Metabolic Enabling and Detoxification by Mammalian Gut Microbes.

The longstanding interactions between mammals and their symbionts enable thousands of mammal species to consume herbivorous diets. The microbial communities in mammals degrade both plant fiber and toxins. Microbial toxin degradation has been repeatedly documented in domestic ruminants, but similar work in wild mammals is more limited due to constraints on sampling and manipulating the microbial communities in these species. In this review, we briefly describe the toxins commonly encountered in mammalian diets, major classes of biotransformation enzymes in microbes and mammals, and the gut chambers that house symbiotic microbes. We next examine evidence for microbial detoxification in domestic ruminants before providing case studies on microbial toxin degradation in both foregut- and hindgut-fermenting wild mammals. We end by discussing species that may be promising for future investigations, and the advantages and limitations of approaches currently available for studying degradation of toxins by mammalian gut microbes.

Differential Effects of Two Common Antiparasitics on Microbiota Resilience.

BACKGROUND: Parasitic infections challenge vertebrate health worldwide, and off-target effects of antiparasitic treatments may be an additional obstacle to recovery. However, there have been few investigations of the effects of antiparasitics on the gut microbiome in the absence of parasites. METHODS: We investigated whether two common antiparasitics-albendazole (ALB) and metronidazole (MTZ)-significantly alter the gut microbiome of parasite-free mice. We treated mice with ALB or MTZ daily for 7 days and sampled the fecal microbiota immediately before and after treatment and again after a two-week recovery period. RESULTS: ALB did not immediately change the gut microbiota, while MTZ decreased microbial richness by 8.5% and significantly changed community structure during treatment. The structural changes caused by MTZ included depletion of the beneficial family Lachnospiraceae, and predictive metagenomic analysis revealed that these losses likely depressed microbiome metabolic function. Separately, we compared the fecal microbiotas of treatment groups after recovery, and there were minor differences in community structure between the ALB, MTZ, and sham-treated control groups. CONCLUSIONS: These results suggest that a healthy microbiome is resilient after MTZ-induced depletions of beneficial gut microbes, and ALB may cause slight, latent shifts in the microbiota but does not deplete healthy gut microbiota diversity.

Microbiome stability and structure is governed by host phylogeny over diet and geography in woodrats (Neotoma spp.).

The microbiome is critical for host survival and fitness, but gaps remain in our understanding of how this symbiotic community is structured. Despite evidence that related hosts often harbor similar bacterial communities, it is unclear whether this pattern is due to genetic similarities between hosts or to common ecological selection pressures. Here, using herbivorous rodents in the genus Neotoma, we quantify how geography, diet, and host genetics, alongside neutral processes, influence microbiome structure and stability under natural and captive conditions. Using bacterial and plant metabarcoding, we first characterized dietary and microbiome compositions for animals from 25 populations, representing seven species from 19 sites across the southwestern United States. We then brought wild animals into captivity, reducing the influence of environmental variation. In nature, geography, diet, and phylogeny collectively explained ∼50% of observed microbiome variation. Diet and microbiome diversity were correlated, with different toxin-enriched diets selecting for distinct microbial symbionts. Although diet and geography influenced natural microbiome structure, the effects of host phylogeny were stronger for both wild and captive animals. In captivity, gut microbiomes were altered; however, responses were species specific, indicating again that host genetic background is the most significant predictor of microbiome composition and stability. In captivity, diet effects declined and the effects of host genetic similarity increased. By bridging a critical divide between studies in wild and captive animals, this work underscores the extent to which genetics shape microbiome structure and stability in closely related hosts.

Demonstrating the role of symbionts in mediating detoxification in herbivores.

Plant toxins constitute an effective defense against herbivorous animals. However, many herbivores have evolved adaptations to cope with dietary toxins through detoxification, excretion, sequestration, target site insensitivity and/or via behavioral avoidance. While these adaptations are often directly encoded in herbivore genomes, evidence is accumulating that microbial symbionts can reduce the dose of plant toxins by metabolizing or sequestering them prior to absorption by the herbivore. Here, we describe a few well-studied examples to assess such symbiont-mediated detoxification and showcase different approaches that have been used for their analyses. These include: (i) a host phenotypic route in which the symbiotic association is manipulated to reveal host fitness costs upon toxin exposure in the presence/absence of detoxifying symbionts, including function restoration after symbiont re-infection, (ii) a molecular microbiological approach that focuses on the identification and characterization of microbial genes involved in plant toxin metabolism, and (iii) an analytical chemical route that aims to characterize the conversion of the toxin to less harmful metabolites in vivo and link conversion to the activities of a detoxifying symbiont. The advantages and challenges of each approach are discussed, and it is argued that a multi-pronged strategy combining phenotypic, molecular, and chemical evidence is needed to unambiguously demonstrate microbial contributions to plant toxin reduction and the importance of these processes for host fitness. Given the interdisciplinary nature of the topic, we aim to provide a guideline to researchers interested in symbiont-mediated detoxification and hope to encourage future studies that contribute to a more comprehensive and mechanistic understanding of detoxification in herbivores and their symbionts.

Wild herbivorous mammals (genus Neotoma) host a diverse but transient assemblage of fungi.

Fungi are often overlooked in microbiome research and, as a result, little is known about the mammalian mycobiome. Although frequently detected in vertebrate guts and known to contribute to digestion in some herbivores, whether these eukaryotes are a persistent part of the mammalian gut microbiome remains contentious. To address this question, we sampled fungi from wild woodrats (Neotoma spp.) collected from 25 populations across the southwestern United States. For each animal, we collected a fecal sample in the wild, and then re-sampled the same individual after a month in captivity on a controlled diet. We characterized and quantified fungi using three techniques: ITS metabarcoding, shotgun metagenomics and qPCR. Wild individuals contained diverse fungal assemblages dominated by plant pathogens, widespread molds, and coprophilous taxa primarily in Ascomycota and Mucoromycota. Fungal abundance, diversity and composition differed between individuals, and was primarily influenced by animal geographic origin. Fungal abundance and diversity significantly declined in captivity, indicating that most fungi in wild hosts came from diet and environmental exposure. While this suggests that these mammals lack a persistent gut mycobiome, natural fungal exposure may still impact fungal dispersal and animal health.

Strategies in herbivory by mammals revisited: The role of liver metabolism in a juniper specialist (Neotoma stephensi) and a generalist (Neotoma albigula).

Although herbivory is widespread among mammals, few species have adopted a strategy of dietary specialization. Feeding on a single plant species often exposes herbivores to high doses of plant secondary metabolites (PSMs), which may exceed the animal's detoxification capacities. Theory predicts that specialists will have unique detoxification mechanisms to process high levels of dietary toxins. To evaluate this hypothesis, we compared liver microsomal metabolism of a juniper specialist, Neotoma stephensi (diet >85% juniper), to a generalist, N. albigula (diet ≤30% juniper). Specifically, we quantified the concentration of a key detoxification enzyme, cytochrome P450 2B (CYP2B) in liver microsomes, and the metabolism of α-pinene, the most abundant terpene in the juniper species consumed by the specialist woodrat. In both species, a 30% juniper diet increased the total CYP2B concentration (2-3×) in microsomes and microsomal α-pinene metabolism rates (4-fold). In N. stephensi, higher levels of dietary juniper (60% and 100%) further induced CYP2B and increased metabolism rates of α-pinene. Although no species-specific differences in metabolism rates were observed at 30% dietary juniper, total microsomal CYP2B concentration was 1.7× higher in N. stephensi than in N. albigula (p < .01), suggesting N. stephensi produces one or more variant of CYP2B that is less efficient at processing α-pinene. In N. stephensi, the rates of α-pinene metabolism increased with dietary juniper and were positively correlated with CYP2B concentration. The ability of N. stephensi to elevate CYP2B concentration and rate of α-pinene metabolism with increasing levels of juniper in the diet may facilitate juniper specialization in this species.

Symbiotic microbes and potential pathogens in the intestine of dead southern right whale (Eubalaena australis) calves.

Between 2003 and 2017, at least 706 southern right whale (Eubalaena australis) calves died at the Península Valdés calving ground in Argentina. Pathogenic microbes are often suggested to be the cause of stranding events in cetaceans; however, to date there is no evidence supporting bacterial infections as a leading cause of right whale calf deaths in Argentina. We used high-throughput sequencing and culture methods to characterize the bacterial communities and to detect potential pathogens from the intestine of stranded calves. We analyzed small and large intestinal contents from 44 dead calves that stranded at Península Valdés from 2005 to 2010 and found 108 bacterial genera, most identified as Firmicutes or Bacteroidetes, and 9 genera that have been previously implicated in diseases of marine mammals. Only one operational taxonomic unit was present in all samples and identified as Clostridium perfringens type A. PCR results showed that all C. perfringens isolates (n = 38) were positive for alpha, 50% for beta 2 (n = 19) and 47% for enterotoxin (CPE) genes (n = 18). The latter is associated with food-poisoning and gastrointestinal diseases in humans and possibly other animals. The prevalence of the cpe gene found in the Valdés' calves is unusually high compared with other mammals. However, insufficient histologic evidence of gastrointestinal inflammation or necrosis (the latter possibly masked by autolysis) in the gut of stranded calves, and absence of enterotoxin detection precludes conclusions about the role of C. perfringens in calf deaths. Further work is required to determine whether C. perfringens or other pathogens detected in this study are causative agents of calf deaths at Península Valdés.

Microbial communities exhibit host species distinguishability and phylosymbiosis along the length of the gastrointestinal tract.

Host-associated microbial communities consist of stable and transient members that can assemble through purely stochastic processes associated with the environment or by interactions with the host. Phylosymbiosis predicts that if host-microbiota interactions impact assembly patterns, then one conceivable outcome is concordance between host evolutionary histories (phylogeny) and the ecological similarities in microbial community structures (microbiota dendrogram). This assembly pattern has been demonstrated in several clades of animal hosts in laboratory and natural populations, but in vertebrates, it has only been investigated using samples from faeces or the distal colon. Here, we collected the contents of five gut regions from seven rodent species and inventoried the bacterial communities by sequencing the 16S rRNA gene. We investigated how community structures varied across gut regions and whether the pattern of phylosymbiosis was present along the length of the gut. Gut communities varied by host species and gut region, with Oscillospira and Ruminococcus being more abundant in the stomach and hindgut regions. Gut microbial communities were highly distinguishable by host species across all gut regions, with the strength of the discrimination increasing along the length of the gut. Last, the pattern of phylosymbiosis was found in all five gut regions, as well as faeces. Aspects of the gut environment, such as oxygen levels, production of antimicrobials or other factors, may shift microbial communities across gut regions. However, regardless of these differences, host species maintain distinguishable, phylosymbiotic assemblages of microbes that may have functional impacts for the host.

Role of cytochrome P450 2B sequence variation and gene copy number in facilitating dietary specialization in mammalian herbivores.

Theory postulates that dietary specialization in mammalian herbivores is enabled by a specialized set of liver enzymes that process the high concentrations of similar plant secondary metabolites (PSMs) in the diets of specialists. To investigate whether qualitative and quantitative differences in detoxification mechanisms distinguish dietary specialists from generalists, we compared the sequence diversity and gene copy number of detoxification enzymes in two woodrat species: a generalist, the white-throated woodrat (Neotoma albigula) and a juniper specialist, Stephens' woodrat (N. stephensi). We focused on enzymes in the cytochrome P450 subfamily 2B (CYP2B), because previous research suggests this subfamily plays a key role in the processing of PSMs. For both woodrat species, we obtained and sequenced CYP2B cDNA, generated CYP2B phylogenies, estimated CYP2B gene copy number and created a homology model of the active site. We found that the specialist possessed on average ~5 more CYP2B gene copies than the generalist, but the specialist's CYP2B sequences were less diverse. Phylogenetic analysis of putative CYP2B homologs resolved woodrat species as reciprocally monophyletic and suggested evolutionary convergence of distinct homologs on similar key amino acid residues in both species. Homology modelling of the CYP2B enzyme suggests that interspecific differences in substrate preference and function likely result from amino acid differences in the enzyme active site. The characteristics of CYP2B in the specialist, that is greater gene copy number coupled with less sequence variation, are consistent with specialization to a narrow range of dietary toxins.

Gut microbial communities of American pikas (Ochotona princeps): Evidence for phylosymbiosis and adaptations to novel diets.

Gut microbial communities provide many physiological functions to their hosts, especially in herbivorous animals. We still lack an understanding of how these microbial communities are structured across hosts in nature, especially within a given host species. Studies on laboratory mice have demonstrated that host genetics can influence microbial community structure, but that diet can overwhelm these genetic effects. We aimed to test these ideas in a natural system, the American pika (Ochotona princeps). First, pikas are high-elevation specialists with significant population structure across various mountain ranges in the USA, allowing us to investigate whether similarities in microbial communities match host genetic differences. Additionally, pikas are herbivorous, with some populations exhibiting remarkable dietary plasticity and consuming high levels of moss, which is exceptionally high in fibre and low in protein. This allows us to investigate adaptations to an herbivorous diet, as well as to the especially challenging diet of moss. Here, we inventoried the microbial communities of pika caecal pellets from various populations using 16S rRNA sequencing to investigate structuring of microbial communities across various populations with different natural diets. Microbial communities varied significantly across populations, and differences in microbial community structure were congruent with genetic differences in host population structure, a pattern known as "phylosymbiosis." Several microbial members (Ruminococcus, Prevotella, Oxalobacter and Coprococcus) were detected across all samples, and thus likely represent a "core microbiome." These genera are known to perform a number of services for herbivorous hosts such as fibre fermentation and the degradation of plant defensive compounds, and thus are likely important for herbivory in pikas. Moreover, pikas that feed on moss harboured microbial communities highly enriched in Melainabacteria. This uncultivable candidate phylum has been proposed to ferment fibre for herbivores, and thus may contribute to the ability of some pika populations to consume high amounts of moss. These findings demonstrate that both host genetics and diet can influence the microbial communities of the American pika. These animals may be novel sources of fibre-degrading microbes. Last, we discuss the implications of population-specific microbial communities for conservation efforts in this species.

Rational Re-Engineering of the O-Dealkylation of 7-Alkoxycoumarin Derivatives by Cytochromes P450 2B from the Desert Woodrat Neotoma lepida.

On the basis of recent functional and structural characterization of cytochromes P450 2B from the desert woodrat (Neotoma lepida), the 7-alkoxycoumarin and 7-alkoxy-4-(trifluoromethyl)coumarin O-dealkylation profiles of CYP2B35 and CYP2B37 were re-engineered. Point mutants interchanging residues at seven positions in the enzyme active sites were created and purified from an Escherichia coli expression system. In screens for O-dealkylation activity, wild-type CYP2B35 metabolized long-chain 7-alkoxycoumarins but not 7-alkoxy-4-(trifluoromethyl)coumarins or short-chain 7-alkoxycoumarins. Wild-type CYP2B37 metabolized short-chain substrates from both series of compounds. CYP2B35 A367V showed maximal activity with 7-butoxycoumarin as opposed to 7-heptoxycoumarin in the parental enzyme, and CYP2B35 A363I/A367V produced an activity profile like that generated by CYP2B37. CYP2B35 A363I/A367V/I477F showed 7-ethoxycoumarin and 7-ethoxy-4-(trifluoromethyl)coumarin O-dealkylation rates similar to those of CYP2B37 and higher than those of the double mutant. A CYP2B35 septuple mutant retained a CYP2B37-like activity profile. In contrast, the CYP2B37 septuple mutant produced very low rates of O-dealkylation of all substrates. As mutating residue 108 in either enzyme was detrimental, this change was removed from both septuple mutants. Remarkably, the CYP2B35 sextuple mutant produced an activity profile that was a hybrid of that of CYP2B35 and CYP2B37, whereas the CYP2B37 sextuple mutant had almost no O-dealkylation activity. Docking of 7-substituted coumarin derivatives into a model of the CYP2B35 sextuple mutant based on a previous crystal structure of the 4-(4-chlorophenyl)imidazole wild-type complex revealed how the mutant can exhibit activities of both CYP2B35 and CYP2B37.

Patterns of host gene expression associated with harboring a foregut microbial community.

BACKGROUND: Harboring foregut microbial communities is considered a key innovation that allows herbivorous mammals to colonize new ecological niches. However, the functions of these chambers have only been well studied at the molecular level in ruminants. Here, we investigate gene expression in the foregut chamber of herbivorous rodents and ask whether these gene expression patterns are consistent with results in ruminants. We compared gene expression in foregut tissues of two rodent species: Stephen's woodrat (Neotoma stephensi), which harbors a dense foregut microbial community, and the lab rat (Rattus norvegicus), which lacks such a community. RESULTS: We found that woodrats have higher abundances of transcripts associated with smooth muscle processes, specifically a higher expression of the smoothelin-like 1 gene, which may assist in contractile properties of this tissue to retain food material in the foregut chamber. The expression of genes associated with keratinization and cornification exhibited a complex pattern of differences between the two species, suggesting distinct molecular mechanisms. Lab rats exhibited higher abundances of transcripts associated with immune function, likely to inhibit microbial growth in the foregut of this species. CONCLUSIONS: Some of our results were consistent with previous findings in ruminants (high expression of facilitative glucose transporters, lower expression of B4galnt2), suggestive of possible convergent evolution, while other results were unclear, and perhaps represent novel host-microbe interactions in rodents. Overall, our results suggest that harboring a foregut microbiota is associated with changes to the functions and host-microbe interactions of the foregut tissues.

Ambient temperature-mediated changes in hepatic gene expression of a mammalian herbivore (Neotoma lepida).

Herbivores regularly ingest natural toxins produced by plants as a defence against herbivory. Recent work suggests that compound toxicity is exacerbated at higher ambient temperatures. This phenomenon, known as temperature-dependent toxicity (TDT), is the likely result of decreased liver function at warmer temperatures; however, the underlying cause of TDT remains speculative. In the present study, we compared the effects of temperature and dietary plant toxins on differential gene expression in the liver of an herbivorous rodent (Neotoma lepida), using species-specific microarrays. Expression profiles revealed a greater number of differentially expressed genes at an ambient temperature below the thermal neutral zone for N. lepida (22°C) compared to one within (27°C). Genes and pathways upregulated at 22°C were related to growth and biosynthesis, whereas those upregulated at 27°C were associated with gluconeogenesis, apoptosis and protein misfolding, suggestive of a stressed state for the liver. Additionally, few genes associated with xenobiotic metabolism were induced when woodrats ingested plant toxins compared to nontoxic diets, regardless of temperature. Taken together, the results highlight the important role of ambient temperature on gene expression profiles in the desert woodrat. Temperatures just below the thermal neutral zone might be a favourable state for liver metabolism. Furthermore, the reduction in the number of genes expressed at a temperature within the thermal neutral zone indicates that liver function may be reduced at temperatures that are not typically considered as thermally stressful. Understanding how herbivorous mammals will respond to ambient temperature is imperative to accurately predict the impacts of climate change.

Intestinal Lymphatic Transport: an Overlooked Pathway for Understanding Absorption of Plant Secondary Compounds in Vertebrate Herbivores.

Herbivores employ numerous strategies to reduce their exposure to toxic plant secondary chemicals (PSCs). However, the physiological mechanisms of PSC absorption have not been extensively explored. In particular, the absorption of PSCs via intestinal lymphatic absorption has been largely overlooked in herbivores, even though this pathway is well recognized for pharmaceutical uptake. Here, we investigated for the first time whether PSCs might be absorbed by lymphatic transport. We fed woodrats (Neotoma albigula) diets with increasing concentrations of terpene-rich juniper (Juniperus monosperma) either with or without a compound that blocks intestinal lymphatic absorption (Pluronic L-81). Woodrats consuming diets that contained the intestinal lymphatic absorption blocker exhibited increased food intakes and maintained higher body masses on juniper diets. Our study represents the first demonstration that PSCs may be absorbed by intestinal lymphatic absorption. This absorption pathway has numerous implications for the metabolism and distribution of PSCs in the systemic circulation, given that compounds absorbed via lymphatic transport bypass first-pass hepatic metabolism. The area of lymphatic transport of PSCs represents an understudied physiological pathway in plant-herbivore interactions.

Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism.

Crystal structures of CYP2B35 and CYP2B37 from the desert woodrat were solved in complex with 4-(4-chlorophenyl)imidazole (4-CPI). The closed conformation of CYP2B35 contained two molecules of 4-CPI within the active site, whereas the CYP2B37 structure demonstrated an open conformation with three 4-CPI molecules, one within the active site and the other two in the substrate access channel. To probe structure-function relationships of CYP2B35, CYP2B37, and the related CYP2B36, we tested the O-dealkylation of three series of related substrates-namely, 7-alkoxycoumarins, 7-alkoxy-4-(trifluoromethyl)coumarins, and 7-alkoxy-4-methylcoumarins-with a C1-C7 side chain. CYP2B35 showed the highest catalytic efficiency (kcat/KM) with 7-heptoxycoumarin as a substrate, followed by 7-hexoxycoumarin. In contrast, CYP2B37 showed the highest catalytic efficiency with 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), followed by 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC). CYP2B35 had no dealkylation activity with 7-MFC or 7-EFC. Furthermore, the new CYP2B-4-CPI-bound structures were used as templates for docking the 7-substituted coumarin derivatives, which revealed orientations consistent with the functional studies. In addition, the observation of multiple -Cl and -NH-π interactions of 4-CPI with the aromatic side chains in the CYP2B35 and CYP2B37 structures provides insight into the influence of such functional groups on CYP2B ligand binding affinity and specificity. To conclude, structural, computational, and functional analysis revealed striking differences between the active sites of CYP2B35 and CYP2B37 that will aid in the elucidation of new structure-activity relationships.

Inoculation of tannin-degrading bacteria into novel hosts increases performance on tannin-rich diets.

It has been hypothesized that herbivores host tannin-degrading bacteria (TDB) to overcome the toxic challenges posed by plant tannins. While TDB have been isolated from the guts of numerous mammals, their functional significance to their hosts has never been explicitly tested. We introduced TDB into lab rats, which do not host TDB, and measured host performance on tannin-rich diets. We first isolated three species of TDB, Escherichia coli, Bacillus subtilis and Enterococcus faecalis, from the guts of the desert woodrat (Neotoma lepida), which regularly feeds on tannin-rich plants. Then, we inoculated isolated TDB, as well as full woodrat microbial communities into laboratory rats. A control group was inoculated with sterilized woodrat faeces. Recipient lab rats were fed increasing concentrations of tannic acid, and we monitored tannic acid intake, body mass and liver damage as measured by serum alanine aminotransferase activity. Lab rats given TDB as isolates or full communities exhibited increased tannic acid intake, higher maintenance of body mass and lower indicators of liver damage compared with control animals. These differences were maintained when the trial was repeated after 6 weeks of feeding on tannin-free diets. Our results are the first to demonstrate that TDB significantly increase host performance on tannin-rich diets.

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

Diet 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 herbivore Neotoma albigula and dietary oxalate, a plant secondary compound (PSC) degraded exclusively by the gut microbiota. We quantified oxalate degradation in N. albigula fed increasing amounts of oxalate over time and tracked the response of the fecal microbiota using high-throughput sequencing. The amount of oxalate degraded in vivo was 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., Oxalobacter spp.). 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.

Microbial Community Transplant Results in Increased and Long-Term Oxalate Degradation.

Gut microbes are essential for the degradation of dietary oxalate, and this function may play a role in decreasing the incidence of kidney stones. However, many oxalate-degrading bacteria are susceptible to antibiotics and the use of oxalate-degrading probiotics has only led to an ephemeral reduction in urinary oxalate. The objective of the current study was to determine the efficacy of using whole-community microbial transplants from a wild mammalian herbivore, Neotoma albigula, to increase oxalate degradation over the long term in the laboratory rat, Rattus norvegicus. We quantified the change in total oxalate degradation in lab rats immediately after microbial transplants and at 2- and 9-month intervals following microbial transplants. Additionally, we tracked the fecal microbiota of the lab rats, with and without microbial transplants, using high-throughput Illumina sequencing of a hyper-variable region of the 16S rRNA gene. Microbial transplants resulted in a significant increase in oxalate degradation, an effect that persisted 9 months after the initial transplants. Functional persistence was corroborated by the transfer, and persistence of a group of bacteria previously correlated with oxalate consumption in N. albigula, including an anaerobic bacterium from the genus Oxalobacter known for its ability to use oxalate as a sole carbon source. The results of this study indicate that whole-community microbial transplants are an effective means for the persistent colonization of oxalate-degrading bacteria in the mammalian gut.

Small intestinal hydrolysis of plant glucosides: higher glucohydrolase activities in rodents than passerine birds.

Glycosides are a major group of plant secondary compounds characterized by one or more sugars conjugated to a lipophilic, possibly toxic aglycone, which is released upon hydrolysis. We compared small intestinal homogenate hydrolysis activity of three rodent and two avian species against four substrates: amygdalin and sinigrin, two plant-derived glucosides, the sugar lactose, whose hydrolysis models some activity against flavonoid and isoflavonoid glucosides, and the disaccharide sugar maltose (from starch), used as a comparator. Three new findings extend our understanding of physiological processing of plant glucosides: (1) the capacity of passerine birds to hydrolyze plant glucosides seems relatively low, compared with rodents; (2) in this first test of vertebrates' enzymic capacity to hydrolyze glucosinolates, sinigrin hydrolytic capacity seems low; (3) in laboratory mice, hydrolytic activity against lactose resides on the enterocytes' apical membrane facing the intestinal lumen, but activity against amygdalin seems to reside inside enterocytes.

Using the Specialization Framework to Determine Degree of Dietary Specialization in a Herbivorous Woodrat.

To be considered a dietary specialist, mammalian herbivores must consume large quantities of a plant species considered "difficult" with respect to nutrient or toxin content, and possess specialized adaptations to deal with plant defensive compounds or low nutritional content. Populations of Neotoma lepida in the Great Basin consume Juniperus osteosperma, a plant heavily defended by terpenes, but a detailed dietary analysis of this population is lacking. Therefore, we investigated the extent of dietary specialization in this species in comparison with the better-studied specialist species, N. stephensi. Microhistological analysis of feces from N. lepida revealed that greater than 90% of their diet in nature was comprised of juniper. In laboratory tolerance trials, N. lepida tolerated a diet of 80% J. osteosperma, similar to that observed for N. stephensi. There was no difference in the abilities of N. lepida and N. stephensi to metabolize hexobarbital, a proxy compound for terpene metabolism. In preference tests of native and non-native juniper species, N. lepida did not exhibit a preference for its native or co-occurring juniper, J. osteosperma, over the non-native species, J. monosperma, whereas N. stephensi preferred its native or co-occurring juniper J. monosperma over non-native J. osteosperma. Behavioral and habitat differences between these woodrat species lead to the categorization of N. stephensi as an obligate juniper specialist with a small range that overlaps that of its preferred food, J. monosperma, and N. lepida as a facultative juniper specialist with a large range, and only a portion of its distribution containing populations that feed extensively on J. osteosperma.