Evolutionary patterns of proteinase activity in attine ant fungus gardens

Abstract

BackgroundAttine 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.ResultsWe 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.ConclusionsProteinase 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.

Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

“…4A). This is consistent with studies showing that both hemicellulases and proteases are produced by this organism (20,50,51) and that activity against these substrates can be consistently detected in whole fungus gardens (52). Moreover, culture-based work has shown that L. gongylophorus can produce enzymes active against a variety of polysaccharides when grown in pure culture (53,54).…”

Ant-fungal species combinations engineer physiological activity of fungus gardens

“…Protease activity was measured using the Azocasein method (Charney and Tomarelli, 1947) used previously in studies on attine protease activity (Semenova et al, 2011). This method uses azocasein (azo-labeled casein; Sigma-Aldrich).…”

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

“…In the 1970s, leaf-cutting ant fecal fluid was shown to contain active proteases (Martin, 1970(Martin, , 1974Martin, 1970a, b, 1971) with similar chemical properties as enzymes originating from the fungal symbiont (Boyd and Martin, 1975a, b), and recent work has shown that fungus garden endo-protease activity in evolutionarily derived leaf-cutting ants is much higher than in sister ant lineages that do not use fresh leaves to make their gardens grow (De Fine Licht et al, 2010), activity that could subsequently be assigned to metalloproteases and serine proteases (Semenova et al, 2011).…”