Investigation of glucosinolates in the desert plant Ochradenus baccatus (Brassicales: Resedaceae). Unveiling glucoochradenin, a new arabinosylated glucosinolate.

Here we describe the structure elucidation and quantification of six glucosinolates (GSLs) from the roots of the desert plant Ochradenus baccatus, Delile 1813 (family Resedaceae; order Brassicales). The structure elucidation was established on the corresponding enzymatically desulfated derivatives of the native GSLs of the plant. Among these GSLs we describe the previously undescribed 2″-O-(α-L-arabinopyranosyloxy)benzylglucosinolate (1a), for which we propose the name glucoochradenin. The other five glucosinolates (2a-6a) were (2S)-2-hydroxy-2-phenylethylglucosinolate (2a; glucobarbarin), 2″-O-(α-L-rhamnopyranosyloxy)benzylglucosinolate (3a), benzylglucosinolate (4a; glucotropaeolin), indol-3-ylmethylglucosinolate (5a; glucobrassicin) and phenethylglucosinolate (6a; gluconasturtiin), all elucidated as their desulfo-derivatives, 2b-6b respectively). Structures were elucidated by MS and 1D and 2D-NMR techniques, the identity of the arabinose verified by ion chromatography, and the absolute configuration of the sugar units determined by hydrolysis, coupling to cysteine methyl-ester and phenyl isothiocyanate followed by HPLC-MS analysis of the resulted diastereomers. Response factors were generated for desulfo-2″-O-(α-L-arabinopyranosyloxy)benzylglucosinolate and for desulfo-2″-O-(α-L-rhamnopyranosyloxy)benzylglucosinolate and all six GSLs were quantified, indicating that the root of O. baccatus is rich in GSLs (Avg. 61.3 ± 10.0 µmol/g DW and up to 337.2 µmol/g DW).

Effects of Fruit Toxins on Intestinal and Microbial ß-Glucosidase Activities of Seed-Predating and Seed-Dispersing Rodents (Acomys spp.).

Plant secondary compounds (PSCs) have profound influence on the ecological interaction between plants and their consumers. Glycosides, a class of PSC, are inert in their intact form and become toxic on activation by either plant ß-glucosidase enzymes or endogenous ß-glucosidases produced by the intestine of the plant-predator or its microbiota. Many insect herbivores decrease activities of endogenous ß-glucosidases to limit toxin exposure. However, such an adaptation has never been investigated in nonmodel mammals. We studied three species of spiny mice (Acomys spp.) that vary in their feeding behavior of the glycoside-rich fruit of Ochradenus baccatus. Two species, the common (Acomys cahirinus) and Crete (Acomys minous) spiny mice, behaviorally avoid activating glycosides, while the golden spiny mouse (Acomys russatus) regularly consumes activated glycosides. We fed each species a nontoxic diet of inert glycosides or a toxic diet of activated fruit toxins and investigated the responses of intestinal and microbial ß-glucosidase activities. We found that individuals feeding on activated toxins had lower intestinal ß-glucosidase activity and that the species that behaviorally avoid activating glycosides also had lower intestinal ß-glucosidase activity regardless of treatment. The microbiota represented a larger source of toxin liberation, and the toxin-adapted species (golden spiny mouse) exhibited almost a fivefold increase in microbial ß-glucosidase when fed activated toxins, while other species showed slight decreases. These results are contrary to those in insects, where glycoside-adapted species have lower ß-glucosidase activity. The glycoside-adapted golden spiny mouse may have evolved tolerance mechanisms such as enhanced detoxification rather than avoidance mechanisms.

Izhakiella capsodis gen. nov., sp. nov., in the family Enterobacteriaceae, isolated from the mirid bug Capsodes infuscatus.

Gram-stain-negative, oxidase-negative, facultatively anaerobic, motile, rod-shaped, non-pigmented bacterial strains (N6PO6T, N8PO1 and N8PI1) were isolated from the mirid bug Capsodes infuscatus captured on Asphodelus aestivus plants. The 16S rRNA gene sequences of the strains shared 94.7-95.7 % similarity with species of the genus Pantoea and 95.6 % or less with species from other genera in the family Enterobacteriaceae. A polyphasic approach that included determination of phenotypic properties and phylogenetic analysis based on 16S rRNA, rpoB, gyrB and atpD gene sequences supported the classification of strains N6PO6T, N8PO1 and N8PI1 as representing a novel species of a new genus in the family Enterobacteriaceae. Strain N6PO6T, and the two reference strains of the novel species, grew at 1-37 °C, and in the presence of NaCl (up to 7.5 %, w/v) and sucrose (up to 60 %). Their major cellular fatty acids were C16 : 0, C17 : 0 cyclo, C18 : 1ω7c, summed feature 2 (C14 : 0 3-OH and/or iso-C16 : 1 I) and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The DNA G+C content of strain N6PO6T was 49.9 mol%. On the basis of phenotypic properties and phylogenetic distinctiveness, the mirid bug isolates are classified as representing a novel species in a new genus Izhakiella, in the family Enterobacteriaceae, for which the name Izhakiella capsodis gen. nov., sp. nov. is proposed. The type strain of Izhakiella capsodis is N6PO6T ( = LMG 28430T = DSM 29293T).

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.

Taste and physiological responses to glucosinolates: seed predator versus seed disperser.

In contrast to most other plant tissues, fleshy fruits are meant to be eaten in order to facilitate seed dispersal. Although fleshy fruits attract consumers, they may also contain toxic secondary metabolites. However, studies that link the effect of fruit toxins with seed dispersal and predation are scarce. Glucosinolates (GLSs) are a family of bitter-tasting compounds. The fleshy fruit pulp of Ochradenus baccatus was previously found to harbor high concentrations of GLSs, whereas the myrosinase enzyme, which breaks down GLSs to produce foul tasting chemicals, was found only in the seeds. Here we show the differential behavioral and physiological responses of three rodent species to high dose (80%) Ochradenus' fruits diets. Acomys russatus, a predator of Ochradenus' seeds, was the least sensitive to the taste of the fruit and the only rodent to exhibit taste-related physiological adaptations to deal with the fruits' toxins. In contrast, Acomys cahirinus, an Ochradenus seed disperser, was more sensitive to a diet containing the hydrolyzed products of the GLSs. A third rodent (Mus musculus) was deterred from Ochradenus fruits consumption by the GLSs and their hydrolyzed products. We were able to alter M. musculus avoidance of whole fruit consumption by soaking Ochradenus fruits in a water solution containing 1% adenosine monophosphate, which blocks the bitter taste receptor in mice. The observed differential responses of these three rodent species may be due to evolutionary pressures that have enhanced or reduced their sensitivity to the taste of GLSs.

The role of abiotic environmental conditions and herbivory in shaping bacterial community composition in floral nectar.

Identifying the processes that drive community assembly has long been a central theme in ecology. For microorganisms, a traditional prevailing hypothesis states that "everything is everywhere, but the environment selects". Although the bacterial community in floral nectar may be affected by both atmosphere (air-borne bacteria) and animals as dispersal vectors, the environmental and geographic factors that shape microbial communities in floral nectar are unknown. We studied culturable bacterial communities in Asphodelus aestivus floral nectar and in its typical herbivorous bug Capsodes infuscatus, along an aridity gradient. Bacteria were sampled from floral nectar and bugs at four sites, spanning a geographical range of 200 km from Mediterranean to semi-arid conditions, under open and bagged flower treatments. In agreement with the niche assembly hypothesis, the differences in bacterial community compositions were explained by differences in abiotic environmental conditions. These results suggest that microbial model systems are useful for addressing macro-ecological questions. In addition, similar bacterial communities were found in the nectar and on the surface of the bugs that were documented visiting the flowers. These similarities imply that floral nectar bacteria dispersal is shaped not only by air borne bacteria and nectar consumers as previously reported, but also by visiting vectors like the mirid bugs.

Physiological and behavioural effects of fruit toxins on seed-predating versus seed-dispersing congeneric rodents.

Fleshy, ripe fruits attract seed dispersers but also seed predators. Although many fruit consumers (legitimate seed dispersers as well as seed predators) are clearly exposed to plant secondary compounds (PSCs), their impact on the consumers' physiology and foraging behaviour has been largely overlooked. Here, we document the divergent behavioural and physiological responses to fruit consumption of three congeneric rodent species in the Middle East, representing both seed dispersers and seed predators. The fruit pulp of the desert plant Ochradenus baccatus contains high concentrations of glucosinolates (GLSs). These GLSs are hydrolyzed into active toxic compounds upon contact with the myrosinase enzyme released from seeds crushed during fruit consumption. Acomys russatus and A. cahirinus share a desert habitat. Acomys russatus acts as an O. baccatus seed predator, and A. cahirinus circumvents the activation of the GLSs by orally expelling vital seeds. We found that between the three species examined, A. russatus was physiologically most tolerant to whole fruit consumption and even A. minous, which is evolutionarily naïve to O. baccatus, exhibits greater tolerance to whole fruit consumption than A. cahirinus. However, like A. cahirinus, A. minous may also behaviourally avoid the activation of the GLSs by making a hole in the pulp and consuming only the seeds. Our findings demonstrate that seed predators have a higher physiological tolerance than seed dispersers when consuming fruits containing toxic PSCs. The findings also demonstrate the extreme ecological/evolutionary lability of this plant-animal symbiosis to shift from predation to mutualism and vice versa.

Effects of anatomy and diet on gastrointestinal pH in rodents.

The pH of the gastrointestinal tract can have profound influences on digestive processes. Rodents exhibit wide variation in both stomach morphology and dietary strategies, both of which may influence gut pH. Various rodent species have evolved bilocular (or semi-segmented) stomachs that may allow for more microbial growth compared to unilocular (single-chambered) stomachs. Additionally, herbivory has evolved multiple times in rodents. The high dietary fiber typical of an herbivorous diet is known to induce secretion of bicarbonate in the gut. We predicted that stomach segmentation might facilitate the separation of contents in the proximal chamber from that of the gastric stomach, facilitating a chemical environment suitable to microbial growth. To investigate the effect of stomach anatomy and diet on gut pH, several species of rodent with varying stomach morphology were fed either a high or low-fiber diet for 7 days, and pH of the proximal stomach, gastric stomach, small intestine, and cecum were measured. We discovered that rodents with bilocular stomach anatomy maintained a larger pH gradient between the proximal and gastric stomach compartments, and were able to achieve a lower absolute gastric pH compared to those with unilocular stomachs. Dietary fiber increased the pH of the small intestine, but not in any other gut regions. The stomach pH data supports the century old hypothesis that bilocular stomach anatomy creates an environment in the proximal stomach that is suitable for microbial growth. Additionally, the alkaline small intestinal pH on a high fiber diet may enhance digestion.

Intraspecific directed deterrence by the mustard oil bomb in a desert plant.

Plant secondary metabolites (SMs) acting as defensive chemicals in reproductive organs such as fruit tissues play roles in both mutualistic and antagonistic interactions between plants and seed dispersers/predators. The directed-deterrence hypothesis states that SMs in ripe fruits deter seed predators but have little or no effect on seed dispersers. Indeed, studies have demonstrated that birds are able to cope with fruit SMs whereas rodents are deterred by them. However, this mechanism was only demonstrated at the class level, i.e., between birds and mammals, based on differences in the vanilloid receptors. Here we present experimental and behavioral data demonstrating the use of the broad-range, class-independent "mustard oil bomb" mechanism in Ochradenus baccatus fruits to force a behavioral change at an ecological timescale, converting rodents from seed predators to seed dispersers. This is achieved by a unique compartmentalization of the mustard oil bomb, causing activation of the system only upon seed and pulp coconsumption, encouraging seed dispersal via seed spitting by rodents. Our findings demonstrate the power of SMs to shift the animal-plant relationship from predation to mutualism and provide support for the directed-deterrence hypothesis at the intraspecific level, in addition to the interspecific level.

Capacity for absorption of water-soluble secondary metabolites greater in birds than in rodents.

Plant secondary metabolites (SMs) are pervasive in animal foods and potentially influence feeding behavior, interspecies interactions, and the distribution and abundance of animals. Some of the major classes of naturally occurring SMs in plants include many water-soluble compounds in the molecular size range that could cross the intestinal epithelium via the paracellular space by diffusion or solvent drag. There are differences among species in paracellular permeability. Using Middle Eastern rodent and avian consumers of fruits containing SMs, we tested the hypothesis that avian species would have significantly higher paracellular permeability than rodent species. Permeability in intact animals was assessed using standard pharmacological methodology to measure absorption of two radiolabeled, inert, neutral water-soluble probes that do not interact with intestinal nutrient transporters, L-arabinose (M(r) = 150.1 Da) and lactulose (M(r) = 342.3 Da). We also measured absorption of labeled 3-O-methyl-D-glucose (3OMD-glucose; M(r) = 194.2 Da), which is a nonmetabolized analogue of D-glucose that is passively absorbed through the paracellular space but also transported across the enterocyte membranes. Most glucose was absorbed by all species, but arabinose fractional absorption (f) was nearly three times higher in birds (1.03±0.17, n = 15 in two species) compared to rodents (0.37±0.06, n = 10 in two species) (P<0.001). Surprisingly, the apparent rates of absorption in birds of arabinose exceeded those of 3OMD-glucose. Our findings are in agreement with previous work showing that the paracellular pathway is more prominent in birds relative to nonflying mammals, and suggests that birds may be challenged by greater absorption of water-soluble, dietary SMs. The increased expression of the paracellular pathway in birds hints at a tradeoff: the free energy birds gain by absorbing water-soluble nutrients passively may be offset by the metabolic demands placed on them to eliminate concomitantly absorbed SMs.