Detailed chemical analysis of honey bee (Apis mellifera) worker brood volatile profile from egg to emergence.

Chemical communication is a widely used mode of communication for social insects and has been demonstrated to be involved in many behaviours and physiological processes such as reproduction, nutrition or the fight against parasites and pathogens. In the honey bee, Apis mellifera, the release of chemical compounds by the brood plays a role in worker behaviour, physiology, and foraging activities and colony health as a whole. Several compounds have already been described as brood pheromones, such as components of the brood ester pheromone and (E)-ß-ocimene. Several other compounds originating from diseased or varroa-infested brood cells have been described as triggering the hygienic behaviour of workers. So far, studies of brood emissions have focused on specific stages of development and little is known about the emission of volatile organic compounds by the brood. In this study, we investigate the semiochemical profile of worker honey bee brood during its whole developmental cycle, from egg to emergence, with a specific focus on volatile organic compounds. We describe variation in emissions of thirty-two volatile organic compounds between brood stages. We highlight candidate compounds that are particularly abundant in specific stages and discuss their potential biological significance.

Erratum: Despinasse et al. Structure of the Chemical and Genetic Diversity of the True Lavender over Its Natural Range. Plants 2020, 9, 1640.

As requested by the Editorial Office, the authors remove the scientific consortium "Camille Nous" from the author list and the Author Contributions section in the published paper [...].

Structure of the Chemical and Genetic Diversity of the True Lavender over Its Natural Range.

The true lavender Lavandula angustifolia Miller is a Mediterranean aromatic shrub widely cultivated for its high quality essential oil used in perfumery and phytotherapy. Despite its economic importance, the intra-specific diversity among wild, non-cultivated plants remains poorly understood. We analyzed the structure of the chemical and genetic diversity of plants from 14 sites sampled over the entire native range of the true lavender. Volatile organic compounds of inflorescences were analyzed using gas chromatography coupled to mass spectrometry. Genotyping was performed with fingerprinting genetic markers. To limit the influence of environmental variability on chemical composition, plants were grown in the same conditions in a common garden. Without prior knowledge, discriminant analysis of principal component identified unambiguously four distinct chemotypes among three genetic populations. Co-inertia analysis and supervised analysis which integrated multiple datasets indicated a strong congruency between chemical and genetic patterns. Two distinct genetic units were located at the edge of the distribution area in the south of Italy and in the northeast of Spain, and were associated with two distinct chemotypes. Our results confirmed the existence of three genetically distinct entities, suggesting speciation. All French populations and the Italian Piedmontese population were genetically homogeneous but separated in two distinct chemotypes. The dominant chemotype was present in the center of the native range in southeastern France and was at the origin of the current most cultivated French varieties. Its main compounds were linalyl acetate, linalool, and caryophyllene oxide. The second French chemotype was found in south of Massif Central and presented high abundance of valuable linalyl and lavandulyl acetates. Linalool, eucalyptol, ß-caryophyllene, borneol, camphor, and cis-sabinene-hydrate were significantly associated with southern latitudes and their role would be worth exploring.

Metabolomic study of volatile compounds emitted by lavender grown under open-field conditions: a potential approach to investigate the yellow decline disease.

INTRODUCTION: Fine lavender and lavandin are perfume and medicinal plants originate from the South of France and are widely cultivated for their essential oils. Recently, cultivated plants suffered from a severe decline in France, due to the propagation of the yellow decline disease. This disease is caused by the stolbur phytoplasma, a bacterium transmitted by a sap-sucking insect, the planthopper. OBJECTIVES: In order to understand the complex relationships between host plant, pest, pathogen and environment responsible for the yellow decline of lavender, we use a metabolomic approach to highlight changes in chemical emissions from asymptomatic ("healthy") and symptomatic ("infected") plants. METHODS: Volatile compounds produced by fine lavender and lavandin were collected in the field using a dynamic headspace extraction approach. Afterwards, compounds trapped on Tenax adsorbent were thermodesorbed and analysed using an automated thermal desorption-gas chromatography-mass spectrometry (ATD-GC-MS). Multivariate statistical analyses was performed using principal component analysis and partial least square discriminant analyses. RESULTS: The untargeted screening of volatiles allowed the separation of asymptomatic and symptomatic plants according to their emissions. The approach was sufficiently accurate so as to separate the emissions according to the different stages of infection. Twelve compounds were found to be deregulated metabolites of yellow disease infection, common to fine lavender (variety 7713) and lavandin (variety abrial). CONCLUSION: The metabolomic approach allowed for the effective identification of chemical variations between infected and healthy plants in a complex field environment.

A Promiscuous CYP706A3 Reduces Terpene Volatile Emission from Arabidopsis Flowers, Affecting Florivores and the Floral Microbiome.

Flowers are essential but vulnerable plant organs, exposed to pollinators and florivores; however, flower chemical defenses are rarely investigated. We show here that two clustered terpene synthase and cytochrome P450 encoding genes (TPS11 and CYP706A3) on chromosome 5 of Arabidopsis (Arabidopsis thaliana) are tightly coexpressed in floral tissues, upon anthesis and during floral bud development. TPS11 was previously reported to generate a blend of sesquiterpenes. By heterologous coexpression of TPS11 and CYP706A3 in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana, we demonstrate that CYP706A3 is active on TPS11 products and also further oxidizes its own primary oxidation products. Analysis of headspace and soluble metabolites in cyp706a3 and 35S:CYP706A3 mutants indicate that CYP706A3-mediated metabolism largely suppresses sesquiterpene and most monoterpene emissions from opening flowers, and generates terpene oxides that are retained in floral tissues. In flower buds, the combined expression of TPS11 and CYP706A3 also suppresses volatile emissions and generates soluble sesquiterpene oxides. Florivory assays with the Brassicaceae specialist Plutella xylostella demonstrate that insect larvae avoid feeding on buds expressing CYP706A3 and accumulating terpene oxides. Composition of the floral microbiome appears also to be modulated by CYP706A3 expression. TPS11 and CYP706A3 simultaneously evolved within Brassicaceae and form the most versatile functional gene cluster described in higher plants so far.plantcell;31/12/2947/FX1F1fx1.

Development of a Headspace Solid-Phase Microextraction Gas Chromatography-Mass Spectrometry Method to Study Volatile Organic Compounds (VOCs) Emitted by Lavender Roots.

A headspace solid-phase microextraction (HS-SPME) method combined with gas chromatography-mass spectrometry (GC/MS) was developed and optimized for the extraction and the analysis of volatile organic compounds (VOCs) from lavandin and fine lavender roots. Optimal parameters to extract volatile molecules from ground and intact roots were determined using a divinylbenzene-carboxen-polydimethylsiloxane (DVB/CAR/PDMS) coating fiber at 70 °C for 60 min. A total of 99 VOCs, including 40 monoterpenoids, 15 sesquiterpenoids, 1 diterpenoid and 2 coumarins were detected. The main compounds detected in lavandin roots were fenchol, borneol, and coumarin. Performances of the optimized SPME GC/MS method were evaluated via the comparison of VOC emissions between roots from different cultivars of fine lavender (7713 and maillette) and lavandin (abrial and grosso). Chemometric analysis, using partial least squares-discriminant analysis (PLS-DA), suggests fifteen significant features as potential discriminatory compounds. Among them, ß-phellandrene allows discrimination between lavender and lavandin varieties.

Multivariate Analysis of Multiple Datasets: a Practical Guide for Chemical Ecology.

Chemical ecology has strong links with metabolomics, the large-scale study of all metabolites detectable in a biological sample. Consequently, chemical ecologists are often challenged by the statistical analyses of such large datasets. This holds especially true when the purpose is to integrate multiple datasets to obtain a holistic view and a better understanding of a biological system under study. The present article provides a comprehensive resource to analyze such complex datasets using multivariate methods. It starts from the necessary pre-treatment of data including data transformations and distance calculations, to the application of both gold standard and novel multivariate methods for the integration of different omics data. We illustrate the process of analysis along with detailed results interpretations for six issues representative of the different types of biological questions encountered by chemical ecologists. We provide the necessary knowledge and tools with reproducible R codes and chemical-ecological datasets to practice and teach multivariate methods.

Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221.

PLANT VOLATILES. Biosynthesis of monoterpene scent compounds in roses.

The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta.

Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia.

In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a (E)-ß-caryophyllene (LaCARS) and a τ-cadinol synthase (LaCADS). τ-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum γ-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum δ-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of τ-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and (E)-ß-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender.

Contributions of covariance: decomposing the components of stochastic population growth in Cypripedium calceolus.

Although correlations between vital rates can have important effects on evolution and demography, few studies have investigated their effects on population dynamics. Here, we extend life-table response experiments (LTREs) to variable environments, showing how to quantify contributions made by (1) mean vital rates, (2) variability driven by environmental fluctuations, (3) correlations implying demographic trade-offs and reflecting stage transition synchrony, and (4) elasticities reflecting local selection pressures. Applying our methods to the lady's slipper orchid Cypripedium calceolus, we found that mean rates accounted for 77.1% of all effects on the stochastic growth rate, variability accounted for 12.6%, elasticities accounted for 6.6%, and correlations accounted for 3.7%. Stochastic effects accounted for 17.6%, 15.3%, and 35.9% of the total in our three populations. Larger elasticities to transitions between dormancy states and stronger correlations between emergence and survival suggest that one population was under greater pressure to remain active while the other two showed survival payoffs for dormancy in poor years. Strong negative correlations between dormancy, emergence, and stasis balanced opposing contributions, resulting in near stationarity in two populations. These new methods provide an additional tool for researchers investigating stochastic population dynamics and should be useful for a broad range of applications in basic ecology and conservation biology.

Extracellular localization of the diterpene sclareol in clary sage (Salvia sclarea L., Lamiaceae).

Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces.

MSeasy: unsupervised and untargeted GC-MS data processing.

UNLABELLED: MSeasy performs unsupervised data mining on gas chromatography-mass spectrometry data. It detects putative compounds within complex metabolic mixtures through the clustering of mass spectra. Retention times or retention indices are used after clustering, together with other validation criteria, for quality control of putative compounds. The package generates a fingerprinting or profiling matrix compatible with NIST mass spectral search program and ARISTO webtool (Automatic Reduction of Ion Spectra To Ontology) for molecule identification. Most commonly used file formats, NetCDF, mzXML and ASCII, are acceptable. A graphical and user-friendly interface, MSeasyTkGUI, is available for R novices. AVAILABILITY: MSeasy and MSeasytkGUI are implemented as R packages available at http://cran.r-project.org/web/packages/MSeasy/index.html and http://cran.r-project.org/web/packages/MSeasyTkGUI/index.html.

Effect of stage-specific vital rates on population growth rates and effective population sizes in an endangered iteroparous plant.

Effective population size (N(e)) determines the strength of genetic drift and can influence the level of genetic diversity a population can maintain. Assessing how changes in demographic rates associated with environmental variables and management actions affect N(e) thus can be crucial to the conservation of endangered species. Calculation of N(e) through demographic models makes it possible to use elasticity analyses to study this issue. The elasticity of N(e) to a given vital rate is the proportional change in N(e) associated with a proportional increase in that vital rate. In addition, demographic models can be used to study N(e) and population growth rate (λ) simultaneously. Simultaneous examination is important because some vital rates differ diametrically in their associations with λ and N(e). For example, in some cases increasing these vital rates increases λ and decreases N(e). We used elasticity analysis to study the effect of stage-specific survival and flowering rates on N(e), annual effective population size (N(a)), and λ in seven populations of the endangered plant Austrian dragonhead (Dracocephalum austriacum). In populations with λ ≥ 1, the elasticities of N(e) and N(a) were similar to those of λ. Survival rates of adults were associated with greater elasticities than survival rates of juveniles, flowering rates, or fecundity. In populations with λ < 1, N(e) and N(a) exhibited greater elasticities to juvenile than to adult vital rates. These patterns are similar to those observed in other species with similar life histories. We did not observe contrasting effects of any vital rate on λ and N(e); thus, management actions that increase the λ of populations of Austrian dragonhead will not increase genetic drift. Our results show that elasticity analyses of N(e) and N(a) can complement elasticity analysis of λ. Moreover, such analyses do not require more data than standard matrix models of population dynamics.

Lavender inflorescence: a model to study regulation of terpenes synthesis.

We analysed VOC composition of complete inflorescences and single flowers of lavender during the flowering period. Our analyses, focused on the 20 most abundant terpenes, showed that three groups of components could be separated according to their patterns of variation during inflorescence ontogeny. These three groups were associated with three developmental stages: flower in bud, flower in bloom and faded flower. The expression of two terpene synthases (TPS) was followed using qPCR during inflorescence ontogeny. A comparison of these chemical and molecular analyses suggested that VOC production in lavender spike is mainly regulated at the transcriptional level. These results highlighted that lavender could be a model plant for future investigations on terpene biosynthesis and regulation, and could be used to explore the functions of terpene metabolites.

Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence development.

Despite the commercial importance of Lavandula angustifolia Mill. and L. x intermedia Emeric ex Loisel floral essential oils (EOs), no information is currently available on potential changes in individual volatile organic compound (VOC) content during inflorescence development. Calyces were found to be the main sites of VOC accumulation. The 20 most abundant VOCs could be separated into three sub-groups according to their patterns of change in concentration The three groups of VOCs sequentially dominated the global scent bouquet of inflorescences, the transition between the first and second groups occurring around the opening of the first flower of the inflorescence and the one between the second and third groups at the start of seed set. Changes in calyx VOC accumulation were linked to the developmental stage of individual flowers. Leaves accumulated a smaller number of VOCs which were a subset of those seen in preflowering inflorescences. Their nature and content remained constant during the growing season. Quantitative real time polymerase chain reaction assessments of the expression of two terpene synthase (TPS) genes, LaLIMS and LaLINS, revealed similar trends between their patterns of expression and those of their VOC products. Molecular and chemical analyses suggest that changes in TPS expression occur during lavender inflorescence development and lead to changes in EO composition. Both molecular data and terpene analysis support the findings that changes in biosynthesis of terpene occurred during inflorescence development.

One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L.

The outermost floral whorl, composed of sepals, is generally thought to function in the protection of reproductive tissues. In the plant family Lamiaceae, sepals are fused into a tube that is densely covered by hairs for mechanical defence and contains secondary metabolites for chemical defence against insects and abiotic stresses. Despite the importance of this tissue in plant fitness, virtually no study has addressed the basic aspects of sepal development and functioning. Because of its large size and its impressive metabolic activity (both in terms of quantity and diversity of secondary metabolites), we have used clary sage calyx as a model system to generate the first high throughput sequencing of the transcriptome of an angiosperm calyx. We applied massive parallel 454 pyrosequencing technology to a normalized cDNA extract and unveiled potential candidate genes for all steps of secondary metabolite pathways (phenylpropanoids and terpenoids). It also proved efficient in predicting the expression of large numbers of transcription factors and, with the use of bioinformatics tools, it predicted in the same sequencing run the presence of a novel class of gene transcription regulatory elements, miRNAs, without the need to generate a separate miRNA library. In our clary sage EST library, 18 conserved miRNAs were predicted. Among them, 15 were present in most studied plant species while the others were only shared with limited or discrete plant lineages. A separate data mining of the same clary sage EST library suggested the presence of 19 potential target genes to the 18 predicted conserved miRNAs. These coded for only 6 transcription factors or F-box proteins, 11 metabolism or abiotic stress response related proteins and 2 products with no known predicted function. All in all, this study provides novel genomic information on an angiosperm calyx and an experimental framework to predict in a single step metabolic pathway enzymes and regulator genes including miRNAs.

Population adaptive index: a new method to help measure intraspecific genetic diversity and prioritize populations for conservation.

In conservation biology genetic diversity is recognized as an important criterion to consider when prioritizing populations for protection. Today, population genomics offers the opportunity to evaluate both neutral and adaptive components of genetic diversity directly at the genome level with molecular tools. By screening the genome with many genetic markers, it is possible to detect loci supposedly under natural selection and thus of adaptive significance. We devised a new diversity index, the population adaptive index (PAI), which accounts for the adaptive value of the population it refers to. To estimate this index, we performed a genome scan with amplified fragment length polymorphism markers to identify neutral and selected loci in several populations of a widespread amphibian (common frog, Rana temporaria) and a threatened plant (Austrian dragonhead, Dracocephalum austriacum L.). We then investigated four different conservation strategies aimed at protecting the maximum amount of genetic diversity (neutral or selected). In particular we explored the relevance of the principle of complementarity, usually applied to the protection of species, in the management of intraspecific diversity. This principle advocates the conservation of sets of units that together maximize the species' or genetic diversity, which is in opposition to the traditional approach of targeting populations that are the most diverse individually. Four major conclusions emerged from these results. First, the PAI seemed to be a valuable index to evaluate the adaptive diversities within populations. Second, in the two species, the neutral and adaptive diversities within and among populations were not correlated, so conservation strategies based on the neutral and adaptive indexes would not select the same populations for protection. Third, because of its efficiency in conserving genetic diversity, the principle of complementarity deserves to be used more often for this purpose. Fourth, when neutral and adaptive results conflict, additional arguments (e.g., demography, ecology, and geographic proximity) should be considered together with levels of genetic diversity to determine a conservation strategy.