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.

Enterotypes of the Gut Microbial Community and Their Response to Plant Secondary Compounds in Plateau Pikas

Animal gut microbiomes can be clustered into “enterotypes” characterized by an abundance of signature genera. The characteristic determinants, stability, and resilience of these community clusters remain poorly understood. We used plateau pika (Ochotona curzoniae) as a model and identified three enterotypes by 16S rDNA sequencing. Among the top 15 genera, 13 showed significantly different levels of abundance between the enterotypes combined with different microbial functions and distinct fecal short-chain fatty acids. We monitored changes in the microbial community associated with the transfer of plateau pikas from field to laboratory and observed that feeding them a single diet reduced microbial diversity, resulting in a single enterotype with an altered composition of the dominant bacteria. However, microbial diversity, an abundance of some changed dominant genera, and enterotypes were partially restored after adding swainsonine (a plant secondary compound found in the natural diet of plateau pikas) to the feed. These results provide strong evidence that gut microbial diversity and enterotypes are directly related to specific diet, thereby indicating that the formation of different enterotypes can help animals adapt to complex food conditions. Additionally, natural plant secondary compounds can maintain dominant bacteria and inter-individual differences of gut microbiota and promote the resilience of enterotypes in small herbivorous mammals.

Bacteria contribute to plant secondary compound degradation in a generalist herbivore system

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, degrading 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. Here, we investigate 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 p450s and genes in geraniol, cumate, cinnamate, and α-pinene/limonene degradation pathways. Using metatranscriptomic analysis, we show that some of these degradation genes are expressed in situ. Most of the bacterial isolates grew unhindered in the presence of PSCs and, using 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 sub-colonies of fungus gardens reduced α-pinene and linalool over a 36-hour period, while L. gongylophorus strains alone only reduced linalool. Overall, our results reveal that the bacterial community in fungus gardens play a pivotal role in alleviating the effect of PSCs on the leaf-cutter ant system.ImportanceLeaf-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 role in the detoxification of plant secondary compounds. Here, we assess if the bacterial community 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 the fungus garden and its associated microbial community influences the generalist foraging abilities of the ants, underscoring the importance of microbial symbionts in plant substrate suitability for herbivores.

Modeling time-series data from microbial communities

As sequencing technologies have advanced, the amount of information regarding the composition of bacterial communities from various environments (e.g. skin, soil) has grown exponentially. To date, most work has focused on cataloging taxa present in samples and determining whether the distribution of taxa shifts with exogenous covariates. However, important questions regarding how taxa interact with each other and their environment remain open, thus preventing in-depth ecological understanding of microbiomes. Time-series data from 16S rDNA amplicon sequencing are becoming more common within microbial ecology, but given the ‘big data’ nature of these studies, there are currently no methods capable of utilizing the breadth of the data to infer ecological interactions from these longitudinal data. We address this gap by presenting a method of analysis using Poisson regression fit with an elastic-net penalty that 1) takes advantage of the fact that the data are time series; 2) constrains estimates to allow for the possibility of many more interactions than data; and 3) is scalable enough to handle data consisting of thousands of taxa. We test the method on gut microbiome data from white-throated woodrats (Neotoma albigula) that were fed varying amounts of the plant secondary compound oxalate over a period of 22 days to estimate interactions between OTUs and their environment.

Effect of Dietary Oxalate on the Gut Microbiota of the Mammalian Herbivore Neotoma albigula

ABSTRACTDiet is one of the primary drivers that sculpts the form and function of the mammalian gut microbiota. However, the enormous taxonomic and metabolic diversity held within the gut microbiota makes it difficult to isolate specific diet-microbe interactions. The objective of the current study was to elucidate interactions between the gut microbiota of the mammalian herbivoreNeotoma albigulaand dietary oxalate, a plant secondary compound (PSC) degraded exclusively by the gut microbiota. We quantified oxalate degradation inN. albigulafed increasing amounts of oxalate over time and tracked the response of the fecal microbiota using high-throughput sequencing. The amount of oxalate degradedin vivowas linearly correlated with the amount of oxalate consumed. The addition of dietary oxalate was found to impact microbial species diversity by increasing the representation of certain taxa, some of which are known to be capable of degrading oxalate (e.g.,Oxalobacterspp.). Furthermore, the relative abundances of 117 operational taxonomic units (OTU) exhibited a significant correlation with oxalate consumption. The results of this study indicate that dietary oxalate induces complex interactions within the gut microbiota that include an increase in the relative abundance of a community of bacteria that may contribute either directly or indirectly to oxalate degradation in mammalian herbivores.

The Gastrointestinal Tract of the White-Throated Woodrat (Neotoma albigula) Harbors Distinct Consortia of Oxalate-Degrading Bacteria

ABSTRACTThe microbiota inhabiting the mammalian gut is a functional organ that provides a number of services for the host. One factor that may regulate the composition and function of gut microbial communities is dietary toxins. Oxalate is a toxic plant secondary compound (PSC) produced in all major taxa of vascular plants and is consumed by a variety of animals. The mammalian herbivoreNeotoma albigulais capable of consuming and degrading large quantities of dietary oxalate. We isolated and characterized oxalate-degrading bacteria from the gut contents of wild-caught animals and used high-throughput sequencing to determine the distribution of potential oxalate-degrading taxa along the gastrointestinal tract. Isolates spanned three genera:Lactobacillus,Clostridium, andEnterococcus. Over half of the isolates exhibited significant oxalate degradationin vitro, and allLactobacillusisolates contained theoxcgene, one of the genes responsible for oxalate degradation. Although diverse potential oxalate-degrading genera were distributed throughout the gastrointestinal tract, they were most concentrated in the foregut, where dietary oxalate first enters the gastrointestinal tract. We hypothesize that unique environmental conditions present in each gut region provide diverse niches that select for particular functional taxa and communities.