Background: Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant
material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific
mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase
activity may have been under selection for efficiency and that different classes of proteinases might be involved.
Results: We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian
species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently
obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts
peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher
proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values
around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH
optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more
derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being
remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific
level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the
ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the
higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the
production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group
of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent
changes in enzyme production may have occurred throughout the domestication history of fungus-garden
symbionts.
Conclusions: Proteinase pH optima and buffering capacities of fungal symbionts appear to have evolved
remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine
and metalloproteinases in fungus gardens are unknown, the differential production of these classes of proteolytic
enzymes suggest that substrate specificity may be important and that trade-offs may prevent the simultaneous
upregulation of both classes of enzymes.