| Herbivorous insects are ecosystem engineers, affecting the trophic structure of natural systems and causing enormous losses in agriculture. I studied the role of symbiotic microorganisms in the ecophysiology of the two main insect herbivores in Neotropical forests: tropical caterpillars (Lepidoptera: Saturniidae) and leaf-cutting ants (LCA, Hymenoptera: Formicidae, Myrmicinae, tribe Attini). Caterpillars rely on a highly specialized digestive system to degrade plant biomass, including an extremely alkaline midgut. To identify potential symbionts I compared midgut bacterial diversity between caterpillars fed on natural host plants and insects fed on artificial diets. Diversity analysis of 16S rRNA gene libraries demonstrated that food source influences midgut bacterial diversity. Furthermore, bacteria in the genera Methylobacterium, Bradyrhizobium and Propionibacterium were detected in all midgut libraries, suggesting that they are able to persist in the midgut and establish symbiotic associations with the insect. Molecular analysis indicated that tropical caterpillar eggs are colonized by symbiotic bacteria, thus establishing the possibility of vertical transmission for these symbionts. LCA use freshly-cut plant tissue to cultivate a mutualistic fungus that makes plant nutrients available to the ants; thus LCA are functionally herbivores and are potentially limited by the low nitrogen concentration of plant tissue. I demonstrated that LCA engage in mutualistic associations with specialized nitrogen-fixing bacteria to complement their nitrogen budget. Nitrogen fixation was detected in all fungus garden samples from eight different species of LCA using the acetylene reduction assay. 15N-enrichment experiments established that nitrogen fixed by symbiotic bacteria is incorporated into fungus garden and ant worker biomass, providing evidence that LCA directly benefit from this association. Extensive microbial isolations from fungus gardens of 80 LCA colonies collected across the Neotropics, coupled with functional assays to measure nitrogen fixation and phylogenetic analyses based on multiple housekeeping genes demonstrated that specialized nitrogen fixers in the genera Klebsiella and Pantoea form stable and consistent associations with LCA colonies. Finally, I provide evidence suggesting that this type of association may also be present in other genera of attine ants, fungus-growing termites and ambrosia beetles. Taken together, these results further confirm the key role of symbiotic microorganisms in the ecological success of herbivorous insects. |
