Insect eggs suppress plant defence against chewing herbivores.

Plants activate direct and indirect defences in response to insect egg deposition. However, whether eggs can manipulate plant defence is unknown. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. In the present study, we found that the plant defence signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls defence against fungal and bacterial pathogens and negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defence against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect-responsive genes after challenge with caterpillars, suggesting that egg-derived elicitors suppress plant defence. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not seen in the SA-deficient mutant sid2-1, indicating that it is SA that mediates this phenomenon. These data reveal an intriguing facet of the cross-talk between SA and JA signalling pathways, and suggest that insects have evolved a way to suppress the induction of defence genes by laying eggs that release elicitors. We show here that egg-induced SA accumulation negatively interferes with the JA pathway, and provides an advantage for generalist herbivores.

Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis.

Wounding in multicellular eukaryotes results in marked changes in gene expression that contribute to tissue defense and repair. Using a cDNA microarray technique, we analyzed the timing, dynamics, and regulation of the expression of 150 genes in mechanically wounded leaves of Arabidopsis. Temporal accumulation of a group of transcripts was correlated with the appearance of oxylipin signals of the jasmonate family. Analysis of the coronatine-insensitive coi1-1 Arabidopsis mutant that is also insensitive to jasmonate allowed us to identify a large number of COI1-dependent and COI1-independent wound-inducible genes. Water stress was found to contribute to the regulation of an unexpectedly large fraction of these genes. Comparing the results of mechanical wounding with damage by feeding larvae of the cabbage butterfly (Pieris rapae) resulted in very different transcript profiles. One gene was specifically induced by insect feeding but not by wounding; moreover, there was a relative lack of water stress-induced gene expression during insect feeding. These results help reveal a feeding strategy of P. rapae that may minimize the activation of a subset of water stress-inducible, defense-related genes.

High host-plant nitrogen content: a prerequisite for the evolution of ant-caterpillar mutualism?

The amount of nitrogen required to complete an insect's life cycle may vary greatly among species that have evolved distinct life history traits. Myrmecophilous caterpillars in the Lycaenidae family produce nitrogen-rich exudates from their dorsal glands to attract ants for protection, and this phenomenon has been postulated to shape the caterpillar's host-plant choice. Accordingly, it was postulated that evolution towards myrmecophily in Lycaenidae is correlated with the utilization of nitrogen-rich host plants. Although our results were consistent with the evolutionary shifts towards high-nutrient host plants serving as exaptation for the evolution of myrmecophily in lycaenids, the selection of nitrogen-rich host plants was not confined to lycaenids. Butterfly species in the nonmyrmecophilous family Pieridae also preferred nitrogen-rich host plants. Thus, we conclude that nitrogen is an overall important component in the caterpillar diet, independent of the level of myrmecophily, as nitrogen can enhance the overall insect fitness and survival. However, when nitrogen can be obtained through alternative means, as in socially parasitic lycaenid species feeding on ant brood, the selective pressure for maintaining the use of nutrient-rich host plants is relaxed, enabling the colonization of nitrogen-poor host plants.

Species distributions and interactions in a changing world : from understanding to forecasting

Les écosystèmes fournissent de nombreuses ressources et services écologiques qui sont utiles à la population humaine. La biodiversité est une composante essentielle des écosystèmes et maintient de nombreux services. Afin d'assurer la permanence des services écosystémiques, des mesures doivent être prises pour conserver la biodiversité. Dans ce but, l'acquisition d'informations détaillées sur la distribution de la biodiversité dans l'espace est essentielle. Les modèles de distribution d'espèces (SDMs) sont des modèles empiriques qui mettent en lien des observations de terrain (présences ou absences d'une espèce) avec des descripteurs de l'environnement, selon des courbes de réponses statistiques qui décrive la niche réalisée des espèces. Ces modèles fournissent des projections spatiales indiquant les lieux les plus favorables pour les espèces considérées. Le principal objectif de cette thèse est de fournir des projections plus réalistes de la distribution des espèces et des communautés en montagne pour le climat présent et futur en considérant non-seulement des variables abiotiques mais aussi biotiques. Les régions de montagne et l'écosystème alpin sont très sensibles aux changements globaux et en même temps assurent de nombreux services écosystémiques. Cette thèse est séparée en trois parties : (i) fournir une meilleure compréhension du rôle des interactions biotiques dans la distribution des espèces et l'assemblage des communautés en montagne (ouest des Alpes Suisses), (ii) permettre le développement d'une nouvelle approche pour modéliser la distribution spatiale de la biodiversité, (iii) fournir des projections plus réalistes de la distribution future des espèces ainsi que de la composition des communautés. En me focalisant sur les papillons, bourdons et plantes vasculaires, j'ai détecté des interactions biotiques importantes qui lient les espèces entre elles. J'ai également identifié la signature du filtre de l'environnement sur les communautés en haute altitude confirmant l'utilité des SDMs pour reproduire ce type de processus. A partir de ces études, j'ai contribué à l'amélioration méthodologique des SDMs dans le but de prédire les communautés en incluant les interactions biotiques et également les processus non-déterministes par une approche probabiliste. Cette approche permet de prédire non-seulement la distribution d'espèces individuelles, mais également celle de communautés dans leur entier en empilant les projections (S-SDMs). Finalement, j'ai utilisé cet outil pour prédire la distribution d'espèces et de communautés dans le passé et le futur. En particulier, j'ai modélisé la migration post-glaciaire de Trollius europaeus qui est à l'origine de la structure génétique intra-spécifique chez cette espèce et évalué les risques de perte face au changement climatique. Finalement, j'ai simulé la distribution des communautés de bourdons pour le 21e siècle afin d'évaluer les changements probables dans ce groupe important de pollinisateurs. La diversité fonctionnelle des bourdons va être altérée par la perte d'espèces spécialistes de haute altitude et ceci va influencer la pollinisation des plantes en haute altitude. - Ecosystems provide a multitude of resources and ecological services, which are useful to human. Biodiversity is an essential component of those ecosystems and guarantee many services. To assure the permanence of ecosystem services for future generation, measure should be applied to conserve biodiversity. For this purpose, the acquisition of detailed information on how biodiversity implicated in ecosystem function is distributed in space is essential. Species distribution models (SDMs) are empirical models relating field observations to environmental predictors based on statistically-derived response surfaces that fit the realized niche. These models result in spatial predictions indicating locations of the most suitable environment for the species and may potentially be applied to predict composition of communities and their functional properties. The main objective of this thesis was to provide more accurate projections of species and communities distribution under current and future climate in mountains by considering not solely abiotic but also biotic drivers of species distribution. Mountain areas and alpine ecosystems are considered as particularly sensitive to global changes and are also sources of essential ecosystem services. This thesis had three main goals: (i) a better ecological understanding of biotic interactions and how they shape the distribution of species and communities, (ii) the development of a novel approach to the spatial modeling of biodiversity, that can account for biotic interactions, and (iii) ecologically more realistic projections of future species distributions, of future composition and structure of communities. Focusing on butterfly and bumblebees in interaction with the vegetation, I detected important biotic interactions for species distribution and community composition of both plant and insects along environmental gradients. I identified the signature of environmental filtering processes at high elevation confirming the suitability of SDMs for reproducing patterns of filtering. Using those case-studies, I improved SDMs by incorporating biotic interaction and accounting for non-deterministic processes and uncertainty using a probabilistic based approach. I used improved modeling to forecast the distribution of species through the past and future climate changes. SDMs hindcasting allowed a better understanding of the spatial range dynamic of Trollius europaeus in Europe at the origin of the species intra-specific genetic diversity and identified the risk of loss of this genetic diversity caused by climate change. By simulating the future distribution of all bumblebee species in the western Swiss Alps under nine climate change scenarios for the 21st century, I found that the functional diversity of this pollinator guild will be largely affected by climate change through the loss of high elevation specialists. In turn, this will have important consequences on alpine plant pollination.

Turnover of plant lineages shapes herbivore phylogenetic beta diversity along ecological gradients.

Understanding drivers of biodiversity patterns is of prime importance in this era of severe environmental crisis. More diverse plant communities have been postulated to represent a larger functional trait-space, more likely to sustain a diverse assembly of herbivore species. Here, we expand this hypothesis to integrate environmental, functional and phylogenetic variation of plant communities as factors explaining the diversity of lepidopteran assemblages along elevation gradients in the Swiss Western Alps. According to expectations, we found that the association between butterflies and their host plants is highly phylogenetically structured. Multiple regression analyses showed the combined effect of climate, functional traits and phylogenetic diversity in structuring butterfly communities. Furthermore, we provide the first evidence that plant phylogenetic beta diversity is the major driver explaining butterfly phylogenetic beta diversity. Along ecological gradients, the bottom up control of herbivore diversity is thus driven by phylogenetically structured turnover of plant traits as well as environmental variables.

Induced responses to herbivory and jasmonate in three milkweed species

We studied constitutive and induced defensive traits (latex exudation, cardenolides, proteases, and C/N ratio) and resistance to monarch caterpillars (Danaus plexippus) in three closely related milkweed species (Asclepias angustifolia, A. barjoniifolia and A. fascicularis). All traits showed significant induction in at least one of the species. Jasmonate application only partially mimicked the effect of monarch feeding. We found some correspondence between latex and cardenolide content and reduced larval growth. Larvae fed cut leaves of A. angustifolia grew better than larvae fed intact plants. Addition of the cardenolide digitoxin to cut leaves reduced larval growth but ouabain (at the same concentration) had no effect. We, thus, confirm that latex and cardenolides are major defenses in milkweeds, effective against a specialist herbivore. Other traits such as proteases and C/N ratio additionally may be integrated in the defense scheme of those plants. Induction seems to play an important role in plants that have an intermediate level of defense, and we advocate incorporating induction as an additional axis of the plant defense syndrome hypothesis.

A probabilistic approach to niche-based community models for spatial forecasts of assemblage properties and their uncertainties

Aim Conservation strategies are in need of predictions that capture spatial community composition and structure. Currently, the methods used to generate these predictions generally focus on deterministic processes and omit important stochastic processes and other unexplained variation in model outputs. Here we test a novel approach of community models that accounts for this variation and determine how well it reproduces observed properties of alpine butterfly communities. Location The western Swiss Alps. Methods We propose a new approach to process probabilistic predictions derived from stacked species distribution models (S-SDMs) in order to predict and assess the uncertainty in the predictions of community properties. We test the utility of our novel approach against a traditional threshold-based approach. We used mountain butterfly communities spanning a large elevation gradient as a case study and evaluated the ability of our approach to model species richness and phylogenetic diversity of communities. Results S-SDMs reproduced the observed decrease in phylogenetic diversity and species richness with elevation, syndromes of environmental filtering. The prediction accuracy of community properties vary along environmental gradient: variability in predictions of species richness was higher at low elevation, while it was lower for phylogenetic diversity. Our approach allowed mapping the variability in species richness and phylogenetic diversity projections. Main conclusion Using our probabilistic approach to process species distribution models outputs to reconstruct communities furnishes an improved picture of the range of possible assemblage realisations under similar environmental conditions given stochastic processes and help inform manager of the uncertainty in the modelling results

Insect eggs induce a systemic acquired resistance in Arabidopsis.

Although they constitute an inert stage of the insect's life, eggs trigger plant defences that lead to egg mortality or attraction of egg parasitoids. We recently found that salicylic acid (SA) accumulates in response to oviposition by the Large White butterfly Pieris brassicae, both in local and systemic leaves, and that plants activate a response that is similar to the recognition of pathogen-associated molecular patterns (PAMPs), which are involved in PAMP-triggered immunity (PTI). Here we discovered that natural oviposition by P. brassicae or treatment with egg extract inhibit growth of different Pseudomonas syringae strains in Arabidopsis through the activation of a systemic acquired resistance (SAR). This egg-induced SAR involves the metabolic SAR signal pipecolic acid, depends on ALD1 and FMO1, and is accompanied by a stronger induction of defence genes upon secondary infection. Although P. brassicae larvae showed a reduced performance when feeding on Pseudomonas syringae-infected plants, this effect was less pronounced when infected plants had been previously oviposited. Altogether, our results indicate that egg-induced SAR might have evolved as a strategy to prevent the detrimental effect of bacterial pathogens on feeding larvae.

Pattern dystrophy with high intrafamilial variability associated with Y141C mutation in the peripherin/RDS gene and successful treatment of subfoveal CNV related to multifocal pattern type with anti-VEGF (ranibizumab) intravitreal injections.

OBJECTIVE: To identify disease causing mutation in three generations of a Swiss family with pattern dystrophy and high intrafamilial variability of phenotype. To assess the effect of intravitreal ranibizumab injections in the treatment of subfoveal choroidal neovascularization associated with pattern dystrophy in one patient. METHODS: Affected family members were ascertained for phenotypic and genotypic characterization. Ophthalmic evaluations included fundus photography, autofluorescence imaging, optical coherence tomography, and International Society for Clinical Electrophysiology of Vision standard full-field electroretinography. When possible family members had genetic testing. The proband presented with choroidal neovascularization and had intravitreal injections as needed according to visual acuity and optical coherence tomography. RESULTS: Proband had a multifocal type pattern dystrophy, and his choroidal neovascularization regressed after four intravitreal injections. The vision improved from 0.8 to 1.0, and optical coherence tomography showed complete anatomical restoration. A butterfly-shaped pattern was observed in her cousin, whereas a fundus pulverulentus pattern was seen in a second cousin. Aunt had a multifocal atrophic appearance, simulating geographic atrophy in age-related macular degeneration. The Y141C mutation was identified in the peripherin/RDS gene and segregated with disease in the family. CONCLUSION: This is the first report of marked intrafamilial variation of pattern dystrophy because of peripherin/RDS Y141C mutation. Intravitreal ranibizumab injections might be a valuable treatment for associated subfoveal choroidal neovascularization.

Traitement de la dépression résistante par l'association citalopram-lithium. Méthodologie d'une étude multicentrique en double aveugle et résultats préliminaires [Treatment of resistant depression with the citalopram-lithium combination. Methodology of a double-blind multicenter study and preliminary results].

Citalopram, a new bicyclic antidepressant, is the most selective serotonin reuptake inhibitor. In a number of double-blind controlled studies, citalopram was compared to placebo and to known tricyclic antidepressants. These studies have shown their efficacy and good safety. The inefficacy of a psychotropic treatment in at least 20% of depressives has led a number of authors to propose original drug combinations and associations, like antidepressant/lithium (Li), antidepressant/sleep deprivation (agrypnia), antidepressant/ECT, or antidepressant/LT3. The aim of this investigation is to evaluate the clinical effectiveness and safety of a combined citalopram/lithium treatment in therapy-resistant patients, taking account of serotonergic functions, as tested by the fenfluramine/prolactin test, and of drug pharmacokinetics and pharmacogenetics of metabolism. DESIGN OF THE STUDY: A washout period of 3 days before initiating the treatment is included. After an open treatment phase of 28 days (D) with citalopram (20 mg D1-D3; 40 mg D4-D14; 40 or 60 mg D15-D28; concomitant medication allowed: chloral, chlorazepate), the nonresponding patients [less than 50% improvement in the total score on the 21 item-Hamilton Depression Rating Scale (HDRS)] are selected and treated with or without Li (randomized in double-blind conditions: citalopram/Li or citalopram/placebo) during the treatment (D29-D35). Thereafter, all patients included in the double-blind phase subsequently receive an open treatment with citalopram/Li for 7 days (D36-D42). The hypothesis of a relationship between serotoninergic functions in patients using the fenfluramine/prolactin test (D1) and the clinical response to citalopram (and Li) is assessed. Moreover, it is evaluated whether the pharmacogenetic status of the patients, as determined by the mephenytoin/dextromethorphan test (D0-D28), is related to the metabolism of fenfluramine and citalopram, and also to the clinical response. CLINICAL ASSESSMENT: Patients with a diagnosis of major depressive disorders according to DSM III are submitted to a clinical assessment of D1, D7, D14, D28, D35, D42: HDRS, CGI (clinical global impression), VAS (visual analog scales for self-rating of depression), HDRS (Hamilton depression rating scale, 21 items), UKU (side effects scale), and to clinical laboratory examens, as well as ECG, control of weight, pulse, blood pressure at D1, D28, D35. Fenfluramine/prolactin test: A butterfly needle is inserted in a forearm vein at 7 h 45 and is kept patent with liquemine. Samples for plasma prolactin, and d- and l-fenfluramine determinations are drawn at 8 h 15 (base line). Patients are given 60 mg fenfluramine (as a racemate) at 8 h 30. Kinetic points are determined at 9 h 30, 10 h 30, 11 h 30, 12 h 30, 13 h 30. Plasma levels of d- and l-fenfluramine are determined by gas chromatography and prolactin by IRNA. Mephenytoin/dextromethorphan test: Patients empty their bladders before the test; they are then given 25 mg dextropethorphan and 100 mg mephenytoin (as a racemate) at 8 h 00. They collect all urines during the following 8 hours. The metabolic ratio is determined by gas chromatography (metabolic ratio dextromethorphan/dextrorphan greater than 0.3 = PM (poor metabolizer); mephenytoin/4-OH-mephenytoin greater than 5.6, or mephenytoin S/R greater than 0.8 = PM). Citalopram plasma levels: Plasma levels of citalopram, desmethylcitalopram and didesmethylcitalopram are determined by gas chromatography--mass spectrometry. RESULTS OF THE PILOT STUDY. The investigation has been preceded by a pilot study including 14 patients, using the abovementioned protocol, except that all nonresponders were medicated with citalopram/Li on D28 to D42. The mean total score (n = 14) on the 21 item Hamilton scale was significantly reduced after the treatment, ie from 26.93 +/- 5.80 on D1 to 8.57 +/- 6.90 on D35 (p less than 0.001). A similar patCitalopram, a new bicyclic antidepressant, is the most selective serotonin reu

Recognition of insect eggs in Arabidopsis modulates defense responses

Plants activate direct and indirect defenses in response to insect egg deposition. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. Even though this innate immune response did not affect egg survival in Arabidopsis, we could show that different insect eggs elicit specific gene expression changes. Additionally, egg- induced necrosis could be observed in a variety of plants from different families ranging from dicotyledonous plants to monocots, suggesting that insect-egg detection by plants is a widespread mechanism and that different insect species contain elicitors of immune responses. Extracts from caterpillars and eggs contain elicitors that co-purified over several extraction steps. Chemical fractionation of caterpillar extracts lead to the characterisation of an active compound that was determined to be a triglyceride by NMR analysis. The exact structure of the side chains as well as the elicitor's presence in insect eggs have yet to be confirmed.We also found that the plant defense signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls the defense against fungal and bacterial pathogens whereas it negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defense against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect-responsive genes after challenge with caterpillars, suggesting that egg-derived elicitors suppress plant defense. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not found in the SA-deficient mutant sid2-l, indicating that SA mediates this phenomenon. These data reveal an intriguing facet of the crosstalk between SA- and JA-signalling pathways and suggest that insects have evolved a way to suppress the induction of defense genes by laying eggs that release elicitors. Additionally, we demonstrated that mutants of known crosstalk regulators, including nprl-1, tga2356, ein2-l and wrky70-l, are not affected in egg-induced suppression of herbivore defenses. JA treatment was not able to alleviate this SA/JA negative crosstalk, suggesting that this suppression operates through a novel mechanism downstream of JA biosynthesis.

Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens.

In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.