Ist β-Glucosidase ein Schlüsselenzym für die Anpassung an Iridoidglycoside? Ein Vergleich zwischen herbivoren Generalisten (Arctiidae, Lepidoptera) und einem Iridoidglycosidspezialist (Nymphalidae, Lepidoptera)

Pankoke H. Ist β-Glucosidase ein Schlüsselenzym für die Anpassung an Iridoidglycoside? Ein Vergleich zwischen herbivoren Generalisten (Arctiidae, Lepidoptera) und einem Iridoidglycosidspezialist (Nymphalidae, Lepidoptera).; 2010

Low rates of iridoid glycoside hydrolysis in two Longitarsus leaf beetles with different feeding specialization confer tolerance to iridoid glycoside containing host plants

Pankoke H, Dobler S. Low rates of iridoid glycoside hydrolysis in two Longitarsus leaf beetles with different feeding specialization confer tolerance to iridoid glycoside containing host plants. Physiological Entomology. 2015;40(1):18-29.Iridoid glycosides are plant defence compounds that are deterrent and/or toxic for unadapted herbivores but are readily sequestered by dietary specialists of different insect orders. Hydrolysis of iridoid glycosides by -glucosidase leads to protein denaturation. Insect digestive -glucosidases thus have the potential to mediate plant-insect interactions. In the present study, mechanisms associated with iridoid glycoside tolerance are investigated in two closely-related leaf beetle species (Coleoptera: Chrysomelidae) that feed on iridoid glycoside containing host plants. The polyphagous Longitarsus luridusScopoli does not sequester iridoid glycosides, whereas the specialist Longitarsus tabidusFabricius sequesters these compounds from its host plants. To study whether the biochemical properties of their -glucosidases correspond to the differences in feeding specialization, the number of -glucosidase isoforms and their kinetic properties are compared between the two beetle species. To examine the impact of iridoid glycosides on the -glucosidase activity of the generalist, L. luridus beetles are kept on host plants with or without iridoid glycosides. Furthermore, -glucosidase activities of both species are examined using an artificial -glucosidase substrate and the iridoid glycoside aucubin present in their host plants. Both species have one or two -glucosidases with different substrate affinities. Interestingly, host plant use does not influence the specific -glucosidase activities of the generalist. Both species hydrolyse aucubin with a much lower affinity than the standard substrate. The neutral pH reduces the -glucosidase activity of the specialist beetles by approximately 60% relative to its pH optimum. These low rates of aucubin hydrolysis suggest that the ability to sequester iridoid glycosides has evolved as a key to potentially preventing iridoid glycoside hydrolysis by plant-derived -glucosidases

Impact of the dual defence system of Plantago lanceolata (Plantaginaceae) on performance, nutrient utilisation and feeding choice behaviour of Amata mogadorensis larvae (Lepidoptera, Erebidae)

Pankoke H, Gehring R, Müller C. Impact of the dual defence system of Plantago lanceolata (Plantaginaceae) on performance, nutrient utilisation and feeding choice behaviour of Amata mogadorensis larvae (Lepidoptera, Erebidae). Journal of Insect Physiology. 2015;82:99-108

Role of plant β-glucosidases in the dual defense system of iridoid glycosides and their hydrolyzing enzymes in Plantago lanceolata and Plantago major

Pankoke H, Buschmann T, Müller C. Role of plant β-glucosidases in the dual defense system of iridoid glycosides and their hydrolyzing enzymes in Plantago lanceolata and Plantago major. Phytochemistry. 2013;94:99-107.The typical defense compounds of Plantaginaceae are the iridoid glycosides, which retard growth and/or enhance mortality of non-adapted herbivores. In plants, glycosidic defense compounds and hydrolytic enzymes often form a dual defense system, in which the glycosides are activated by the enzymes to exert biological effects. Yet, little is known about the activating enzymes in iridoid glycoside-containing plants. To examine the role of plant-derived β-glucosidases in the dual defense system of two common plantain species, Plantago lanceolata and Plantago major, we determined the concentration of iridoid glycosides as well as the β-glucosidase activity in leaves of different age. To investigate the presence of other leaf metabolites potentially involved in plant defense, we used a metabolic fingerprinting approach with ultra-high performance liquid chromatography coupled with time-of-flight-mass spectrometry. According to the optimal defense hypothesis, more valuable parts such as young leaves should be better protected than less valuable parts. Therefore, we expected that both, the concentrations of defense compounds as well as the β-glucosidase activity, should be highest in younger leaves and decrease with increasing leaf age. Both species possessed β-glucosidase activity, which hydrolyzed aucubin, one of the two most abundant iridoid glycosides in both plant species, with high activity. In line with the optimal defense hypothesis, the β-glucosidase activity in both Plantago species as well as the concentration of defense-related metabolites such as iridoid glycosides correlated negatively to leaf age. When leaf extracts were incubated with bovine serum albumin and aucubin, SDS–PAGE revealed a protein-denaturing effect of the leaf extracts of both plantain species, suggesting that iridoid glycosides and plant β-glucosidase interact in a dual defense system

Coping with toxic plant compounds – The insect’s perspective on iridoid glycosides and cardenolides

Dobler S, Petschenka G, Pankoke H. Coping with toxic plant compounds – The insect’s perspective on iridoid glycosides and cardenolides. Phytochemistry. 2011;72(13):1593-1604.Specializing on host plants with toxic secondary compounds enforces specific adaptation in insect herbivores. In this review, we focus on two compound classes, iridoid glycosides and cardenolides, which can be found in the food plants of a large number of insect species that display various degrees of adaptation to them. These secondary compounds have very different modes of action: Iridoid glycosides are usually activated in the gut of the herbivores by β-glucosidases that may either stem from the food plant or be present in the gut as standard digestive enzymes. Upon cleaving, the unstable aglycone is released that unspecifically acts by crosslinking proteins and inhibiting enzymes. Cardenolides, on the other hand, are highly specific inhibitors of an essential ion carrier, the sodium pump. In insects exposed to both kinds of toxins, carriers either enabling the safe storage of the compounds away from the activating enzymes or excluding the toxins from sensitive tissues, play an important role that deserves further analysis. To avoid toxicity of iridoid glycosides, repression of activating enzymes emerges as a possible alternative strategy. Cardenolides, on the other hand, may lose their toxicity if their target site is modified and this strategy has evolved multiple times independently in cardenolide-adapted insects

The interplay between toxin-releasing β-glucosidase and plant iridoid glycosides impairs larval development in a generalist caterpillar, Grammia incorrupta (Arctiidae)

Pankoke H, Bowers MD, Dobler S. The interplay between toxin-releasing β-glucosidase and plant iridoid glycosides impairs larval development in a generalist caterpillar, Grammia incorrupta (Arctiidae). Insect Biochemistry and Molecular Biology. 2012;42(6):426-434.Herbivores with polyphagous feeding habits must cope with a diet that varies in quality. One of the most important sources of this variation in host plant suitability is plant secondary chemistry. We examined how feeding on plants containing one such group of compounds, the iridoid glycosides, might affect the growth and enzymatic activity in a polyphagous caterpillar that feeds on over 80 plant species in 50 different families. Larvae of the polyphagous arctiid, Grammia incorrupta, were reared exclusively on one of two plant species, one of which contains iridoid glycosides (Plantago lanceolata, Plantaginaceae) while the other does not (Taraxacum officinale, Asteraceae). Larval weight was measured on the two host plants, and midgut homogenates of last instar larvae were then assayed for activity and kinetic properties of β-glucosidases, using both a standard substrate, 4-nitrophenyl-β-d-glucose (NPβGlc), and the iridoid glycoside aucubin, one of the two main iridoid glycosides in P. lanceolata. Larvae feeding on P. lanceolata weighed significantly less and developed more slowly compared to larvae on T. officinale. While the larval midgut β-glucosidase activity determined with NPβGlc was significantly decreased when fed on P. lanceolata, aucubin was substantially hydrolyzed and the larval β-glucosidase activity towards both substrates correlated negatively with larval weight. Our results demonstrate that host plants containing high concentrations of iridoid glycosides have a negative impact on larval development of this generalist insect herbivore. This is most likely due to the hydrolysis of plant glycosides in the larval midgut which results in the release of toxic aglycones. Linking the reduced larval weight to the toxin-releasing action of an iridoid glycoside cleaving β-glucosidase, our results thus support the detoxification limitation hypothesis, suggesting fitness costs for the larvae feeding solely on P. lanceolata. Thus, in addition to the adaptive regulation of midgut β-glucosidase activity, host plant switching as a behavioral adaptation might be a prerequisite for generalist herbivores that allows them to circumvent the negative effects of plant secondary compounds

Influence of iridoid glycoside containing host plants on midgut β-glucosidase activity in a polyphagous caterpillar, Spilosoma virginica Fabricius (Arctiidae)

Pankoke H, Bowers MD, Dobler S. Influence of iridoid glycoside containing host plants on midgut β-glucosidase activity in a polyphagous caterpillar, Spilosoma virginica Fabricius (Arctiidae). Journal of Insect Physiology. 2010;56(12):1907-1912.Iridoid glycosides are secondary plant compounds that have deterrent, growth reducing or even toxic effects on non-adapted herbivorous insects. To investigate the effects of iridoid glycoside containing plants on the digestive metabolism of a generalist herbivore, larvae of Spilosoma virginica (Lepidoptera: Arctiidae) were reared on three plant species that differ in their secondary plant chemistry: Taraxacum officinale (no iridoid glycosides), Plantago major (low iridoid glycoside content), and P. lanceolata (high iridoid glycoside content). Midguts of fifth instar larvae were assayed for the activity and kinetic properties of β-glucosidase using different substrates. Compared to the larvae on T. officinale, the β-glucosidase activity of larvae feeding on P. lanceolata was significantly lower measured with 4-nitrophenyl-β-d-glucopyranoside. Using the iridoid glycoside aucubin as a substrate, we did not find differences in the β-glucosidase activity of the larvae reared on the three plants. Heat inactivation experiments revealed the existence of a heat-labile and a more heat-stable β-glucosidase with similar Michaelis constants for 4-nitrophenyl-β-d-glucopyranoside. We discuss possible mechanisms leading to the observed decrease of β-glucosidase activity for larvae reared on P. lanceolata and its relevance for generalist herbivores in adapting to iridoid glycoside containing plant species and their use as potential host plants

Evidence for a deterrent effect of cardenolides on Nephila spiders

Petschenka G, Bramer C, Pankoke H, Dobler S. Evidence for a deterrent effect of cardenolides on Nephila spiders. Basic and Applied Ecology. 2011;12(3):260-267.Sequestration of toxic plant compounds by herbivorous insects as a defence against predators has been observed in many tritrophic systems. In this study, we focused on the potential benefit of sequestered cardenolides, potent toxins that block the Na+K+-ATPase against an invertebrate predator. To evaluate the effect of cardenolides we used three different types of cardenolide-containing prey, which were offered to the orb-weaving spider Nephila senegalensis. As a natural system spiders were presented with the cardenolide-sequestering moth Empyreuma pugione. To reduce the complexity of the predator–prey interaction spiders were furthermore fed with ouabain-laced non-toxic flies. Lastly, we developed a new reductionistic assay involving gelatine capsules as insect dummies. Our results show that ouabain elicits significant rejection behaviour and suggest cardenolides to be the basis for rejection of E. pugione by N. senegalensis

The effects of mineral nitrogen limitation, competition, arbuscular mycorrhiza, and their respective interactions, on morphological and chemical plant traits of Plantago lanceolata

Pankoke H, Höpfner I, Matuszak A, Beyschlag W, Müller C. The effects of mineral nitrogen limitation, competition, arbuscular mycorrhiza, and their respective interactions, on morphological and chemical plant traits of Plantago lanceolata. Phytochemistry. 2015;118:149-161

A Synbiotic Formulation Comprising Bacillus subtilis DSM 32315 and L-Alanyl-L-Glutamine Improves Intestinal Butyrate Levels and Lipid Metabolism in Healthy Humans

The gut microbiota is a crucial modulator of health effects elicited by food components, with SCFA (short chain fatty acids), especially butyrate, acting as important mediators thereof. We therefore developed a nutritional synbiotic composition targeted at shifting microbiome composition and activity towards butyrate production. An intestinal screening model was applied to identify probiotic Bacillus strains plus various amino acids and peptides with suitable effects on microbial butyrate producers and levels. A pilot study was performed to test if the synbiotic formulation could improve fecal butyrate levels in healthy humans. A combination of Bacillus subtilis DSM (Number of German Collection of Microorganisms and Cell Cultures) 32315 plus L-alanyl-L-glutamine resulted in distinctly increased levels of butyrate and butyrate-producing taxa (Clostridium group XIVa, e.g., Faecalibacterium prausnitzii), both in vitro and in humans. Moreover, circulating lipid parameters (LDL-, and total cholesterol and LDL/HDL cholesterol ratio) were significantly decreased and further metabolic effects such as glucose-modulation were observed. Fasting levels of PYY (Peptide YY) and GLP-1 (Glucagon-like Peptide 1) were significantly reduced. In conclusion, our study indicates that this synbiotic composition may provide an effective and safe tool for stimulation of intestinal butyrate production with effects on e.g., lipid and glucose homeostasis. Further investigations in larger cohorts are warranted to confirm and expand these findings