A specialist herbivore (Neotoma stephensi) absorbs fewer plant toxins than does a generalist (Neotoma albigula)

Detoxification capacity of enzymes in the liver is thought to be the primary factor governing dietary toxin intake by mammalian herbivores. Recently, toxin absorption in the gut was proposed as an alternative process that also influences toxin intake. We examined the role of the gut in regulating toxin absorption by quantifying excretion of a plant secondary compound in the feces. We hypothesized that specialists have a greater capacity to reduce intestinal absorption of toxins than do generalists. To test this hypothesis, we compared fecal excretion of alpha-pinene in specialist (Neotoma stephensi) and generalist (Neotoma albigula) woodrats. Alpha-pinene is the most abundant monoterpene in Juniperus monosperma, which occurs in the natural diet of both woodrat species. Woodrats were fed alpha-pinene in diets containing juniper foliage for 3 wk and, in a separate experiment, were given a single oral dose of alpha-pinene. Feces were collected from animals at the end of each experiment and analyzed for alpha-pinene concentration using gas chromatography. Both woodrat species excreted unchanged alpha-pinene in the feces. However, specialist woodrats excreted 40% more alpha-pinene per unit ingested from a juniper diet and excreted nearly four times a greater percentage of an oral dose of alpha-pinene compared with generalists.     

Diet breadth of mammalian herbivores: nutrient versus detoxification constraints

Two hypotheses, nutrient constraints and detoxification limitation, have been proposed to explain the lack of specialists among mammalian herbivores. The nutrient constraint hypothesis proposes that dietary specialization in mammalian herbivores is rare because no one plant can provide all requisite nutrients. The detoxification limitation hypothesis suggests that the mammalian detoxification system is incapable of detoxifying high doses of similar secondary compounds present in a diet of a single plant species. We experimentally tested these hypotheses by comparing the performance of specialist and generalist woodrats (Neotoma) on a variety of dietary challenges. Neotoma stephensi is a narrow dietary specialist with a single species, one-seeded juniper, Juniperus monosperma, comprising 85–95% of its diet. Compared with other plants available in the habitat, juniper is low in nitrogen and high in fiber, phenolics, and monoterpenes. The generalist woodrat, N. albigula, also consumes one-seeded juniper, but to a lesser degree. The nutrient constraint hypothesis was examined by feeding both species of woodrats a low-nitrogen, high-fiber diet similar to that found in juniper. We found no differences in body mass change, or apparent digestibility of dry matter or nitrogen between the two species of woodrats after 35 days on this diet. Moreover, both species were in positive nitrogen balance. We tested the detoxification limitation hypothesis by comparing the performance of the generalist and specialist on diets with and without juniper leaves, the preferred foliage of the specialist, as well as on diets with and without α-pinene, the predominant monoterpene in juniper. We found that on the juniper diet, compared with the specialist, the generalist consumed less juniper and lost more mass. Urine pH, a general indicator of overall detoxification processes, declined in both groups on the juniper diet. The generalist consumed half the toxin load of the specialist yet its urine pH was slightly lower. Moreover, the generalist consumed significantly less of the treatment with high concentrations of α-pinene compared to the control treatment, while the specialist consumed the same amount of food regardless of α-pinene concentration. For both groups, urine pH declined as levels of α-pinene in the diet increased. The generalist produced a significantly more acidic urine than the specialist on the treatment with the highest α-pinene concentration. Our results suggest that in this system, specialists detoxify plant secondary compounds differently than generalists and plant secondary compounds may be more important than low nutrient levels in maintaining dietary diversity in generalist herbivores. Received: 5 May 1999 / Accepted: 14 November 1999     

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.     

Evidence for local specialization in a generalist mammalian herbivore, Neotoma fuscipes

Herbivores with very plastic dietary requirements, or so-called generalist species, can include individuals that develop specialized feeding habits through their experience with local chemically-defended plants. Local specialization has important implications for understanding a variety of ecological and evolutionary dynamics. However, the extent to which individuals within a generalist species specialize on local plants and the consequences of such specialization remain poorly understood, especially in non-insect herbivores. To better understand this phenomenon, we determined the diet and food preferences of a generalist mammalian herbivore, the dusky-footed woodrat ( Neotoma fuscipes ), in two adjacent but distinct plant communities. Based on a combination of cafeteria trials and stable isotope analyses (δ¹³C and δ¹⁵N), our results indicate that woodrats display preferences for local plants and tend to avoid novel chemically-defended plants. Moreover, both methodologies support the conclusion that individual woodrats are dietary specialists restricting their diets to only a few (2-3) of the available plant species. In juniper woodland, woodrats prefer western juniper ( Juniperus occidentalis ), while less than one km away in mixed-coniferous forest, woodrats prefer incense cedar ( Calocedrus decurrens ). Both plants contain high levels of plant secondary compounds that require detoxification mechanisms within consumers. Therefore, preferences are likely indicative of underlying physiological adaptations that could promote further behavioral, physiological and ultimately genetic differences between woodrats in different habitats. This study provides additional evidence for local specialization and urges caution when using the term generalist to characterize feeding behaviors at the individual level.     

Xenobiotic metabolism of plant secondary compounds in juniper (Juniperus monosperma) by specialist and generalist woodrat herbivores, genus Neotoma

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.     

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.     

Detoxification rates of wild herbivorous woodrats (Neotoma)

The detoxification systems of mammalian herbivores are thought to have evolved in response to the ingestion of plant secondary compounds. Specialist herbivores consume high quantities of secondary compounds and are predicted to have faster rates of Phase 1 detoxification compared to generalist herbivores. We tested this hypothesis by comparing the performances of a specialist (Neotoma fuscipes) and generalist (Neotoma lepida) herbivore using hypnotic state assays. Herbivores foraging in nature were live trapped and injected with hexobarbital (100 mg/kg). We measured the length of time in the hypnotic state as the time in which the animal was unable to right itself twice in 30 s. The specialist metabolized hexobarbital 1.7 times faster than the generalist (F(1, 19) = 9.31, P = 0.007) as revealed by its significantly shorter time spent in the hypnotic state (56+/-9 min vs. 87+/-8 min, respectively). The results are consistent with the hypothesis that specialists have faster rates of Phase 1 detoxification. This is the first evaluation of the detoxification capability of mammalian herbivores foraging under natural conditions. Hypnotic state assays have broad potential applications to the study of vertebrate-plant interactions.     

Testing the diet-breadth trade-off hypothesis: differential regulation of novel plant secondary compounds by a specialist and a generalist herbivore

Specialist herbivores are predicted to have evolved biotransformation pathways that can process large doses of secondary compounds from the plant species on which they specialize. It is hypothesized that this physiological specialization results in a trade-off such that specialists may be limited in ability to ingest novel plant secondary compounds (PSCs). In contrast, the generalist foraging strategy requires that herbivores alternate consumption of plant species and PSC types to reduce the possibility of over-ingestion of any particular PSC. The ability to behaviorally regulate is a key component of this strategy. These ideas underpin the prediction that in the face of novel PSCs, generalists should be better able to maintain body mass and avoid toxic consequences compared to specialists. We explored these predictions by comparing the feeding behavior of two herbivorous rodents: a juniper specialist, Neotoma stephensi, and a generalist, Neotoma albigula, fed diets with increasing concentrations of phenolic resin extracted from the creosote bush (Larrea tridentata), which produces a suite of PSCs novel to both species. The specialist lost more mass than the generalist during the 15-day trial. In addition, although the specialist and generalist both regulated phenolic resin intake by reducing meal size while on the highest resin concentration (4%), the generalist began to regulate intake on the 2% diet. The ability of the generalist to regulate intake at a lower PSC concentration may be the source of the generalist’s performance advantage over the specialist. These data provide evidence for the hypothesis that the specialist’s foraging strategy may result in behavioral as well as physiological trade-offs in the ability to consume novel PSCs.     

Ingestion of plant secondary compounds causes diuresis in desert herbivores

Plant secondary compounds are recognized deterrents and toxins to a variety of herbivores. The effect of secondary compounds on water balance of herbivores is virtually unexplored, yet secondary compounds could potentially cause a decrease in an animal's ability to maintain water balance. We investigated the effects of secondary compounds, alpha-pinene and creosote resin, on water balance in three species of herbivorous woodrats (Neotoma stephensi, N. albigula, N. lepida). In separate experiments, we measured the effect of these secondary compounds on voluntary water consumption, urine volume and urine osmolarity. In both experiments, water intake and urine volume increased and urine osmolarity decreased compared to controls. Water balance of specialist or experienced woodrats was less affected than generalists and woodrats with less prior experience with particular secondary compounds. Our results suggest that secondary compounds have diuretic-like effects on herbivores. Woodrats live in arid habitats with limited access to freestanding water; thus an increase in water requirements may have profound consequences on foraging behavior and fitness.     

Liver biotransforming enzymes in woodrats Neotoma stephensi (Muridae)

Mammalian herbivores are exposed to extremely high levels of plant secondary compounds naturally present in their diet. It has been speculated that specialist herbivores should express a unique pattern of biotransforming enzymes to permit the consumption of a single species of toxic plant. Specifically, specialists should rely on pathways that effectively biotransform the toxins they routinely encounter in their diet. We examined the hepatic mRNA expression and activity or content of biotransforming enzymes in the specialist herbivorous woodrat, Neotoma stephensi, and compared results to those of laboratory rats (Sprague-Dawley strain Rattus norvegicus). In addition, we investigated the role of alpha-pinene, a specific plant toxin present in the diet of N. stephensi on the mRNA expression pattern and activity or content of biotransforming enzymes in Sprague-Dawley rats. Overall, the levels of functionalization enzyme activity and mRNA were found to be higher in specialists, while glucuronidation enzyme activity and mRNA were lower. These results support predictions that specialist herbivores rely more on functionalization biotransformation pathways rather than glucuronidation pathways.     

Response of pest control by generalist predators to local‐scale plant diversity: a meta‐analysis

Disentangling the effects of plant diversity on the control of herbivores is important for understanding agricultural sustainability. Recent studies have investigated the relationships between plant diversity and arthropod communities at the landscape scale, but few have done so at the local scale. We conducted a meta‐analysis of 32 papers containing 175 independent measures of the relationship between plant diversity and arthropod communities. We found that generalist predators had a strong positive response to plant diversity, that is, their abundance increased as plant diversity increased. Herbivores, in contrast, had an overall weak and negative response to plant diversity. However, specialist and generalist herbivores differed in their response to plant diversity, that is, the response was negative for specialists and not significant for generalists. While the effects of scale remain unclear, the response to plant diversity tended to increase for specialist herbivores, but decrease for generalist herbivores as the scale increased. There was no clear effect of scale on the response of generalist predators to plant diversity. Our results suggest that the response of herbivores to plant diversity at the local scale is a balance between habitat and trophic effects that vary according to arthropod specialization and habitat type. Synthesis and applications. Positive effects of plant diversity on generalist predators confirm that, at the local scale, plant diversification of agroecosystems is a credible and promising option for increasing pest regulation. Results from our meta‐analysis suggest that natural control in plant‐diversified systems is more likely to occur for specialist than for generalist herbivores. In terms of pest management, our results indicate that small‐scale plant diversification (via the planting of cover crops or intercrops and reduced weed management) is likely to increase the control of specialist herbivores by generalist predators.     

Specialist and generalist herbivores exert opposing selection on a chemical defense

* Plant defense traits often show high levels of genetic variation, despite clear impacts on plant fitness. This variation may be partly maintained by trade-offs in the defense against multiple herbivore species, for example between generalists and coevolved specialists. Despite a long-standing discussion in the literature on the subject, no study to date has specifically manipulated specialist and generalist herbivores independently of one another to determine whether the two guilds exert opposing selection pressures on specific defensive traits. * In two separate experiments, the dominant specialist and generalist herbivores of Brassica nigra were independently manipulated to test whether the composition of the herbivore community altered the direction of selection on a major defensive trait of the plant, sinigrin concentration. * It was found that generalist damage was negatively correlated but specialist loads were positively correlated with increasing sinigrin concentrations; and sinigrin concentration was favored when specialists were removed, disfavored (past an intermediate point) when generalists were removed and selectively neutral when plants faced both generalists and specialists.     

Revisiting the dietary niche: When is a mammalian herbivore a specialist?

Understanding dietary specialization in herbivores has theoretical and practical implications in ecology, yet defining niche breadth consistently has been problematic. To increase clarity and communication among ecologists and among disciplines (i.e., chemists, pharmacologists), we propose a specialization key for mammalian herbivores that assigns "obligatory" and "facultative" modifiers to the terms "specialist" and "generalist". These modifiers are assigned based on (1) relative breadth of the animal's realized niche and diet (what it eats), (2) relative breadth of the fundamental niche and available diet (what it could eat), (3) the extent of chemical or physical characteristics, termed "difficulty", that make food items either low in value or unpalatable to most herbivores, and (4) relevant temporal and spatial scales at which diets and niche breadth were measured. Obligatory specialists always have a narrow realized niche consisting of difficult food items, and morphological adaptations and/or the loss of redundant behavioral flexibility that effectively limit their fundamental niches, precluding them from expanding their diet under changed environmental conditions. Facultative specialists have a consistently narrow realized niche for difficult foods during at least one spatial or temporal scale, but have a broad enough fundamental niche to allow them to expand their diet to include less difficult foods when environmental conditions allow. Facultative generalists have the broadest fundamental niche, allowing them to consume a wide variety of foods. However, they may occasionally demonstrate a narrow realized niche, focused on less difficult plants than is the case with specialists. Finally, the obligatory generalists always have a wide realized niche because of a relatively narrow fundamental niche, precluding them from eating much of any difficult plant. We summarize hypothesized characteristics of mammalian herbivores in each of the four categories of specialization. We demonstrate the need for further work on defining the realized and fundamental niches, comparing among herbivores across categories conducted under similar conditions, and understanding the nature of trade-offs required for specialization and generalization for both community and phylogenetically based analyses.     

Enemy release but no evolutionary loss of defence in a plant invasion: an inter-continental reciprocal transplant experiment

Plant chemical defenses and escape from natural enemies have been postulated to select for dietary specialization in herbivorous insects. In field and laboratory bioassays, we evaluated the effectiveness of intact and chemically modified larval shield defenses of the generalist Chelymorpha alternans and the specialists Acromis sparsa and Stolas plagiata (Chrysomelidae: Cassidinae) against three natural predators, using larvae reared on two morning glory (Convolvulaceae) species. We assessed whether: (1) specialists were better defended than generalists when both were fed and assayed on the same plant; (2) larval shield defenses were chemical, physical, or both; and (3) specialists exploit chemistry better than generalists. Live specialist larvae survived at higher rates than did generalists in predator bioassays with the bug Montina nigripes (Reduviidae), but there were no differences among groups against two species of Azteca ants (Hymenoptera: Dolichoderinae). Solvent leaching by H₂O or MeOH significantly reduced shield efficacy for all species compared to larvae with intact shields. In contrast, freshly killed specialist larvae exhibited significantly lower capture rates and frequencies than the generalists. Although solvent leaching significantly reduced overall shield efficacy for freshly killed larvae of all species, the pattern of leaching effects differed between specialists and generalists, with H₂O-leaching having a greater impact on the specialists. The overall vulnerability of the generalists appears due to lower chemical protection, which is ameliorated by increased escape behaviors, suggesting a selective trade-off between these defensive components. These experiments indicate that shield defenses are essential for larval survival and that specialists are superior at exploiting plant compounds residing in the aqueous fraction. Our results support the hypothesis that diet-specialized herbivorous insects have more effective defenses than generalists when both feed on the same plant due to the differential ability to exploit defensive precursors obtained from the host. The evolution of dietary specialization may therefore confer the advantage of enhanced enemy-free space.     

The tri-trophic interactions hypothesis: interactive effects of host plant quality, diet breadth and natural enemies on herbivores

Several influential hypotheses in plant-herbivore and herbivore-predator interactions consider the interactive effects of plant quality, herbivore diet breadth, and predation on herbivore performance. Yet individually and collectively, these hypotheses fail to address the simultaneous influence of all three factors. Here we review existing hypotheses, and propose the tri-trophic interactions (TTI) hypothesis to consolidate and integrate their predictions. The TTI hypothesis predicts that dietary specialist herbivores (as compared to generalists) should escape predators and be competitively dominant due to faster growth rates, and that such differences should be greater on low quality (as compared to high quality) host plants. To provide a preliminary test of these predictions, we conducted an empirical study comparing the effects of plant (Baccharis salicifolia) quality and predators between a specialist (Uroleucon macolai) and a generalist (Aphis gossypii) aphid herbivore. Consistent with predictions, these three factors interactively determine herbivore performance in ways not addressed by existing hypotheses. Compared to the specialist, the generalist was less fecund, competitively inferior, and more sensitive to low plant quality. Correspondingly, predator effects were contingent upon plant quality only for the generalist. Contrary to predictions, predator effects were weaker for the generalist and on low-quality plants, likely due to density-dependent benefits provided to the generalist by mutualist ants. Because the TTI hypothesis predicts the superior performance of specialists, mutualist ants may be critical to A. gossypii persistence under competition from U. macolai. In summary, the integrative nature of the TTI hypothesis offers novel insight into the determinants of plant-herbivore and herbivore-predator interactions and the coexistence of specialist and generalist herbivores.     

The energetic advantages of slug specialization in garter snakes (genus Thamnophis)

We tested the hypothesis that dietary specialization by foraging garter snakes is accompanied by increased assimilation efficiency on specialist prey items. Our comparison included two closely related garter snake species considered to be slug specialists (Thamnophis ordinoides and Thamnophis elegans terrestris), one fish specialist (Thamnophis couchii), and one diet generalist (Thamnophis elegans elegans). Our results suggest that slug specialists have an energetic advantage over non-slug-eating snakes when both eat slugs. Slug specialists T. ordinoides and T. e. terrestris both have higher assimilation and net assimilation efficiencies when eating slugs than do generalists T. e. elegans and T. couchii. The slug specialists did not experience decreased efficiency when eating fish. Therefore, there was no apparent digestive trade-off for the slug specialists when eating other prey.     

Comparing responses of generalist and specialist herbivores to various cyanogenic plant features

Plants are obliged to defend themselves against multiple generalist and specialist herbivores. Whereas plant cyanogenesis is considered an efficient defence against generalists, it is thought to affect specialists less. In the present study, we analysed the function of various cyanogenic features of lima bean [Phaseolus lunatus L. (Fabaceae)] during interaction with different herbivores. Three cyanogenic features were analysed, i.e., cyanogenic potential (HCNp; concentration of cyanogenic precursors), β‐glucosidase activity, and cyanogenic capacity (HCNc; release of cyanide per unit time). In no‐choice and free‐choice feeding trials, five lima bean accessions were offered to generalist desert locust [Schistocerca gregaria Forskål (Orthoptera: Acrididae)] and specialist Mexican bean beetle [Epilachna varivestis Mulsant (Coleoptera: Coccinellidae)]. The HCNc was the most important parameter determining host plant selection by generalists, whereas choice behaviour of specialists was strongly affected by HCNp. Although locusts were effectively repelled by high HCNc, this cue was misleading for the detection of suitable host plants, as extensive consumption of low HCNc plant material resulted in strong intoxication of locusts. Balancing cyanide in consumed leaf area, the quantitative release of gaseous cyanide during feeding, and cyanide in faeces suggested that specialists metabolized significantly lower rates of cyanide per consumed leaf material than generalists. We hypothesize that specialists are able to avoid toxic concentrations of cyanide by using HCNp rather than HCNc as a cue for host plant quality, and that they exhibit mechanisms that reduce incorporation of host plant cyanide.     

Specificity of induced plant responses to specialist herbivores of the common milkweed Asclepias syriaca

Induced plant responses to herbivory appear to be universal, yet the degree to which they are specific to sets of herbivores is poorly understood. The generalist/specialist hypothesis predicts that generalist herbivores are more often negatively affected by host plant defenses, wheras specialists may be either unaffected by or attracted to these same "plant defenses". Therefore, specialists should be less predictable than generalists in their responses to induced plant resistance traits. To better understand the variation in plant responses to herbivore attack, and the impacts these responses have on specialist herbivores, we conducted a series of experiments examining pairwise interactinos between two specialaist herbivores of the common milkweed ( Asclepias syriaca ). We damaged plants mechnically, with swamp milkweed beetles ( Labidomera clivicollis ), or with monarchs ( Danaus plexippus ), and then asessed specificity of elicitation, both by measuring a putative defensive trait (latex volume) and by challenging plants with insects of both species in bioasays. Latex production increased by 34% and 13% following beetle and monarch herbivory, respectively, but only beetles significantly elevated latex production compared to undamaged controls. While beetle growth was negatively affected by latex across all experiments, beetles were not affected by previous damage caused by conspecifies or by monarchs. In contrast, monarchs feeding on previously damaged plants were 20% smaller, and their response was the same on plants damaged mechnically or by either herbivore. Therefore, these specialist herbivores exhibit both specificity of elicitation in plant responses and specificity of effects in response to prior damage.