Bacteria Contribute to Plant Secondary Compound Degradation in a Generalist Herbivore System

Abstract

Herbivores must overcome a variety of plant defenses, including coping with plant secondary compounds (PSCs). To help detoxify these defensive chemicals, several insect herbivores are known to harbor gut microbiota with the metabolic capacity to degrade PSCs. Leaf-cutter ants are generalist herbivores, obtaining sustenance from specialized fungus gardens that act as external digestive systems and which degrade the diverse collection of plants foraged by the ants. There is in vitro evidence that certain PSCs harm Leucoagaricus gongylophorus, the fungal cultivar of leaf-cutter ants, suggesting a role for the Proteobacteria-dominant bacterial community present within fungus gardens. In this study, we investigated the ability of symbiotic bacteria present within fungus gardens of leaf-cutter ants to degrade PSCs. We cultured fungus garden bacteria, sequenced the genomes of 42 isolates, and identified genes involved in PSC degradation, including genes encoding cytochrome P450 enzymes and genes in geraniol, cumate, cinnamate, and α-pinene/limonene degradation pathways. Using metatranscriptomic analysis, we showed that some of these degradation genes are expressed in situ. Most of the bacterial isolates grew unhindered in the presence of PSCs and, using gas chromatography-mass spectrometry (GC-MS), we determined that isolates from the genera Bacillus, Burkholderia, Enterobacter, Klebsiella, and Pseudomonas degrade α-pinene, β-caryophyllene, or linalool. Using a headspace sampler, we show that subcolonies of fungus gardens reduced α-pinene and linalool over a 36-h period, while L. gongylophorus strains alone reduced only linalool. Overall, our results reveal that the bacterial communities in fungus gardens play a pivotal role in alleviating the effect of PSCs on the leaf-cutter ant system. IMPORTANCE Leaf-cutter ants are dominant neotropical herbivores capable of deriving energy from a wide range of plant substrates. The success of leaf-cutter ants is largely due to their external gut, composed of key microbial symbionts, specifically, the fungal mutualist L. gongylophorus and a consistent bacterial community. Both symbionts are known to have critical roles in extracting energy from plant material, yet comparatively little is known about their roles in the detoxification of plant secondary compounds. In this study, we assessed if the bacterial communities associated with leaf-cutter ant fungus gardens can degrade harmful plant chemicals. We identify plant secondary compound detoxification in leaf-cutter ant gardens as a process that depends on the degradative potential of both the bacterial community and L. gongylophorus. Our findings suggest that the fungus garden and its associated microbial community influence the generalist foraging abilities of the ants, underscoring the importance of microbial symbionts in plant substrate suitability for herbivores.

Chemical Gradients of Plant Substrates in an Atta texana Fungus Garden

“…Our study enabled us to detect molecular families and chemical transformations occurring in a fungus garden. These results will enable further investigations concerning the roles of these metabolites, as has been recently demonstrated for fatty acids in fungus gardens ( 24 ) and for plant volatile compounds that are modified by fungus garden-associated bacteria ( 26 ). Because previous reports have shown that the enzymes produced in the fungus garden vary depending on the available substrates ( 53 ), we expect that the chemical modifications and the types of transformations observed in this study will also vary based on changes in the substrates available for the colony.…”

“…Because previous reports have shown that the enzymes produced in the fungus garden vary depending on the available substrates ( 53 ), we expect that the chemical modifications and the types of transformations observed in this study will also vary based on changes in the substrates available for the colony. Additionally, environmental factors such as temperature or humidity and the composition of microbiomes that are associated with ants and their fungus gardens ( 26 ) will likely also influence these modifications in natural ecosystems.…”

“…Fungus gardens from leaf-cutter ants have been compared to bioreactors ( 21 ) and human compost ( 23 ) and have even been described as external ant guts ( 24 ) due to their capacity to process plant constituents. Few studies have directly assayed small molecules from fungus gardens ( 24 – 26 ), though some have shown the differential distribution of fungal metabolic enzymes in different regions of the fungus garden ( 25 , 27 ). That distribution of enzymes reflects the incorporation of fresh plant material at the top of leaf-cutting ant fungus gardens followed by its sequential degradation while moving through the garden, with recalcitrant biomass being removed by ants from the bottom of the fungus garden as trash ( 28 , 29 ).…”

Metagenomics Reveals Diet-Specific Specialization of Bacterial Communities in Fungus Gardens of Grass- and Dicot-Cutter Ants

“…It is possible that we would see an enrichment for plant defense compound detoxification genes in the top of the fungus garden, where fresh plant material is deposited. To elucidate the role of bacteria in plant defense compound remediation, closely controlled experiments with particular defense compounds of interest applied to bacterial cultures and to fungus gardens would be necessary, and have been pursued as a follow-up to this study (Francoeur et al, 2020). Pinto-Tomás et al (2009) established that Pantoea and Klebsiella bacteria in Central American leaf-cutter ant fungus gardens supplement the ant diet through nitrogen fixation.…”

Gut Bacterial and Fungal Communities of the Wild and Laboratory-Reared Thitarodes Larvae, Host of the Chinese Medicinal Fungus Ophiocordyceps sinensis on Tibetan Plateau

“…Insect gut is the house of diverse microbiomes, which are evolving to play important roles in insect physiology and ecology, supporting growth, development, and survival of their hosts [ 22 , 23 , 24 ]. In general, gut microbiota can contribute to the nutrition provision for host [ 25 ], host immunity [ 26 ], host behavior [ 27 ], and mediate detoxification of plant secondary compounds [ 28 , 29 ] and insecticides [ 30 ]. Conversely, multiple factors such as host, environment, and diet can shape the structure of gut microbial communities [ 31 , 32 , 33 , 34 , 35 ].…”