Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts

Abstract

Leaf-cutting ants combine large-scale herbivory with fungus farming to sustain advanced societies. Their stratified colonies are major evolutionary achievements and serious agricultural pests, but the crucial adaptations that allowed this mutualism to become the prime herbivorous component of neotropical ecosystems has remained elusive. Here we show how coevolutionary adaptation of a specific enzyme in the fungal symbiont has helped leaf-cutting ants overcome plant defensive phenolic compounds. We identify nine putative laccase-coding genes in the fungal genome of Leucocoprinus gongylophorus cultivated by the leaf-cutting ant Acromyrmex echinatior. One of these laccases (LgLcc1) is highly expressed in the specialized hyphal tips (gongylidia) that the ants preferentially eat, and we confirm that these ingested laccase molecules pass through the ant guts and remain active when defecated on the leaf pulp that the ants add to their gardens. This accurate deposition ensures that laccase activity is highest where new leaf material enters the fungus garden, but where fungal mycelium is too sparse to produce extracellular enzymes in sufficient quantities to detoxify phenolic compounds. Phylogenetic analysis of LgLcc1 ortholog sequences from symbiotic and free-living fungi revealed significant positive selection in the ancestral lineage that gave rise to the gongylidia-producing symbionts of leaf-cutting ants and their non-leaf-cutting ant sister group. Our results are consistent with fungal preadaptation and subsequent modification of a particular laccase enzyme for the detoxification of secondary plant compounds during the transition to active herbivory in the ancestor of leaf-cutting ants between 8 and 12 Mya.

Fig. 1.

Diagram illustrating the dynamics of fungal enzyme transfer and substrate processing in leaf-cutting ant fungus gardens, which grow at the top or periphery and are cropped and discarded by the ants at the bottom (19). (A) Ants deposit chewed-up fresh leaf fragments combined with fecal fluid at the top of their garden (green shade). Fecal fluid vectoring of fungal enzymes resolves the problem that low fungal biomass in the top sections of gardens would delay substrate decomposition. (B) Fungal gongylidia (Inset Left) concentrate fungal enzymes to be eaten by the ants, but are most abundant in the central layer of the garden where fungal growth is most vigorous [mean number of gongylidia clusters (staphylae)/cm² ± SE from four A. echinatior colonies]. Leaf-pulp decomposition rate is therefore enhanced when the farming ants transfer fungal enzymes from the middle to the top of a garden by deposition of fecal droplets that vector these gongylidia-produced enzymes.

Fig. 2.

Fungus garden laccase activity in units per milligram of fungus garden (units = unit oxidizing 1 nmol syringaldazine per minute) + SE for representative attine ants from Panama. (A) Simplified fungal phylogeny showing laccase activity in newly built sections of laboratory fungus gardens of 14 ant species representing 8 of the 15 attine ant genera. Dark brown: pterulaceous fungi cultivated by debris-foraging Apterostigma (almost no activity); light brown: lower-attine ants (Myrmicocrypta, Mycocepurus, and Cyphomyrmex) cultivating either clade 1 or clade 2 leucocoprinaceous fungi on a substrate of mostly dead plant material (very low activity); blue: higher attine ants (Trachymyrmex and Sericomyrmex) cultivating fungi with gongylidia on a substrate of mostly flowers and soft shed leaves (very low activity); green: leaf-cutting ants (Acromyrmex and Atta) cultivating L. gongylophorus (high activity)(n = number of colonies; A and B indicate significantly different means in post hoc tests following ANOVA, F_13,39 = 19.48, P < 0.0001). (B) Laccase activity (units per milligram + SE) in the top, middle, and bottom sections of laboratory fungus gardens of three A. echinatior colonies (see SI Appendix, Fig. S2 for similar data on other leaf-cutting ants) showing that laccase activity decreases from top to bottom (ANOVA: F_2,6 = 6.33, P = 0.0332). (C) Fecal droplet laccase activity (units per microliter + SE) of medium-sized A. echinatior workers from laboratory colonies with a fungus garden and from workers kept on sugar water for 20 d without a fungus garden (ANOVA: F_1,4 = 8.93, P = 0.0404, five workers measured from each of three A. echinatior colonies).

Fig. 3.

Laccase gene expression and protein/cDNA sequence comparison demonstrating the transfer of active laccases via the ant gut. (A) Partial alignment of fecal droplet protein sequences and the corresponding translated cDNA of LgLcc1 from gongylidia showing 96% sequence identity (see SI Appendix for details). Numbers refer to amino acid positions in LgLcc1. (B) Recombinant expression of the LgLcc1 gene in yeast (INVSc1) transformed with the vector pYES2 and grown in media supplemented with 20 mM gallic acid. Dark brown staining indicates that LgLcc1 was expressed, secreted into the medium, and able to oxidize a phenolic compound such as gallic acid. Left and Right pictures are identical, but illustrate contrasts with black and white background, respectively. (C) Ratio of laccase gene expression (mean ± SE) (normalized relative to housekeeping genes) between gongylidia and mycelium, measured for nine laccase-coding genes from A. echinatior fungus gardens (green) and the LgLcc1 homolog TcLcc1 from Trachymyrmex cornetzi fungus gardens (blue), a sympatric non–leaf-cutting ant sister group representative. Values above 1 show an up-regulation of laccase gene expression in gongylidia, whereas values below 1 show a down-regulation of laccase gene expression in gongylidia relative to undifferentiated mycelium (based on 10,000 random permutations in reallocation tests of four A. echinatior and three T. cornetzi fungus gardens, respectively). (D) Absolute gene expression (mean number of transcripts + SE per nanogram of total RNA) of TcLcc1 and LgLcc1 in gongylidia and undifferentiated mycelium from three T. cornetzi and four A. echinatior fungus gardens, respectively. (E) Unrooted maximum likelihood phylogeny of ant-cultivated fungal laccase sequences showing positive selection on the branch leading to the clade of gongylidia-producing fungi (L. gongylophorus and Leucocoprinus sp. “T. cornetzi”). Branch-site tests identified the branch (highlighted in bold) containing codon positions that are significantly positively selected (2ΔlnL = 7.44, P = 0.025). The orthologous laccase from the free-living fungi Cyathus bulleri (ABW75771) and Lac2 from Coprinus comatus (JQ228449) are included as outgroups to the fungus-growing ant cultivars. dN/dS ratios (ω) and branch lengths (substitutions per codon) are given above and below each branch, respectively. The nonsignificant ω = 4.204 for the branch leading to ClLcc1 is due to very few synonymous substitutions (see SI Appendix for details).