Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald R. Reeck / Armet is a bifunctional protein that is apparently universally distributed among multicellular animal species, vertebrate and invertebrate alike. A member of the Unfolded Protein Response, (UPR) Armet promotes survival in cells that are under endoplasmic-reticulum (ER) stress. I have carried out biophysical studies on human Armet looking for compounds that bind to Armet and hence could reduce its anti-apoptotic function, thus potentially joining the growing class of pro-apoptotic drugs. Performed primarily with 1H-15N HSQC NMR, ligand studies showed that approximately 60 of the 158 residues are potentially involved with binding. The 60 residues are distributed throughout both domains and the linker suggesting multi-domain interaction with the ligand. Circular dichroism studies showed heat denaturation in a two-step unfolding process with independent unfolding of both domains of Armet with Tm values near 68°C and 83 C with the C-terminal domain unfolding first, as verified by 1H-15N HSQC NMR measurements. I also provide the first identification of UPR transcripts in pea aphids, Acyrthosiphon pisum, the genetic model among aphids. I measured transcript abundance with hope of finding future transcriptional targets for pest mitigation. I identified 74 putative pea aphid UPR components, and all but three of the components have higher transcript levels in aphids feeding on plants than those that fed on diets. This activated UPR state is attributed to the need for saliva proteins for plant feeding. Because aphids are agriculturally significant pests, and saliva is pivotal to their feeding on host plants, genes that encode saliva proteins may be targets for pest mitigation. Here I have sought the aphid’s saliva proteome by combining results obtained in several laboratories by proteomic and transcriptomic approaches on several aphid species. With these data I constructed a tentative saliva proteome for the pea aphid by compiling, collating, and annotating the data from several laboratories. I used RNA-seq to verify the transcripts in pea aphid salivary glands, thus expanding the proposed saliva proteome from approximately 50 components to around 130 components, I found that transcripts of saliva proteins are upregulated during plant feeding compared to diet feeding.

The maternal rearing environment (MRE) of an organism can be a key determinant of an organism's host choice decisions, its own fitness, or the fitness of its offspring. Here, it is investigated if the MRE of an organism can influence lower or higher trophic levels. A series of reciprocal cross-over experiments was conducted using pea aphids (Acyrthosiphon pisum), bean (Vicia faba) or pea (Pisum sativum) plants, and an aphid natural enemy, the parasitoid wasp Aphidius ervi, as model organisms. In each experiment, pea aphid offspring experienced either the same or an alternative plant host to that experienced by their mothers. This PhD showed that the MRE of pea aphids and parasitoid wasps was not a main contributory factor of host choice decisions or offspring fitness but influenced mother parasitoid wasp fecundity. Additionally, the MRE of pea aphids influenced the foliar nutrient concentration of pea plants when infested with the aphid's offspring. First, over shorter infestation periods, variation in foliar nitrogen and essential amino acid concentrations of pea leaves could be explained by pea aphid MRE. Over longer infestation periods, variation in foliar nitrogen and essential amino acid concentrations of pea leaves was explained by a combination of pea aphid MRE and aphid genotype. Second, the 13C concentration of pea leaf tissue, an indicator of stomatal aperture and leaf water stress, varied with pea aphid MREs over longer infestation periods. However, stomatal conductance and the expression of abscisic acid-responsive genes did not vary in a manner that was consistent with leaf water stress. Additional components of an organism's maternal rearing conditions are considered, including symbioses, as a more realistic MRE compared with that observed in nature. Taking account of MREs could provide a better understanding of the factors influencing the fitness of many organisms interacting in natural and managed ecosystems.

The use of stable nitrogen isotope data in ecological and physiological studies is based in the assumption that nitrogen fractionates predictably during metabolism, leading to a broadly conserved pattern whereby consumers are isotopically enriched with respect to their diets. The application of stable isotope data to such studies is limited is by our understanding of the factors in that cause variability in the Δ15N values of consumers. In particular, parasites and fluid-feeders have been shown to demonstrate isotopic depletion with respect to their food sources. One factor that has been suggested to influence the Δ15N values seen in fluid-feeding consumers is the presence of endosymbionts and their contribution to nitrogen metabolism. The experiments described in this thesis directly test the hypothesis that the endosymbiotic bacteria Buchnera aphidicola is influencing the Δ15N value of the pea aphid on host alfalfa plants. Here I find that although aphids cured of their bacterial symbionts are less isotopically depleted than untreated aphids, they are still not enriched with respect to their phloem sap diet, indicating that endosymbiont metabolism alone is not responsible for the isotopic depletion observed in pea aphids. Metabolism of nitrogen in the pea aphid-Buchnera symbiosis has been well described with decades of physiological studies and with the publication of the pea aphid and Buchnera genomes. The two key features of metabolism in the pea aphid-Buchnera symbiosis are the recycling of waste ammonia by the aphid and the upgrading of the nonessential amino acids found in phloem sap to essential amino acids through collaborative metabolism between the pea aphid and Buchnera. Consistent with the described role of Buchnera in nitrogen metabolism, amino acid analyses of symbiotic and aposymbiotic aphids demonstrates an accumulation of the nonessential amino acids glutamine and glutamate and lower amounts of essential amino acids in the aposymbiotic aphids. I tested the influence of dietary amino acid profile on the Δ15N value of pea aphids and found that aphids are only isotopically depleted when they feed on diets with unbalanced amino acid compositions and are isotopically enriched when fed on a diet with a balanced profile of amino acids. I used isotopically labeled fructose to determine whether the difference in Δ15N value of pea aphids on diets of varying amino acid profiles is correlated to the amount of de novo amino acid synthesis occurring in the aphid. I found that there was a significantly higher incorporation of the labeled carbon backbone in the protein of pea aphids feeding on the unbalanced diets, supporting the idea that increased de novo amino acid synthesis are responsible for the differences in Δ15N values among aphids feeding on the two diets. The findings of this study highlight the influence of endosymbionts on the Δ15N values for pea aphids, demonstrate that dietary amino acid composition can influence the Δ15N value of pea aphids through the demand for metabolic upgrading of amino acids, and provide a model for the study of Δ15N values in systems where metabolism has been well characterized by experimental and genomic data.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald Reeck / Pea aphids (Acyrthosiphon pisum) are sap-sucking insects that feed on the phloem sap of some plants of the family Fabaceae (legumes). Aphids feed on host plants by inserting their stylets between plant cells to feed from phloem sap in sieve elements. Their feeding is of major agronomical importance, as aphids cause hundreds of millions of dollars in crop damage worldwide, annually. Salivary gland transcripts from plant-fed and diet-fed pea aphids were studied by RNASeq to analyze their expression. Most transcripts had higher expression in plant-fed pea aphids, likely due to the need for saliva protein in the aphid/plant interaction. Numerous salivary gland transcripts and saliva proteins have been identified in aphids, including a glutathione peroxidase. Glutathione peroxidases are a group of enzymes with the purpose of protecting organisms from oxidative damage. Here, I present a bioinformatic analysis of pea aphid expressed sequence tag libraries that identified four unique glutathione peroxidases in pea aphids. One glutathione peroxidase, ApGPx1 has high expression in the pea aphid salivary gland. Two glutathione peroxidase genes are present in the current annotation of the pea aphid genome. My work indicates that the two genes need to be revised.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are two well-established analytical separation techniques that are continuously being adapted for performing distinctive separations and analyses of multitudes of complex and/or unique samples. Since their introduction, these techniques have been pivotal in the discovery, analysis, and understanding of a variety of samples and still prove to be key analytical tools for biological investigation. Using these techniques, one can obtain a wide-range of valuable sample information from the hydrophobicity and molecular weights to size and charge distributions. Furthermore, these techniques allow for sample analysis, purification, and collection for additional sample analysis, such as mass spectrometry analysis. My doctoral dissertation encompasses the full scope of these two techniques and novel approaches for the investigation of distinct, relevant samples. Described herein is the fabrication of glass microfluidic devices used for CE and their diversity for numerous investigations. Chapter 2 shows that the resolution of the photomasks used in microchip fabrication does not alter the separation efficiency of the devices, as the separations remain diffusion-limited. Using an in-house built capillary electrophoresis system, wheat proteins were separated more than 25% faster than previously reported in literature, and the electropherograms used for sample varietal identification. The fabrication of a robust, portable CE system capable of performing biological analysis in microgravity and hypergravity environments is also discussed. The need for and features necessary to achieve a reliable, robust, automated system is further described in Chapter 4. Isolation and analysis of the pea aphid (Acyrthosiphon pisum) salivary secretions was completed for the first time using HPLC. By altering the aphid environment and the sample treatment parameters, sample concentrations were increased above the limit of detection. Coupled with mass spectrometry, identification of pea aphid salivary proteins such as exopeptidase, angiotensin converting enzyme, and Buchnera proteins has been achieved. Finally, a simplified contact conductivity detection system for the detection of jurkat cells was developed that surpasses current, complex optical systems. The experiments described in this dissertation demonstrate novel approaches for the preparation, separation, analysis, and identification of a wide variety of common, and uncommon, samples.

Les effecteurs jouent un rôle fondamental lors des interactions antagonistes plantes-pathogènes en supprimant les défenses de la plante, permettant ainsi aux parasites de se développer. De tels effecteurs ont été caractérisés chez les insectes herbivores mais leur rôle dans la spécialisation à la plante reste méconnu. Les pucerons se nourrissent de la sève du phloème et injectent dans la plante des effecteurs salivaires. L'étude des patrons d’évolution des effecteurs, ainsi que la caractérisation de leurs fonctions sont nécessaires à la compréhension des mécanismes de spécialisation chez les pucerons. Au cours de ces travaux, nous avons cherché à identifier les effecteurs salivaires impliqués dans l'adaptation du puceron du pois, Acyrthosiphon pisum, à ses hôtes.Des approches évolutives, basées sur un nouveau catalogue de 740 effecteurs candidats surexprimés dans les glandes salivaires de A. pisum, ont révélé que certains d'entre eux évoluent rapidement et que l'expansion de familles multigéniques apparaît comme une source importante de diversité des effecteurs. En parallèle, ces travaux ont permis d'optimiser l'expression transitoire médiée par Agrobacterium dans le pois. Ce nouvel outil d'analyse fonctionnelle permet maintenant l'étude des effecteurs candidats afin d'identifier les effecteurs du puceron du pois impliqués dans l'adaptation à la plante hôte. / Effectors play fundamental roles in antagonistic plant-pathogen interactions mainly by suppressing plant defense and allow parasites to multiply on the plant. Some effectors have been characterized in herbivorous insects; however, their role to the evolution in plant specialization remains unknown. Aphids feed from phloem sap and inject salivary effectors into the host plant. Studying evolutionary patterns and characterizing functions of effectors appear as important steps toward unveiling the mechanisms of host plant specialization in aphids. This work sought to identify salivary effectors that are involved in plant specialization of the pea aphid, Acyrthosiphon pisum. Evolutionary approaches based on a new catalogue of 740 putative effectors that are up-regulated in salivary glands of A. pisum revealed that some of them evolve rapidly.Moreover, gene family expansion appear as an important source of novel effectors. In parallel, this work optimized Agrobacterium-mediated transient gene expression in pea to provide a new tool for functional analyses of pea aphid effectors. The construction of a comprehensive catalogue of A. pisum salivary effectors and evolutionary analysis of them provide new candidates in host plant adaptation. By using the gene expression tool now available in pea, functional characterization of candidates will help to identify the effectors that are involved in plant specialization of the pea aphid.

Doctor of Philosophy / Department of Entomology / Gerald R. Reeck / John C. Reese / Salivary secretions are a key component of aphid-plant interactions. Aphids’ salivary proteins interact with plant tissues, gaining access to phloem sap and eliciting responses which may benefit the insect. In an effort to isolate and identify key components in salivary secretions, we created a salivary gland cDNA library. Several thousand randomly selected cDNA clones were sequenced. We grouped these sequences into 1769 sets of essentially identical sequences, or clusters. About 22% of the clusters matched clearly to (non-aphid) proteins of known function. Among our cDNAs, we have identified putative oxido-reductases and hydrolases that may be involved in the insect's attack on plant tissue. C002 represents an abundant transcript among the genes expressed in the salivary glands. This cDNA encodes a novel protein that fails to match to proteins outside of aphids and is of unknown function. In situ hybridization and immunohistochemistry localized C002 in the same sub-set of cells within the principal salivary gland. C002 protein was detected in fava beans that were exposed to aphids, verifying that C002 protein is a secreted protein. Injection of siC002-RNA caused depletion of C002 transcript levels dramatically over a 3 day period after injection. With a lag of 1 – 2 days, the siC002-RNA injected insects died, on average 8 days before the death of control insects injected with siRNA for green fluorescent protein. It appears, therefore, that siRNA injections of adults will be a useful tool in studying the roles of individual transcripts in aphid salivary glands.

Les pucerons sont des hémiptères ravageurs des cultures agronomiques particulièrement adaptés à leur environnement. Acyrthosiphon pisum (le puceron du pois) présente un cycle de vie basé sur l’alternance d’une reproduction sexuée ou asexuée en réponse à la photopériode. Ils présentent ainsi un polyphénisme de reproduction aboutissant à la formation de trois phénotypes distincts : femelles asexuées, femelles sexuées, et mâles. Ces derniers étant obtenus par élimination d’un chromosome X, A. pisum est une espèce hétérogamétique mâle présentant un système chromosomique XX chez les femelles et X0 chez les mâles. Le déséquilibre du nombre de chromosome X entre mâles et femelles engendré par cette hétérogamétie nécessite chez certains organismes d’être corrigé par des mécanismes de compensation de dose. Les polyphénismes et compensation de dose impliquent chez d’autres organismes des régulations transcriptionnelles notamment régulées par l’accessibilité de la chromatine.Ma thèse vise ainsi à étudier le polyphénisme de reproduction et la compensation de dose des pucerons sous l’angle d’analyses bio-informatiques de données d’expression des gènes (RNA-seq) et d’accessibilité de la chromatine (FAIRE-seq) dans le but de caractériser l’impact des mécanismes épigénétiques dans ces deux processus biologiques fondamentaux du cycle de vie des pucerons. Les résultats développés dans ma thèse ont permis de montrer d’une part la présence d’une compensation de dose chez le puceron du pois au niveau transcriptomique, supportée par une accessibilité accrue de la chromatine de l’unique X des / Aphids are hemipterous crops pests that are particularly adapted to their environment. Acyrthosiphon pisum (pea aphid) displays a life cycle based on the alternation of sexual or asexual reproduction in response to photoperiod. They thus exhibit a reproductive polyphenism resulting in the formation of three distinct phenotypes: asexual females, sexual females, and males. The latter being obtained by elimination of an X chromosome, A. pisum is a male heterogametic species with a XX chromosomal system in females and X0 in males. The X chromosome number between males and females caused by this heterogamy requires in some organisms to be corrected by dosage compensation mechanisms. Polyphenisms and dosage compensation both involve in other organisms transcriptional regulations that are notably regulated by the chromatin accessibility regulations. My thesis aims to study the reproductive polyphenism and dosage compensation in aphids in the context of bioinformatic analyzes of gene expressioThe results developed in my thesis have shown, on one hand, the presence of dose compensation in pea aphid at the transcriptomic level, which is supported by increased chromatin accessibility of the males’ single X in somatic cells. On the other hand, specific sites of chromatin opening between sexual and asexual embryos seem to participate in the definition of their reproduction mode by modulating the expression of certain genes and by allowing the fixation of transcription factors. Their analysis shows the involvement of ecdysone as a new hormonal pathway that may trigger sexual reproducti

4 pp. / The alfalfa aphid discussed in this publication includes blue alfalfa aphid, pea aphid, and the spotted alfalfa aphid. This publication discusses the biology of these alfalfa aphids, the damages they cause, the resistant varieties and biological control, and their monitoring and treatments.

Community genetics research investigates the influence of intra-specific genetic variation on species interactions. This rapidly growing research field consists of more than one approach to explore how a significant portion of the environment of a focal species is differentially defined by the expressed genomes of other interacting species. While the basic concept of community genetics is well supported empirically, there is still a set of pertinent issues in need of further investigation. The initial research addressed herein focused on the extent to which the magnitude of a community genetic effect can be moderated when acting in concert with other forces in nature, i.e. the interaction between community genetic effects and the effects of other eco-evolutionary processes such as competition and parasitism. Subsequent research investigated the impact of genetic variation of host plants in agro-ecosystems on the performance (reproductive success) and behaviour (distribution and feeding-site choice) of plant-associated pests such as aphids, especially when pests and their hosts were subject to plant-mediated interactions. In addition, the differential effects of Indirect Ecological Effects (IEEs) and Indirect Genetic effects (IGEs) on the emergence of shared (extended) phenotypes between natural enemies (i.e. biological control agents and phytophagous insects) were examined. I provide clear evidence for significant effects of the genetic variation of host plant on aphid performance, behaviour and intra- and inter-specific competition. My findings also give credence to the concept of reciprocal moderation between plant genotype and aphid competition. I also provide observations on competition that segues into less antagonistic and possibly into a more cooperative form of interaction. In addition, I establish novel systems of economically important crop genotypes, noxious sap-feeding aphid species and root-galling nematodes. I also devise an amalgamated approach to interpret the interwoven set of mechanisms that underpin the observations presented and conclusions drawn. I also provide further investigation on the role of Indirect Ecological Effects (IEEs) between root-knot nematodes and sap-feeding aphids, and demonstrate the influence of in-plant variation on the interaction between the spatially separated plant consumers. Furthermore, I use a quantitative genetic experimental design in order to demonstrate a differential impact of parasitoid genotype on the behaviour of its aphid host. As such, I provide some of the clearest evidence to date that the phenotype of an organism can be the product of the genes expressed in another organism via Inter-specific Indirect Genetic Effects (IIGEs). Finally, I conducted research on epiphytic bromeliads and their associated faunal communities in the tropics. Here I demonstrate that the influence of intra-specific genetic variation of the host plant on the associated ecological communities may be more universal than previously conceived, with a plausible role for such variation in the maintenance of biological diversity. My research provides evidence for the genetic basis of species interactions and, interestingly, a genetic basis for the evolutionary arms-race between foragers and their hosts. My doctoral work adds new evidence to the increasing literature on the evolutionary importance of (Genotype x Genotype) interactions and (Genotype x Genotype x Environment) interactions in shaping the dynamics of pest communities, which in turn can affect plant phenotype and can influence the properties and services of the focal ecosystem in which the inter-players live and interact.