Search

Abstract Gut microbiota are intrinsic to an herbivorous lifestyle, but very little is known about how plant secondary compounds (PSCs), which are often toxic, influence these symbiotic partners. Here we interrogated the possibility of unique functional core microbiomes in populations of two species of woodrat (Neotoma lepida and bryanti) that have independently converged to feed on the same toxic diet (creosote bush; Larrea tridentata) and compared them to populations that do not feed on creosote bush. Leveraging this natural experiment, we collected samples across a large geographic region in the U.S. desert southwest from 20 populations (~ 150 individuals) with differential ingestion of creosote bush and analyzed three gut regions (foregut, cecum, hindgut) using16S sequencing and shotgun metagenomics. In each gut region sampled, we found a distinctive set of microbes in individuals feeding on creosote bush that were more abundant than other ASVs, enriched in creosote feeding woodrats, and occurred more frequently than would be predicted by chance. Creosote core members were from microbial families e.g., Eggerthellaceae, known to metabolize plant secondary compounds and three of the identified core KEGG orthologs (4-hydroxybenzoate decarboxylase, benzoyl-CoA reductase subunit B, and 2-pyrone-4, 6-dicarboxylate lactonase) coded for enzymes that play important roles in metabolism of plant secondary compounds. The results support the hypothesis that the ingestion of creosote bush sculpts the microbiome across all major gut regions to select for functional characteristics associated with the degradation of the PSCs in this unique diet.

Abstract Climate change is increasing temperature, decreasing precipitation, and increasing atmospheric CO2 concentrations in many ecosystems. As atmospheric carbon rises, plants may increase carbon‐based defenses, such as phenolics, thereby potentially affecting food quality, foraging habits, and habitat suitability for mammalian herbivores. In alpine habitats, the American pika (Ochotona princeps) is a model species for studying effects of changing plant chemistry on mammals. To survive between growing seasons, pikas cache “haypiles” of plants rich in phenolics. Although they are toxic to pikas, phenolic compounds facilitate retention of plant biomass and nutrition during storage, and they degrade over time. Alpine avens (Geum rossii, Rosales: Rosaceae) is a high‐phenolic plant species that comprises up to 75% of pika haypiles in Colorado. Here, we tested the hypothesis that contemporary climate change has affected the nutritional value of alpine avens to pikas in the last 30 years. Specifically, we compared phenolic activity, nutritional quality, and overwinter preservation of plants collected at Niwot Ridge, Colorado (USA), in 1992 to those collected between 2010 and 2018, spanning nearly three decades of climate change. Phenolic activity increased in alpine avens since 1992, while fiber and nitrogen content decreased. Importantly, overwinter preservation of plant biomass also increased, particularly on windblown slopes without long‐lasting snow cover. Previous studies indicate that pikas at this site still depend on alpine avens for their winter food caches. Higher phenolic content in alpine avens could therefore enhance the preservation of haypiles over winter; however, if pikas must further delay consuming these plants to avoid toxicity or invest extra energy in detoxification, then the nutritional gains from enhanced preservation may not be beneficial. This study provides important insights into how climate‐driven changes in plant chemistry will affect mammalian herbivores in the future.

Ambient temperature is an underappreciated determinant of foraging behaviour in wild endotherms, and the requirement to thermoregulate likely influences food intake through multiple interacting mechanisms. We investigated relationships between ambient temperature and hepatic detoxification capacity in two herbivorous marsupials, the common ringtail possum (Pseudocheirus peregrinus) and common brushtail possum (Trichosurus vulpecula) that regularly feed on diets rich in plant toxins. As an indicator of hepatic detoxification capacity, we determined the functional clearance rate of an anaesthetic agent, Alfaxalone, after possums were acclimated to 10°C [below the thermoneutral zone (TNZ)], 18°C [approximately lower critical temperature (LCT)], and 26°C [approximately upper critical temperature (UCT)] for either 7 days or less than 24 h. We then measured intake of foods with high or low plant secondary metabolite (PSM) concentrations under the same temperature regimes. After 7 days of acclimation, we found a positive correlation between the functional clearance rate of Alfaxalone and ambient temperature, and a negative relationship between ambient temperature and intake of foods with high or low PSM concentrations for both species. The effect of ambient temperature on intake of diets rich in PSMs was absent or reduced when possums were kept at temperatures for less than 24 h. Our results underscore the effects of ambient temperature in hepatic metabolism particularly with respect intake of diets containing PSMs. Given that the planet is warming, it is vital that effects of ambient temperature on metabolism, nutrition and foraging by mammalian herbivores is taken into account to predict range changes of species and their impact on ecosystems.

Abstract 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.

ABSTRACT Oxalate, broadly found in both dietary and endogenous sources, is a primary constituent in 80% of kidney stones, an affliction that has tripled in prevalence over the last 40 years. Oxalate-degrading bacteria within the gut microbiota can mitigate the effects of oxalate and are negatively correlated with kidney stone formation, but bacteriotherapies involving oxalate-degrading bacteria have met with mixed results. To inform the development of more effective and consistent bacteriotherapies, we sought to quantify the interactions and limits between oxalate and an oxalate-adapted microbiota from the wild mammalian herbivore Neotoma albigula (woodrat), which consumes a high-oxalate diet in the wild. We tracked the microbiota over a variable-oxalate diet ranging from 0.2% to 12%, with the upper limit approximating 10× the level of human consumption. The N. albigula microbiota was capable of degrading ~100% of dietary oxalate regardless of the amount consumed. However, the microbiota exhibited significant changes in diversity dynamically at the operational taxonomic unit (OTU), family, and community levels in accordance with oxalate input. Furthermore, a cohesive microbial network was stimulated by the consumption of oxalate and exhibited some resistance to the effects of prolonged exposure. This study demonstrates that the oxalate-adapted microbiota of N. albigula exhibits a very high level of degradation and tolerance for oxalate. IMPORTANCE The bacteria associated with mammalian hosts exhibit extensive interactions with overall host physiology and contribute significantly to the health of the host. Bacteria are vital to the mitigation of the toxic effects of oxalate specifically as mammals do not possess the enzymes to degrade this compound, which is present in the majority of kidney stones. Contrary to the body of literature on a few oxalate-degrading specialists, our work illustrates that oxalate stimulates a broad but cohesive microbial network in a dose-dependent manner. The unique characteristics of the N. albigula microbiota make it an excellent source for the development of bacteriotherapies to inhibit kidney stone formation. Furthermore, this work successfully demonstrates methods to identify microbial networks responsive to specific toxins, their limits, and important elements such as microbial network cohesivity and architecture. These are necessary steps in the development of targeted bacteriotherapies.

ABSTRACT For mammals, oxalate enters the body through the diet or is endogenously produced by the liver; it is removed by microbial oxalate metabolism in the gut and/or excretion in feces or urine. Deficiencies in any one of the these pathways can lead to complications, such as calcium oxalate urinary stones. While considerable research has been conducted on individual oxalate-degrading bacterial isolates, interactions between oxalate and the gut microbiota as a whole are unknown. We examined the reduction in oxalate excretion in a rat model following oral administration of fecal microbes from a mammalian herbivore adapted to a high oxalate diet or to fecal transplants consisting of two different formulations of mixed oxalate-degrading isolates. While all transplants elicited a significant reduction in oxalate excretion initially, the greatest effect was seen with fecal microbial transplants, which persisted even in the absence of dietary oxalate. The reduction in oxalate excretion in animals given fecal transplants corresponded with the establishment of diverse bacteria, including known oxalate-degrading bacteria and a cohesive network of bacteria centered on oxalate-degrading specialists from the Oxalobacteraceae family. Results suggested that the administration of a complete community of bacteria facilitates a cohesive balance in terms of microbial interactions. Our work offers important insights into the development of targeted bacteriotherapies intended to reduce urinary oxalate excretion in patients at risk for recurrent calcium oxalate stones as well as bacteriotherapies targeting other toxins for elimination. IMPORTANCE Oxalate is a central component in 80% of kidney stones. While mammals do not possess the enzymes to degrade oxalate, many gastrointestinal bacteria are efficient oxalate degraders. We examined the role of cohesive microbial networks for oxalate metabolism, using Sprague-Dawley rats as a model host. While the transplantation of oxalate-degrading bacteria alone to the Sprague-Dawley hosts did increase oxalate metabolism, fecal transplants from a wild mammalian herbivore, Neotoma albigula, had a significantly greater effect. Furthermore, the boost for oxalate metabolism persisted only in animals that received fecal transplants. Animals receiving fecal transplants had a more diverse and cohesive network of bacteria associated with the Oxalobacteraceae, a family known to consist of specialist oxalate-degrading bacteria, than did animals that received oxalate-degrading bacteria alone. Our results indicate that fecal transplants are more effective at transferring specific functions than are microbial specialists alone, which has broad implications for the development of bacteriotherapies.

Dipodomys microps, the chisel-toothed kangaroo rat, is heralded as one of few mammalian herbivores capable of dietary specialization. Throughout its range, the diet of D. microps is thought to consist primarily of Atriplex confertifolia (saltbush), a C4 plant, and sparing amounts of C3 plants. Using stable isotopes of carbon and nitrogen as natural diet tracers, we asked whether D. microps is an obligate specialist on saltbush. We analyzed hair samples of D. microps for isotopes from historic and recent museum specimens (N = 66). A subset of samples (N = 17) from 2017 that were associated with field notes on plant abundances were further evaluated to test how local saltbush abundance affects its inclusion in the diet of D. microps. Overall, we found that the chisel-toothed kangaroo rat facultatively specializes on saltbush and that the degree of specialization has varied over time and space. Moreover, saltbush abundance dictates its inclusion in the diet. Furthermore, roughly a quarter of the diet is comprised of insects, and over the past century, insects have become more prevalent and saltbush less prevalent in the diet. We suggest that environmental factors such as climate change and rangeland expansion have caused D. microps to include more C3 plants and insects.

The microbial communities inhabiting the alimentary tracts of mammals, particularly those of herbivores, are estimated to be one of the densest microbial reservoirs on Earth. The significance of these gut microbes in influencing the physiology, ecology and evolution of their hosts is only beginning to be realized. To understand the microbiome of herbivores with a focus on nutritional ecology, while evaluating the roles of host evolution and environment in sculpting microbial diversity, we have developed an experimental system consisting of the microbial communities of several species of herbivorous woodrats (genus Neotoma) that naturally feed on a variety of dietary toxins. We designed this system to investigate the long-standing, but experimentally neglected hypothesis that ingestion of toxic diets by herbivores is facilitated by the gut microbiota. Like several other rodent species, the woodrat stomach has a sacculated, nongastric foregut portion. We have documented a dense and diverse community of microbes in the woodrat foregut, with several genera potentially capable of degrading dietary toxins and/or playing a role in stimulating hepatic detoxification enzymes of the host. The biodiversity of these gut microbes appears to be a function of host evolution, ecological experience and diet, such that dietary toxins increase microbial diversity in hosts with experience with these toxins while novel toxins depress microbial diversity. These microbial communities are critical to the ingestion of a toxic diet as reducing the microbial community with antibiotics impairs the host’s ability to feed on dietary toxins. Furthermore, the detoxification capacity of gut microbes can be transferred from Neotoma both intra and interspecifically to naïve animals that lack ecological and evolutionary history with these toxins. In addition to advancing our knowledge of complex host-microbes interactions, this system holds promise for identifying microbes that could be useful in the treatment of diseases in humans and domestic animals.

Although many plant secondary compounds are known to have serious consequences for herbivores, the costs of processing them are generally unknown. Two potential costs of ingestion and detoxification of secondary compounds are elevation of the minimum drinking water requirement and excretion of energetically expensive metabolites (i.e., glucuronides) in the urine. To address these impacts, we studied the costs of ingestion of resin from creosote bush (Larrea tridentata) on desert woodrats (Neotoma lepida). The following hypotheses were tested: ingestion of creosote resin by woodrats (1) increases minimum water requirement and (2) reduces energy available by increasing fecal and urinary energy losses. We tested the first hypothesis, by measuring the minimum water requirement of woodrats fed a control diet with and without creosote resin. Drinking water was given in decreasing amounts until woodrats could no longer maintain constant body mass. In two separate experiments, the minimum drinking water requirement of woodrats fed resin was higher than that of controls by 18–30% (about 1–1.7 ml/d). We tested several potential mechanisms of increased water loss associated with the increase in water requirement. The rate of fecal water loss was higher in woodrats consuming resin. Neither urinary water nor evaporative water loss was affected by ingestion of resin. Hypothesis 2 was tested by measuring energy fluxes of woodrats consuming control vs. resin-treated diets. Woodrats on a resin diet had higher urinary energy losses and, thus, metabolized a lower proportion of the dietary energy than did woodrats on control diet. Fecal energy excretion was not affected by resin. The excretion of glucuronic acid represented almost half of the energy lost as a consequence of resin ingestion. The increased water requirement and energy losses of woodrats consuming a diet with resin could have notable ecological consequences. [ABSTRACT FROM AUTHOR]

Ingestion of plant secondary compounds by herbivores is predicted to increase resting or basal metabolic rates. We tested this hypothesis with two species of woodrat herbivores, Neotoma stephensi and Neotoma albigula, consuming diets of juniper (Juniperus monosperma), which is rich in plant secondary compounds. In nature, N. stephensi specializes on juniper, whereas N. albigula consumes a variety of plant species including juniper. We measured resting metabolic rates (RMR) of woodrats on control, 25% juniper and a treatment containing the maximum tolerable dose of juniper (50% juniper for N. albigula and 70% juniper for N. stephensi). Ingestion of a juniper diet resulted in decreased RMR in both species of woodrats. We propose several potential mechanisms for metabolic depression of Neotoma on juniper diets. Our novel results underscore the need for more studies utilizing plant-based diets to determine the general effect of plant secondary compounds on metabolic rates of herbivores. [ABSTRACT FROM AUTHOR]

Predicting habitat suitability under climate change is vital to conserving biodiversity. However, current species distribution models rely on coarse scale climate data, whereas fine scale microclimate data may be necessary to assess habitat suitability and generate predictive models. Here, we evaluate disparities between temperature data at the coarse scale from weather stations versus fine-scale data measured in microhabitats required for a climate-sensitive mammal, the American pika (Ochotona princeps). We collected two years of temperature data in occupied talus habitats predicted to be suitable (high elevation) and unsuitable (low elevation) by the bioclimatic envelope approach. At low elevations, talus surface and interstitial microclimates drastically differed from ambient temperatures measured on-site and at a nearby weather station. Interstitial talus temperatures were frequently decoupled from high ambient temperatures, resulting in instantaneous disparities of over 30 °C between these two measurements. Microhabitat temperatures were also highly heterogeneous, such that temperature measurements within the same patch of talus were not more correlated than measurements at distant patches. An experimental manipulation revealed that vegetation cover may cool the talus surface by up to 10 °C during the summer, which may contribute to this spatial heterogeneity. Finally, low elevation microclimates were milder and less variable than typical alpine habitat, suggesting that, counter to species distribution model predictions, these seemingly unsuitable habitats may actually be better refugia for this species under climate change. These results highlight the importance of fine-scale microhabitat data in habitat assessments and underscore the notion that some critical refugia may be counterintuitive.

Detoxification enzymes play a key role in plant-herbivore interactions, contributing to the on-going evolution of ecosystem functional diversity. Mammalian detoxification systems have been well studied by the medical and pharmacological industries to understand human drug metabolism; however, little is known of the mechanisms employed by wild herbivores to metabolize toxic plant secondary compounds. Using a wild rodent herbivore, the desert woodrat (Neotoma lepida), we investigated genomic structural variation, sequence variability, and expression patterns in a multigene subfamily involved in xenobiotic metabolism, cytochrome P450 2B (CYP2B). We hypothesized that differences in CYP2B expression and sequence diversity could explain differential abilities of woodrat populations to consume native plant toxins. Woodrats from two distinct populations were fed diets supplemented with either juniper (Juniperus osteosperma) or creosote bush (Larrea tridentata), plants consumed by woodrats in their respective desert habitats. We used Southern blot and quantitative PCR to determine that the genomic copy number of CYP2B in both populations was equivalent, and similar in number to known rodent copy number. We compared CYP2B expression patterns and sequence diversity using cloned hepatic CYP2B cDNA. The resulting sequences were very diverse, and clustered into four major clades by amino acid similarity. Sequences from the experimental treatments were distributed non-randomly across a CYP2B tree, indicating unique expression patterns from woodrats on different diets and from different habitats. Furthermore, within each major CYP2B clade, sequences shared a unique combination of amino acid residues at 13 sites throughout the protein known to be important for CYP2B enzyme function, implying differences in the function of each major CYP2B variant. This work is the most comprehensive investigation of the genetic diversity of a detoxification enzyme subfamily in a wild mammalian herbivore, and contributes an initial genetic framework to our understanding of how a wild herbivore responds to critical changes in its diet.

BACKGROUND:Species diversity is proposed to greatly impact the prevalence of pathogens. Two predominant hypotheses, the "Dilution Effect" and the "Amplification Effect", predict divergent outcomes with respect to the impact of species diversity. The Dilution Effect predicts that pathogen prevalence will be negatively correlated with increased species diversity, while the Amplification Effect predicts that pathogen prevalence will be positively correlated with diversity. For many host-pathogen systems, the relationship between diversity and pathogen prevalence has not be empirically examined. METHODOLOGY/PRINCIPAL FINDINGS:We tested the Dilution and Amplification Effect hypotheses by examining the prevalence of Sin Nombre virus (SNV) with respect to diversity of the nocturnal rodent community. SNV is directly transmitted primarily between deer mice (Peromyscus maniculatus). Using mark-recapture sampling in the Spring and Fall of 2003-2005, we measured SNV prevalence in deer mice at 16 landscape level sites (3.1 hectares each) that varied in rodent species diversity. We explored several mechanisms by which species diversity may affect SNV prevalence, including reduced host density, reduced host persistence, the presence of secondary reservoirs and community composition. We found a negative relationship between species diversity and SNV prevalence in deer mice, thereby supporting the Dilution Effect hypothesis. Deer mouse density and persistence were lower at sites with greater species diversity; however, only deer mouse persistence was positively correlated with SNV prevalence. Pinyon mice (P. truei) may serve as dilution agents, having a negative effect on prevalence, while kangaroo rats (Dipodomys ordii), may have a positive effect on the prevalence of SNV, perhaps through effects on deer mouse behavior. CONCLUSIONS/SIGNIFICANCE:While previous studies on host-pathogen systems have found patterns of diversity consistent with either the Dilution or Amplification Effects, the mechanisms by which species diversity influences prevalence have not been investigated. Our study indicates that changes in host persistence, coupled with interspecific interactions, are important mechanisms through which diversity may influence patterns of pathogens. Our results reveal the complexity of rodent community interactions with respect to SNV dynamics.

Wild animals entering captivity experience radical lifestyle changes resulting in microbiome alterations. However, little is known about the factors that drive microbial community shifts in captivity, and what actions could mitigate microbial changes. Using white-throated woodrats (Neotoma albigula), we tested whether offering natural diets in captivity facilitates retention of native microbial communities of captive animals. Wild-caught woodrats were brought to laboratory conditions. Woodrats received either a natural diet of Opuntia cactus or an artificial diet of commercial chow over three weeks. Microbial inventories from woodrat feces at the time of capture and in captivity were generated using Illumina 16S rRNA sequencing. We found that providing woodrats with wild-natural diets significantly mitigated alterations in their microbiota, promoting a 90% retention of native microbial communities across the experiment. In contrast, the artificial diet significantly impacted microbial structure to the extent that 38% of the natural microflora was lost. Core bacteria including Bifidobacterium and Allobaculum were lost, and abundances of microbes related to oxalate degradation decreased in individuals fed artificial but not natural diets. These results highlight the importance of supplementing captive diets with natural foods to maintain native microbiomes of animals kept in artificial conditions for scientific or conservation purposes.

An in vivo assay using the cytochrome P450 (P450) suicide inhibitor 1-aminobenzotriazole (ABT) and 24-h food intake was developed to determine the relative importance of P450s in two populations of Neotoma lepida with respect to biotransformation of plant secondary compounds in the animals' natural diets. The efficacy of ABT as a P450 inhibitor was first validated using hypnotic-state assays with and without pretreatment with ABT. Pretreatment with 100 mg/kg ABT by gavage increased hexobarbital sleep times 3-4-fold in both populations, showing effective inhibition of P450s in woodrats. Next, the Great Basin population was fed a terpene-rich juniper diet, and the Mojave population was fed a phenolic-rich creosote diet, with rabbit chow serving as the control diet in each group. Treatment with ABT inhibited food intake in the Great Basin population fed the juniper diet to a greater extent (35%) than the Great Basin population fed the control diet (19%) or the Mojave population fed the creosote diet (16%). The food intake of the Mojave population fed the control diet was not significantly inhibited by ABT. The findings suggest that the biotransformation of terpenes in juniper relies more heavily on P450s than that of phenolics in creosote. This assay provides an inexpensive and noninvasive method to explore the relative importance of P450s in the biotransformation strategies of wild mammalian herbivores.

Mammalian herbivores routinely consume diets laden with often-toxic xenobiotics, yet the manner in which mammalian herbivores detoxify these plant secondary compounds (PSC) is largely unknown. Theory predicts that specialists rely more heavily on functionalization pathways whereas generalists rely on conjugation pathways to metabolize PSC in their diet. We took a pharmacological approach to determine how a specialist (Neotoma stephensi) of juniper foliage (Juniperus monosperma) and a generalist (N. albigula) may process the same dietary PSC. We investigated the xenobiotic metabolizing enzymes of the specialist and generalist on a control diet and a low (25%) juniper diet. We also examined enzyme activities in the specialist on a high (70%) juniper diet. We assayed for cytochrome P450 concentration and biotransformation activities of three specific cytochrome P450 isozymes (CYP1A, CYP2B, CYP3A), NAD(P)H:quinone oxidoreductase, glutathione conjugation, sulfation and glucuronidation. Results provide partial evidence for the hypothesis in that the specialist and generalist consuming juniper at a level similar to their natural diet, differ in the level of conjugation enzyme activity with generalists having higher activity overall than specialists.