Résultat de la recherche
Field evidence for the role of plant volatiles induced by caterpillar-derived elicitors in the prey location behavior of predatory social wasps
1. One assumed function of herbivore-induced plant volatiles (HIPVs) is to attract natural enemies of the inducing herbivores. Field evidence for this is scarce and often indirect. Also, the assumption that elicitors in insect oral secretions that trigger the volatile emissions are essential for attraction of natural enemies has not yet been demonstrated under field conditions. 2. After observing social wasps removing caterpillars from maize plants in an agricultural field, we hypothesized that these wasps use HIPVs to locate their prey. To test this, we conducted an experiment that simultaneously explored the importance of caterpillar oral secretions in the interaction. 3. We found that Spodoptera caterpillars placed on mechanically damaged plants treated with oral secretion were more likely to be attacked by wasps compared to caterpillars on plants that were only mechanically wounded. Both of the the latter treatments were considerably more attractive than plants that were only treated with oral secretion or left untreated. Subsequent analyses of headspace volatiles confirmed differences in emitted volatiles that likely account for the differential predation events across the treatments. 4. These findings highlight the importance of HIPVs in prey location by social wasps and provide evidence for the role that elicitors play in inducing attractive odor blends.
The consequences of squash domestication on the chemical ecology of plant-insect interactions
La domestication des plantes ainsi que le début de l’agriculture ont été des innovations clés qui ont modelé la société et l’écosystème que nous connaissons aujourd’hui. La plupart des fruits et légumes que nous consommons ont été sélectionnés par l’homme et divergent des plantes sauvages ancestrales en de nombreux aspects que nous nommons syndrome de domestication. Il s’agit en général d’une augmentation de la taille du fruit, d’un complément en nutriment et, dans 60% des cultures, la réduction de la toxicité dans le but de leur consommation. Cette perte de composé chimique est intimement liée à l’augmentation et la prolifération des ravageurs de cultures. Cependant cette affirmation n’est pas ubiquitaire et dépendant de la plante étudiée. Souvent, la sélection de l’organe d’une plante a lieux en vue de sa consommation alimentaire. D’autres motifs de sélection comme l’utilisation des fibres, fabrication d’huile, décoration comme c’est le cas pour les courges, existent également. Les courges (genre Cucurbita) sont d’origine mexicaine et font partie des premières plantes à avoir été domestiquées sur le continent américain avec le maïs et le haricot. Leur domestication daterait d’il y a 10'000 ans selon des preuves archéologiques. Une de leurs premières utilisations a été en tant que récipient car leur chaire était très amère et toxique à cause d’une molécule nommée cucurbitacine. Il existe cinq espèces différentes de courges, toutes ont été domestiquées principalement pour la consommation de leur fruit, mais également pour leur beauté. La domestication des courges et son impact sur les insectes herbivores n’ayant pas été encore étudié, je me suis lancé le défi de comprendre ses conséquences. J’ai précisément voulu étudier les effets de la domestication sur les défenses physiques (trichomes) et chimiques (cucurbitacines) de la plante de courge et comment ces changements ont impacté les insectes sur plusieurs niveaux trophiques. Pour y répondre, j’ai travaillé sous 4 angles différents : 1) J’ai testé l’hypothèse que le but de domestication de la courge (alimentation ou ornementation) influencerait le contenu en cucurbitacines et trichomes des variétés. Pour cela, j’ai utilisé plusieurs espèces de courges par but de domestication. J’ai étudié l’effet de ces changements de défenses sur deux insectes généralistes se nourrissant soit sur les feuilles (Spodoptera latifascia), soit sur les racines (Diabrotica balteata). J’ai trouvé que les buts de domestication n’expliquaient pas les contenus en cucurbitacines. Cependant, les trichomes ont été diminués sur les variétés sélectionnées pour la consommation de leur fruit. Les cucurbitacines n’ont pas d’impact sur la croissance du stade larvaire des insectes étudiés, en revanche elles attireraient fortement les larves de D. balteata. Cette étude a démontré l’importance de la phylogénie lorsqu’on étudie l’impact de la domestication. 2) J’ai analysé les différences en termes de défense de la plante, entre les courges sauvages et domestiquées de l’espèce Cucurbita argyrosperma. Parmi les courges domestiquées, j’ai gardé le concept de but de domestication en étudiant des variétés sélectionnées pour la consommation du fruit ou l’ornementation. J’ai étudié l’impact de la domestication des courges sur un insecte généraliste (Diabrotica balteata) et un insecte spécialiste des courges (Acalymma vittatum) en laboratoire et dans leur milieu naturel (Mexique). J’ai trouvé que toutes les courges domestiquées ont perdu les cucurbitacines dans leurs racines. Les cotylédons des variétés sélectionnées pour la consommation du fruit contiennent toujours des cucurbitacines mais beaucoup moins que les courges sauvages. Le contenu en cucurbitacine des courges sauvages réduit la croissance des insectes généralistes mais pas des spécialistes. La cucurbitacine est un attractif très fort pour les deux types d’insectes. Au Mexique, les dommages sur les plantes domestiquées étaient plus importants. 3) Je me suis intéressée à l’impact indirect de la domestication des courges sur le troisième niveau trophique (ennemi naturel de l’herbivore). Pour cela j’ai étudié la survie, la préférence et la performance du prédateur Atheta coriaria pour des proies ayant mangé des courges soit sauvages, soit domestiquées. En d’autres termes des larves de Diabrotica balteata qui avaient consommé des racines contenant des cucurbitacines versus des larves qui se sont nourries de racines sans cucurbitacines. J’ai trouvé que la cucurbitacine des plantes sauvages n’a pas impacté la survie ou la préférence du prédateur. La séquestration de cucurbitacine par les herbivores ne les protège en rien contre le prédateur étudié. 4) J’ai voulu comprendre pourquoi les variétés de courge domestiquées ne contiennent pas de cucurbitacine. Plus précisément, j’ai étudié l’impact de la domestication des courges sur l’expression des gènes liés à la biosynthèse de la cucurbitacine. J’ai voulu savoir si les courges domestiquées avaient perdu le gène ou perdu l’expression du gène permettant la production de cucurbitacines. J’ai commencé par trouver des gènes orthologues aux gènes connus chez le concombre. Puis j’ai découvert un candidat pour le gène de la première enzyme responsable de la production de cucurbitacine (cucurbitadienol synthase) et six gènes du cytochrome P-450 responsable d’oxydation permettant la production de cucurbitacines. Ensuite j’ai analysé l’expression de ces gènes dans les racines, cotylédons et feuilles des courges sauvages et domestiquées. J’ai trouvé que les variétés de courge domestiquées possédaient toujours les gènes responsables de la production de cucurbitacine, mais que c’était leur expression qui était perturbée par la domestication. Dans les racines, l’expression des gènes est plus élevée chez les courges sauvages. L’expression de ces gènes est nulle dans les feuilles et très faible dans les cotylédons. ABSTRACT Since the start of agriculture, crop domestication has induced significant changes in both plants and human societies. Plant domestication has generally resulted in decreased chemical and physical defenses in crop plants compared to their wild ancestors. A reduction in plant defensive traits is often expected to result in increased insect performance. By selecting (modifying the genetics) desirable traits for humans, it may favor the pest insects that we have to face today in agriculture. However, recent studies have shown that the impact of crop domestication on plant-insect interactions is not ubiquitous. Surprisingly, there is still much discussion on the way by which crop domestication has influenced insect pests. In my PhD project, I hypothesize that the outcome of plant-insect interactions may be influenced by the type of organ targeted during domestication, as well as the by purpose of domestication. Squash (genus Cucurbita) is one of the earliest and most important domesticated plants in the Americas, along with maize and the common bean. The genus Cucurbita was domesticated on several occasions leading to five different species throughout the Americas, beginning around 10,000 years ago. Wild forms of squash are very bitter and toxic to humans and other mammals because they contain cucurbitacins (toxic secondary metabolites). Squash was domesticated at different times and for different purposes (including soap, oil, music instrument, food and beverage container). As a result, we have great variation on fruits size, color and shape. The aim of my PhD project was to examine the overall consequences of squash domestication on plant defenses and its impact on plant-insect interactions. To date, no studies with these specific questions were done on this important world-wide crop. To meet the challenge, I had four main axes: 1) Study the impact of purpose of domestication on the squash defenses (trichomes and cucurbitacins) and how it altered the interaction with generalist insects (Spodoptera latifascia that eat aboveground tissue and Diabrotica balteata, whose adults feed on aboveground tissues and larvae feed on belowground tissues). To test that, I had squash varieties from different species selected for consumption (fruit and seeds) and varieties selected for an ornamental purpose. I found that the varietal selection rather than the purpose of domestication explained the differences in cucurbitacin content. However, trichome density was reduced on varieties selected for consumption. The herbivore performance was not negatively affected by high cucurbitacin content nor by trichome density. Instead, the root herbivore D. balteata larvae preferred to feed on the varieties with high levels of cucurbitacins. This study highlighted the idea that D. balteata is adapted to cucurbits and the importance of phylogeny while studying plant domestication. 2) Study the consequences of Cucurbita argyrosperma domestication on plant chemical (cucurbitacins) and physical (trichomes) defenses and extent to which altered defenses impacted the interaction with two beetle species, the generalist Diabrotica balteata and the squash specialist, Acalymma spp. (Coleoptera: Chrysomelidae). These plants and insects have a long co-evolutionary history. To add to the behavioral bioassays in the lab, I tested the susceptibility of Cucurbita argyrosperma wild and domesticated varieties in a common garden during a field season in Mexico. I found that cucurbitacins were selected out of the domesticated varieties. Trichome density was not reduced through domestication. Larvae of both insects preferred to feed on roots of wild squash, but this negatively affected the performance of the generalist. In the field, leaves of wild and domesticated plants were mainly attacked by adults of the specialist. 3) Study the indirect consequence of Cucurbita argyrosperma domestication on the third trophic level. One of the main results from previous axes was the reduction of cucurbitacins in domesticated squash varieties. It was hypothesized that D. balteata sequester this compound for its own defense against natural enemies. Then, deprived of cucurbitacin by eating domesticated squash, larvae should be more attractive to soil predators. To test this hypothesis, I used the rove beetle predator (Atheta coriaria), which is a generalist soil predator with no coevolutionary history with D. balteata or squash. Overall, I found no evidence that cucurbitacins serve as a protection against predators. The assumed lethal and deterrent impacts of sequestered cucurbitacin by the herbivore on the predator were not observed. However, the survival of Diabrotica balteata larvae was lower when fed on wild squash populations. 4) Study how squash domestication changed the expression of cucurbitacin biosynthesis genes. From Cucurbita argyrosperma genome and literature on genes involved in the cucurbitacins pathway in cucumber, I targeted seven candidates for cucurbitacins biosynthetic genes. One candidate for the very first enzyme involved in the cucurbitacin biosynthesis: cucurbitadienol synthase, and six cytochrome P-450 enzyme. I analyzed the expression of those genes in roots, cotyledons and leaves to explain the differences in cucurbitacin concentration among plant tissues and among wild and domesticated squash. Overall, gene expression was higher in roots compared to cotyledons and for some genes, wild squash populations had a higher expression than the domesticated varieties.
Assessing the role of native plant growth-promoting Rhizobacteria as bio-inoculants for Yerba Mate ("Ilex paraguariensis")
The accelerated process of soil degradation due to long-term inadequate agricultural practices in Misiones, the major productive region of Yerba Mate (Ilex paraguariensis St. Hill.) in Argentina, has led to an urgent need for research and development of more sustainable agricultural practices. Yerba Mate is an emblematic crop in southern South America due to its leaves are used to prepare an energizing beverage called “mate” consumed as an alternative to coffee. The main goal of this study was to evaluate the role of native plant growth promoting rhizobacteria (PGPR) as potential bio-inoculants for Yerba Mate seedlings.
This thesis has shown that biomass yields of Yerba Mate seedlings can be increased up to 183% through bio-inoculation with native PGPR strains. Interestingly, the biomass yield increase was obtained through bio-inoculation in a less fertile soil. Kosakonia radicincitans YD4 was the most effective PGPR strain in enhancing the growth of Yerba Mate seedlings. Bio-inoculation with this strain in soil led to higher yields than those obtained in compost, even though higher yields can be expected in the latter due its high fertility. These results suggested that this strain represents a promising candidate to evaluate its potential as bio-inoculant for this crop in low-productive plantations.
The whole genome of K. radicincitans YD4 was sequenced and annotated to analyze the genetic potential ability to colonize the rhizosphere, and if virulence-associated genes are present in this strain. The presence of genes for chemotaxis, adherence structures (fimbriae) and anti-microbial activities reflected the genetic potential of this strain to colonize and compete successfully in the rhizosphere. Regarding the safety of using YD4 as a bio-inoculant, two gene clusters (Type III and Type VI secretions systems) often associated to pathogenicity were identified. Comparative genomic analysis using closely related pathogenic and PGPR strains suggested that these gene clusters might confer adaptative advantages to YD4 to compete against other microbes and survive in the rhizosphere. This hypothesis has been also proposed for other beneficial plant associated bacteria possessing these gene clusters.
In order to evaluate the ability of the bio-inoculant YD4 to colonize the rhizosphere of Yerba Mate seedlings, a strain-specific real time PCR approach was designed. The dynamics of YD4 was monitored in the rhizosphere of inoculated seedlings during five weeks in nursery. The plant growth-promoting effect of YD4 was confirmed by higher biomass yields and the introduced strain was detected in the rhizospheric soil until the end of the experiment (five weeks). These results suggested that the strain YD4 once inoculated, colonize and remain associated to the rhizosphere exerting its plant growth-promoting effect. Combining these results we can confirm that the isolate YD4 is a PGPR strain with a promising potential to be used as a bio-inoculant for Yerba Mate seedlings.
In the last chapter, the bacterial and fungal root-associated microbiome of Yerba Mate was analyzed for the first time using a pyrosequencing approach. This study aimed at the description of the enriched microbial taxa potentially involved in plant growth promotion inhabiting the roots of this crop. In addition, we analyzed if the agricultural historical management and location sites of Yerba Mate plantations have an effect on the alpha and beta diversity. Our results have shown that the bacterial genera Burkholderia and Enterobacter (containing well-known PGPR representatives) were enriched in Yerba Mate roots, whereas endomycorrhizal fungi were enriched particularly in one low-productive plantation with low soil P content. Yerba Mate trees might be exerting a selective pressure for mycorrhization in this type of plantations. The bacterial community composition, genetic diversity and phylogenetic diversity were significantly influenced by the geographical location of the plantations; whereas the fungal community composition and genetic diversity were significantly affected by the geographical location and soil pH. No effect of the historical agricultural management or location of the plantations was observed on the alpha diversity.
This thesis has contributed with an eco-friendly strategy to improve Yerba Mate growth in nursery, and highlighted the fact that better yields can be obtained thought bio-inoculation in less fertile soils. We are optimistic that this strategy could be exploited in field conditions in the future.
Population genetic structure of two primary parasitoids of Spodoptera frugiperda (Lepidoptera), Chelonus insularis and Campoletis sonorensis (Hymenoptera): to what extent is the host plant important?
Plant chemistry can strongly influence interactions between herbivores and their natural enemies, either by providing volatile compounds that serve as foraging cues for parasitoids or predators, or by affecting the quality of herbivores as hosts or prey. Through these effects plants may influence parasitoid population genetic structure. We tested for a possible specialization on specific crop plants in Chelonus insularis and Campoletis sonorensis, two primary parasitoids of the fall armyworm, Spodoptera frugiperda. Throughout Mexico, S. frugiperda larvae were collected from their main host plants, maize and sorghum and parasitoids that emerged from the larvae were used for subsequent comparison by molecular analysis. Genetic variation at eight and 11 microsatellites were respectively assayed for C. insularis and C. sonorensis to examine isolation by distance, host plant and regional effects. Kinship analyses were also performed to assess female migration among host-plants. The analyses showed considerable within population variation and revealed a significant regional effect. No effect of host plant on population structure of either of the two parasitoid species was found. Isolation by distance was observed at the individual level, but not at the population level. Kinship analyses revealed significantly more genetically related—or kin—individuals on the same plant species than on different plant species, suggesting that locally, mothers preferentially stay on the same plant species. Although the standard population genetics parameters showed no effect of plant species on population structure, the kinship analyses revealed that mothers exhibit plant species fidelity, which may speed up divergence if adaptation were to occur.
Soil salinization disrupts plant–plant signaling effects on extra-floral nectar induction in wild cotton
Plant–plant interactions via volatile organic compounds (VOCs) have received much attention, but how abiotic stresses affect these interactions is poorly understood. We tested the effect of VOCs exposure from damaged conspecifics on the production of extra-floral nectar (EFN) in wild cotton plants (Gossypium hirsutum), a coastal species in northern Yucatan (Mexico), and whether soil salinization affected these responses. We placed plants in mesh cages, and within each cage assigned plants as emitters or receivers. We exposed emitters to either ambient or augmented soil salinity to simulate a salinity shock, and within each group subjected half of the emitters to no damage or artificial leaf damage with caterpillar regurgitant. Damage increased the emission of sesquiterpenes and aromatic compounds under ambient but not under augmented salinity. Cor- respondingly, exposure to VOCs from damaged emitters had effect on receiver EFN induction, but this effect was contingent on salinization. Receivers produced more EFN in response to damage after being exposed to VOCs from damaged emitters when the latter were grown under ambient salinity, but not when they were subjected to salinization. These results suggest complex effects of abiotic factors on VOC-mediated plant interactions.
Consequences of the domestication of chili pepper on multitrophic interactions
La domestication des plantes vise à réduire leurs défenses et à sélectionner des variétés ayant un rendement plus élevé que leurs ancêtres sauvages. On pense que ces caractéristiques modifiées ont un impact sur les interactions plantes-insectes. Cependant, dans le cas du piment, ces effets restent peu clair. Par conséquent, cette thèse de doctorat visait à étudier les conséquences de la domestication du piment sur les interactions multitrophiques, en utilisant des variétés de piment et leur ancêtre sauvage Chiltepin comme modèles d'étude. Le premier chapitre se concentre sur l'effet des niveaux de capsaïcine dans les fruits domestiques sur la performance de l'herbivore généraliste S. latifascia et de son ectoparasitoïde E. platyhypenae. Nous avons constaté que la domestication du piment a augmenté et diminué la capsaïcine dans les fruits. Seulement à une concentration élevée de ces métabolites secondaires, la capsaïcine a eu un effet négatif sur les insectes herbivores, et leur ectoparasitoïde. Par conséquent, la réponse de la plante à l'herbivorie dépend du niveau de piquant des fruits. Le chapitre deux explore les insectes nuisibles et leur virus associé sur les piments sauvages et domestiques dans leur environnement naturel. Nos résultats montrent que la domestication du piment a altéré les défenses physiques et chimiques des feuilles. En conséquence, les piments sauvages étaient plus résistants aux insectes broyeurs et aux virus, mais pas à leurs vecteurs, les aleurodes. Le chapitre trois est consacré à l'étude de l'alimentation et de l’oviposition du charançon spécialiste du piment (A. eugenii) sur les piments sauvages et domestiques comme ornement et pour la consommation. Globalement, la domestication a modifié les caractéristiques morphologiques et chimiques (capsaïcine) des fruits avec des conséquences directes sur l'alimentation et l’oviposition du charançon du piment. Ce travail a des implications importantes pour l'agriculture durable qui repose sur une résistance accrue des plantes et une utilisation réduite des pesticides. Enfin, cette étude permet de comprendre l'impact des pressions de sélection à court terme exercées par l'homme sur nos plantes cultivées et les forces de sélection naturelle qui ont façonné les interactions plantes-insectes au cours de l'évolution. ABSTRACT Plant domestication aims to reduce plant defenses and select varieties with a higher yield than their wild ancestors. It is believed that these altered traits have an impact on plant-insect interactions. However, in the case of chili pepper, these effects remain unclear. Therefore, This Ph.D. thesis aimed to study the consequences of domestication of chili pepper on multitrophic interactions, using chili pepper varieties and their wild ancestor Chiltepin as study models. Chapter one focuses on the effect of altered levels of capsaicin in domesticated chili on the performance of the generalist herbivore S. latifascia and its ectoparasitoid E. platyhypenae. We found that chili domestication has increased and decreased capsaicinoids in fruits. Only at a high concentration of these secondary metabolites, capsaicin had a negative effect on insect herbivores, and their ectoparasitoid. Therefore, the response of the plant to herbivory depends on the pungency level in fruits. Chapter two explores the pest insects and their associated virus on wild and domesticated chili peppers in their natural environment. Our results showed chili domestication has altered physical and chemical defenses in leaves. As consequence wild chili were more resistant to chewing insect and viruses’ infection but not to their vector, whiteflies. Chapter three is devoted to studying the feeding and oviposition of the specialist pepper weevil (A. eugenii) on wild and domesticated chiles as ornamental and for consumption. Overall, domestication has altered morphological and chemical (capsaicin) traits in fruits with direct consequences for the feeding and oviposition of the pepper weevil. This work has important implications for sustainable agriculture that relies on enhanced plant resistance and reduced pesticide use. Finally, this study allows understanding the impact of short-term human-mediated selection pressures applied to our crop plants and the natural selection forces that have molded plant-insect interactions over evolutionary time.
Direct and indirect defense response of Lima bean ("Phaseolus lunatus") to herbivory: implications for tritrophic interactions
In the nature plants are exposed to a huge amount of interactions with species from different guilds from mutualistic to antagonistic, and during the last 10 years researchers have been trying to underlying the ecological and evolutionary mechanisms that plants use to respond against this complex web of interactions. Plant traits such as chemical defenses after damage can affect not only the performance of the insect herbivores that feed on the plants but also the availability of them to their predators / parasitoids. The aim of my Phd thesis was in one side, to test whether in each specific community herbivores and their natural enemies will have adapted to the plant defenses induced by earlier herbivores. We tested that by determining the extent to which damage on leaves and seeds of Lima bean plants (Phaseolus lunatus) by the leaf beetle (Cerotoma ruficornis) and the bean pod weevil (Apion godmani) affects the performance and choice behavior of the Mexican bean weevil (Zabrotes subfasciatus) which arrive later in the season. In parallel we also studied how such effects affected the next trophic level (parasitoids). In the other side, we showed that in Lima bean there is a potential conflict between attracting defending ants and pollinators. This conflict could only be detected in situations of an increased production of extrafloral and floral nectar by plants induced with JA. By integrating the results regarding both direct and indirect defenses in Lima bean we can conclude that the complexity of the plant responses is driven by hormonal control. As soon as the first visitor arrives to the plant it triggers a series of responses that will affect not only the next visitor’s choice but also will affect the plant defense in the next generation.
Experimental Growth Conditions affect Direct and Indirect Defences in two Cotton Species
Cotton has been used as a model plant to study direct and indirect plant defence against herbivorous insects. However, the plant growing conditions could have an important effect on the outcome of such plant defence studies. We examined how common experimental growth conditions influence constitutive and inducible defences in two species of cotton, Gossypium hirsutum and G. herbaceum. We induced plants by applying caterpillar regurgitant to mechanical wounds to compare the induction levels between plants of both species grown in greenhouse or phytotron conditions. For this we measured defence metabolites (gossypol and heliocides) and performance of Spodoptera frugiperda caterpillars on different leaves, the emission of plant volatiles, and their attractiveness to parasitic wasps. Induction increased the levels of defence metabolites, which in turn decreased the performance of S. frugiperda larvae. Constitutive and induced defence levels were the highest in plants grown in the phytotron (compared to greenhouse plants), G. hirsutum and young leaves. Defence induction was more pronounced in plants grown in the phytotron and in young leaves. Also, the differences between growing conditions were more evident for metabolites in the youngest leaves, indicating an interaction with plant ontogeny. The composition of emitted volatiles was different between plants from the two growth conditions, with greenhouse-grown plants showing more variation than phytotron-grown plants. Also, G. hirsutum released higher amounts of volatiles and attracted more parasitic wasps than G. herbaceum. Overall, these results highlight the importance of experimental abiotic factors in plant defence induction and ontogeny of defences. We therefore suggest careful consideration in selecting the appropriate experimental growing conditions for studies on plant defences.
Plant species variation in bottom-up effects across three trophic levels: a test of traits and mechanisms
Colour polymorphism in the leaf beetle genus 'Oreina'
This dissertation investigates the maintenance of colour polymorphism in the leaf beetle genus Oreina CHEVROLAT (Coleoptera; Chrysomelidae). The remarkable colour variation in this seemingly aposematic and chemically defended genus forms a paradox with current scientific views concerning predator learning and mimicry. Using a variety of approaches we explore the selective forces that influence the dynamic colour polymorphic equilibrium. On the basis of a literature review we argue that there isn’t just one selective force of overriding importance but a multitude of factors in a heterogeneous landscape producing a geographic mosaic of coevolution in space and time. Using a phylogenetic approach we analysed the genetic structure and habitat use of Oreina speciosissima populations from the Swiss Alps. Specimens grouped according to their habitat, which was in turn mostly defined by plant associations. Via field experiments we test Müller’s theory of warning colour and mimicry in the wild and prove frequency-dependent selection using tethered beetles exposed to natural predators. By making use of TEM microscopy we prove that colour in Oreina gloriosa is structural and produced by microstructures in the epicuticle. Differences in colour are the result of minute differences in thickness of the electron lucent layers within the chirped multilayer reflector in the beetle’s elytra. I conclude by arguing that there is a multiplicity of factors driving the persisting prevalence of colour polymorphism in Oreina and provide suggestions for further investigation.