The dynamics of plant cell-wall polysaccharide decomposition in leaf-cutting ant fungus gardens

Abstract

The degradation of live plant biomass in fungus gardens of leaf-cutting ants is poorly characterised but fundamental for understanding the mutual advantages and efficiency of this obligate nutritional symbiosis. Controversies about the extent to which the garden-symbiont Leucocoprinus gongylophorus degrades cellulose have hampered our understanding of the selection forces that induced large scale herbivory and of the ensuing ecological footprint of these ants. Here we use a recently established technique, based on polysaccharide microarrays probed with antibodies and carbohydrate binding modules, to map the occurrence of cell wall polymers in consecutive sections of the fungus garden of the leaf-cutting ant Acromyrmex echinatior. We show that pectin, xyloglucan and some xylan epitopes are degraded, whereas more highly substituted xylan and cellulose epitopes remain as residuals in the waste material that the ants remove from their fungus garden. These results demonstrate that biomass entering leaf-cutting ant fungus gardens is only partially utilized and explain why disproportionally large amounts of plant material are needed to sustain colony growth. They also explain why substantial communities of microbial and invertebrate symbionts have evolved associations with the dump material from leaf-cutting ant nests, to exploit decomposition niches that the ant garden-fungus does not utilize. Our approach thus provides detailed insight into the nutritional benefits and shortcomings associated with fungus-farming in ants.

Figure 1. Analysis of plant degradation in leaf-cutting ant fungus gardens.

Comprehensive Microarray Polymer Profiling (CoMPP) (A–G) and enzymatic assays (H–J) were used to assess plant degradation in Acromyrmex echinatior fungus gardens. (A) Leaf-cutting ants collect and transport fragments of fresh leaves back to the fungus garden where they are further fragmented and deposited in the top layer. The gradual degradation of cell wall polysaccharides, as the fungus garden grows upwards into the new substrate material, results in the plant material moving downwards as it is degraded because debris consisting of old fungus and exhausted substrate material is removed from the bottom of the fungus garden and discarded by the ants. The main steps in our CoMPP technique were: (A) collection of replicate material from leaves, top, middle and bottom layers of fungus gardens, and debris; (B) sample preparation by homogenization and precipitation of cell wall polymers; (C) sequential extraction of cell wall components with CDTA, NaOH and cadoxen; (D) printing of polysaccharides as microarrays using a robot, three concentrations, and four replicates (E); (F) probing of microarrays with monoclonal antibodies (mAbs) or carbohydrate binding modules (CBMs); (G) spot quantification and analysis. The activity of enzymes in the corresponding samples was approximated with azurine dyed and cross-linked (AZCL) polysaccharides substrates: (H) Protein extraction in tris buffer, (I) Substrate incubation, and (K) quantification of the area of blue halo (see Text S1 for detailed methods).

Figure 2. Comprehensive microarray polymer profiling (CoMPP) of Acromyrmex echinatior fungus gardens.

(A) An example of a CoMPP microarray populated with material from two colonies and probed with the anti-xyloglucan mAb LM15. (B) Change in average fungal biomass during the processing of leaf material in A. echinatior fungus gardens (±SE). Fungal biomass was determined by measuring the amount of chitin present in fungal cell walls from five different sections: leaves, top, middle and bottom of the fungus garden, and debris pile from the four replicate colonies. (C) The distribution of cell-wall polysaccharides in fungus gardens represented as a heatmap where mean CoMPP spot signals (numbered values, corrected for changes in fungal biomass) are correlated to colour intensity. Sample locations and extraction conditions are shown on the left, polysaccharide epitopes and corresponding monoclonal antibodies mAbs and carbohydrate binding modules (CBM), are shown on the top. The highest corrected mean signal value in the data set was set to 100 and all other values adjusted accordingly. All data are averages of four experiments.

Figure 3. Overall changes in polysaccharide occurrence over five sampling locations.

The relative amount of pectins, hemicelluloses and cellulose across five different sampling locations (leaves, top, middle, and bottom layers of the fungus garden, and debris) were determined by CoMPP analysis. Data are sums that combine the mean signals for all three extractions (CDTA, NaOH and Cadoxen), averaged across the four colonies and scaled relative to 100 for each polysaccharide epitope corresponding to a specific mAb or CBM. Error bars represent standard errors (±SE).

Figure 4. A simplified model of polysaccharide degradation in Acromyrmex echinatior fungus gardens.

The diagram is a synthesis of the CoMPP results obtained in our study. It illustrates three major classes of polysaccharides (pectins, hemicelluloses and cellulose) that are typically present in leaves and the fate of individual polysaccharides when they are processed by the fungus gardens.