It has been almost 150 years since Darwin’s “The Origin of Species” was published, yet the mechanisms that lead to the creation of new species are still a topic of fervent research in Evolutionary Biology. In the last several decades, there has been considerable interest in how genetic drift and adaptive processes internal to populations may lead to population divergence. It has been recognized that intersexual co-evolution may drive the rapid evolution of reproductive traits, leading to reproductive isolation between populations in allopatry. To explore the nature of this process, I conducted a series of interpopulation interaction experiments using a set of laboratory-evolved Drosophila melanogaster populations. These “B” (for baseline) populations share a common ancestor and had evolved in identical habitats in allopatry for 650-700 generations prior to my experiments. When different populations were combined in competitive fitness assays, I found that foreign males usually suffer depressed fitness when competing with males who share a common evolutionary history with the focal females. I selected a subset of B populations to investigate potential sources of variation that could explain this result. I examined; 1) characters involved in post-copulatory interactions between mates and 2) traits involved in mating behaviour. I found that sperm precedence patterns varied significantly depending on female population of origin and suggestive evidence that males between populations vary in ability to compete in sperm offence. Males also varied in mating behaviour between populations, however we found no evidence that divergence of these traits was correlated to that of traits involved in post-copulatory interactions, suggesting that understanding how intersexual co-evolution drives population divergence requires considering independent components of the reproductive phenotype and their contributions to overall fitness differences. / Thesis (Master, Biology) -- Queen's University, 2008-08-13 12:29:52.602

Governments have a choice whether to intervene in the transport infrastructure sector to manage, finance and own the assets of the sector, or to rely on markets and private sector actors for the provision of these systems. In this article the development of rail and road infrastructure in Sweden and the choice between government and market provision of these systems are analyzed from a co-evolutionary perspective. Technology, economics, and politics have influenced the government’s policy formation and decisions on organizational models over time, such as the nationalization of roads and railroads in the 1930s-40s. The aim for improved economic efficiency and reduction of cost differences between different parts of the country rather than political ideology explain why roads and railroads were nationalized. Through adjusting its ownership role and policy content the government has, over time, accommodated to the different influences from technology, economics and politics. The government’s ownership of transport infrastructure has not been challenged since the nationalization. This could be seen as a sign of a successful gradual policy adjustment from the government’s side, thereby avoiding private sector solutions. / <p>QC 20130614</p>

Over the past few years information technology (IT) and business alignment has become a great concern to organizations. To achieve alignment has become a daunting task for organizations due to rapid changes in business environment and lack of IT support. In business organizations business processes and IT are interrelated and interact with each other where one entity influences to another entity i.e. evolution in business processes requires evolution in IT and vice versa. When this co-evolution is not well aligned, a gap is created due to wrong configuration between business requirements and IT deployment. Organizations usually strive to bridge the gap by implementing business and IT strategies (i.e. top-down planning) and tend to ignore other aspects of the co-evolution. Alignment is a continuous co-evolutionary process in which all components of business and IT are interrelated and enhance organization performance. The co-evolution between business and IT is not restricted to a level but it occurs at all levels and therefore, it is necessary to understand and study co-evolution at all levels within organizations. This thesis presents a co-evolutionary framework that helps to study and understand the co-evolution at three levels i.e. strategic level, operational level and individual level in an integrated fashion. The three levels need to co-evolve so that all components at each level co-evolve. This framework will speed up the alignment in organizations. We argue that the lack of knowledge of business among IT people and IT knowledge among business executives may cause the gap; therefore, a need arises to have a knowledgeable mediator between the domains that could help in the co-evolution. A K-mediator (i.e. knowledge mediator) has been used in the proposed co-evolutionary framework that facilitates the co-evolution at each level. Finally the thesis presents a case study in financial domain in order to evaluate and validate the framework.

MHC genes encode proteins that are responsible for the recognition of foreign antigens and the triggering of a subsequent, adequate immune response of the organism. Thus they hold a key position in the immune system of vertebrates. It is believed that the extraordinary genetic diversity of MHC genes is shaped by adaptive selectional processes in response to the reoccurring adaptations of parasites and pathogens. A large number of MHC studies were performed in a wide range of wildlife species aiming to understand the role of immune gene diversity in parasite resistance under natural selection conditions. Methodically, most of this work with very few exceptions has focussed only upon the structural, i.e. sequence diversity of regions responsible for antigen binding and presentation. Most of these studies found evidence that MHC gene variation did indeed underlie adaptive processes and that an individual’s allelic diversity explains parasite and pathogen resistance to a large extent. Nevertheless, our understanding of the effective mechanisms is incomplete. A neglected, but potentially highly relevant component concerns the transcriptional differences of MHC alleles. Indeed, differences in the expression levels MHC alleles and their potential functional importance have remained unstudied. The idea that also transcriptional differences might play an important role relies on the fact that lower MHC gene expression is tantamount with reduced induction of CD4+ T helper cells and thus with a reduced immune response. Hence, I studied the expression of MHC genes and of immune regulative cytokines as additional factors to reveal the functional importance of MHC diversity in two free-ranging rodent species (Delomys sublineatus, Apodemus flavicollis) in association with their gastrointestinal helminths under natural selection conditions. I established the method of relative quantification of mRNA on liver and spleen samples of both species in our laboratory. As there was no available information on nucleic sequences of potential reference genes in both species, PCR primer systems that were established in laboratory mice have to be tested and adapted for both non-model organisms. In the due course, sets of stable reference genes for both species were found and thus the preconditions for reliable measurements of mRNA levels established. For D. sublineatus it could be demonstrated that helminth infection elicits aspects of a typical Th2 immune response. Whereas mRNA levels of the cytokine interleukin Il4 increased with infection intensity by strongyle nematodes neither MHC nor cytokine expression played a significant role in D. sublineatus. For A. flavicollis I found a negative association between the parasitic nematode Heligmosomoides polygyrus and hepatic MHC mRNA levels. As a lower MHC expression entails a lower immune response, this could be evidence for an immune evasive strategy of the nematode, as it has been suggested for many micro-parasites. This implies that H. polygyrus is capable to interfere actively with the MHC transcription. Indeed, this parasite species has long been suspected to be immunosuppressive, e.g. by induction of regulatory T-helper cells that respond with a higher interleukin Il10 and tumor necrosis factor Tgfb production. Both cytokines in turn cause an abated MHC expression. By disabling recognition by the MHC molecule H. polygyrus might be able to prevent an activation of the immune system. Indeed, I found a strong tendency in animals carrying the allele Apfl-DRB*23 to have an increased infection intensity with H. polygyrus. Furthermore, I found positive and negative associations between specific MHC alleles and other helminth species, as well as typical signs of positive selection acting on the nucleic sequences of the MHC. The latter was evident by an elevated rate of non-synonymous to synonymous substitutions in the MHC sequences of exon 2 encoding the functionally important antigen binding sites whereas the first and third exons of the MHC DRB gene were highly conserved. In conclusion, the studies in this thesis demonstrate that valid procedures to quantify expression of immune relevant genes are also feasible in non-model wildlife organisms. In addition to structural MHC diversity, also MHC gene expression should be considered to obtain a more complete picture on host-pathogen coevolutionary selection processes. This is especially true if parasites are able to interfere with systemic MHC expression. In this case advantageous or disadvantageous effects of allelic binding motifs are abated. The studies could not define the role of MHC gene expression in antagonistic coevolution as such but the results suggest that it depends strongly on the specific parasite species that is involved. / Die Hauptaufgabe von MHC-kodierten Proteinen ist die Erkennung von körperfremden Molekülen sowie das Einleiten einer adäquaten Immunantwort, womit sie eine Schlüsselrolle im Immunsystem der Wirbeltiere einnehmen. Man nimmt an, dass ihre außergewöhnliche Vielfalt eine Antwort auf die sich ständig anpassenden Parasiten und Krankheitserreger ist, durch adaptive Selektion erhalten wird und dass die individuelle Allelausstattung einen Großteil der Parasitenbelastung erklärt, wofür bereits zahlreiche MHC-Studien Hinweise gefunden haben. Trotzdem ist unser Verständnis über die wirkenden Mechanismen teilweise noch lückenhaft. Ein stark vernachlässigter Aspekt hierbei sind z.B. eventuelle Unterschiede in der Genexpression der MHC-Allele und eine geringere Expression wäre gleichbedeutend mit einer geringeren Aktivierung des Immunsystems. Ich habe hierzu zwei frei lebende Kleinsäugerarten (Delomys sublineatus, Apodemus flavicollis) unter natürlichen Selektionsbedingungen untersucht. Dabei habe ich neben der genotypischen Diversität von MHC-Genen auch deren Expression, sowie die Genexpression immunregulativer Zytokine mit in Betracht gezogen und in Relation zur individuellen Belastung mit gastrointestinalen Helminthen gesetzt. Anhand von Leber und Milzproben beider Arten habe ich die Methode der ‚real-time PCR‘ zur relativen Quantifizierung von mRNA im Labor etabliert. Bereits für die Labormaus etablierte PCR-Primersysteme wurden an beiden Arten getestet und so konnten stabile Referenzgene gefunden werden, die Grundvoraussetzung für zuverlässige Genexpressionsmessungen. Für D. sublineatus konnte gezeigt werden, dass Helminthenbefall eine typische Th2 Immunantwort induziert, und dass der Zytokin Il4 Gehalt mit Befallsintensität strongyler Nematoden zunimmt. Es wurde für D. sublineatus kein signifikanter Zusammenhang zwischen MHC Expression oder anderen Zytokinen mit Helminthenbefall gefunden. In A. flavicollis wurde ein negativer Zusammenhang zwischen haptischer MHC-Expression und dem parasitären Nematoden Heligmosomoides polygyrus festgestellt, was auf eine Immunvermeidungsstrategie des Nematoden hindeutet. Ich fand typische positive und negative Assoziationen zwischen MHC-Allelen und anderen Helminthenarten, sowie Zeichen eines positiven Selektionsdruckes auf den MHC-Sequenzen, was sich durch eine erhöhte Rate aminosäureverändernder Mutationen zeigte. Diese nicht-synonymen Veränderungen waren auf Positionen innerhalb des zweiten Exons des DRB-Genes beschränkt, wohingegen die untersuchten Bereiche des ersten und dritten Exons stark konserviert vorlagen. Diese variablen Positionen kodieren Schlüsselstellen im Bereich der Antigenbindungsstelle im MHC Molekül. Zusammenfassend zeigt diese Arbeit, dass Genexpressionsstudien auch an Wildtieren durchgeführt und verlässliche Daten erzeugt werden können. Zusätzlich zur strukturellen Vielfalt sollten zukünftig auch mögliche Genexpressionsunterschiede bei MHC-Studien berücksichtigt werden, um ein kompletteres Bild der koevolutiven Wirt-Parasiten-Beziehungen zeichnen zu können. Dies ist vor allem dann von evolutiver Bedeutung, wenn die Parasiten in der Lage sind die MHC Expression aktiv zu beeinflussen. Die Studien konnten nicht die exakte Bedeutung von MHC-Genexpression in der antagonistischen Koevolution definieren, aber sie konnten zeigen dass diese Bedeutung stark von den jeweils beteiligten Partnern abzuhängen vermag.

Estudos co-evolutivos requerem uma base taxonômica e filogenética robusta para estabelecerem de forma inequívoca as relações entre as linhagens envolvidas. Neste sentido, a presente contribuição identificou as linhagens de parasitas marinhos das espécies anfi-Americanas de Himantura Müller & Henle, considerado o suposto grupo-irmão dos potamotrigonídeos - arraias Neotropicais restritas ao sistemas fluviais da America do Sul. O objetivo do presente estudo foi contribuir com o alicerce taxonômico necessário para a elucidar as associações históricas entre as arraias de água doce, seu suposto grupo-irmão marinho, e suas faunas helmintológicas. Neste sentido, este estudo aborda a diversidade de três gêneros de cestóideos, cujas linhagens são compartilhadas entre arraias marinhas e potamotrigonídeos: Acanthobothrium Blanchard, 1948, Anindobothrium Marques, Brooks & Lasso, 2001 e Rhinebothrium Linton, 1890. Cada um destes grupos é abordado em um capítulo individualmente. Os resultados deste estudo incluem a descrição de 11 espécies novas, dentre as quais, sete são parasitas de H. schmardae (Werner) e 4 parasitas de H. pacifica (Beebe & Tee-Van), além da redescrição de três espécies previamente conhecidas para H. schmardae. Todas as descrições e redescrições foram baseadas em um número de indivíduos sem precedentes na taxonomia dos grupos e incluíram dados sobre microscopia eletrônica de varredura dentro dos padrões atuais de descrições taxonômicas. Este estudo também abordou as relações filogenéticas das linhagens de Acanthobothrium e Anindobothrium. Em ambos os casos, dados moleculares revelam congruência entre as relações de parentesco de seus membros e aqueles evidenciados para seus hospedeiros. Os dados parasitológicos apresentados corroboram hipóteses recentes que postulam que potamotrigonídeos compartilham um ancestral comum com as linhagens anfi-Americanas de Himantura. Por fim, algumas abordagens utilizadas neste estudo ilustram os benefícios de integrar diferentes bases de dados no refinamento taxonômico destes grupos dentro do conceito do que hoje reconhecemos como taxonomia integrativa / Studies on the co-evolution require accurate taxonomic and phylogenetic information to unambiguously establish associations within the lineages involved. Therefore, the present study identified marine parasite lineages from amphi-American species of Himantura Müller & Henle, H. schmardae (Werner) and H. pacifica (Beebe & Tee-Van). These hosts are considered the sister-group of potamotrygonids, which are Neotropical freshwater stingrays restricted to river systems in South America. Our motivation was the contribution on sound taxonomic grounds, in order to elucidate the historical associations among freshwater batoids, their alleged marine sister-group and their cestode parasites. To achieve this goal, we documented the fauna of three genera of cestodes, whose lineages can be found both in marine and freshwater stingrays, namely Acanthobothrium Blanchard, 1948, Anindobothrium Marques, Brooks & Lasso, 2001 and Rhinebothrium Linton, 1890. Each chapter addresses each genus separately. Our results consist of descriptions of 11 species new to science, among which seven are found parasitizing H. schmardae and 4 infecting H. pacifica. Furthermore, redescriptions are provided for three species detected in H. schmardae. All descriptions and redescriptions were based on an unprecedented number of specimens and included data obtained from histology, ligth microscopy and scanning electron microscopy. In addition to the taxonomic approach, we evaluated the phylogenetic relationships of Acanthobothrium and Anindobothrium. Molecular data from both genera revealed the congruence between the known patterns of host relationships and their parasites. The parasitological data presented in this study supports the recent hypothesis that potamotrygonids and amphi-American species of Himantura share a common ancestor. Moreover, the combined approach applied in this study illustrates the benefits of integrating different data sources for the taxonomic refinement of these groups within the concept of integrative taxonomy

In this thesis, I study a radiation of brood-parasitic finch species, the indigobirds and whydahs (genus Vidua), that occur across Africa. Host colonisation is tightly linked to speciation in Vidua because of their remarkable capacity to imprint on their hosts, with mating traits and host preferences being influenced by the parasite's early environment. The challenge of explaining why the radiation has diversified to the extent it has therefore simplifies to understanding why only certain potential host species have been successfully colonised. Following on from the introduction (Chapter 1), I begin by critically examining the logic with which mimicry in the natural world can be conceptually organized (Chapter 2). This creates a “mimicry landscape” in which to situate the mimetic adaptations of hosts exhibited by Vidua. The framework can be used to contrast and draw parallels between these and other mimetic adaptations present in the natural world. In Chapter 3, I review the literature on begging call mimicry and development across all avian brood parasite species. I outline the conditions under which we expect begging call mimicry to evolve, and when we expect it to develop primarily through genetic or environmental cues. This provides clear predictions for what we expect to occur in Vidua finches, which are tested in Chapters 4 and 5. In Chapter 4, I quantify the mimicry of host nestlings by Vidua in detail. I provide the first quantitative evidence that Vidua nestlings mimic the begging calls and show for the first time that Vidua are imperfect mimics of their hosts. In Chapter 5, I simulate the colonisation of a new host by transferring Vidua eggs into the nest of a new host species. I monitor Vidua survival in the foreign host environment and test several hypotheses about what explains differences in chick survival. I find that Vidua survive poorly in the new nest environment and that they do not show adaptive plasticity in begging calls or head movements. This poor survival occurs despite there being minimal differences in the diets each host species feeds their young. Finally, in Chapter 6, I carry out a comparative analysis on the evolution of estrildid mouth markings. Estrildid finches are the hosts of Vidua and so provide the landscape of potential ecological niches that Vidua may colonise and adapt to. I demonstrate that the host family shows strong phylogenetic signal in mouth marking traits, and find no evidence that ecological factors such as light environment or predation pressure has shaped estrildid mouth marking evolution. The work in this thesis highlights how difficult successfully colonising new hosts is for Vidua finches. Vidua must mimic hosts in multiple traits (mouth markings, begging calls, head movements) to obtain sufficient amounts of food from host parents. Overall, habitat filters, the complex and diverse begging displays of estrildid nestlings, the discriminatory behaviour of estrildid parents against mismatching chicks and the lack of adaptive plasticity in begging displays by Vidua together help explain why the Vidua radiation consists of only 19 species rather than many more or fewer.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第19000号 / 工博第4042号 / 新制||工||1622 / 31951 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 田中 庸裕, 教授 今堀 博, 教授 阿部 竜 / 学位規則第4条第1項該当

abstract: Molybdenum (Mo) is a key trace nutrient for biological assimilation of nitrogen, either as nitrogen gas (N2) or nitrate (NO3-). Although Mo is the most abundant metal in seawater (105 nM), its concentration is low (<5 nM) in most freshwaters today, and it was scarce in the ocean before 600 million years ago. The use of Mo for nitrogen assimilation can be understood in terms of the changing Mo availability through time; for instance, the higher Mo content of eukaryotic vs. prokaryotic nitrate reductase may have stalled proliferation of eukaryotes in low-Mo Proterozoic oceans. Field and laboratory experiments were performed to study Mo requirements for NO3- assimilation and N2 fixation, respectively. Molybdenum-nitrate addition experiments at Castle Lake, California revealed interannual and depth variability in plankton community response, perhaps resulting from differences in species composition and/or ammonium availability. Furthermore, lake sediments were elevated in Mo compared to soils and bedrock in the watershed. Box modeling suggested that the largest source of Mo to the lake was particulate matter from the watershed. Month-long laboratory experiments with heterocystous cyanobacteria (HC) showed that <1 nM Mo led to low N2 fixation rates, while 10 nM Mo was sufficient for optimal rates. At 1500 nM Mo, freshwater HC hyperaccumulated Mo intercellularly, whereas coastal HC did not. These differences in storage capacity were likely due to the presence in freshwater HC of the small molybdate-binding protein, Mop, and its absence in coastal and marine cyanobacterial species. Expression of the mop gene was regulated by Mo availability in the freshwater HC species Nostoc sp. PCC 7120. Under low Mo (<1 nM) conditions, mop gene expression was up-regulated compared to higher Mo (150 and 3000 nM) treatments, but the subunit composition of the Mop protein changed, suggesting that Mop does not bind Mo in the same manner at <1 nM Mo that it can at higher Mo concentrations. These findings support a role for Mop as a Mo storage protein in HC and suggest that freshwater HC control Mo cellular homeostasis at the post-translational level. Mop's widespread distribution in prokaryotes lends support to the theory that it may be an ancient protein inherited from low-Mo Precambrian oceans. / Dissertation/Thesis / Ph.D. Geological Sciences 2011

Doctor of Philosophy / Biology / Stefan Rothenburg / Poxviruses are large double-stranded DNA viruses that collectively exhibit a broad host range. Whereas many members of the poxvirus family are capable of infecting various host species, others are restricted to only one or a very limited numbers of species, such as variola virus, which is the causative agent of smallpox and is restricted to humans. Since the entry of poxviruses is not dependent upon any specific receptors, the cell tropism is therefore fully determined by the virus’ ability to manipulate the cellular signaling networks that are responsible for antagonizing viral infections. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a unique antiviral protein found in most vertebrates, which serves both as a virus sensor by detecting the presence of dsRNA and an antiviral effector by suppressing cap-dependent translation during virus infection. Many viruses, including poxviruses, have therefore evolved genes that encode for PKR inhibitors, such as vaccinia virus K3L, which shows sequence homology to the N-terminal region of the eukaryotic translation initiation factor 2α (eIF2α), the substrate of PKR. K3L is able to inhibit PKR-mediated eIF2α phosphorylation in vitro and in vivo. Because K3L was shown to be indispensable for virus replication in Syrian hamster cells but not in human cells, it was categorized as a host range factor. However, the molecular basis for K3L’s host range function is not fully understood. We examined the interactions of poxvirus K3L orthologs, especially vaccinia virus K3L and M156R, the K3L ortholog in the rabbit-specific myxoma virus, and PKR from a variety of host species in multiple assays, and found that K3L and M156R inhibit PKR in a species-specific manner, which likely contributes to the cell tropism and host range for both viruses. Inactivation of M156R or K3L led to virus attenuation in cells, which could be rescued by ectopic expression of viral PKR inhibitors. We also identified the helix αG region as the main molecular determinant for PKR’s sensitivity to inhibition by K3L orthologs. In conclusion, the research summarized here indicates that the interactions of PKR and poxvirus pseudosubstrate inhibitors play important roles in virus host range and virulence.

Los terpenos constituyen el mayor grupo de metabolitos secundarios, siendo componentes de las glándulas de aceites esenciales, de las flores y de las resinas defensivas de plantas aromáticas, a los que proporcionan sus aromas y sabores característicos. Los terpenos volátiles se asocian a la defensa de muchas especies de plantas, animales y microorganismos contra depredadores, patógenos y competidores. Por otra parte, estos compuestos parecen servir como señales para atraer a los polinizadores y agentes dispersores de semillas, así como a depredadores de plagas. El estudio de compuestos orgánicos volátiles emitidos durante el desarrollo del fruto y después del desafío con diferentes agentes bióticos puede ayudar a conocer las interacciones de los frutos carnosos no sólo con vertebrados dispersores y depredadores, sino también con insectos y microorganismos. Los frutos carnosos son particularmente ricos en volátiles. En los frutos cítricos, los monoterpenos son los principales componentes de las glándulas del aceite esencial de la cáscara (flavedo), siendo el D-limoneno el más abundante (hasta 95% en la naranja). Esta característica hace que los cítricos sean un buen sistema modelo para el estudio de la función de los terpenos en los frutos. La biología molecular moderna permite la realización de experimentos para comprobar la función de terpenos por medio del uso de organismos transformados genéticamente en los que se han manipulado los niveles de acumulación de dichos compuestos. En este trabajo, se ha utilizado un plásmido que alberga el cDNA completo del gen de una limoneno sintasa de cítricos (CiTMTSE1) en orientación antisentido (AS) o sentido (S) para modificar la expresión y la acumulación de D-limoneno en plantas de naranjo dulce (Citrus sinensis L. Osb.). La acumulación de D-limoneno en las frutas AS se redujo drásticamente pero la acumulación de otros terpenos también se modificó, afectando a compuestos tales como alcoholes monoterpenos, cuya concentración se incrementó en la cáscara de las frutas. Las plantas transformadas fueron morfológicamente indistinguibles de las plantas control (WT) y de las plantas transformadas con el vector vacío (EV). Los frutos transgénicos fueron desafiados con un insecto plaga y con diferentes patógenos para probar si la alteración de los niveles de acumulación de estos volátiles daba como resultado una mejora en la respuesta del flavedo frente a plagas y patógenos. Los machos de la mosca mediterránea de la fruta (Ceratitis capitata) expuestos a las frutas AS y EV en ensayos en túnel de viento fueron significativamente más atraídos por el aroma de los frutos control EV. En otros experimentos de desafío con el hongo de la podredumbre verde Penicillium digitatum y la bacteria causante de la cancrosis de los cítricos Xanthomonas axonopodis subsp. citri, las frutas transgénicas con un contenido reducido de D-limoneno mostraron elevada resistencia a estos patógenos. El alto contenido en D-limoneno en la cáscara de naranjas maduras puede ser una señal para la atracción de plagas y microorganismos que podrían estar involucrados en la facilitación del acceso a la pulpa de los frugívoros dispersores de semillas. El análisis de la expresión génica global en el flavedo de las frutas transgénicas vinculó la disminución de D-limoneno y la reducción de la expresión de genes del metabolismo de monoterpenos con la activación de la expresión de genes implicados en inmunidad innata, incluyendo factores de transcripción, genes de quinasas implicadas en la entrada de Ca2+ en la célula y genes implicados en la activación de las cascadas de MAPKs, con la consiguiente activación de la ruta de señalización de ácido jasmónico (JA), lo que provocó la activación del metabolismo de JA y un aumentó drástico de la acumulación de JA en la cáscara de la naranja tras el desafío con P. digitatum, lo que explicaría la resistencia al menos a hongos necrotrofos observada en las frutas. Estos resultados indican que la acumulación de D-limoneno en la cáscara de la naranja estaría implicada en la interacción trófica entre las frutas, insectos y microorganismos, lo cual proporciona una visión mucho más amplia de las funciones de los terpenos en la naturaleza. También representa una alternativa muy prometedora para incrementar la resistencia o tolerancia de las plantas frente a patógenos y plagas. / Terpenes, the largest group of secondary metabolites, are well known as constituents of essential oils, floral scents and defensive resins of aromatic plants, to which they impart their characteristic aromas and flavors. Terpene volatiles defend many species of plants, animals and microorganisms against predators, pathogens and competitors. Moreover, those compounds seem to serve as advertisements to attract pollinators and seed-dispersal agents as well as pest predators. The study of VOCs emitted during fruit development and after challenge with different biotic agents may help to determine the interactions of fleshy fruits not only with legitimate vertebrate dispersers and predators, but also with insects and microorganisms. Fleshy fruits are particularly rich in volatiles. In citrus fruits, monoterpenes are the main components of the essential oil glands of the peel, being D-limonene the most abundant one (up to 95% in orange fruits). This characteristic makes citrus a good model system for studying the function of terpenes in plants. Modern molecular biology now enable experiments to test terpenoid function by the use of genetically transformed organisms in which terpene levels have been manipulated. In this work, a plasmid harboring the complete cDNA of a citrus limonene synthase gene (CiTMTSE1) in antisense (AS) or sense (S) orientation was used to modify the expression and accumulation of D-limonene of sweet orange (Citrus sinensis L. Osb) plants. D-limonene accumulation in AS fruits was dramatically reduced but the accumulation of other terpenoids was also modified, such as monoterpene alcohols, whose concentration increased in the peel of fruits. Genetically transformed plants were morphologically indistinguishable from wild-type (WT) and empty vector (EV) control plants. Transgenic fruits were challenged against a pest and different pathogens to test whether volatile profile alteration results in an improvement in the response of the fruit flavedo against them. Males of the Mediterranean fruit fly (Ceratitis capitata) exposed to AS fruits versus EV in wind tunnel assays were significantly more attracted to the odor of EV control fruits. In separate experiments with the green mould rot of citrus fruits and citrus canker caused by Penicillium digitatum and Xanthomonas axonopodis subsp. citri, respectively, transgenic fruits with a reduced content in D-limonene showed resistance to both pathogens. High D-limonene content in mature orange peels may be a signal for attractiveness of pests and microorganisms which might be likely involved in facilitating the access to the pulp of seed dispersal frugivores. A global gene expression analysis of the flavedo of AS transgenic fruits linked the decrease of D-limonene and monoterpene metabolism to the up-regulation of genes involved in the innate immunity response, including transcription factors together with Ca2+ entry into the cell and activation of MAPK cascades, contributing to activation of jasmonic acid (JA) signaling, which triggered the up-regulation of JA metabolism and drastically increased the accumulation of JA in orange peels upon fungal challenge, explaining the resistance to necrotrophic fungi observed in AS fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms and provide a much more comprehensive view of roles of terpenes in nature. It also represents a very promising alternative for increasing resistance or tolerance of plants to pathogens. / Rodríguez Baixauli, AM. (2013). Genetic engineering of plant volatiles in fleshy fruits: pest repellency and disease resistance through D-limonene downregulation in transgenic orange plants [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31655 / TESIS