Influences of chemotype and parental genotype on metabolic fingerprints of tansy plants uncovered by predictive metabolomics.

Intraspecific plant chemodiversity shapes plant-environment interactions. Within species, chemotypes can be defined according to variation in dominant specialised metabolites belonging to certain classes. Different ecological functions could be assigned to these distinct chemotypes. However, the roles of other metabolic variation and the parental origin (or genotype) of the chemotypes remain poorly explored. Here, we first compared the capacity of terpenoid profiles and metabolic fingerprints to distinguish five chemotypes of common tansy (Tanacetum vulgare) and depict metabolic differences. Metabolic fingerprints captured higher variation in metabolites while preserving the ability to define chemotypes. These differences might influence plant performance and interactions with the environment. Next, to characterise the influence of the maternal origin on chemodiversity, we performed variation partitioning and generalised linear modelling. Our findings revealed that maternal origin was a higher source of chemical variation than chemotype. Predictive metabolomics unveiled 184 markers predicting maternal origin with 89% accuracy. These markers included, among others, phenolics, whose functions in plant-environment interactions are well established. Hence, these findings place parental genotype at the forefront of intraspecific chemodiversity. We recommend considering this factor when comparing the ecology of various chemotypes. Additionally, the combined inclusion of inherited variation in main terpenoids and other metabolites in computational models may help connect chemodiversity and evolutionary principles.

Fungicides and strawberry pollination-Effects on floral scent, pollen attributes and bumblebee behavior.

Fungicides are used in agriculture to protect crops from various fungal diseases. However, they may modulate the plants metabolism. Moreover, fungicides can accumulate in the environment and may cause toxic effects on non-target organisms such as nectar microbes and pollinators. Nectar microbes contribute to the volatile profile of flowers and can influence pollinators behaviour. Thus, fungicide treatment could potentially affect the pollination. In this study, we investigated the influence of fungicide treatment on floral attributes as well as the behavioural impact on bumblebees. In separate experiments, we used one or both strawberry cultivars (Fragaria × ananassa var. Darselect and Malwina), which were either kept untreated (control) or treated with either Cuprozin® progress or SWITCH® fungicide. We analysed various flower traits including volatiles, pollen weight, pollen protein, and the attraction of bumblebees towards the flowers in the greenhouse. Additionally, we analysed the viability of pollen and pollen live-to-dead ratio, as well as the composition of nectar fungi in the field. A treatment with Cuprozin® progress led to a lower emission of floral volatiles and a slightly lower pollen protein content. This had no impact on the visit latency of bumblebees but on the overall visit frequency of these flowers. The treatment with the fungicide SWITCH® resulted in a higher emission of floral volatiles as well as a delayed first visit by bumblebees. Furthermore, flowers of control plants were visited more often than those treated with the two fungicides. Plant-pollinator interactions are highly complex, with many contributing factors. Fungicides can have an impact on the pollen quality and pollinator attraction, potentially leading to an altered pollen dispersal by pollinators and a change in fruit quality.

Inter-laboratory comparison of plant volatile analyses in the light of intra-specific chemodiversity.

INTRODUCTION: Assessing intraspecific variation in plant volatile organic compounds (VOCs) involves pitfalls that may bias biological interpretation, particularly when several laboratories collaborate on joint projects. Comparative, inter-laboratory ring trials can inform on the reproducibility of such analyses. OBJECTIVES: In a ring trial involving five laboratories, we investigated the reproducibility of VOC collections with polydimethylsiloxane (PDMS) and analyses by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). As model plant we used Tanacetum vulgare, which shows a remarkable diversity in terpenoids, forming so-called chemotypes. We performed our ring-trial with two chemotypes to examine the sources of technical variation in plant VOC measurements during pre-analytical, analytical, and post-analytical steps. METHODS: Monoclonal root cuttings were generated in one laboratory and distributed to five laboratories, in which plants were grown under laboratory-specific conditions. VOCs were collected on PDMS tubes from all plants before and after a jasmonic acid (JA) treatment. Thereafter, each laboratory (donors) sent a subset of tubes to four of the other laboratories (recipients), which performed TD-GC-MS with their own established procedures. RESULTS: Chemotype-specific differences in VOC profiles were detected but with an overall high variation both across donor and recipient laboratories. JA-induced changes in VOC profiles were not reproducible. Laboratory-specific growth conditions led to phenotypic variation that affected the resulting VOC profiles. CONCLUSION: Our ring trial shows that despite large efforts to standardise each VOC measurement step, the outcomes differed both qualitatively and quantitatively. Our results reveal sources of variation in plant VOC research and may help to avoid systematic errors in similar experiments.

Floral volatiles evoke partially similar responses in both florivores and pollinators and are correlated with non-volatile reward chemicals.

BACKGROUND: Plants often use floral displays to attract mutualists and prevent antagonist attacks. Chemical displays detectable from a distance include attractive or repellent floral volatile organic compounds (FVOCs). Locally, visitors perceive contact chemicals including nutrients but also deterrent or toxic constituents of pollen and nectar. The FVOC and pollen chemical composition can vary intra- and interspecifically. For certain pollinator and florivore species, responses to these compounds are studied in specific plant systems, yet we lack a synthesis of general patterns comparing these two groups and insights into potential correlations between FVOC and pollen chemodiversity. SCOPE: We reviewed how FVOCs and non-volatile floral chemical displays, i.e. pollen nutrients and toxins, vary in composition and affect the detection by and behaviour of insect visitors. Moreover, we used meta-analyses to evaluate the detection of and responses to FVOCs by pollinators vs. florivores within the same plant genera. We also tested whether the chemodiversity of FVOCs, pollen nutrients and toxins is correlated, hence mutually informative. KEY RESULTS: According to available data, florivores could detect more FVOCs than pollinators. Frequently tested FVOCs were often reported as pollinator-attractive and florivore-repellent. Among FVOCs tested on both visitor groups, there was a higher number of attractive than repellent compounds. FVOC and pollen toxin richness were negatively correlated, indicating trade-offs, whereas a marginal positive correlation between the amount of pollen protein and toxin richness was observed. CONCLUSIONS: Plants face critical trade-offs, because floral chemicals mediate similar information to both mutualists and antagonists, particularly through attractive FVOCs, with fewer repellent FVOCs. Furthermore, florivores might detect more FVOCs, whose richness is correlated with the chemical richness of rewards. Chemodiversity of FVOCs is potentially informative of reward traits. To gain a better understanding of the ecological processes shaping floral chemical displays, more research is needed on floral antagonists of diverse plant species and on the role of floral chemodiversity in visitor responses.

Fungicides Cuprozin Progress and SWITCH Modulate Primary and Specialized Metabolites of Strawberry Fruits.

Numerous pesticides, including fungicides, are applied every year to crop plants. However, such application may affect the plant metabolism and thus crop quality. Strawberry is an economically important crop, but the fruits are highly susceptible, especially to fungal diseases. In the present study, the effects of two fungicides, Cuprozin progress and SWITCH, on the metabolism of two cultivars and the wild strawberry were tested, focusing on primary (amino acids, (in)organic acids, sugars, total phenolics) and specialized metabolites (aroma volatiles), which determine the fruit flavor. The fungicide treatment significantly affected 11 out of 57 metabolites, while 20 of those differed between strawberry types and 27 were affected by the interaction of both factors. Given these modifications in metabolites in response to the treatments, the taste and quality of the strawberries may pronouncedly change when plants are treated with fungicides.

Flower Production, Headspace Volatiles, Pollen Nutrients, and Florivory in Tanacetum vulgare Chemotypes.

Floral volatiles and reward traits are major drivers for the behavior of mutualistic as well as antagonistic flower visitors, i.e., pollinators and florivores. These floral traits differ tremendously between species, but intraspecific differences and their consequences on organism interactions remain largely unknown. Floral volatile compounds, such as terpenoids, function as cues to advertise rewards to pollinators, but should at the same time also repel florivores. The reward composition, e.g., protein and lipid contents in pollen, differs between individuals of distinct plant families. Whether the nutritional value of rewards within the same plant species is linked to their chemotypes, which differ in their pattern of specialized metabolites, has yet not been investigated. In the present study, we compared Tanacetum vulgare plants of five terpenoid chemotypes with regard to flower production, floral headspace volatiles, pollen macronutrient and terpenoid content, and floral attractiveness to florivorous beetles. Our analyses revealed remarkable differences between the chemotypes in the amount and diameter of flower heads, duration of bloom period, and pollen nutritional quality. The floral headspace composition of pollen-producing mature flowers, but not of premature flowers, was correlated to that of pollen and leaves in the same plant individual. For two chemotypes, florivorous beetles discriminated between the scent of mature and premature flower heads and preferred the latter. In semi-field experiments, the abundance of florivorous beetles and flower tissue miners differed between T. vulgare chemotypes. Moreover, the scent environment affected the choice and beetles were more abundant in homogenous plots composed of one single chemotype than in plots with different neighboring chemotypes. In conclusion, flower production, floral metabolic composition and pollen quality varied to a remarkable extend within the species T. vulgare, and the attractiveness of floral scent differed also intra-individually with floral ontogeny. We found evidence for a trade-off between pollen lipid content and pollen amount on a per-plant-level. Our study highlights that chemotypes which are more susceptible to florivory are less attacked when they grow in the neighborhood of other chemotypes and thus gain a benefit from high overall chemodiversity.

Pollination Requirements of Almond (Prunus dulcis): Combining Laboratory and Field Experiments.

Almond (Prunus dulcis (Mill.) D. A. Webb; Rosales: Rosaceae) is a cash crop with an estimated global value of over seven billion U.S. dollars annually and commercial varieties are highly dependent on insect pollination. Therefore, the understanding of basic pollination requirements of the main varieties including pollination efficiency of honey bees (Apis mellifera, Linnaeus, Hymenoptera: Apidae) and wild pollinators is essential for almond production. We first conducted two lab experiments to examine the threshold number of pollen grains needed for successful pollination and to determine if varietal identity or diversity promotes fruit set and weight. Further, we examined stigma and ovules of flowers visited by Apis and non-Apis pollinators in the field to study the proportion of almond to non-almond pollen grains deposited, visitation time per flower visit, and tube set. Results indicate that the threshold for successful fertilization is around 60 pollen grains, but pollen can be from any compatible variety as neither pollen varietal identity nor diversity enhanced fruit set or weight. Andrena cerasifolii Cockerell (Hymenoptera: Andrenidae) was a more effective pollinator on a per single visit basis than Apis and syrphid flies. Nevertheless, Apis was more efficient than A. cerasifolii and syrphid flies as they spent less time on a flower during a single visit. Hence, planting with two compatible varieties and managing for both Apis and non-Apis pollinators is likely to be an optimal strategy for farmers to secure high and stable pollination success.

Elm leaves 'warned' by insect egg deposition reduce survival of hatching larvae by a shift in their quantitative leaf metabolite pattern.

Plants may take insect eggs on their leaves as a warning of future herbivory and intensify their defence against feeding larvae. Responsible agents are, however, largely unknown, and little knowledge is available on this phenomenon in perennial plants. We investigated how egg deposition affects the anti-herbivore defence of elm against the multivoltine elm leaf beetle. Prior egg deposition caused changes in the quality of feeding-damaged leaves that resulted in increased larval mortality and reduced reproductive capacity of the herbivore by harming especially female larvae. Chemical analyses of primary and secondary leaf metabolites in feeding-damaged, egg-free (F) and feeding-damaged, egg-deposited (EF)-leaves revealed only small differences in concentrations when comparing metabolites singly. However, a pattern-focused analysis showed clearly separable patterns of (F) and (EF)-leaves because of concentration differences in especially nitrogen and phenolics, of which robinin was consumed in greater amounts by larvae on (EF) than on (F)-leaves. Our study shows that insect egg deposition mediates a shift in the quantitative nutritional pattern of feeding-damaged leaves, and thus might limit the herbivore's population growth by reducing the number of especially female herbivores. This may be a strategy that pays off in a long run particularly in perennial plants against multivoltine herbivores.

Novel Set-Up for Low-Disturbance Sampling of Volatile and Non-volatile Compounds from Plant Roots.

Most studies on rhizosphere chemicals are carried out in substrate-free set-ups or in artificial substrates using sampling methods that require an air flow and may thus cause disturbance to the rhizosphere. Our study aimed to develop a simplified and inexpensive system that allows analysis of rhizosphere chemicals at experimentally less disturbed conditions. We designed a mesocosm in which volatile rhizosphere chemicals were sampled passively (by diffusion) without air- and water flow on polydimethylsiloxane-(PDMS) tubes. Dandelion (Taraxacum sect. ruderalia) was used as model plant; roots were left undamaged. Fifteen volatiles were retrieved from the sorptive material by thermal desorption for analysis by gas chromatography/mass spectrometry (GC/MS). Furthermore, three sugars were collected from the rhizosphere substrate by aqueous extraction and derivatized prior to GC/MS analysis. In order to study how the quantity of detected rhizosphere compounds depends on the type of soil or substrate, we determined the matrix-dependent recovery of synthetic rhizosphere chemicals. Furthermore, we compared sorption of volatiles on PDMS tubes with and without direct contact to the substrate. The results show that the newly designed mesocosm is suitable for low-invasive extraction of volatile and non-volatile compounds from rhizospheres. We further highlight how strongly the type of substrate and contact of PDMS tubes to the substrate affect the detectability of compounds from rhizospheres.

Application of Silicone Tubing for Robust, Simple, High-throughput, and Time-resolved Analysis of Plant Volatiles in Field Experiments.

Plant volatiles (PVs) mediate manifold interactions between plants and their biotic and abiotic environments (Dicke and Baldwin, 2010; Holopainen and Gershenzon, 2010). An understanding of the physiological and ecological functions of PVs must therefore be based on measurements of PV emissions under natural conditions. Yet sampling PVs in natural environments is difficult, limited by the need to transport, maintain, and power instruments, or else to employ expensive sorbent devices in replicate. Thus PVs are usually measured in the artificial environments of laboratories or climate chambers. However, polydimethysiloxane (PDMS), a sorbent commonly used for PV sampling (Van Pinxteren et al., 2010; Seethapathy and Górecki, 2012), is available as silicone tubing (ST) for as little as 0.60 €/m (versus 100-550 € apiece for standard PDMS sorbent devices). Small (mm-cm) ST pieces can be placed in any experimental setting and used for headspace sampling with little manipulation of the organism or headspace. ST pieces have absorption kinetics and capacities sufficient to sample plant headspaces on a timescale of minutes to hours, producing biologically meaningful "snapshots" of PV blends. When combined with thermal desorption (TD)-GC-MS analysis - a 40-year-old and widely available technology - ST pieces yield reproducible, sensitive, spatiotemporally resolved, quantitative data from headspace samples taken in natural environments (Kallenbach et al., 2014).

A robust, simple, high-throughput technique for time-resolved plant volatile analysis in field experiments.

Plant volatiles (PVs) mediate interactions between plants and arthropods, microbes and other plants, and are involved in responses to abiotic stress. PV emissions are therefore influenced by many environmental factors, including herbivore damage, microbial invasion, and cues from neighboring plants, and also light regime, temperature, humidity and nutrient availability. Thus, an understanding of the physiological and ecological functions of PVs must be based on measurements reflecting PV emissions under natural conditions. However, PVs are usually sampled in the artificial environments of laboratories or climate chambers. Sampling of PVs in natural environments is difficult, being limited by the need to transport, maintain and provide power to instruments, or use expensive sorbent devices in replicate. Ideally, PVs should be measured in natural settings with high replication, spatio-temporal resolution and sensitivity, and modest costs. Polydimethylsiloxane (PDMS), a sorbent commonly used for PV sampling, is available as silicone tubing for as little as 0.60 € m(-1) (versus 100-550 € each for standard PDMS sorbent devices). Small pieces of silicone tubing (STs) of various lengths from millimeters to centimeters may be added to any experimental setting and used for headspace sampling, with little manipulation of the organism or headspace. STs have sufficiently fast absorption kinetics and large capacity to sample plant headspaces over a timescale of minutes to hours, and thus can produce biologically meaningful 'snapshots' of PV blends. When combined with thermal desorption coupled to GC-MS (a 40-year-old widely available technology), use of STs yields reproducible, sensitive, spatio-temporally resolved quantitative data from headspace samples taken in natural environments.

Sensing the underground--ultrastructure and function of sensory organs in root-feeding Melolontha melolontha (Coleoptera: Scarabaeinae) larvae.

INTRODUCTION: Below ground orientation in insects relies mainly on olfaction and taste. The economic impact of plant root feeding scarab beetle larvae gave rise to numerous phylogenetic and ecological studies. Detailed knowledge of the sensory capacities of these larvae is nevertheless lacking. Here, we present an atlas of the sensory organs on larval head appendages of Melolontha melolontha. Our ultrastructural and electrophysiological investigations allow annotation of functions to various sensory structures. RESULTS: Three out of 17 ascertained sensillum types have olfactory, and 7 gustatory function. These sensillum types are unevenly distributed between antennae and palps. The most prominent chemosensory organs are antennal pore plates that in total are innervated by approximately one thousand olfactory sensory neurons grouped into functional units of three-to-four. In contrast, only two olfactory sensory neurons innervate one sensillum basiconicum on each of the palps. Gustatory sensilla chaetica dominate the apices of all head appendages, while only the palps bear thermo-/hygroreceptors. Electrophysiological responses to CO(2), an attractant for many root feeders, are exclusively observed in the antennae. Out of 54 relevant volatile compounds, various alcohols, acids, amines, esters, aldehydes, ketones and monoterpenes elicit responses in antennae and palps. All head appendages are characterized by distinct olfactory response profiles that are even enantiomer specific for some compounds. CONCLUSIONS: Chemosensory capacities in M. melolontha larvae are as highly developed as in many adult insects. We interpret the functional sensory units underneath the antennal pore plates as cryptic sensilla placodea and suggest that these perceive a broad range of secondary plant metabolites together with CO(2). Responses to olfactory stimulation of the labial and maxillary palps indicate that typical contact chemo-sensilla have a dual gustatory and olfactory function.

Contribution of pollinator-mediated crops to nutrients in the human food supply.

The contribution of nutrients from animal pollinated world crops has not previously been evaluated as a biophysical measure for the value of pollination services. This study evaluates the nutritional composition of animal-pollinated world crops. We calculated pollinator dependent and independent proportions of different nutrients of world crops, employing FAO data for crop production, USDA data for nutritional composition, and pollinator dependency data according to Klein et al. (2007). Crop plants that depend fully or partially on animal pollinators contain more than 90% of vitamin C, the whole quantity of Lycopene and almost the full quantity of the antioxidants ß-cryptoxanthin and ß-tocopherol, the majority of the lipid, vitamin A and related carotenoids, calcium and fluoride, and a large portion of folic acid. Ongoing pollinator decline may thus exacerbate current difficulties of providing a nutritionally adequate diet for the global human population.