Grayanotoxin I variation across tissues and species of Rhododendron suggests pollinator-herbivore defence trade-offs

Grayanotoxin I (GTX I) is a major toxin in leaves of Rhododendron species, where it provides a defence against insect and vertebrate herbivores. Surprisingly, it is also present in R. ponticum nectar, and this can hold important implications for plant-pollinator mutualisms. However, knowledge of GTX I distributions across the genus Rhododendron and in different plant materials is currently limited, despite the important ecological function of this toxin. Here we characterise GTX I expression in the leaves, petals, and nectar of seven Rhododendron species. Our results indicated interspecific variation in GTX I concentration across all species. GTX I concentrations were consistently higher in leaves compared to petals and nectar. Our findings provide preliminary evidence for phenotypic correlation between GTX I concentrations in defensive tissues (leaves and petals) and floral rewards (nectar), suggesting that Rhododendron species may commonly experience functional trade-offs between herbivore defence and pollinator attraction

Grayanotoxin I variation across different tissues and species of Rhododendron suggest herbivore defence trade-offs

Grayanotoxin I (GTX I) is a major toxin in leaves of Rhododendron species, where it provides a defence against insect and vertebrate herbivores. Surprisingly, it is also present in R. ponticum nectar, and this can hold important implications for plant-pollinator mutualisms. However, knowledge of GTX I distributions across the genus Rhododendron and in different plant materials is currently limited, despite the important ecological function of this toxin. Here we characterise GTX I expression in the leaves, petals, and nectar of seven Rhododendron species. Our results indicated interspecific variation in GTX I concentration across all species.GTX I concentrations were consistently higher in leaves compared to petals and nectar. Our findings provide preliminary evidence for phenotypic correlation between GTX I concentrations in defensive tissues (leaves and petals) and floral rewards (nectar), suggesting that Rhododendron species may commonly experience functional trade-offs between herbivore defence and pollinator attraction

Insecticidal activity in Tithonia diversifolia and Vernonia amygdalina

The diversity of synthetic pesticides has been reduced through regulation especially in the European Union leading to a resurgence of interest in natural plant products for pest control. Here we investigated two Asteraceae species, Tithonia diversifolia and Vernonia amygdalina that are used by farmers in Africa in bio rational pest control to determine the chemical basis of activity against pests of stored legumes and identify plant compounds with commercial potential. The cowpea beetle, Callosobruchus maculatus, an ubiquitous pest of African stored grain legumes, was exposed to extracts of both plant species at 10, 1 and 0.1% w/v and fractions of these extracts at representative concentrations. Extracts and fractions were toxic to recently emerged adults, but did not reduce oviposition by those females that survived. The sesquiterpene, Tagitinin A, was isolated from one of the active fractions and identified using H1 and C13-NMR and shown also be toxic to C. maculatus and so partially explains the activity of the whole plant. Other compounds in the active fractions were identified, at least to structural class, using high resolution mass spectroscopy (HRESI-MS). Sequiterpenes and flavones were common to fractions from both plants. Stigmostane steroidal saponins were the most abundant secondary metabolites in V. amygdalina

Agri-environment scheme nectar chemistry can suppress the social epidemiology of parasites in an important pollinator

Emergent infectious diseases are one of the main drivers of species loss. Emergent infection with the microsporidian Nosema bombi has been implicated in the population and range declines of a suite of North American bumblebees, a group of important pollinators. Previous work has shown that phytochemicals found in pollen and nectar can negatively impact parasites in individuals, but how this relates to social epidemiology and by extension whether plants can be effectively used as pollinator disease management strategies remains unexplored. Here we undertook a comprehensive screen of UK agri-environment scheme plants, a programme designed to benefit pollinators and wider biodiversity in agricultural settings, for phytochemicals in pollen and nectar using liquid chromatography and mass spectrometry. Caffeine, which occurs across a range of plant families, was identified in the nectar of Sainfoin (Onobrychis viciifolia), a component of UK agri-environment schemes and a major global crop. We showed that caffeine significantly reduces N. bombi infection intensity, both prophylactically and therapeutically, in individual bumblebees (Bombus terrestris), and, for the first time, that such effects impact social epidemiology, with colonies reared from wild caught queens having both lower prevalence and intensity of infection. Furthermore, infection prevalence was lower in foraging bumblebees from caffeine treated colonies, suggesting a likely reduction in population-level transmission. Combined, these results show that N. bombi is less likely to be transmitted intra-colonially when bumblebees consume naturally-available caffeine, and that this may in turn reduce environmental prevalence. Consequently, our results demonstrate that floral phytochemicals at ecologically relevant concentrations can impact pollinator disease epidemiology and that planting strategies that increase floral abundance to support biodiversity could be co-opted as disease management tools

Nor-hopanes from Zanha africana root bark with toxicity to bruchid beetles

Zanha africana (Radlk.) Exell (Sapindaceae) root bark is used by farmers throughout sub-Saharan Africa to protect stored grain from bruchid beetles, such as Callosobruchus maculatus. Chloroform, methanol and water extracts of Z. africana root bark inhibited oviposition and caused significantly higher mortality of C. maculatus at a rate of application equivalent to that applied by farmers compared to control insects. The chloroform extract contained nor-hopanes rarely found in plants of which seven were isolated, one of which was previously known. Two of the most abundant nor-hopanes 3β,6β-dihydroxy-7β-[(4-hydroxybenzoyl)oxy]-21αH-24-norhopa-4(23),22(29)-diene and 3β,6β-dihydroxy-7β-[(4-hydroxybenzoyl)oxy]-24-norhopa-4(23),17(21)-diene were toxic to and reduced oviposition of C. maculatus in a dose dependent manner. Z. africana root bark is rich in insecticidal compounds that account for its effective use by smallholder farmers as an alternative to conventional insecticides

Chemistry of floral rewards: intra- and interspecific variability of nectar and pollen secondary metabolites across taxa

Floral chemistry mediates plant interactions with pollinators, pathogens, and herbivores, with major consequences for fitness of both plants and flower visitors. The outcome of such interactions often depends on compound dose and chemical context. However, chemical diversity and intraspecific variation of nectar and pollen secondary chemistry are known for very few species, precluding general statements about their composition. We analyzed methanol extracts of flowers, nectar, and pollen from 31 cultivated and wild plant species, including multiple sites and cultivars, by liquid chromatography-mass spectrometry. To depict the 29 chemical niche of each tissue type, we analyzed differences in nectar and pollen chemical richness, absolute and proportional concentrations, and intraspecific variability. We hypothesized that pollen would have higher concentrations and more compounds than nectar, consistent with Optimal Defense Theory and pollen’s importance as a male gamete. To investigate chemical correlations across and within tissues, which could reflect physiological constraints, we quantified chemical overlap between conspecific nectar and pollen, and phenotypic integration of individual compounds within tissue types Nectar and pollen were chemically differentiated both across and within species. Of 102 compounds identified, most occurred in only one species. Machine-learning algorithms assigned samples to the correct species and tissue type with 98.6% accuracy. Consistent with our hypothesis, pollen had 23.8- to 235-fold higher secondary chemical concentrations and 63% higher chemical richness than nectar. The most common secondary compound classes were flavonoids, alkaloids, terpenoids, and phenolics (primarily phenylpropanoids including chlorogenic acid). The most common specific compound types were quercetin and kaempferol glycosides, known to mediate biotic and abiotic effects. Pollens were distinguished from nectar by high concentrations of hydroxycinnamoyl-spermidine conjugates, which affect plant development, abiotic stress tolerance, and herbivore resistance. Although chemistry was qualitatively consistent within species and tissue types, concentrations varied across cultivars and sites, which could influence pollination, herbivory, and disease in wild and agricultural plants. Analyses of multivariate trait space showed greater overlap across sites and cultivars in nectar than pollen chemistry; this overlap reflected greater within-site and within-cultivar variability of nectar. Our analyses suggest different ecological roles of nectar and pollen mediated by chemical concentration, composition, and variability

Secondary metabolites from nectar and pollen: A resource for ecological and evolutionary studies

Floral chemistry mediates plant interactions with herbivores, pathogens, and pollinators. The chemistry of floral nectar and pollen—the primary food rewards for pollinators—can affect both plant reproduction and pollinator health. Although the existence and functional significance of nectar and pollen secondary metabolites has long been known, comprehensive quantitative characterizations of secondary chemistry exist for only a few species. Moreover, little is known about intraspecific variation in nectar and pollen chemical profiles. Because the ecological effects of secondary chemicals are dose-dependent, heterogeneity across genotypes and populations could influence floral trait evolution and pollinator foraging ecology. To better understand within- and across species heterogeneity in nectar and pollen secondary chemistry, we undertook exhaustive LC-MS and LC-UV-based chemical characterizations of nectar and pollen methanol extracts from 31 cultivated and wild plant species. Nectar and pollen were collected from farms and natural areas in Massachusetts, Vermont, and California, USA, in 2013 and 2014. For wild species, we aimed to collect 10 samples from each of 3 sites. For agricultural and horticultural species, we aimed for 10 samples from each of 3 cultivars. Our dataset (1535 samples, 102 identified compounds) identifies and quantifies each compound recorded in methanolic extracts, and includes chemical metadata that describe the molecular mass, retention time, and chemical classification of each compound. A reference phylogeny is included for comparative analyses. We found that each species possessed a distinct chemical profile; moreover, within species, few compounds were found in both nectar and pollen. The most common secondary chemical classes were flavonoids, terpenoids, alkaloids and amines, and chlorogenic acids. The most common compounds were quercetin and kaempferol glycosides. Pollens contained high concentrations of hydroxycinnamoyl-spermidine conjugates, mainly triscoumaroyl and trisferuloyl spermidine, found in 71% of species. When present, pollen alkaloids and spermidines had median nonzero concentrations of 23,000 μM (median 52% of recorded micromolar composition). Although secondary chemistry was qualitatively consistent within each species and sample type, we found significant quantitative heterogeneity across cultivars and sites. These data provide a standard reference for future ecological and evolutionary research on nectar and pollen secondary chemistry, including its role in pollinator health and plant reproduction

Distasteful nectar deters floral robbery

Toxic nectar is an ecological paradox[1,2]. Plants divert substantial resources to produce nectar that attracts pollinators [3], but toxins in this reward could disrupt the mutualism and reduce plant fitness [4]. Alternatively, such compounds could protect nectar from robbers [2], provided they do not significantly alter pollinator visitation to the detriment of plant fitness [1,5–8]. Indeed, very few studies have investigated the role of plant toxins in nectar for defence against nectar robbers [4,9,10]. Here, we compared two Aconitum species (A. napellus and A. lycoctonum) that have flowers specialized for long-tongued bumblebee pollinators (Bombus hortorum) but are occasionally robbed by short-tongued bumblebees (B. terrestris) [6,11–13]. Pollinator visits to flowers were much more frequent than by robbers but visits correlated negatively with nectar alkaloid concentration and declined sharply between 200-380ppm. However, alkaloid concentrations of > 20ppm were deterrent to B. terrestris suggesting robbers were less tolerant of nectar alkaloids. Nectar of both plant species contained similar concentrations of carbohydrates and toxic alkaloids, but A. lycoctonum was more likely to secrete nectar in each flower and was also visited more frequently by pollinators and robbers.  We conclude that alkaloids in Aconitum sp. nectar affect rates of both pollinator visitation and robbery but may have co-evolved with nectar availability to maintain the fitness benefits of specialized plant-pollinator relationships. Chemical defence of nectar is, however, ultimately constrained by pollinator gustatory sensitivity

Phytochemical analysis of Tephrosia vogelii across East Africa reveals three chemotypes that influence its use as a pesticidal plant

Tephrosia vogelii is a plant species chemically characterized by the presence of entomotoxic rotenoids and used widely across Africa as a botanical pesticide. Phytochemical analysis was conducted to establish the presence and abundance of the bioactive principles in this species across three countries in East Africa: Tanzania, Kenya, and Malawi. Analysis of methanolic extracts of foliar parts of T. vogelii revealed the occurrence of two distinct chemotypes that were separated by the presence of rotenoids in one, and flavanones and flavones that are not bioactive against insects on the other. Specifically, chemotype 1 contained deguelin as the major rotenoid along with tephrosin, and rotenone as a minor component, while these compounds were absent from chemotype 2, which contained previously reported flavanones and flavones including obovatin‐3‐O‐methylether. Chemotype 3 contained a combination of the chemical profiles of both chemotype 1 and 2 suggesting a chemical hybrid. Plant samples identified as chemotype 1 showed chemical consistency across seasons and altitudes, except in the wet season where a significant difference was observed for samples in Tanzania. Since farmers are unable to determine the chemical content of material available care must be taken in promoting this species for pest management without first establishing efficacy. While phytochemical analysis serves as an important tool for quality control of pesticidal plants, where analytical facilities are not available simple bioassays could be developed to enable extension staff and farmers to determine the efficacy of their plants and ensure only effective materials are adopted

Pollen sterols are associated with phylogenetics and environment but not with pollinators

Phytosterols are primary plant metabolites that have fundamental structural and regulatory functions. They are also essential nutrients for phytophagous insects, including pollinators, that cannot synthesize sterols. Despite the well-described composition and diversity in vegetative plant tissues, few studies have examined phytosterol diversity in pollen. We quantified 25 pollen phytosterols in 122 plant species (105 genera, 51 families) to determine their composition and diversity across plant taxa. We searched literature and databases for plant phylogeny, environmental conditions, and pollinator guilds of the species to examine the relationships with pollen sterols. 24-methylenecholesterol, sitosterol and isofucosterol were the most common and abundant pollen sterols. We found phylogenetic clustering of twelve individual sterols, total sterol content and sterol diversity, and of sterol groupings that reflect their underlying biosynthesis pathway (24 carbon alkylation, ring B desaturation). Plants originating in tropical-like climates (higher mean annual temperature, lower temperature seasonality, higher precipitation in wettest quarter) were more likely to record higher pollen sterol content. However, pollen sterol composition and content showed no clear relationship with pollinator guilds. Our study is the first to show that pollen sterol diversity is phylogenetically clustered and that pollen sterol content may adapt to environmental conditions