybe (Basidiomycota, Hymenogastraceae) are widely distributed around the world and number between 277 and 300 species (Guzmán et al. 1998; Guzmán 2005; Kirk et al. 2008). Species of Psilocybe contain the psychoactive alkaloids psilocybin, psilocin, and baeocystin, among others (Beug and Bigwood 1981; Koike et al. 1981; Ott 1993; Gartz 1994). The levels of these alkaloids between species of psilocybin producing fungi can be highly variable (Beug and Bigwood 1981). While psilocybin is synapomorphic within Psilocybe, psilocybin has multiple origins within Agaricales (Stamets 1997; Guzmán et al. 1998; Kosentka et al. 2013).
Given the current data on the subject, it is likely that Psilocybe cubensis (Earle) Singer, a species which often grow on and near cattle feces (Ramírez-Cruz et al. 2013), produces its unique alkaloids for defense. Despite the multitude of studies investigating the psychopharmacological effects of psilocybin on humans and higher mammals, no studies have investigated the potential ecological role of psilocybin producing mushrooms containing these serotonergic alkaloids on likely natural fungivores or competitors.
This study explores whether the assemblage of serotonergic compounds within psilocybin-producing mushrooms function as a defense against other biotic antagonists. More specifically, does the assemblage of serotonergic compounds within P. cubensis produce antagonistic effects on potential antagonistic groups of organisms? To answer this, extracts of P. cubensis were tested on brine shrimp (Artemia spp. as a representative of Arthropoda), bacteria (Staphylococcus aureus and Escherichia coli as representatives of gram positive and negative bacteria), soil microbes, and nematodes (Caenorhabditis elegans as representative of Nematoda), and were compared to the effects of extracts of non-psilocybin-producing mushrooms.