Ophiocordyceps unilateralis

Ophiocordyceps unilateralis, commonly known as zombie-ant fungus, is an insect-pathogenic fungus, discovered by the British naturalist Alfred Russel Wallace in 1859, and currently found predominantly in tropical forest ecosystems. O. unilateralis infects ants of the tribe Camponotini, with the full pathogenesis being characterized by alteration of the behavioral patterns of the infected ant. Infected hosts leave their canopy nests and foraging trails for the forest floor, an area with a temperature and humidity suitable for fungal growth; they then use their mandibles to attach themselves to a major vein on the underside of a leaf, where the host remains after its eventual death. The process, leading up to mortality, takes 4–10 days, and includes a reproductive stage where fruiting bodies grow from the ant's head, rupturing to release the fungus's spores. O. unilateralis is, in turn, also susceptible to fungal infection itself, an occurrence that can limit its impact on ant populations, which has otherwise been known to devastate ant colonies.
Ophiocordyceps unilateralis and related species are known to engage in an active secondary metabolism for, among other reasons, the production of substances active as antibacterial agents that protect the fungus-host ecosystem against further pathogenesis during fungal reproduction. Because of this secondary metabolism, an interest in the species has been taken by natural products chemists, with corresponding discovery of small molecule agents (e.g. of the polyketide family) of potential interest for use as human immunomodulatory, anti-infective, and anticancer agents.
== Systematics[edit] ==
After years of research, the taxonomy of Ophiocordyceps unilateralis is becoming increasingly clear.
=== Cordyceps vs Ophiocordyceps[edit] ===
Throughout history there has been confusion about the distinction between the genera Cordyceps and Ophiocordyceps. There have been many debates about whether the zombie-ant fungus (and other fungi) belonged to one or to the other as Ophiocordyceps was only recently brought forward.
The genus Cordyceps comprises over 400 species, historically classified in the family Clavicipitaceae within the order Hypocreales. The classification was based on different morphological characteristics such as filiform ascospores and cylindrical asci. When Cordyceps were first classified, there was no concrete evidence for the genus Ophiocordyceps. However, in 2007, important new molecular data was tested, and enabled them to reorganize the family Clavicipitaceae. It was found that Clavicipitaceae was in fact three distinct monophyletic families: the Clavicipitaceae, the Cordycipitaceae and the Ophiocordycipitaceae.
The new molecular phylogenetics studies contradicted the older classification and moved all Cordyceps species forming a sister group with Tolypocladium, into Ophiocordycipitaceae. Fungi able to parasitize ants were also included in the transfer, such as Cordyceps unilateralis which was later renamed Ophiocordyceps unilateralis. Following this study, multiple traits such as the production of darkly pigmented, hard to flexible stromata were defined as characteristics of the family Ophiocordycipitaceae.
=== Ophiocordyceps unilateralis sensu lato[edit] ===
The fungus's scientific name is sometimes written as Ophiocordyceps unilateralis sensu lato, which means 'in the broad sense', because the species actually represents a complex of many species within O. unilateralis.
Support for this term has become increasingly important. In 2011, it was hypothesized that the zombie-ant fungus could actually be described as a complex of species which are host-specific, meaning that one O. unilateralis species can only successfully infect and manipulate one host ant species. There is a possibility that this resulted in or reinforced the reproductive isolation of the fungi, leading to its speciation. Following this, a study conducted in Brazil delimited, using morphological comparisons of the ascospores, germination processes, and asexual morphs, four different Ophiocordyceps species. Afterwards, three new species were described in the Brazilian Amazon, six in Thailand, and one in Japan.
More recently in 2018, 15 new O. unilateralis species were described based on classic taxonomic criteria, and macro-morphological data with a deeper focus on ascospore and asexual morphology. The asexual morphologies made it possible to distinguish two different clades mainly composed of species associated with ants which they termed "O. unilateralis core clade" and "O. kniphofioides subclade."
Further analyses were conducted using a set of different traits. Morphological traits were used and included both macro-morphological characters (e.g. typical single stroma arising from the host's dorsal pronotum, the ascoma (perithecia) growing from the stroma) and microscopic traits (e.g. the morphology of the ascospores in terms of size, shape, septation and germination). Moreover, other traits such as the host and the location of the death grip were added to the analyses. The morphological study led to 15 new identified species, with 14 which were distributed in the core clade, and one in the subclade. Moreover, it was found that species in the O. kniphofioides subclade specialise on neotropical ants, whereas species in the core clade specialise on Camponotini species.
Species within the O. unilateralis core clade as described in 2018:
* O. albacongiuae
* O. blakebarnesii
* O. camponoti-atricipis
* O. camponoti-balzani
* O. camponoti-bispinosi
* O. camponoti-chartificis
* O. camponoti-femorati
* O. camponoti-floridani
* O. camponoti-hippocrepidis
* O. camponoti-indiani
* O. camponoti-leonardi
* O. camponoti- melanotic
* O. camponoti-nidulantis
* O. camponoti-novogranadensis
* O. camponoti-renggeri
* O. camponoti-saundersi
* O. halabalaensis
* O. kimflemingiae
* O. naomipierceae
* O. ootakii
* O. polyrhachis-furcata
* O. pulvinata
* O. rami
* O. satoi
Species within the O. kniphofioides subclade as described in 2018:
* O. daceti
* O. kniphofioides
== Morphology ==
=== Typical morphology ===
The zombie-ant fungus is easily identifiable when its reproductive structure becomes apparent on its dead host, usually a carpenter ant. At the end of its life cycle, O. unilateralis typically generates a single, wiry yet pliant, darkly pigmented stroma which arises from the dorsal pronotum region of the ant once it is dead. Moreover, perithecia, the spore-bearing sexual structure, can be observed on the stalk, just below its tip. This complex forms the fungus' fruiting body.
Most species within the O. unilateralis s.l. species complex have both a sexual (teleomorph) and an asexual morph (anamorph). These are different in terms of their function and characteristics. Generally, the asexual morphs identified for Ophiocordyceps are Hirsutella and Hymenostilbe, two genera of asexually reproducing fungi.
=== Morphological variation ===
O. unilateralis species exhibit morphological variations which are most certainly due to their wide geographic range, from Japan to the Americas. Moreover, it has been hypothesized that their morphological variations may also be a result of one fungus species maximizing its infection on one specific host ant species (host-specific infections). Different subspecies of ant can occur within the same area, which means that in order to coexist they have to occupy different ecological niches. Consequently, the fungi may have evolved at the subspecies level in order to maximize its fitness.
==== O. unilateralis core clade morphological characteristics ====
The O. unilateralis core clade, as described in 2018, has distinct morphological characteristics. It exhibits a single stroma with a Hirsutella asexual morph, which arises from the dorsal neck region of the dead ant and produces a dark brown perithecia attached to its stalk. These species are also recognizable through the host species they infect, which are only Camponotini species. Once the host is killed by the fungus, it is commonly found fixed through their mandibles onto the surfaces of leaves.
==== O. kniphofioides subclade morphological characteristics ====
The O. kniphofioides subclade, as described in 2018, also has distinct morphological characteristics. Its species produce a stroma that grows laterally from the host's thorax which itself generates an orange ascoma. Moreover, species within this subclade share a Hirsutella asexual morph. As for the core clade, these species are also recognizable through the hosts they infect, which are usually neotropical ant species. The subclade does not present the same extended phenotype with the famous "death grip" that O. unilateralis species typically exhibit. Their hosts usually die at the base of large trees in the Amazonian rainforest, among the moss carpets.
== Life cycle ==
In tropical forests, the ant species Camponotus leonardi lives in the high canopy and has an extensive network of aerial trails. Sometimes the canopy gaps are too difficult to cross, so the ants' trails descend to the forest floor where they are exposed to O. unilateralis spores. The spores attach to their exoskeletons and eventually break through using mechanical pressure and enzymes. Like other fungi pathogenic to insects in the genus Ophiocordyceps, the fungus targets a specific host species, Camponotus leonardi; despite this, the fungus may parasitize other closely related species of ants with lesser degrees of host manipulation and reproductive success.
Yeast stages of the fungus spread in the ant's body and presumably produce compounds that affect the ant's hemocoel, using the evolutionary trait of an extended phenotype to manipulate the behavioral patterns exhibited by the ant. An infected ant exhibits irregularly timed full-body convulsions that dislodge it from its canopy nest to the forest floor.
The changes in the behavior of the infected ants are very specific, giving rise to the popular term "zombie ants." Behaviors are tuned for the benefit of the fungus in terms of its growth and its transmission, thereby increasing its fitness. The ant climbs up the stem of a plant and uses its mandibles with abnormal force to secure itself to a leaf vein, leaving dumbbell-shaped marks on it. The ants generally clamp to a leaf's vein at a height of 26 cm above the forest floor, on the northern side of the plant, in an environment with 94–95% humidity and temperatures between 20 and 30 °C (68 and 86 °F). Infections may lead to 20 to 30 dead ants per square meter. When the dead ants are moved to other places and positions, further vegetative growth and sporulation either fails to occur or results in undersized and abnormal reproductive structures. In temperate forests, the typical behavior of zombie ants is to attach themselves to the lower side of twigs, not leaves.
A search of plant-fossil databases revealed similar marks on a fossil leaf from the Messel Pit, which is 48 million years old. Once the mandibles of the ant are secured to the leaf vein, atrophy quickly sets in, destroying the sarcomere connections in the muscle fibers and reducing the mitochondria and sarcoplasmic reticular. The ant is no longer able to control the muscles of the mandible and remains fixed in place, hanging upside-down on the leaf. This lockjaw trait is popularly known as the death grip and is essential in the fungus's lifecycle. A study led in Thailand revealed that there is a synchronization of this manipulated biting behavior at solar noon.
The fungus then kills the ant and continues to grow as its hyphae invade more soft tissues and structurally fortify the ant's exoskeleton. More mycelia then sprout out of the ant, securely anchoring it to the plant substrate while secreting antimicrobials to ward off competition. When the fungus is ready to reproduce, its fruiting bodies grow from the ant's head and rupture, releasing the spores. This process takes 4–10 days. Dead ants are found in areas termed "graveyards" which contain high densities of dead ants previously infected by the same fungus.
The term "zombie ants" has been used in popular media as well as scientific articles, but has also been described as "catchy, yet misleading."
== Natural products ==
O. unilateralis' life cycle includes and depends on the infection and the manipulation of a carpenter ant, principally C. leonardi. The behavioral manipulation of the ant, which gives rise to the name "zombie-ant", is an extended phenotype of the fungus. It first affects the ant's behavior through convulsions that make it fall from its high canopy nest onto the forest floor. This is followed by the fungus controlling the climbing of the ant and the locking of its jaw (and subsequent death) onto a leaf around 25 centimetres above the ground, which is thought to be the optimal height for fungal spore growth and dispersion.
Throughout the lifecycle, unique challenges must be met by equally unique metabolic activities. The fungal pathogen must attach securely to the arthropod exoskeleton and penetrate it—avoiding or suppressing host defenses—then, control the behavior of the host before killing it; and finally, it must protect the carcass from microbial and scavenger attack.
The behavioral manipulation of the ant would not be possible without the presence of huge fungal cell populations beside the host's brain and within muscles because these lead to the secretion of various metabolites known to have important behavioral consequences. During the infection the parasite comes across an array of environments such as different host tissues or the immune response. Studies have shown that O. unilateralis reacts heterogeneously by secreting different metabolites according to the host tissue it encounters and whether they are live or dead. The identification of these natural products is important in order to understand which aspects of the ants are under control and consequently how O. unilateralis manipulates the ant.
# Attachment of O. unilateralis spores onto the ant's exoskeleton: The first step O. unilateralis has to overcome to have a successful infection is to attach itself onto the ant's cuticle and then infiltrate it. For this purpose, the fungus' hypha pierces the exoskeleton using enzymes such as chitinase, lipase and protease, combined with mechanical pressure.
# Convulsions and climbing behavior: After the fungus enters the ant, it propagates, and fungal cells are found beside the host's brain. Once the population is of sufficient size, the fungus secretes compounds and takes over the central nervous system (CNS), which enables it to manipulate the ant to reach the forest floor and climb up the vegetation. Two candidate compounds, sphingosine and guanidinobutyric acid (GBA), have been identified as responsible for the manipulation of the host brain. Both compounds are known to be involved in various neurological disorders. However, more research is needed to determine whether other fungal metabolites interact with the host brain to cause higher levels of sphingosine and GBA. Some studies identified another compound, hypoxanthine, present at high extracellular concentrations. Hypoxanthine has deleterious effects on neural tissues of the cerebral cortex, which in the context of zombie ants may indicate a way for the fungus to alter the motor neurons of the ant, consequently affecting its behavior.
# Death grip: The famous "death grip" exhibited by the ant is also a result of fungus-induced manipulation. This behavior consists of an infected ant locking its mandibles onto (i.e. biting) a leaf so tightly that the ant is prevented from falling as it dies hanging upside down, consequently enabling the proper growth of the fungus' fruiting body. This is possibly a result of the atrophy of the ant's mandibular muscles caused by the secretion of fungal compounds. In multiple studies, fungal cell populations were found within atrophied mandibular muscle tissues. These fungal cell populations have been found to be interconnected through anastomosing tubules. Researchers have proposed that these hyperconnected fungal networks may indicate cooperativity between fungal cells to control the actions of the ant's mandibular muscle. Another common hypothesis amongst researchers is that fungal cells infiltrate between the muscle fibers and then secrete chemicals which cause the muscles to atrophy. Significant decreases in leucine concentration and mitochondria number were identified in infected ants. A deficit in leucine results in the prevention of muscle regeneration because the amino acid is a nutrient regulator of muscle protein synthesis. A decrease in mitochondria ultimately results in a reduction of energy and calcium levels due to the lack of ATP and sarcoplasmic reticulum which provides calcium for actin–myosin binding which is essential for muscle cells.
More in-depth research is needed for the identification of other fungal compounds which act to atrophy the mandibular muscles, and for the understanding of their exact effects on the ant.