By Sabino Méndez Pastor
The time is around solar noon. In the Thai rainforest, a worker ant is wandering alone on low vegetation, far away from its dry, hot nest located 3-5 m above the forest floor and from the well-defined trails that lead to it. It is displaying irregularly spaced whole-body convulsions that make it fall from the vegetation onto the ground. Suddenly, the ant climbs a small plant and at the precise height of 25 centimetres, it bites the major vein underneath a leaf on the northern side of the plant. The ant’s mandibular muscles atrophy and it dies, remaining attached to the leaf. During the three weeks that follow, a large stalk with a bulbous capsule at the end grows from the back of the ant’s head. If a closer look is taken around the dead ant, it becomes remarkable that there are many others in the same state, even more than 25/m2, creating a graveyard of Ophiocordyceps infected ants (Hughes et al., 2011).
Ophiocordyceps unilateralis sensu lato (O. unilateralis s.l.) is a fungal parasite that infects ants from the Camponotini tribe and exerts behavioural manipulation to induce the actions described above. The first step of the infection is the attachment of a spore to the cuticle surface of an ant. The spore uses nutrients available on the cuticle to germinate. It produces enzymes (such as endoprotease, lipase and chitinase) to degrade and pass through the cuticle’s multiple layers to penetrate the haemolymph of the insect (Whipps et al., 1989).
The parasite is now a small free-floating cell in the open circulatory system of its host. From there it multiplies and invades all major body regions of the ant by forming hyphae and hyphal bodies. Hyphae are filaments that explore and invade new tissues. Hyphal bodies develop from the end of hyphae and are ‘yeast-like cells that grow inside the insect body and multiply by budding’ (Fredericksen et al., 2017:p.12591). Interestingly, no fungal cells have been observed inside the ants’ brains. Therefore, against the reasonable idea that a behaviour manipulating parasite takes over its host’s brain, O. unilateralis s.l. does not infect it (Fredericksen et al., 2017).
The parasite’s cells grow and multiply between muscle fibres forming hyphal bodies specially in the head and leg regions. There they occupy 10.06% of the tissue volume and cause muscular atrophy as they create large spaces between muscle fibres (Fredericksen et al., 2017). Most of these hyphal bodies are connected by short tubes (~1 µm long) called conidial anastomosis tubes (CATs). In fungi, CATs connect neighbouring cells to allow transfers. This allow O. unilateralis s.l. to create extensive 3D networks of hyphal bodies that surround muscle fibres. For example, Fredericksen et al. (2017) observed in ants infected by the parasite that 59% of the hyphal bodies were connected to at least another one and they could be attached to up to six others. They also discovered that in five of the eight ants sampled, hyphae had penetrated the membrane and entered muscle cells. This, combined with the numerous connections between fungal cells, resulted in 75% of these cells being in direct or indirect contact with muscle fibres.
While hyphae and hyphal bodies develop between muscle fibres, they release an array of compounds that cause the described behavioural manipulation. First, fungal cells secrete bacterial-like enterotoxins which are known to reduce the production of signalling molecules in insects. Enterotoxins down-regulate the ant’s genes encoding for receptors and binding proteins involved in odorant and gustatory perception. This sensorial impairment makes the infected individuals unresponsive to external stimuli. In consequence, ants walk erratically until they find the optimal microenvironment for fungal growth and spore dispersion (de Bekker et al., 2015). Besides this, large amounts of kynurenine formamidase are produced by O. unilateralis s.l. cells. Kynurenine formamidase causes serotonin depletion, which prevents ants from following foraging trails. The parasite also up-regulates the ant’s homologs of clock genes per and cycle. This modifies the ant’s circadian clock to make them switch from a primarily nocturnal behaviour to diurnal activity, which in turn explains why infected ants perform their characteristic behaviour always around solar noon (de Bekker et al., 2015).
In order to manipulate the nervous system, hyphal bodies secrete neuromodulatory agents. These include ergot, indole and tropane alkaloids that act as agonists or antagonists in the central and peripheral nervous systems. Sphingomyelinase changes neuron cell membranes. Polyketides, non-ribosomal peptides, and protein-tyrosine phosphatases induce enhanced locomotion activity which makes the ants move to an elevated position outside the nest before biting the major vein of a leaf and dying. O. unilateralis s.l. also causes the up-regulation of genes encoding for enzymes involved in dopamine metabolism and protein-tyrosine phosphatases. This allows manipulation of locomotion and mandible movement and induces the biting behaviour of the host to a leaf before dying (de Bekker et al., 2015).
Once the ant has bitten a leaf of a small plant, the fungal cells release acid sphingomyelinase which combined with the enterotoxin secretion mentioned before, cause cell death. In addition, the parasite down-regulates the genes coding for collagen and muscle LIM protein during the manipulated biting. Down-regulation of collagen and LIM genes cause muscle fibre detachment from the head capsule and break of the Z-lines. This leads to muscular atrophy, leaving the ant attached to the leaf. During the six following hours, O. unilateralis s.l. induces neural apoptosis and releases hydrolytic enzymes, causing tissue degradation in the host which ultimately kills it (de Bekker et al., 2015). Then, the hyphal bodies located in the ant’s head grow until they form a large stalk that develops from the back of the head with a fruiting body that releases spores. These in turn will infect new hosts, repeating the process and ensuring the reproduction and continuity of Ophiocordyceps unilateralis sensu lato.
References:
de Bekker, C. et al. (2015) Gene expression during zombie ant biting behavior reflects the complexity underlying fungal parasitic behavioral manipulation. BMC Genomics. 16 (1), 620. Available from: doi: 10.1186/s12864-015-1812-x.
Fredericksen, M. A. et al. (2017) ‘Three-dimensional visualization and a deep-learning model reveal complex fungal parasite networks in behaviorally manipulated ants’, Proceedings of the National Academy of Sciences of the United States of America. 114 (47), 12590–12595. Available from: doi: 10.1073/pnas.1711673114.
Hughes, D. P. et al. (2011) ‘Behavioral mechanisms and morphological symptoms of zombie ants dying from fungal infection’, BMC Ecology. 11 (1), 13. Available from: doi: 10.1186/1472-6785-11-13.
Lu J. (2019) How a parasitic fungus turns ants into ‘zombies’. Available from: https://www.nationalgeographic.com/animals/2019/04/cordyceps-zombie-fungus-takes-over-ants/ [Accessed 4th September 2020]
Yong E. (2017) How the Zombie Fungus Takes Over Ants’ Bodies to Control Their Minds. Available from: https://www.theatlantic.com/science/archive/2017/11/how-the-zombie-fungus-takes-over-ants-bodies-to-control-their-minds/545864/ [Accessed 2nd September 2020]
Imperial Bioscience Review 