Metabolomic evidence of independent biotransformation pathways for terpenes in two specialist mammalian herbivores (genus Neotoma).

Herbivory is common in mammals, yet our understanding of detoxification processes used by mammals to biotransform plant secondary compounds (PSCs) is limited. Specialist herbivores are thought to have evolved detoxification mechanisms that rely more heavily on energetically cheap Phase I biotransformation reactions to process high levels of PSCs in their diets. We explored this hypothesis by comparing the urinary metabolite patterns of two specialist herbivores (genus Neotoma). Neotoma stephensi is an obligate specialist on one-seeded juniper (Juniperus monosperma). Neotoma lepida is a generalist forager across its range, yet populations in the Great Basin specialize on Utah juniper (J. osteosperma). While both juniper species have high levels of terpenes, the terpene profiles and quantities differ between the two. Individuals from both woodrat species were fed diets of each juniper in a cross-over design. Urine, collected over a 24-h period, was extracted and analyzed in an untargeted metabolomics approach using both GC-MS and HPLC-MS/MS. The obligate specialist N. stephensi excreted a unique pattern of Phase I metabolites when fed its native juniper, while N. lepida excreted a unique pattern of Phase II metabolites when fed its native juniper. Both woodrat species utilized the Phase II metabolic pathway of glucuronidation more heavily when consuming the more chemically diverse J. osteosperma, and N. stephensi utilized less glucuronidation than N. lepida when consuming J. monosperma. These results are consistent with the hypothesis that obligate specialists may have evolved unique and efficient biotransformation mechanisms for dealing with PSCs in their diet.

Mammalian cytochrome P450 biodiversity: Physiological importance, function, and protein and genomic structures of cytochromes P4502B in multiple species of woodrats with different dietary preferences.

The vast diversity of cytochrome P450 enzymes in mammals has been proposed to result in large measure from plant-animal warfare, whereby evolution of chemical defenses such as phenolics and terpenoids in plants led to duplication and divergence of P450 genes in herbivores. Over evolutionary time, natural selection is predicted to have produced P450s with high affinity and enhanced metabolism of substrates that are ingested regularly by herbivores. Interestingly, however, almost all knowledge of the interactions of mammalian P450 enzymes with substrates stems from studies of the metabolism of drugs and model compounds rather than studies on wild mammalian herbivores and their respective PSMs. A question of particular interest centers on the role of individual P450 enzymes in the ability of certain herbivores to specialize on plants that are lethal to most other species, including those from the same genus as the specialists. We tackled this intricate problem using a tractable natural system (herbivorous woodrats, genus Neotoma) focusing on comparisons of the specialist N. stephensi, the facultative specialist N. lepida, and the generalist N. albigula, and employing a cross-disciplinary approach involving ecology, biochemistry, pharmacology, structural biology, and genomics. Based on multiple findings suggesting the importance of CYP2B enzymes for ingestion of juniper and a major constituent, α-pinene, we characterized the structure, function and activity of several CYP2B enzymes in woodrats with different dietary habits. Results to date suggest that differences in CYP2B gene copy number may contribute to differential tolerance of PSMs among woodrat species, although additional work is warranted to firmly link gene copy number to juniper tolerance.

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.

Terpenes May Serve as Feeding Deterrents and Foraging Cues for Mammalian Herbivores.

Terpenes, volatile plant secondary compounds produced by woody plants, have historically been thought to act as feeding deterrents for mammalian herbivores. However, three species of woodrats, Neotoma stephensi, N. lepida, and N. albigula, regularly consume juniper, which is high in terpenes, and N. stephensi and N. lepida are considered juniper specialists. By investigating the terpene profiles in Juniperus monosperma and J. osteosperma, which are browsed or avoided by woodrats in the field, and recording the caching and consumption of juniper foliage by woodrats in the lab, we have evidence that terpenes may serve as feeding and/or foraging cues. The obligate specialist N. stephensi chose to forage on trees higher in p-cymene and preferred to consume juniper rather than caching it in a laboratory setting. These observations provide evidence that terpenes serve as a feeding cue and that the obligate specialist's physiological mechanism for metabolizing the terpenes present in juniper may negate the need for caching. The facultative specialist N. lepida chose to forage on trees lower in four terpenes and cached more juniper than the obligate specialist N. stephensi, providing evidence that terpenes serve as a feeding deterrent for N. lepida and that this woodrat species relies on behavioral mechanisms to minimize terpene intake. The generalist N. albigula foraged on trees with higher terpenes levels but consumed the least amount of juniper in the lab and preferred to cache juniper rather than consume it, evidence that terpenes act as foraging but not feeding cues in the generalist. Our findings suggest that volatile plant secondary compounds can act as feeding and/or foraging cues and not just feeding deterrents in mammalian herbivores.

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.

Evidence for functional convergence in genes upregulated by herbivores ingesting plant secondary compounds.

BACKGROUND: Nearly 40 years ago, Freeland and Janzen predicted that liver biotransformation enzymes dictated diet selection by herbivores. Despite decades of research on model species and humans, little is known about the biotransformation mechanisms used by mammalian herbivores to metabolize plant secondary compounds (PSCs). We investigated the independent evolution of PSC biotransformation mechanisms by capitalizing on a dramatic diet change event-the dietary inclusion of creosote bush (Larrea tridentata)-that occurred in the recent evolutionary history of two species of woodrats (Neotoma lepida and N. bryanti). RESULTS: By comparing gene expression profiles of two populations of woodrats with evolutionary experience to creosote and one population naïve to creosote, we identified genes either induced by a diet containing creosote PSCs or constitutively higher in populations with evolutionary experience of creosote. Although only one detoxification gene (an aldo-keto reductase) was induced by both experienced populations, these populations converged upon functionally equivalent strategies to biotransform the PSCs of creosote bush by constitutively expressing aldehyde and alcohol dehydrogenases, Cytochromes P450s, methyltransferases, glutathione S-transferases and sulfotransferases. The response of the naïve woodrat population to creosote bush was indicative of extreme physiological stress. CONCLUSIONS: The hepatic detoxification system of mammals is notoriously complex, with hundreds of known biotransformation enzymes. The comparison herein of woodrat taxa that differ in evolutionary and ecological experience with toxins in creosote bush reveals convergence in the overall strategies used by independent species after a historical shift in diet. In addition, remarkably few genes seemed to be important in this dietary shift. The research lays the requisite groundwork for future studies of specific biotransformation pathways used by woodrats to metabolize the toxins in creosote and the evolution of diet switching in woodrats. On a larger level, this work advances our understanding of the mechanisms used by mammalian herbivores to process toxic diets and illustrates the importance of the selective relationship of PSCs in shaping herbivore diversity.

Captivity results in disparate loss of gut microbial diversity in closely related hosts.

The gastrointestinal tracts of animals contain diverse communities of microbes that provide a number of services to their hosts. There is recent concern that these communities may be lost as animals enter captive breeding programmes, due to changes in diet and/or exposure to environmental sources. However, empirical evidence documenting the effects of captivity and captive birth on gut communities is lacking. We conducted three studies to advance our knowledge in this area. First, we compared changes in microbial diversity of the gut communities of two species of woodrats (Neotoma albigula, a dietary generalist, and Neotoma stephensi, which specializes on juniper) before and after 6-9 months in captivity. Second, we investigated whether reintroduction of the natural diet of N. stephensi could restore microbial diversity. Third, we compared the microbial communities between offspring born in captivity and their mothers. We found that the dietary specialist, N. stephensi, lost a greater proportion of its native gut microbiota and overall diversity in response to captivity compared with N. albigula. Addition of the natural diet increased the proportion of the original microbiota but did not restore overall diversity in N. stephensi. Offspring of N. albigula more closely resembled their mothers compared with offspring-mother pairs of N. stephensi. This research suggests that the microbiota of dietary specialists may be more susceptible to captivity. Furthermore, this work highlights the need for further studies investigating the mechanisms underlying how loss of microbial diversity may vary between hosts and what an acceptable level of diversity loss may be to a host. This knowledge will aid conservation biologists in designing captive breeding programmes effective at maintaining microbial diversity. Sequence Accession Numbers: NCBI's Sequence Read Archive (SRA) - SRP033616.

An in vivo assay for elucidating the importance of cytochromes P450 for the ability of a wild mammalian herbivore (Neotoma lepida) to consume toxic plants.

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.

Biotransformation enzyme expression in the nasal epithelium of woodrats.

When herbivores come in contact with volatile plant secondary compounds (PSC) that enter the nasal passages the only barrier between the nasal cavity and the brain is the nasal epithelium and the biotransformation enzymes present there. The expression of two biotransformation enzymes Cytochrome P450 2B (CYP2B) and glutathione-S-transferase (GST) was investigated in the nasal epithelia and livers of three populations of woodrats. One population of Neotoma albigula was fed juniper that contains volatile terpenes. Juniper caused upregulation of CYP2B and GST in the nasal epithelium and the expression of CYP2B and GST in the nasal epithelium was correlated to liver expression, showing that the nasal epithelia responds to PSC and the response is similar to the liver. Two populations of Neotoma bryanti were fed creosote that contains less volatile phenolics. The creosote naive animals upregulated CYP2B in their nasal epithelia while the creosote experienced animals upregulated GST. There was no correlation between CYP2B and GST expression in the nasal epithelia and livers of either population. The response of the nasal epithelium to PSC seems to be an evolved response that is PSC and experience dependent.

Differential expression and activity of catechol-O-methyl transferase (COMT) in a generalist (Neotoma albigula) and juniper specialist (Neotoma stephensi) woodrat.

Mammalian herbivores, particularly dietary specialists must have an efficient means to metabolize the high doses of plant secondary compounds they consume. We found previously that Neotoma stephensi, a juniper specialist, upregulated catechol-O-methyl transferase (COMT) mRNA almost seven fold in response to an ecologically relevant diet (70% juniper). To further investigate the relevance of this enzyme with respect to juniper metabolism, we compared the protein expression, activity and kinetics of the two forms of COMT, soluble (S-COMT) and membrane bound (MB-COMT), in the blood, kidneys and liver of N. stephensi on its natural juniper diet to that of N. stephensi fed an experimental diet of 70% juniper as well as a non-toxic control diet under laboratory conditions. In addition, we compared these results to that of Neotoma albigula, a generalist species, which consumes a diet of 25% juniper in the wild. The specialist consuming juniper under both field and laboratory conditions had increased S-COMT expression and activity in their livers and kidneys, and increased S-COMT activity in their blood compared to the specialist and generalist fed the control diet. The specialist showed expression and activity of S-COMT in their kidneys that was as high as or higher than that in their livers. The generalist had an elevated V(max) for MB-COMT compared to the specialist that resulted in higher activity for MB-COMT than the specialist despite lower expression of MB-COMT in the generalist's livers and kidneys. This high activity MB-COMT may be in part responsible for differences in the behaviors of the generalist compared to the specialist. We conclude that S-COMT is important in the specialist's ability to consume high levels of juniper.

Flavonoids have differential effects on glucose absorption in rats (Rattus norvegicus) and American robins (Turdis migratorius).

Mounting evidence suggests that small birds rely largely on non-mediated intestinal absorption of glucose through the paracellular pathway, while non-flying mammals rely on mediated absorption across the enterocyte membranes by using glucose transporters SGLT-1 and GLUT-2. Relying on non-mediated transport of glucose may decrease its absorption rate at low glucose concentrations but may release small birds from the effects of glucose transport inhibitors. We evaluated transport by using flavonoids known to inhibit glucose transport in vitro. Quercetin, isoquercetrin, and phloridzin were tested in rats (Rattus norvegicus) and robins (Turdis migratirius), and naringenin, naringenin-7-glucoside, genistein, epigallocatechin gallate (EGCG), and phloretin were used only in rats. By using a pharmacokinetic approach that involves serial blood collection and area under the curve calculations, we determined the bioavailability of 3-0-methyl D-glucose, the non-metabolized analogue of D-glucose. Six of the eight flavonoids tested in rats significantly decreased the absorption of 3-0-methyl D-glucose, while none of the flavonoids tested in robins significantly decreased the bioavailability of 3-0-methyl D-glucose. We conclude that flavonoids effectively decrease glucose absorption in rats, which rely on mediated absorption of glucose, but that flavonoids do not have an effect in robins, which rely on non-mediated absorption of glucose.

An oak (Quercus agrifolia) specialist (Neotoma macrotis) and a sympatric generalist (Neotoma lepida) show similar intakes and digestibilities of oak.

Dietary specialization is thought to be rare in mammalian herbivores as a result of either a limitation in their detoxification system to metabolize higher doses of plant secondary compounds or deficiencies in nutrients present in a diet composed of a single species of plant. Neotoma macrotis is an oak specialist, whereas Neotoma lepida is a dietary generalist when sympatric with N. macrotis. We hypothesized that N. macrotis would have a higher tolerance for and digestibility of oak. We determined the two species' tolerances for oak by feeding them increasing concentrations of ground oak leaves until they could no longer maintain body mass. The highest concentration on which both species maintained body mass was 75% oak. There were no differences between the species in their abilities to digest dry matter, nitrogen, or fiber in the oak diets. The species' similar tolerances for oak were probably due to their similar abilities to digest and potentially assimilate the ground oak leaves.

Differential hepatic gene expression of a dietary specialist (Neotoma stephensi) and generalist (Neotoma albigula) in response to juniper (Juniperus monosperma) ingestion.

Dietary specialization is thought to be rare in mammalian herbivores because of limitations of their detoxification system in processing large doses of a single type of plant secondary compound (PSC). Therefore, in order to specialize on a single species of plant, mammalian herbivores must have a highly efficient detoxification system for the particular types of PSCs they ingest. Using microarray technology, we looked at the expression of hepatic genes of a dietary specialist, Neotoma stephensi, and a sympatric generalist, Neotoma albigula, in response to diets containing different levels of one-seeded juniper (Juniperus monosperma). We found large between species differences in gene expression, as well as large within species differences when specialists fed a low juniper diet (25% juniper) were compared to specialists fed their ecologically relevant level of juniper (70% juniper). We also tested the hypothesis that the specialist relies on less costly phase I detoxification enzymes more than phase II compared to the generalist. Although we found that the specialist had higher cumulative as well as average expression of phase I versus phase II enzymes, the generalist had a similar pattern of expression for phase I versus phase II enzymes.

Application of pharmacological approaches to plant-mammal interactions.

The dominant theory in the field of mammalian herbivore-plant interactions is that intake, and therefore tolerance, of plant secondary metabolites (PSMs) is regulated by mechanisms that reduce absorption and increase detoxification of PSMs. Methods designed by pharmacologists to measure detoxification enzyme activity, metabolite excretion, and most recently, drug absorption, have been successfully applied by ecologists to study PSM intake in a variety of mammalian study systems. Here, we describe several pharmacological and molecular techniques used to investigate the fate of drugs in human that have potential to further advance knowledge of mammalian herbivore-plant interactions.

Do salivary proline-rich proteins counteract dietary hydrolyzable tannin in laboratory rats?

We hypothesized that dietary hydrolyzable tannins would not act as digestibility reducing substances but would be excreted in the feces if the tannin were ingested by rats producing salivary proline-rich proteins (PRPs). To test that hypothesis we used two groups of Sprague-Dawley rats: tannin-naïve rats that were secreting basal levels of salivary PRPs and tannin-habituated rats that were secreting elevated levels of PRPs. The animals were fed for 10-18 d on diets containing 3% (w/w) purified hydrolyzable tannin [pentagalloyl glucose (PGG)] that was periodically spiked with chemically synthesized, radiolabeled 1,2,3,4,6-penta-O-galloyl-[U-14C]-D-glucopyranose (1 microCi per gram diet). The PGG-habituated rats excreted three times more of the consumed 14C in their feces than did the PGG-naive rats (11.4% for PGG-habituated rats vs. 3.5% for PGG-naïve rats, P < 0.05). The addition of 3% PGG to the diet of the PGG-naïve rats had no significant effect on apparent dry matter or nitrogen digestibility (P > 0.05). However, dry matter digestibility and nitrogen digestibility were significantly decreased by PGG in the diets of the PGG-habituated rats (7 and 25%, P < 0.001, respectively). Production of PRPs increased the amount of PGG excreted intact in the feces but at the cost of diminishing apparent dry matter and nitrogen digestibility.