An Evo­lu­tion­ary Tale of Zom­bie Ants...

...and Fun­gal Vil­lains & Knights

by Gemma Reguera

In a re­cent post I shared with you some amaz­ing things I had learnt about co­prophilous ('dung-lov­ing') fungi that spit their spores like pros. What I did not tell you then is that my six-year-old son also fell in love with the spit­ting fungi (dung + spit = child's in­ter­est!) and wanted to learn more. So we spent hours watch­ing on­line videos un­til we stum­bled upon a BBC's Planet Earth video nar­rated by the great David At­ten­bor­ough about ant par­a­sitic fungi in the genus Cordy­ceps. The video shows a car­pen­ter ant (genus Cam­pono­tus) that has been in­fected by spores of the fun­gus Ophio­cordy­ceps uni­lat­er­alis. The spores ger­mi­nate in­side the ant's res­pi­ra­tory track and the mycelia grow to­wards the brain while feed­ing on soft tis­sues. Once the fun­gus reaches the brain, it in­duces be­hav­ioral changes such that the ant climbs up veg­e­ta­tion and bites the un­der­side of the leaves. There the ant awaits its death while the fun­gus con­tin­ues to grow within. The stroma stalk of the fun­gus even­tu­ally pro­trudes from the back of the ant's head and a fruit­ing body bear­ing a cap­sule filled with spores forms near its tip (Fig. 1). Once the spores are sex­u­ally ma­ture, the cap­sule is re­leased, and then ex­plodes, ei­ther in the air or upon hit­ting the ground. This de­liv­ers the spores into the path of healthy ants, there to start a new cy­cle of in­fec­tion.

Fig­ure 1. An ant in­fected by O. uni­lat­er­alis bites the un­der­side of a leaf. The fun­gal stroma emerges from the back of the ant's head and de­vel­ops a fruit­ing body with a cap­sule full of spores. Once ma­tured, the cap­sule is re­leased and the spores dis­perse on the for­est floor. Cour­tesy of David P. Hughes. Source

Right away, I searched the fun­gus name in the STC blog and found a beau­ti­ful post by Il­ham Naili that de­scribes the life cy­cle of this par­a­sitic fun­gus in more de­tail. I also started my own search and learned that the fun­gal spores can at­tach to the ant's body and se­crete en­zymes to pen­e­trate through the cu­ti­cle. These are in­deed some ag­gres­sive par­a­sites ca­pa­ble of a gen­er­al­ized body in­va­sion. In fact, some pic­tures of dead ants show the fruit­ing body pro­trud­ing through their ab­domen, rather than the back of the head. But spores also en­ter the ant though its res­pi­ra­tory spir­a­cles (aka the ex­oskele­ton open­ings that al­low the air to en­ter the tra­chea). This ap­pears to be the most ef­fi­cient route be­cause the spore can use the nu­tri­ents pro­vided by non-es­sen­tial soft tis­sues within the res­pi­ra­tory track to grow its mycelia in close prox­im­ity to the ant's brain. There, bioac­tive com­pounds se­creted by the mycelia en­ter the ant's brain and ef­fec­tively block pheromone re­cep­tion. This dis­ori­ents the ant an­dleaves it un­der com­plete fun­gal con­trol. Like a zom­bie, the ant climbs up the trees and bites a vein on the un­der­side of a leave with great force. In­ter­est­ingly, the shape of the grip bite mark is very dis­tinc­tive and is sim­i­lar to marks iden­ti­fied in 48 mil­lion year old fos­sil leaves!!!!! This bit­ing be­hav­ior ef­fec­tively im­mo­bi­lizes the dy­ing ant up­side down un­der a leaf, but also low­ers its head (Fig. 1). It ap­pears as if, in do­ing so, the ant sub­mis­sively bows to its des­tiny and ex­poses the soft tis­sue be­tween the head and the trunk to fa­cil­i­tate the pro­tru­sion of the fun­gal stroma stalk.

Fig­ure 2. O. uni­lat­er­alis grow­ing on potato dex­trose agar for 7 days se­cretes six type of naph­thoquinones. The for­mu­las are shown in (a) and the growth of the fun­gus on the agar in (b). Af­ter 12 days of in­cu­ba­tion the naph­thoquinone se­cre­tion is so co­pi­ous that it forms drop­lets on the plate ©. Source

Con­cerned as I was about pre­ma­turely de­stroy­ing my son's love for mi­crobes, I set out to learn more about Cordy­ceps, avidly search­ing for that 'pos­i­tive some­thing' about them that could al­le­vi­ate the bad mem­ory of the brain-eat­ing fun­gus. Cordy­ceps, I learned, is a fun­gal group (in ad­di­tion to a genus) in the As­comy­cota that com­prises hun­dreds of species. All are en­dopar­a­sitic; most par­a­sitize arthro­pods, though a few in­fect other fungi. Amaz­ingly, each fun­gus spe­cial­izes in in­fect­ing a sin­gle type of host. While some can in­fect other hosts, re­pro­duc­tive suc­cess is of­ten com­pro­mised. Not all af­fect the host's be­hav­ior, but those that do are known to pro­duce co­pi­ous amounts of bioac­tive com­pounds. O. uni­lat­er­alis, for ex­am­ple, se­cretes at least six types of sec­ondary metabo­lites broadly known as naph­tho­quinones (Fig. 2). All six have an­tibac­te­r­ial, an­ti­cancer, and an­ti­malar­ial ac­tiv­ity. Well, there goes some­thing pos­i­tive...

The ques­tion that lin­gered in my mind was what mech­a­nism, if any, could pro­tect the ants from the deadly fun­gus. The ants build their nests in the above-ground veg­e­ta­tion. But the par­a­sitic fun­gus con­trols the be­hav­ior of the in­fected ants and forces them to climb up­wards so they die close to the colony's nest. This max­i­mizes the chances of in­fect­ing healthy in­di­vid­u­als. Fur­ther­more, dead ants are of­ten found at high den­si­ties (ap­prox­i­mately 26 per m²) in lo­cal­ized ar­eas of the veg­e­ta­tion termed grave­yards. This helps con­cen­trate the re­leased fun­gal spores in the ants' path on the for­est floor be­neath. Healthy ants are known to ac­tively avoid the zom­bie ant grave­yards and only ven­ture to the for­est floor occasionally—strategies that pre­vent in­fec­tion. This spa­tially lim­its their re­sources but, hey, giv­ing up some food sources seems like a good evo­lu­tion­ary trade off to en­sure the sur­vival of the colony.

Fig­ure 3. A stroma of Ophio­cordy­ceps nu­tans in­fected by Poly­cephalomyces sp. is shown on the left. As a com­parison, a healthy, non­par­a­sitized stroma with its red fruit­ing body is shown on the right. Credit Al­isha Owensby. Source

Just when I thought this was it for the de­fense­less ants, I stum­bled upon a pa­per by David P. Hughes's group pub­lished in PLoS One about a white fun­gus that comes to the ant's res­cue. Who is this mys­te­ri­ous knight? None other than a cousin of the Cordy­ceps! The fun­gal fam­ily Clavicip­i­taceae in­cludes gen­era with sex­ual cy­cles such as the Cordy­ceps as well as asex­ual gen­era such as the Poly­cephalomyces. Cordy­ceps are of­ten par­a­sitized by Poly­cephalomyces spp. This makes the Poly­cephalomyces white knight a hy­per­par­a­site (aka a par­a­site of a par­a­site). Cordy­ceps are es­pe­cially sus­cep­ti­ble to in­fec­tion dur­ing the stroma stage. Once the hy­per­par­a­site in­fects a stroma, it feeds from it and pre­vents the for­ma­tion of its fruit­ing body (Fig. 3). More than half of the dead ants in the grave­yards sur­veyed by the re­searchers were hy­per­par­a­sitized. The brain-eat­ing fun­gus has a slow mat­u­ra­tion process, need­ing ap­prox­i­mately 4 weeks to form the stroma and 4 more weeks to pro­duce sex­u­ally ma­ture fruit­ing bod­ies. This al­lows suf­fi­cient time for the white fungi to in­fect the Cordy­ceps stroma. As a re­sult, the white fungi pre­vent the for­ma­tion of a sex­u­ally ma­ture fruit­ing body and 'cas­trate' the brain-eat­ing fun­gus on the spot.

The lengthy mat­u­ra­tion process of the brain-eat­ing fun­gus also ex­poses the fun­gal struc­tures to en­vi­ron­men­tal in­sults for pro­longed pe­ri­ods of time. In fact, as many as 13% of the fun­gal struc­tures in the dead ants sur­veyed in the study were dam­aged. Fur­ther­more, in 25% of the dead ants, the fun­gus had not com­pleted the mat­u­ra­tion of their fruit­ing bod­ies, pos­si­bly be­cause of in­ad­e­quate tem­per­a­ture and hu­mid­ity. The com­bined ac­tion of the white fun­gus and en­vi­ron­men­tal in­sults ren­dered most (93–94%) of the dead ants ster­ile and pos­ing no risk of in­fec­tion. To make things worse for the Cordy­ceps fun­gus, more than half of the re­main­ing in­fec­tive fruit­ing bod­ies suf­fered from some sort of 'sex­ual dys­func­tion' and could not shoot their spores when tested un­der lab­o­ra­tory con­di­tions. Yet, once ma­tured, the fruit­ing bod­ies were ro­bust; they were im­mune to the white fungi and able to sur­vive in the en­vi­ron­ment for pro­longed pe­ri­ods of time. This ca­pa­bil­ity for long-term sur­vival com­pen­sates, at least par­tially, for the low dis­per­sal po­ten­tial and in­creases the re­pro­duc­tive suc­cess of the par­a­sitic fun­gus.

In the end, the white knight, with a lit­tle help from the en­vi­ron­ment, en­sures that only a few, but very ro­bust, fruit­ing bod­ies are pro­duced over time. This is enough to in­crease the size of the ant grave­yards slowly, but steadily, so sex­u­ally ma­ture spores can be pro­duced at reg­u­lar in­ter­vals for longer pe­ri­ods of time. The re­searchers com­pared this evo­lu­tion­ary trade-off to a phe­nom­e­non called iteropar­ity, a re­pro­duc­tive strat­egy in which an or­gan­ism pro­duces off­spring in suc­ces­sive cy­cles, rather than in a sin­gle event, to en­sure long-term sur­vival. Every zom­bie ant that joins the grave­yard is like a new 'preg­nancy' for the fun­gal vil­lain but also food and shel­ter for the white knight. This along with the ants' pre­ven­tive mea­sures to avoid con­tact with in­fected in­di­vid­u­als and ar­eas of high spore con­cen­tra­tion en­sures a per­fect bal­ance be­tween the sur­vival of the ant colony and the par­a­sitic and hy­per­par­a­sitic fungi, so none goes ex­tinct. And al­though the video of the brain-eat­ing fun­gus should be rated PG-13 for the an­i­mal-lov­ing au­di­ence, I can't help but ad­mire how beau­ti­fully na­ture in­ter­twines the lives of an­i­mals and mi­crobes to keep a har­mo­nious bal­ance. This is in­deed a true tale of co-evo­lu­tion, with zom­bies, vil­lains, and white knights in­cluded. I just need to wait a few more years un­til I can help my son un­der­stand that this is, in­deed, a story with a happy end­ing.

Ref­er­ences

Pon­top­p­i­dan MB, Hi­ma­man W, Hy­wel-Jones NL, Boomsma JJ, & Hughes DP (2009). Grave­yards on the Move: The Spa­tio-Tem­po­ral Dis­tri­b­u­tion of Dead Ophio­cordy­ceps-In­fected Ants. PLoS One, 4 (3) PMID: 19279680

Gemma is as­so­ciate pro­fes­sor in the De­part­ment of Mi­cro­bi­ol­ogy and Mol­e­c­u­lar Ge­net­ics, Michi­gan State Uni­ver­sity and an As­so­ciate Blog­ger at STC.