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The use of glyphosate-based herbicides (GBHs) to control weeds has increased exponentially in recent decades, and their residues and degradation products have been found in soils across the globe. GBH residues in soil have been shown to affect plant physiology and specialised metabolite biosynthesis, which, in turn, may impact plant resistance to biotic stressors. In a greenhouse study, we investigated the interactive effects between soil GBH residues and herbivory on the performance, phytohormone concentrations, phenolic compound concentrations and volatile organic compound (VOC) emissions of two woodland strawberry (Fragaria vesca) genotypes, which were classified as herbivore resistant and herbivore susceptible. Plants were subjected to herbivory by strawberry leaf beetle (Galerucella tenella) larvae, and to GBH residues by growing in soil collected from a field site with GBH treatments twice a year over the past eight years. Soil GBH residues reduced the belowground biomass of the susceptible genotype and the aboveground biomass of both woodland strawberry genotypes. Herbivory increased the belowground biomass of the resistant genotype and the root-shoot ratio of both genotypes. At the metabolite level, herbivory induced the emission of several VOCs. Jasmonic acid, abscisic acid and auxin concentrations were induced by herbivory, in contrast to salicylic acid, which was only induced by herbivory in combination with soil GBH residues in the resistant genotype. The concentrations of phenolic compounds were higher in the resistant genotype compared to the susceptible genotype and were induced by soil GBH residues in the resistant genotype. Our results indicate that soil GBH residues can differentially affect plant performance, phytohormone concentrations and phenolic compound concentrations under herbivore attack, in a genotype-dependent manner. Soil GBH altered plant responses to herbivory, which may impact plant resistance traits and species interactions. With ongoing agrochemical pollution, we need to consider plant cultivars with better resistance to polluted soils while maintaining plant resilience under challenging environmental conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Plants employ diverse anti-herbivore defences that can covary to form syndromes consisting of multiple traits. Such syndromes are hypothesized to impact herbivores more than individual defences. We studied 16 species of lowland willows occurring in central Europe and explored if their chemical and physical traits form detectable syndromes. We tested for phylogenetic trends in the syndromes and explored whether three herbivore guilds (i.e., generalist leaf-chewers, specialist leaf-chewers, and gallers) are affected more by the detected syndromes or individual traits. The recovered syndromes showed low phylogenetic signal and were mainly defined by investment in concentration, richness, or uniqueness of structurally related phenolic metabolites. Resource acquisition traits or inducible volatile organic compounds exhibited a limited correlation with the syndromes. Individual traits composing the syndromes showed various correlations to the assemblages of herbivores from the three studied guilds. In turn, we found some support for the hypothesis that defence syndromes are composed of traits that provide defence against various herbivores. However, individual traits rather than trait syndromes explained more variation for all studied herbivore assemblages. The detected negative correlations between various phenolics suggest that investment trade-offs may occur primarily among plant metabolites with shared metabolic pathways that may compete for their precursors. Moreover, several traits characterizing the recovered syndromes play additional roles in willows other than defence from herbivory. Taken together, our findings suggest that the detected syndromes did not solely evolve as an anti-herbivore defence., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The study investigated how the concentration and composition of purified tannin extracts, at various inclusion rates, affect the ruminal in vitro fermentation parameters. Tannin extracts were isolated from four different forage species: birdsfoot trefoil (Lotus corniculatus), sulla (Hedysarum coronarium), big trefoil (Lotus pedunculatus), and salad burnet (Sanguisorba minor). Plants extracts were purified by Sephadex LH-20 gel chromatography and analyzed by UPLC-ESI-MS/MS. The results showed a large variation among the extracts from different species in terms of tannin composition and structural features. The extracts from salad burnet were dominated by hydrolysable tannins, comprising mainly ellagitannins. The extracts derived from sulla and big trefoil contained predominantly proanthocyanidins (PA), primarily composed of prodelphinidins with high mean degree of polymerisation (mDP). Birdsfoot trefoil extracts comprised procyanidin-rich PAs with low mDP. To determine whether the combined presence of tannins and flavonoid together lead to synergistic or antagonistic effects, the tannin extracts were incubated both with or without rutin at concentrations of 10, 20, and 30 g/kg DM, using a base substrate of perennial ryegrass (Lolium perenne, control). In general, all the tannin extracts decreased methane (CH 4 ) production compared to the control, while no significant effect of rutin was observed on both gas (GP) and CH 4 production, neither pure, nor in the simultaneous presence of tannins. The highest CH 4 reduction (15%, at 30 g/kg DM) was observed from sulla and big trefoil extracts compared to control, but this was also supplemented with a concomitant reduction in GP (11%) indicating a reduction in feed digestibility. The extracts from birdsfoot trefoil and salad burnet reduced CH 4 by up to 12% without significantly reducing GP, indicating the importance of tannin composition on ruminal fermentation., (© 2024. The Author(s).)

Calendula officinalis L. is a well-known plant widely used in traditional medicine due to the presence of various biologically active compounds. The main raw material for the production of medicinal preparations is the inflorescence, which consists of ligulate and tubular flowers. However, the characteristics of the metabolome of these flowers are not fully understood. This study identified and compared the levels of major metabolites in the ligulate and tubular flowers of two C. officinalis cultivars, 'Golden Sea' (GS) and 'Paradise Garden' (PG). The metabolome was analysed using ultra-performance liquid chromatography with photodiode array detection and a Q Exactive Orbitrap high-resolution mass spectrometer. It was found that the tubular flowers of both PG and GS cultivars had higher levels of lipids, phenolamides and caffeoylquinic acids and lower levels of triterpenoid glycosides than the ligulate flowers. It was also shown that the inflorescences of the GS, which had a 35% higher proportion of tubular flowers, contained 30% more phenolic compounds and 50% more lipids than the PG. Thus, the results obtained extend our understanding of the features in the metabolomes of ligulate and tubular flowers and suggest that the quality of inflorescences of C. officinalis cultivars, as a source of medicinal preparations, is strongly influenced by the proportion of ligulate and tubular flowers.

Polyphagous insect herbivores feed on multiple host-plant species and face a highly variable chemical landscape. Comparative studies of polyphagous herbivore metabolism across a range of plants is an ideal approach for exploring how intra- and interspecific chemical variation shapes species interactions. We used polyphagous caterpillars of Lymantria mathura (Erebidae, Lepidoptera) to explore mechanisms that may contribute to its ability to feed on various hosts. We focused on intraspecific variation in polyphenol metabolism, the fates of individual polyphenols, and the role of previous feeding experience on polyphenol metabolism and leaf consumption. We collected the caterpillars from Acer amoenum (Sapindaceae), Carpinus cordata (Betulaceae), and Quercus crispula (Fagaceae). We first fed the larvae with the leaves of their original host and characterized the polyphenol profiles in leaves and frass. We then transferred a subset of larvae to a different host species and quantified how host shifting affected their leaf consumption and polyphenol metabolism. There was high intraspecific variation in frass composition, even among caterpillars fed with one host. While polyphenols had various fates when ingested by the caterpillars, most of them were passively excreted. When we transferred the caterpillars to a new host, their previous experience influenced how they metabolized polyphenols. The one-host larvae metabolized a larger quantity of ingested polyphenols than two-host caterpillars. Some of these metabolites could have been sequestered, others were probably activated in the gut. One-host caterpillars retained more of the ingested leaf biomass than transferred caterpillars. The pronounced intraspecific variation in polyphenol metabolism, an ability to excrete ingested metabolites and potential dietary habituation are factors that may contribute to the ability of L. mathura to feed across multiple hosts. Further comparative studies can help identify if these mechanisms are related to differential host-choice and response to host-plant traits in specialist and generalist insect herbivores., Competing Interests: We declare that we have no conflict of interest., (© 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

The analysis of proanthocyanidins (PA) in red wine has typically been conducted using few key methods, such as phloroglucinolysis or precipitation assays. Here, the content of PAs and other common polyphenol groups in commercial red wines were analyzed with a group-specific liquid chromatography-tandem mass spectrometry method. Besides concentrations, the method provides qualitative information about the detected compound groups in the form of two-dimensional (2D) chromatographic fingerprints. The 2D fingerprints of PAs have not been utilized in analysis of red wine before. For instance, 2D chromatographic fingerprints revealed that the complex PA compositions were qualitatively notably similar between many wine types, even when there were considerable differences in concentrations. Finally, 201 commercial red wines had been categorized as either tannic or medium tannic based on their sensorial evaluations. The content of PAs and three different groups of oligomeric adducts of malvidin glycosides and PAs were measured from these wines. The compositional features of the PAs and PA-malvidin glycoside adducts were more important than concentrations in explaining the perceived tannicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gastrointestinal nematode (GIN) infections impact small ruminant health, welfare, and production across farming systems. Rising anthelmintic resistance and regulation of synthetic drug use in organic farming is driving research and development of sustainable alternatives for GIN control. One alternative is the feeding of plants that contain secondary metabolites (PSMs) e.g., proanthocyanidins (PA, syn. condensed tannins) that have shown anthelmintic potential. However, PSMs can potentially impair performance, arising from reduced palatability and thus intake, digestibility or even toxicity effects. In this study, we tested the trade-off between the antiparasitic and anti-nutritional effects of heather consumption by lambs. The impact of additional feeding of a nematophagous fungus (Duddingtonia flagrans) on larval development was also explored. Lambs infected with Teladorsagia circumcincta or uninfected controls, were offered ad libitum heather, or a control chopped hay for 22 days during the infection patent period. Eight days into the patent period, parasitised lambs were supplemented (or remained unsupplemented) with D. flagrans for a 5-day period. Performance and infection metrics were recorded, and polyphenol levels in the heather and control hay were measured to investigate their association with activity. The lambs consumed heather at approximately 20% of their dry matter intake, which was sufficient to exhibit significant anthelmintic effects via a reduction in total egg output (P = 0.007), compared to hay-fed lambs; the magnitude of the reduction over time in heather fed lambs was almost 10-fold compared to control lambs. Negative effects on production were shown, as heather-fed lambs weighed 6% less than hay-fed lambs (P < 0.001), even though dry matter intake (DMI) of heather increased over time. D. flagrans supplementation lowered larval recovery in the faeces of infected lambs by 31.8% (P = 0.003), although no interactions between feeding heather and D. flagrans were observed (P = 0.337). There was no significant correlation between PA, or other polyphenol subgroups in the diet and egg output, which suggests that any association between heather feeding and anthelmintic effect is not simply and directly attributable to the measured polyphenols. The level of heather intake in this study showed no antagonistic effects on D. flagrans, demonstrating the methods can be used in combination, but provide no additive effect on overall anthelmintic efficacies. In conclusion, heather feeding can assist to reduce egg outputs in infected sheep, but at 20% of DMI negative effects on lamb performance can be expected which may outweigh any antiparasitic benefits., Competing Interests: Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Polyphenols are phytochemicals commonly found in plant-based diets which have demonstrated immunomodulatory and anti-inflammatory properties. However, the interplay between polyphenols and pathogens at mucosal barrier surfaces has not yet been elucidated in detail. Here, we show that proanthocyanidin (PAC) polyphenols interact with gut parasites to influence immune function and gut microbial-derived metabolites in mice. PAC intake inhibited mastocytosis during infection with the small intestinal roundworm Heligmosomoides polygyrus , and altered the host tissue transcriptome at the site of infection with the large intestinal whipworm Trichuris muris , with a notable enhancement of type-1 inflammatory and interferon-driven gene pathways. In the absence of infection, PAC intake promoted the expansion of Turicibacter within the gut microbiota, increased fecal short chain fatty acids, and enriched phenolic metabolites such as phenyl-γ-valerolactones in the cecum. However, these putatively beneficial effects were reduced in PAC-fed mice infected with T. muris , suggesting concomitant parasite infection can attenuate gut microbial-mediated PAC catabolism. Collectively, our results suggest an inter-relationship between a phytonutrient and infection, whereby PAC may augment parasite-induced inflammation (most prominently with the cecum dwelling T. muris ), and infection may abrogate the beneficial effects of health-promoting phytochemicals.

Ten dimeric procyanidin (PC) analogs were hemisynthesized from catechin or epicatechin and from five different aldehydes using the same mechanism that produces the important acetaldehyde-mediated adducts of proanthocyanidins (PAs) and anthocyanins in red wine. Protein precipitation capacity (PPC), octanol-water partition coefficient (log P ) and stability of the PC analogs were determined. The emphasis was on the PPC because it has been shown to correlate with anthelmintic activity against gastrointestinal nematodes in ruminants and with other beneficial bioactivities in animals, as well. The PPC of PC analogs was greatly improved compared to natural PC dimers, but the capacity was not as great as that of a PC trimer or epigallocatechin gallate. The log P of PC analogs varied from hydrophobic to hydrophilic depending on the intramolecular linkage. Great variation was observed in stabilities of PC analogs in phosphate buffered saline, and the mixtures of degradation products were characterized using high-resolution mass spectrometry.

Plant species show large variation in the composition and content of their tannins and other polyphenols. These large metabolites are not easy to measure accurately, but they are important factors for species bioactivity and chemotaxonomy. Here, we used an automated group-specific UHPLC-DAD-MS/MS tool to detect and quantify eight most common polyphenol groups in 31 chemically diverse plant species representing many types of growth forms and evolutionary ages. Ten replicate plants were used for each species and two polyphenol-related bioactivities, i.e., protein precipitation capacity and oxidative activity were measured in all samples as well. By the help of a novel 2D fingerprint mapping tool we were able to visualize the qualitative and quantitative differences between the species in hydrolysable tannins (galloyl and hexahydroxydiphenoyl derivatives), proanthocyanidins (procyanidins and prodelphinidins), flavonols (kaempferol, quercetin and myricetin derivatives) and quinic acid derivatives together with the two bioactivities. The highest oxidative activities were found with species containing ellagitannins (e.g., Quercus robur , Geranium sylvaticum , Lythrum salicaria and Chamaenerion angustifolium ) or prodelphinidin-rich proanthocyanidins (e.g., Ribes alpinum , Salix phylicifolia and Lysimachia vulgaris ). The best species with high protein precipitation capacity were rich in gallotannins ( Acer platanoides and Paeonia lactiflora ) or oligomeric ellagitannins (e.g., Comarum palustre , Lythrum salicaria and Chamaenerion angustifolium ). These types of tools could prove their use in many types of screening experiments and might reveal even unusually active polyphenol types directly from the crude plant extracts.

The currently changing climates and environments place plants under many types of stresses that affect both their survival and levels of chemical defenses. The gradual induction of defenses in stressed plant populations could be monitored on a yearly basis unless a seasonal and yearly variation in natural defense levels obscures such monitoring schemes. Here, we studied the stability of the species-specific polyphenol composition and content of 10 tree species over three growing seasons using five replicate trees per species. We specifically measured hydrolyzable tannins (galloyl and hexahydroxydiphenoyl derivatives), proanthocyanidins (procyanidins and prodelphinidins), flavonols (kaempferol, quercetin and kaempferol derivatives) and quinic acid derivatives with the group-specific UHPLC-DAD-MS/MS tool, together with two bioactivities, the protein precipitation capacity and oxidative activity. With the help of a fingerprint mapping tool, we found out that species differed a lot in their seasonal and between-year variation in polyphenols and that the variation was also partially specific to compound groups. Especially ellagitannins tended to have declining seasonal patterns while the opposite was true for proanthocyanidins. Some of the species showed minimal variation in all measured variables, while others showed even induced levels of certain polyphenol groups during the 3-year study. For every species, we found either species-specific baseline levels in qualitative and quantitative polyphenol chemistry or the compound groups with the most plasticity in their production. The used tools could thus form a good combination for future studies attempting to monitor the overall changes in polyphenol chemistry due to various biotic or abiotic stress factors in plant populations or in more controlled environments.

Plants produce diverse chemical defenses with contrasting effects on different insect herbivores. Deploying herbivore-specific responses can help plants increase their defensive efficiency. Here, we explore how variation in induced plant responses correlates with herbivore species, order, feeding guild, and level of specialization. In a greenhouse experiment, we exposed 149 plants of Salix fragilis (Linnaeus, 1753) to 22 herbivore species naturally associated with this host. The insects belonged to four orders (Coleoptera, Lepidoptera, Hemiptera, and Hymenoptera), three feeding guilds (external leaf-chewers, leaf-tying chewers, and sap-sucking), and included both dietary specialists and generalists. Following herbivory, we quantified induced changes in volatiles and nonvolatile leaf metabolites. We performed multivariate analyses to assess the correlation between herbivore order, feeding guild, dietary specialization, chewing damage by herbivores, and induced responses. The volatile composition was best explained by chewing damage and insect order, with Coleoptera and Lepidoptera eliciting significantly different responses. Furthermore, we recorded significant differences in elicited volatiles among some species within the two orders. Variation in nonvolatile leaf metabolites was mainly explained by the presence of insects, as plants exposed to herbivores showed significantly different metabolites from controls. Herbivore order also played a role to some extent, with beetles eliciting different responses than other herbivores. The induction of volatile and nonvolatile leaf metabolites shows different levels of specificity. The specificity in volatiles could potentially serve as an important cue to specialized predators or parasitoids, increasing the efficacy of volatiles as indirect defenses. By contrast, the induction of nonvolatile leaf metabolites was largely unaffected by herbivore identity. Most nonvolatile metabolites were downregulated, possibly indicating that plants redirected their resources from leaves in response to herbivory. Our results demonstrate how diverse responses to herbivores can contribute to the diversity of plant defensive strategies., Competing Interests: There are no conflicts of interest to declare., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Proanthocyanidins (PAs) are a class of plant specialized metabolites with well-documented bioactivities such as antiparasitic effects. However, little is known about how the modification of PAs influences their bioactivity. The objective of this study was to investigate a wide range of PA-containing plant samples to determine if extracts containing PAs modified by oxidation had altered antiparasitic activities, compared to the original extracts that had not been modified in alkaline conditions. We extracted and analyzed samples from 61 proanthocyanidin-rich plants. The extracts were then oxidized under alkaline conditions. We used these non-oxidized and oxidized proanthocyanidin-rich extracts to conduct a detailed analysis of direct antiparasitic effects against the intestinal parasite Ascaris suum in vitro. These tests showed that the proanthocyanidin-rich extracts had antiparasitic activity. Modification of these extracts significantly increased the antiparasitic activity for the majority the extracts, suggesting that the oxidation procedure enhanced the bioactivity of the samples. Some samples that showed no antiparasitic activity before oxidation showed very high activity after the oxidation. High levels of other polyphenols in the extracts, such as flavonoids, was found to be associated with increased antiparasitic activity following oxidation. Thus, our in vitro screening opens up the opportunity for future research to better understand the mechanism of action how alkaline treatment of PA-rich plant extracts increases their biological activity and potential as novel anthelmintics., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Conventional agricultural practices favoring the use of glyphosate-based herbicides (GBHs) increase the risk of GBH residues ending up in animal feed, feces, and, eventually, manure. The use of poultry manure as organic fertilizer in the circular food economy increases the unintentional introduction of GBH residues into horticultural and agricultural systems, with reportedly negative effects on the growth and reproduction of crop plants. To understand the potential lasting effects of exposure to GBH residues via organic manure fertilizers, we studied strawberry (Fragaria x vescana) plant performance, yield quantity, biochemistry, folivory, phytochemistry, and soil elemental composition the year after exposure to GBH. Although plants exposed to GBH residues via manure fertilizer were, on average, 23% smaller in the year of exposure, they were able to compensate for their growth during the following growing season. Interestingly, GBH residue exposure in the previous growing season led to a trend in altered plant size preferences of folivores during the following growing season. Furthermore, the plants that had been exposed to GBH residues in the previous growing season produced 20% heavier fruits with an altered composition of phenolic compounds compared to non-exposed plants. Our results indicate that GBHs introduced via manure fertilizer following circular economy practices in one year can have effects on perennial crop plants in the following year, although GBH residues in soil have largely vanished., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Gastrointestinal nematode (GIN) control is traditionally achieved with the use of anthelmintic drugs, however due to regulations in organic farming and the rise in anthelmintic resistance, alternatives are sought after. A promising alternative is the use of bioactive plant feeding due to the presence of plant secondary metabolites (PSMs) such as proanthocyanidins (PAs). This study focussed on the perennial shrub heather (Ericaceae family), a plant rich in PAs, highly abundant across Europe and with previously demonstrated anthelmintic potential., Methods: In vitro assays were used to investigate heather's anthelmintic efficacy against egg hatching and larval motility. Heather samples were collected from five European countries across two seasons, and extracts were tested against two GIN species: Teladorsagia circumcincta and Trichostrongylus colubriformis. Polyphenol group-specific ultraperformance liquid chromatography-tandem mass spectrometry analysis was performed to identify relevant polyphenol subgroups present, including the PA concentration and size and ratio of the subunits. Partial least squares analysis was performed to associate efficacy with variation in PSM composition., Results: Heather extracts reduced egg hatching of both GIN species in a dose-dependent manner by up to 100%, while three extracts at the highest concentration (10 mg/ml) reduced larval motility to levels that were not significantly different from dead larvae controls. PAs, particularly the procyanidin type, and flavonol derivatives were associated with anthelmintic activity, and the particular subgroup of polyphenols associated with the efficacy was dependent on the GIN species and life stage., Conclusions: Our results provide in vitro evidence that heather, a widely available plant often managed as a weed in grazing systems, has anthelmintic properties attributed to various groups of PSMs and could contribute to sustainable GIN control in ruminant production systems across Europe., (© 2022. The Author(s).)

Precipitation of bovine serum albumin (BSA) by 21 hydrolyzable tannins (HTs) and the characteristics of the insoluble complexes were studied stoichiometrically by ultra-performance liquid chromatography. With regard to HT monomers, the protein precipitation and the characteristic of the formed precipitates were unique for each studied HT and depended upon the functional groups present in the structures. The monomeric units comprising the oligomers formed the functional units important for the protein precipitation capacity, and small structural differences among the monomer units were less important than the overall oligomer size and flexibility. In addition, the greater tendency of certain HTs to form insoluble complexes when mixed with BSA was partially linked to the higher self-association and consequent stronger cooperative binding of these HTs with BSA.

In this study, a chemotaxonomic tool was created on the basis of ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) for the identification of 13 common Finnish deciduous trees and shrubs from their leaf bud metabolites. The bud extracts were screened with UHPLC-ESI-QqQ-MS and UHPLC-ESI-Q-Orbitrap-MS to discover suitable markers for each species. Two approaches were tested in the marker selection: (1) unique species-specific markers to obtain selective fingerprints per species and (2) major markers to maximise the sensitivity of the fingerprints. The markers were used to create two selected ion-recording-based fingerprinting tools with UHPLC-ESI-QqQ-MS. The methods were evaluated for their selectivity, repeatability, and robustness in plant species identification by analysing leaf buds from several replicates of each species. The created chemotaxonomic tools were shown to provide unique chromatographic profiles for the studied species in less than 6 min. A variety of plant metabolites, such as flavonoids, triterpenoids, and hydroxycinnamic acid derivatives, were found to serve as good chemotaxonomic markers for the studied species. In 10 out of 13 cases, species-specific markers were superior in creating selective and repeatable fingerprints., Competing Interests: The authors declare no conflict of interest.

Insect herbivores have evolved a broad spectrum of adaptations in response to the diversity of chemical defences employed by plants. Here we focus on two species of New Guinean Asota and determine how these specialist moths deal with the leaf alkaloids of their fig (Ficus) hosts. As each focal Asota species is restricted to one of three chemically distinct species of Ficus, we also test whether these specialized interactions lead to similar alkaloid profiles in both Asota species. We reared Asota caterpillars on their respective Ficus hosts in natural conditions and analyzed the alkaloid profiles of leaf, frass, caterpillar, and adult moth samples using UHPLC-MS/MS analyses. We identified 43 alkaloids in our samples. Leaf alkaloids showed various fates. Some were excreted in frass or found in caterpillars and adult moths. We also found two apparently novel indole alkaloids-likely synthesized de novo by the moths or their microbiota-in both caterpillar and adult tissue but not in leaves or frass. Overall, alkaloids unique or largely restricted to insect tissue were shared across moth species despite feeding on different hosts. This indicates that a limited number of plant compounds have a direct ecological function that is conserved among the studied species. Our results provide evidence for the importance of phytochemistry and metabolic strategies in the formation of plant-insect interactions and food webs in general. Furthermore, we provide a new potential example of insects acquiring chemicals for their benefit in an ecologically relevant insect genus., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

An in vitro Hohenheim gas test was conducted to analyze the fermentation end-products from 17 cultivars of eight polyphenol containing forage species. The polyphenol composition and proanthocyanidin (PA) structural features of all the cultivars were analyzed with UPLC-MS/MS in leaves of vegetative or generative plants. The samples were incubated with and without polyethylene glycol (PEG, a tannin-binding agent) to separate the tannin-effect on methane (CH 4, ml/200 mg DM) production from that of forage quality. Sulla and big trefoil, two particularly PA rich species, were found to have the highest CH 4 reduction potential of up to 47% when compared to the samples without PEG. However, concomitant reduction in gas production (GP, ml/200 mg DM) of up to 44% was also observed. An increase in both GP and CH 4 production under PEG treatments, confirms the role of tannins in CH 4 reduction. Moreover, PA structural features and concentration were found to be an important source of variation for CH 4 production from PA containing species. Despite having low polyphenol concentrations, chicory and plantain were found to reduce CH 4 production without reducing GP. Additionally, interspecies variability was found to be higher than intraspecies variability, and these results were consistent across growth stages, indicating the findings' representativeness., (© 2022. The Author(s).)

Proanthocyanidins (PAC) are dietary polyphenols with putative anti-inflammatory and immunomodulatory effects. However, whether dietary PAC can regulate type-2 immune function and inflammation at mucosal surfaces remains unclear. Here, we investigated if diets supplemented with purified PAC modulated pulmonary and intestinal mucosal immune responses during infection with the helminth parasite Ascaris suum in pigs. A. suum infection induced a type-2 biased immune response in lung and intestinal tissues, characterized by pulmonary granulocytosis, increased Th2/Th1 T cell ratios in tracheal-bronchial lymph nodes, intestinal eosinophilia, and modulation of genes involved in mucosal barrier function and immunity. Whilst PAC had only minor effects on pulmonary immune responses, RNA-sequencing of intestinal tissues revealed that dietary PAC significantly enhanced transcriptional responses related to immune function and antioxidant responses in the gut of both naïve and A. suum-infected animals. A. suum infection and dietary PAC induced distinct changes in gut microbiota composition, primarily in the jejunum and colon, respectively. Notably, PAC consumption substantially increased the abundance of Limosilactobacillus reuteri. In vitro experiments with porcine macrophages and intestinal epithelial cells supported a role for both PAC polymers and PAC-derived microbial metabolites in regulating oxidative stress responses in host tissues. Thus, dietary PAC may have distinct beneficial effects on intestinal health during infection with mucosal pathogens, while having a limited activity to modulate naturally-induced type-2 pulmonary inflammation. Our results shed further light on the mechanisms underlying the health-promoting properties of PAC-rich foods, and may aid in the design of novel dietary supplements to regulate mucosal inflammatory responses in the gastrointestinal tract., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Grazing ruminants contribute to global climate change through enteric methane and nitrous oxide emissions. However, animal consumption of the plant polyphenolics, proanthocyanidins, or condensed tannins (CTs) can decrease both methane emissions and urine nitrogen levels, leading to reduced nitrous oxide emissions, and concomitantly increase animal health and production. CTs are largely absent in the foliage of important temperate pasture legumes, such as white clover ( Trifolium repens ), but found in flowers and seed coats. Attempts at enhancing levels of CT expression in white clover leaves by mutagenesis and breeding have not been successful. However, the transformation of white clover with the TaMYB14-1 transcription factor from Trifolium arvense has resulted in the production of CTs in leaves up to 1.2% of dry matter (DM). In this study, two generations of breeding elevated foliar CTs to >2% of DM. The CTs consisted predominantly of prodelphinidins (PD, 75-93%) and procyanidins (PC, 17-25%) and had a mean degree of polymerization (mDP) of approximately 10 flavan-3-ol subunits. In vitro studies showed that foliar CTs were bound to bovine serum albumin and white clover proteins at pH 6.5 and were released at pH 2.-2.5. Using rumen in vitro assays, white clover leaves containing soluble CTs of 1.6-2.4% of DM significantly reduced methane production by 19% ( p ≤0.01) and ammonia production by 60% ( p ≤ 0.01) relative to non-transformed wild type (WT) controls after 6 h of incubation. These results provide valuable information for further studies using CT expressing white clover leaves for bloat prevention and reduced greenhouse gas emissions in vivo ., Competing Interests: JC is employed by Grasslanz Technology Ltd., who owns the patent for the TaMYB14 transcription factor. DW was the primary breeder involved with the development of the cultivar “Grasslands Mainstay” and is employed by PGG Wrightson Seeds Ltd., who will have the exclusive license from Grasslanz Technology Ltd for use of TaMYB14 in white clover. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Roldan, Cousins, Muetzel, Zeller, Fraser, Salminen, Blanc, Kaur, Richardson, Maher, Jahufer, Woodfield, Caradus and Voisey.)

Previous studies have illustrated that different bioactive legume fodders containing condensed tannins might represent one of the options for integrated sustainable control of gastrointestinal nematodes (GIN) in ruminants, which may help address the worldwide development of resistance to synthetic anthelmintics. More recently, impetus has been given to assess the potential antiparasitic activity of less conventional resources, represented by different agro-industrial by-products (AIBPs). This review presents in vitro and in vivo results obtained with a range of tannin-containing AIBPs of various geographical and botanical origins, namely AIBP of nuts, temperate and tropical barks, carob, coffee and cocoa. They tend to confirm the "proof of concept" for their antiparasitic effects and also for other aspects of ruminant production in an agro-ecological context. Socio-economic aspects of the exploitation of such non-conventional resources are also discussed as potential models of the circular economy, by using waste. The different modes of use of these resources are presented in this review, as well as strengths, weaknesses, opportunities, and threats (SWOT) analyses to illustrate the advantages and limitations of on-farm use., (© H. Hoste et al., published by EDP Sciences, 2022.)

Staphylococcus aureus is the most common cause of surgical site infections and its treatment is challenging due to the emergence of multi-drug resistant strains such as methicillin-resistant S. aureus (MRSA). Natural berry-derived compounds have shown antimicrobial potential, e.g., ellagitannins such as sanguiin H-6 and lambertianin C, the main phenolic compounds in Rubus seeds, have shown antimicrobial activity. The aim of this study was to evaluate the effect of sanguiin H-6 and lambertianin C fractionated from cloudberry seeds, on the MRSA growth, and as treatment of a MRSA biofilm development in different growth media in vitro and in vivo by using a murine wound infection model where sanguiin H-6 and lambertianin C were used to prevent the MRSA infection. Sanguiin H-6 and lambertianin C inhibited the in vitro biofilm development and growth of MRSA. Furthermore, sanguiin H-6 showed significant anti-MRSA effect in the in vivo wound model. Our study shows the possible use of sanguiin H-6 as a preventive measure in surgical sites to avoid postoperative infections, whilst lambertianin C showed no anti-MRSA activity.

Substantial efforts have been made in incorporating tannin-rich forages into grassland-based livestock production systems. However, the structural and functional diversity of tannins in different species limits their potential use at the field scale. We conducted a greenhouse experiment with 17 cultivars from 8 forage species and their cultivars. Ultraperformance liquid chromatography tandem mass spectrometry was used to analyze their polyphenolic profile and proanthocyanidin (PA) structural features in leaves. Our results highlight large inter- and intraspecies variability of plants in terms of polyphenol and tannin concentrations in the leaves. A concomitant and significant variation was also registered in the structural features of PA-rich forages such as the mean degree of polymerization and prodelphinidin percentage. The concentration of PA also varied within plant organs; the highest concentration was in flowers, but leaves had the highest contribution to harvestable PA biomass. Our research highlights that identifying these variations helps in identifying the representativeness of bioactivity and provides the basis for targeted breeding programs.

Proanthocyanidins (PAC) are dietary compounds that have been extensively studied for beneficial health effects due to their anti-inflammatory properties. However, the structure-function relationships of PAC and their mode-of-action remain obscure. Here, we isolated a wide range of diverse PAC polymer mixtures of high purity from plant material. Polymer size was a key factor in determining the ability of PAC to regulate inflammatory cytokine responses in murine macrophages. PAC polymers with a medium (9.1) mean degree of polymerization (mDP) induced substantial transcriptomic changes, whereas PAC with either low (2.6) or high (12.3) mDP were significantly less active. Short-term oral treatment of mice with PAC modulated gene pathways connected to nutrient metabolism and inflammation in ileal tissue in a polymerization-dependent manner. Mechanistically, the bioactive PAC polymers modulated autophagic flux and inhibited lipopolysaccharide-induced autophagy in macrophages. Collectively, our results highlight the importance of defined structural features in the health-promoting effects of PAC-rich foods., (© 2021. The Author(s).)

Seeds collected from trees, shrubs and lianas growing on Barro Colorado Island, Panama, were analyzed for their content of phenolic compounds, oxidative activities and protein precipitation capacities. Proanthocyanidins and hydrolysable tannins were detected in one-third of 189 studied species. The most oxidatively active group of species were the ones containing prodelphinidins and ellagitannins whereas the species that had the highest protein precipitation capacity in relation to their total phenolics were the ones containing punicalagin. In addition, the oxidative activity and relative protein precipitation capacity were exceptionally high in the proanthocyanidin-rich genus Psychotria. This study offers a comprehensive overview on the tannin composition and the alkaline oxidative activities and protein precipitation capacities of the seeds of tropical plants., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Flavonoids, such as anthocyanins, proanthocyanidins, and flavonols, are widespread plant secondary metabolites and important for plant adaptation to diverse abiotic and biotic stresses. Flavonoids can be variously hydroxylated and decorated; their biological activity is partly dependent on the degree of hydroxylation of the B-ring. Flavonoid biosynthesis is regulated by MYB transcription factors, which have been identified and characterized in a diversity of plants. Here we characterize a new MYB activator, MYB117, in hybrid poplar (Populus tremula×tremuloides). When overexpressed in transgenic poplar plants, MYB117 enhanced anthocyanin accumulation in all tissues. Transcriptome analysis of MYB117-overexpressing poplars confirmed the up-regulation of flavonoid and anthocyanin biosynthesis genes, as well as two flavonoid 3',5'-hydroxylase (F3'5'H) genes. We also identified up-regulated cytochrome b5 genes, required for full activity of F3'5'H . Phytochemical analysis demonstrated a corresponding increase in B-ring hydroxylation of anthocyanins, proanthocyanidins, and flavonols in these transgenics. Similarly, overexpression of F3'5'H1 directly in hybrid poplar also resulted in increased B-ring hydroxylation, but without affecting overall flavonoid content. However, the overexpression of the cytochrome b5 gene in F3'5'H1-overexpressing plants did not further increase B-ring hydroxylation. Our data indicate that MYB117 regulates the biosynthesis of anthocyanins in poplar, but also enhances B-ring hydroxylation by up-regulating F3'5'H1., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In this study, we analyzed the proanthocyanidin (PA) composition of 55 plant extracts before and after alkaline oxidation by ultrahigh-resolution UHPLC-MS/MS. We characterized the natural PA structures in detail and studied the sophisticated changes in the modified PA structures and the typical patterns and models of reactions within different PA classes due to the oxidation. The natural PAs were A- and B-type PCs, PDs and PC/PD mixtures. In addition, we detected galloylated PAs. B-type PCs in different plant extracts were rather stable and showed no or minor modification due to the alkaline oxidation. For some samples, we detected the intramolecular reactions of PCs producing A-type ether linkages. A-type PCs were also rather stable with no or minor modification, but in some plants, the formation of additional ether linkages was detected. PAs containing PD units were more reactive. After alkaline oxidation, these PAs or their oxidation products were no longer detected by MS even though a different type and/or delayed PA hump was still detected by UV at 280 nm. Galloylated PAs were rather stable under alkaline oxidation if they were PC-based, but we detected the intramolecular conversion from B-type to A-type. Galloylated PDs were more reactive and reacted similarly to nongalloylated PDs.

We tested the susceptibility of 102 proanthocyanidin (PA)-rich plant extracts to oxidation under alkaline conditions and the possibility to produce chemically modified PAs via oxidation. Both the nonoxidized and the oxidized extracts were analyzed using group-specific ultrahigh-performance liquid chromatography-diode array detection-tandem mass spectrometry (UHPLC-DAD-MS/MS) methods capable of detecting procyanidin (PC) and prodelphinidin (PD) moieties along the two-dimensional (2D) chromatographic fingerprints of plant PAs. The results indicated different reactivities for PCs and PDs. When detected by UHPLC-DAD only, most of the PC-rich samples exhibited only a subtle change in their PA content, but the UHPLC-MS/MS quantitation showed that the decrease in the PC content varied by 0-100%. The main reaction route was concluded to be intramolecular. The PD-rich and galloylated PAs showed a different pattern with high reductions in the original PA content by both ultraviolet (UV) and MS/MS quantitation, accompanied by the shifted retention times of the chromatographic PA humps. In these samples, both intra- and intermolecular reactions were indicated., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)