The Occasional Hymenoptera: Leaf-cutters spread fungus’s own enzyme
Back in 1966 Neal A. Weber, then at Swarthmore, described how fungus growing ants cultivated their crop in the important article “Fungus-growing ants,” 135 Science 587-604 (August 5, 1966) (abstract; article requires registration). The “higher attine ants,” those species of the genera Atta and Acromyrmex (the true leaf-cutters), center their colony on a fungus garden (where the queen lays eggs), made from recently cut leaves brought by workers from a site where workers cut leaves to sizes that permit transportation back to the nest. Once inside other ants mulch the leaves into 1 millimeter sized bits and mash them into pulp with their own saliva and feces. The ants then plant new fungus by placing small amounts of mycelium (the vegetable part of the fungus) on the newly formed substrate. Weber noted that by their constant attention they were able to cultivate the fungus free of contaminants, but when the ants were removed other fungi and other invaders overtake the garden. (We looked at a study showing the ants used an antibiotic cocktail to prevent pathogens from attacking their fungus cultivar.)
In 1994 Ignacio H. Chapela and others from the US Department of Agriculture’s Systematic Botany and Mycology Laboratory and the Section of Neurobiology and Behavior at Cornell published “Evolutionary History of the Symbiosis Between Fungus-Growing Ants and Their Fungi,” 266 Science 1691-94 (December 9, 1994) (abstract; registration required for article) which showed that while the fungus-growing ants were monophyletic, the fungi they cultivated were not. Most were from the basidiomycete family Lepiotaceae. What was surprising, however, was the finding that with respect to the higher attine ants the fungal lineage they cultivated was clonally propagated for at least 23 million years. This means that the symbiosis is remarkably successful.
It is not simply the historical length of this relationship that makes it remarkable. The growth rate of the fungus itself is very high. Neal showed in “The Fungus-culturing Behavior of Ants,” 12 American Zoologist 577-87 (1972) (full article), that 60-65% of the workers concerned themselves with brood care and cultivation. He also noted that the excretions of these care takers are disproportionately large.
It appears that the fecal excretions are key to solving the following problem: How can such rapid growth of fungus occur among the higher attine ants (1) given that they use principally fungus from the clade which are decomposers (from the subfamily Leucocoprinae) which do not ordinarily feed on live plants and (2) given that the small dots of mycelium are placed on the mulch of live leaves which are not as high a source of nitrogen as decomposed vegetation (i.e., that the nitrogen/carbon ratio of live leaves is not as high as compost)?
A number of studies, principally by the Martins of University of Michigan in the 1970s, showed that the feces of the ants contained a number of enzymes that would break down proteins, (see, e.g., Michael M. Martin & Joan Stadler Martin, “Presence of protease activity in rectal fluid of primitive attine ants,” 17 J. Insect Physiol. 1897-1906 (1971) (pdf file); Joan Stadler Martin & Michael M. Martin, “The presence of protease activity in the rectal fluid of attine ants,” 16 J. Insect Physiol. 227-32 (1970)). It was then thought that the fungus itself broke down the cellulose of the plant cell wall. See, e.g., Michael M. Martin, “The Biochemical Basis of the Fungus-Attine Ant Symbiosis: A complex symbiosis is based upon integration of the carbon and nitrogen metabolisms of the two organisms,” 169 Science 16-20 (July 3, 1970) (abstract; registration necessary for full article). In 1975 Michael Martin showed that the fecal fluid of ants also contained the enzymes to degrade pectin, sodium polypectate, xylan, and carboxy-methylcellulose. M.M. Martin, et al., “Activity of fecal fluid of a leaf-cutting ant toward plant-cell wall polysaccharides,” 21 J. Insect Physiol. 1887-92 (1975) (pdf file). Studies of the proteases and the cellulaces, pectinaces and laccases in the fecal fluid of ants showed their similarity to those produced by the fungus itself. This of course suggested that the ants consumed the enzymes with the fungus and the enzymes passed through the body of the ant unharmed to be used as an aid to the decomposition of the live plant material for the benefit of the fungus.
In a paper published on New Year’s Eve: Morten Schiott, et al., “Leaf-cutting ant fungi produce cell wall degrading pectinase complexes reminiscent of phytopathogenic fungi,” 8 BMC Biology 156 (2010) (provisional pdf), Morten Schiott of the University of Copenhagen together with a colleague from there and co-authors from University of Southern Denmark report on their results of using proteomics methods to determine the gene sequences of fecal proteins in Acromyrmex echinatior leaf-cutting ants.
The result was that of the 33 proteins identified in the ant fecal fluid seven were pectinolytic enzymes that originated from the fungal symbiont and which were still active in the fecal droplets produced by the ants. They also showed that these seven enzymes were present only when the ants had access to the fungus. Moreover, by using quantitative polymerase chain reaction analysis the authors were able to show that six of these enzymes were increased (i.e., their expression was upregulated) in the gongylidia of the fungus. Gongylidia are swellings in the hyphae (the filamentous branches of fungi) which constitute nutritional packages eaten by ant larvae and cultivators. Gongylidia are found only on fungus cultivated by the higher attine ants (the leaf-cutters).
The enzymes found by the authors are generally only found in phytopathogens (organisms that parasitize living plants) and are used to break down cell walls. Among fungus this would permit the fungal mycelium access to the cell contents. Since these enzymes are not a primitive feature of the clade of fungus cultivated by the leaf-cutter ants, the authors come to two conclusions about the co-evolution of the leaf-cutters and their fungus cultivar: 1) the production of pectinolytic enzymes by the fungus cultivated by the leaf-cutters is an adaptation convergent with the same evolutionary development in phytopathogens; and 2) the method of consuming the enzymes by the ants without digesting them is also an adaptation selected to benefit the growth of the mycelia planted by the ants on the leaf mulch. The overproduction of the enzymes by the gongylidia allow the ants to spread it on the recently cut leaves to make the nitrogen inside the leaves much more quickly available to the fungus. Thus the gardens are able to grow more quickly than without the use of the enzymes. This must be at least part of the reason why this form of mutualism survived for such an exceedingly long time.