Gaétan Glauser

Is non-host pollen suitable for generalist bumblebees?

Current evidence suggests that pollen is both chemically and structurally protected. Despite increasing interest in studying bee–flower networks, the constraints for bee development related to pollen nutritional content, toxicity and digestibility as well as their role in the shaping of bee–flower interactions have been poorly studied. In this study we combined bioassays of the generalist bee Bombus terrestris on pollen of Cirsium, Trifolium, Salix, and Cistus genera with an assessment of nutritional content, toxicity, and digestibility of pollen. Microcolonies showed significant differences in their development, non‐host pollen of Cirsium being the most unfavorable. This pollen was characterized by the presence of quite rare δ7‐sterols and a low digestibility. Cirsium consumption seemed increase syrup collection, which is probably related to a detoxification mixing behavior. These results strongly suggest that pollen traits may act as drivers of plant selection by bees and partly explain why Asteraceae pollen is rare in bee generalist diet.

Latitudinal variation in plant chemical defences drives latitudinal patterns of leaf herbivory

A long-standing paradigm in ecology holds that herbivore pressure and thus plant defences increase towards lower latitudes. However, recent work has challenged this prediction where studies have found no relationship or opposite trends where herbivory or plant defences increase at higher latitudes. Here we tested for latitudinal variation in herbivory, chemical defences (phenolic compounds), and nutritional traits (phosphorus and nitrogen) in leaves of a long-lived tree species, the English oak Quercus robur. We further investigated the underlying climatic and soil factors associated with such variation. Across 38 populations of Q. robur distributed along an 18° latitudinal gradient, covering almost the entire latitudinal and climatic range of this species, we observed strong but divergent latitudinal gradients in leaf herbivory and leaf chemical defences and nutrients. As expected, there was a negative relationship between latitude and leaf herbivory where oak populations from lower latitudes exhibited higher levels of leaf herbivory. However, counter to predictions there was a positive relationship between leaf chemical defences and latitude where populations at higher latitudes were better defended. Similarly, leaf phosphorus and nitrogen increased with latitude. Path analysis indicated a significant (negative) effect of plant chemical defences (condensed tannins) on leaf herbivory, suggesting that the latitudinal gradient in leaf herbivory was driven by an inverse gradient in defensive investment. Leaf nutrients had no independent influence on herbivory. Further, we found significant indirect effects of precipitation and soil porosity on leaf herbivory, which were mediated by plant chemical defences. These findings suggest that abiotic factors shape latitudinal variation in plant defences and that these defences in turn underlie latitudinal variation in leaf herbivory. Overall, this study contributes to a better understanding of latitudinal variation in plant–herbivore interactions by determining the identity and modus operandi of abiotic factors concurrently shaping plant defences and herbivory.

Plant physical and chemical defence variation along elevation gradients: a functional trait-based approach

Predicting variation in plant functional traits related to anti-herbivore defences remains a major challenge in ecological research, considering that multiple traits have evolved in response to both abiotic and biotic conditions. Therefore, understanding variation in plant anti-herbivore defence traits requires studyingxa0their expression along steep environmental gradients, such as along elevation,xa0where multiple biotic and abiotic factors co-vary. We expand on plant defence theory and propose a novel conceptual framework to address the sources of variations of plant resistance traits at the community level. We analysed elevation patterns of within-community trait dissimilarity using the RaoQ index, and the community-weighted-mean (CWM) index, on several plant functional traits: plant height, specific leaf area (SLA), leaf-dry-matter-content (LDMC), silicium content, presence of trichomes, carbon-to-nitrogen ratio (CN) and total secondary metabolite richness. We found that at high elevation, where harsh environmental conditions persist, community functional convergence is dictated by traits relating to plant growth (plant height and SLA), while divergence arises for traits relating resource-use (LDMC). At low elevation, where greater biotic pressure occurs, we found a combination of random (plant height), convergence (metabolite richness) and divergence patterns (silicium content). This framework thus combines community assembly rules of ecological filtering and niche partition with plant defence hypotheses to unravel the relationship between environmental variations, biotic pressure and the average phenotype of plants within a community.

Nursing protects honeybee larvae from secondary metabolites of pollen

The pollen of many plants contains toxic secondary compounds, sometimes in concentrations higher than those found in the flowers or leaves. The ecological significance of these compounds remains unclear, and their impact on bees is largely unexplored. Here, we studied the impact of pyrrolizidine alkaloids (PAs) found in the pollen of Echium vulgare on honeybee adults and larvae. Echimidine, a PA present in E. vulgare pollen, was isolated and added to the honeybee diets in order to perform toxicity bioassays. While adult bees showed relatively high tolerance to PAs, larvae were much more sensitive. In contrast to other bees, the honeybee larval diet typically contains only traces of pollen and consists predominantly of hypopharyngeal and mandibular secretions produced by nurse bees, which feed on large quantities of pollen-containing bee bread. We quantified the transfer of PAs to nursing secretions produced by bees that had previously consumed bee bread supplemented with PAs. The PA concentration in these secretions was reduced by three orders of magnitude as compared to the PA content in the nurse diet and was well below the toxicity threshold for larvae. Our results suggest that larval nursing protects honeybee larvae from the toxic effect of secondary metabolites of pollen.

Integration of non-targeted metabolomics and automated determination of elemental compositions for comprehensive alkaloid profiling in plants

Plants produce a large array of specialized metabolites to protect themselves. Among these allelochemicals, alkaloids display highly diverse and complex structures that are directly related to their biological activities. Plant alkaloid profiling traditionally requires extensive and time-consuming sample preparation and analysis. Herein, we developed a rapid and efficient approach for the comprehensive profiling of alkaloids in plants using ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based metabolomics. Using automated compound extraction and elemental composition assignment, our method achieved >83% correct alkaloid identification and even >90% for medium to high intensity peaks. This represented a significant improvement in identification rate compared to generic methods used for EC determination with no a priori, such as in untargeted metabolomics studies. The developed approach was then applied to identify specific alkaloids of Aconitum lycoctonum L. and A. napellus L. (Ranunculaceae) using different parts of the plant (leaf, perianth and pollen). Significant differences in alkaloid profiles between the two species were highlighted and discussed under taxonomic and evolutionary perspectives. Taken together, the presented approach constitutes a valuable chemotaxonomic tool in the search for known and unknown alkaloids from plants.

Interspecific variation in leaf functional and defensive traits in oak species and its underlying climatic drivers

Plants exhibit a diverse set of functional traits and ecological strategies which reflect an adaptation process to the biotic and abiotic components of the environment. The Plant Economic Spectrum organizes these traits along a continuum from conservative to acquisitive resource use strategies and shows how the abiotic environment governs a species’ position along the continuum. However, this framework does not typically account for leaf traits associated with herbivore resistance, despite fundamental metabolic links (and therefore co-variance) between resource use traits and defensive traits. Here we analyzed a suite of leaf traits associated with either resource use (specific leaf area [SLA], nutrients and water content) or defenses (phenolic compounds) for saplings of 11 species of oaks (Quercus spp.), and further investigated whether climatic variables underlie patterns of trait interspecific variation. An ordination of leaf traits revealed the primary axis of trait variation to be leaf economic spectrum traits associated with resource use (SLA, nitrogen, water content) in conjunction with a defensive trait (condensed tannins). Secondary and tertiary axes of trait variation were mainly associated with other defensive traits (lignins, flavonoids, and hydrolysable tannins). Within the primary axis we found a trade-off between resource use traits and both water content and condensed tannins; species with high SLA and leaf N values invested less in condensed tannins and viceversa. Moreover, temperature and precipitation mediated the trait space occupied by species, such that species distributed in warmer and drier climates had less leaf N, lower SLA, and more defenses (condensed tannins, lignins and flavonoids), whereas opposite values were observed for species distributed in colder and wetter climates. These results emphasize the role of abiotic controls over all-inclusive axes of trait variation and contribute to a more complete understanding of interspecific variation in plant functional strategies.

Pleiotropic effect of the Flowering Locus C on plant resistance and defence against insect herbivores

Summary 1. Plants vary widely in the extent to which they defend themselves against herbivores. Because the resources available to plants are often site-specific, variation among sites dictates investment into defence, and may reveal a growth-defence trade-off. Moreover, plants that have evolved different life-history strategies in different environments may situate themselves on this trade-off curve differently. For instance, plants that flower later have a longer vegetative lifespan, and may accordingly defend themselves differently than those that flower earlier. 2. Here, we tested whether late-flowering plants, with a longer vegetative lifespan, invest more in defence than early-flowering plants, using recombinant genotypes of the annual herb Cardamine hirsuta that differ in flowering time as a result of differences in the activity of the major floral repressor Flowering Locus C (FLC). 3. We found that variation at FLC was mainly responsible for regulating flowering time and allocation to reproduction, but this partially depended on where the plants grew. We also found that variation at FLC mediated plant allocation to defence, with late- flowering plants producing higher levels of total glucosinolates and stress-related phytohormones. Nonetheless, plant growth and the qualitative values of plant defence and plant resistance against specialist herbivores were mainly independent from FLC. 4. Synthesis - Our results highlight pleiotropic effects associated with flowering-time genes that might influence plant defence and plant-herbivore interactions.

Improved separation by at-column dilution in preparative hydrophilic interaction chromatography

We evaluated at-column dilution (ACD) as a possible approach to reduce peak distortion caused by the injection of strong solvents in preparative hydrophilic interaction chromatography (HILIC). Three model compounds, namely uridine, cytidine and guanosine were selected and injected either in conventional or ACD conditions, using various proportions of water and acetonitrile in the sample diluent. Plate number and peak capacity were systematically investigated under both isocratic and gradient elution conditions, respectively. In isocratic conditions, ACD was found to provide higher plate numbers with sample diluent containing more than 40% water, but lower plate numbers with injections of less than 40% water, in comparison to a conventional injection system at the preparative scale. These contrasting results were attributed to the fact that i) efficiency was fundamentally reduced in ACD, due to the low make-up flow rate that was used to flush the injection loop, but ii) the trend was reversed for highly aqueous injection thanks to the capacity of ACD to maintain similar efficiency regardless of the injection solvent. On the other hand, peak capacity was comparable between ACD and conventional systems in gradient elution for injections containing low amounts of water. However, ACD became increasingly advantageous as the proportion of water in the injection solvent and/or the injection volume increased. This was confirmed by the separation of a plant root aqueous extract. Altogether, our findings suggest that ACD is beneficial both in isocratic and gradient modes when strongly aqueous solutions are injected, offering an attractive approach to increase loadability, while preserving peak shapes in preparative HILIC.

Development and Validation of an Ultra-Sensitive UHPLC–MS/MS Method for Neonicotinoid Analysis in Milk

A very sensitive ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the quantitation of the most common neonicotinoids (thiamethoxam, clothianidin, imidacloprid, acetamiprid, and thiacloprid) at trace levels in milk. Using fast and selective liquid-liquid extraction (LLE) starting from 0.5 mL of milk, lowest limits of quantitation (LLOQ) equal or lower than 10 pg/mL for all analytes were achieved. Precision and accuracy were evaluated at four different concentrations (5, 10, 500, and 10000 pg/mL) and ranged between 2 and 16% (RSD) and 77-125%, respectively. Extraction recoveries and matrix effects ranged between 64 and 76% and 88-98%, respectively. The method was applied to measure neonicotinoid levels in a series of conventional and organic Swiss milks as well as in human breast milk and commercial powdered milk. More than 90% of the samples tested positive for at least one neonicotinoid. However, all animal samples were far below the maximum residue limits authorized for human consumption with average total neonicotinoid levels of 16.1 ± 13.1 pg/mL. Human breast milks and powdered milks contained similar amounts of neonicotinoids. Taken together, our results demonstrate the high prevalence of neonicotinoids in milk from all origins, albeit at levels considered to be safe for human consumption.

Fagopyrum esculentum Alters Its Root Exudation after Amaranthus retroflexus Recognition and Suppresses Weed Growth

Weed control by crops through growth suppressive root exudates is a promising alternative to herbicides. Buckwheat (Fagopyrum esculentum) is known for its weed suppression and redroot pigweed (Amaranthus retroflexus) control is probably partly due to allelopathic root exudates. This work studies whether other weeds are also suppressed by buckwheat and if the presence of weeds is necessary to induce growth repression. Buckwheat and different weeds were co-cultivated in soil, separating roots by a mesh allowing to study effects due to diffusion. Buckwheat suppressed growth of pigweed, goosefoot and barnyard grass by 53, 42, and 77% respectively without physical root interactions, probably through allelopathic compounds. Root exudates were obtained from sand cultures of buckwheat (BK), pigweed (P), and a buckwheat/pigweed mixed culture (BK-P). BK-P root exudates inhibited pigweed root growth by 49%. Characterization of root exudates by UHPLC-HRMS and principal component analysis revealed that BK and BK-P had a different metabolic profile suggesting that buckwheat changes its root exudation in the presence of pigweed indicating heterospecific recognition. Among the 15 different markers, which were more abundant in BK-P, tryptophan was identified and four others were tentatively identified. Our findings might contribute to the selection of crops with weed suppressive effects.