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.

Master of Science / Department of Entomology / James R. Nechols / Temperature has an important indirect impact on pest populations. Direct effects occur, but also may result from temperature-induced changes in plant quality, including the expression of host plant resistance traits. Therefore, I examined both indirect and direct effects of temperature on biotype 1 soybean aphids (SBA), Aphis glycines, on a Rag1-resistant soybean variety and compared the effects with a susceptible variety to gain a better understanding of how temperature impacts SBA. Four aphid responses were evaluated: preimaginal development, survival to adulthood, number of progeny produced, and adult longevity. In the first experiment, I grew soybean seedlings to the V-0 stage at 25°C and then conditioned them for 0, 3 or 5 days at 20° or 30°C before infesting with a single first instar SBA at each of the two experimental temperatures. Based on previous literature for SBA, I hypothesized that conditioning plants at the lower temperature would cause resistance to break down and that longer exposure would exacerbate the effect. Results showed that conditioning soybeans to 20°C significantly reduced SBA survival, and the effect on survival increased with longer conditioning. Conditioning plants to 30°C had no significant effect on SBA survival. However, estimated population growth decreased as conditioning time increased at 30°C and this effect was also observed at 20°C. Thus, plant resistance may have increased at both temperatures. The second experiment compared SBA responses, including population growth, at four temperatures (15, 20, 25, and 30⁰C) on a Rag1-resistant and susceptible soybean variety. I predicted that SBA fitness would be lower at all temperatures on resistant soybeans, but the magnitude of differences between cultivars would not be uniform across temperatures. Results indicated that both temperature (highest and lowest) and plant resistance detrimentally affected SBA fitness. There was also a significant interaction between the two variables with respect to SBA survival. Survival was lower and development rates were slower on the resistant cultivar. SBA required more degree-days to develop on resistant soybeans compared to the susceptible cultivar. This information will aid soybean producers in implementing a cost-efficient IPM strategy involving Rag1 resistant soybeans to combat SBA under a range of temperatures.

2 pp. / This publication describes the common insects found in Arizona lettuce through the use of pictures. The insects include; lepidopterous larva, striped flea beetle, leafminer fly, leafminer mine, adult western flower thrips, winged adult aphid, false chinch bug, lygus bug, potato leafhopper, and threecornered alfalfa hopper.

Intimate, mutualistic, association with microbes is a common mechanism for organisms to utilize certain niches. Insects are a particularly well-studied group in this respect, frequently forming long-term, obligate associations with symbiotic microbes. These symbioses are often nutritional in nature, with the symbiont providing the host with nutrients that are otherwise unavailable. Aphids are notable for their well-defined relationship with the symbiotic Bacteria Buchnera aphidicola. By synthesizing the amino acids the aphid is unable to produce itself, Buchnera permits its host to feed on plant phloem, which lacks sufficient quantities of these essential nutrients. Buchnera, as with many obligate intracellular symbionts, has a reduced effective population size (Nₑ) due to asexual reproduction and severe population bottlenecks experienced during transmission between generations. The reduction in Nₑ has facilitated the degradation of the symbiont genome through fixation of deleterious mutations via drift. The consequences of accelerated evolutionary rates has been examined primarily through genome sequencing and comparative studies of symbionts from different host species. The work detailed in this dissertation examines the role of deleterious mutations and drift at multiple taxonomic levels. Analysis of aphid amino acid requirements utilizing an artificial diet assay revealed variation in clones of the pea aphid, Acyrthosiphon pisum. In one clone, a mutation in the arginine biosynthesis pathway appears to underlie a host dietary requirement for arginine. Examination of the number of Buchnera within an A. pisum clone also revealed variation in symbiont titer between clones. When compared across F₁ offspring of cross between a low- and a high-titer clone, extensive variation was observed in titer that exceeded variation observed in field-collected clones. No maternal effects were observed, suggesting that Buchnera is not in control of its replication. At a broader taxonomic scale, the replacement of Buchnera in the aphid Cerataphis brasiliensis was examined by sequencing the genome of its fungal symbiont (YLS). The genome of the YLS revealed a much greater metabolic capacity than Buchnera, possibly due to its extracellular habitat. The YLS exhibited signatures of elevated evolutionary rates and intron gain consistent with a reduction in Nₑ due to its symbiotic niche.

Evidence is accumulating that climate change is having a significant effect on a wide range of organisms spanning the full range of biodiversity found on this planet. This study investigates the ecological role of climate change, the North Atlantic Oscillation (NAO) and habitat change on British insect populations. Despite the NAO having a considerable effect on British weather, the role of the NAO on British insects has not previously been studied in great detail. The World's two best entomological time series datasets were used – the United Kingdom Butterfly Monitoring Scheme (UKBMS) and the Rothamsted Insect Survey of aphids – both surveys with very large sample sizes and high quality data. Summary of main findings: 1. Warm weather associated with a positive NAO index caused the spring migration of the green spruce aphid (Elatobium abietinum), a pest species of spruce trees (Picea) to start earlier, continue for longer and contain more aphids. An upward trend in the NAO index during the period 1966-2006 is associated with an increasing population size of E. abietinum. 2. The NAO does not affect the overall UK butterfly population size. However, the abundance of bivoltine butterfly species, which have a longer flight season, were more likely to respond positively to the NAO compared to univoltine species, which show little or a negative response. 3. A positive winter NAO index was associated with warmer weather and earlier butterfly flight dates. For bivoltine (two generations in a year) species, the NAO affects the phenology of the first generation, and then the timing of the second generation is indirectly controlled by the timing of the first generation. The NAO influences the timing of the butterfly flight seasons more strongly than it influences population size. 4. Butterfly data from Monks Wood National Nature Reserve in Cambridgeshire showed that the NAO does not affect the abundance of the whole butterfly community, but it does affect the population size of some species. The NAO does not affect butterfly diversity, but there were decreases in butterfly diversity and number of species with time. 5. The total number of butterflies counted at Monks Wood was constant for most of the time series. However, the population size of the ringlet (Aphantopus hyperantus) increased from very low numbers to more than half the total number of butterflies counted each year. Therefore the total population size of all the other species has decreased considerably. 6. The NAO was more important than climate change in determining the flight phenology of the meadow brown butterfly (Maniola jurtina) at Monks Wood. In conclusion, the NAO affects the abundance of some species of British butterfly, and an aphid species, with a stronger effect on the timing of flight rather than abundance. There was evidence for a long-term decrease in the biodiversity of butterflies at Monks Wood and this decrease is likely to continue.

As cucurbitáceas no Brasil podem ser infectadas por diferentes vírus, tais como o Papaya ringspot virus - type W (PRSV-W); o Zucchini yellow mosaic virus (ZYMV) e o Cucumber mosaic virus (CMV). Os dois primeiros pertencem ao gênero Potyvirus e no geral ocorrem com maior freqüência do que o CMV, que é uma espécie do gênero Cucumovirus. Os dois potyvirus e o cucumovirus são transmitidos por afídeos de maneira não persistente. O principal objetivo desse trabalho foi o de obter subsídios que possam explicar a menor incidência do CMV em espécies de cucurbitáceas, estudando: (a) a interferência dos potyvirus PRSV-W e ZYMV na transmissão do CMV por Aphis gossypii e Myzus persicae para plantas de abobrinha de moita (Cucurbita pepo ‘Caserta’) e (b) o efeito de isolados do CMV provenientes de maracujazeiro (Passiflora edulis f. flavicarpa), de pimentão (Capsicum annuum), de pepineiro (Cucumis sativus), de meloeiro (Cucumis melo) e de trapoeraba (Commelina virginica) na infectividade de plantas de abobrinha de moita por meio da transmissão por afídeos. Para avaliar a possível interferência dos potyvirus na transmissão do CMV, as plantas de abobrinha de moita foram inoculadas com afídeos que adquiriram cada um dos vírus isoladamente; o CMV simultaneamente com cada um dos potyvirus; um dos potyvirus seguido pelo CMV e vice-versa. Os resultados mostraram, na maioria das vezes, que a transmissão dos vírus isoladamente foi mais eficiente do que em mistura, tanto através de aquisição simultânea como seqüencial. Os potyvirus no geral foram mais eficientemente transmitidos por ambas espécies de afídeos. Quando em mistura (aquisição simultânea ou sequencial), de uma maneira geral, houve uma redução na taxa de transmissão do CMV e do potyvirus presente na mistura. As avaliações sobre o efeito da origem dos isolados do CMV na infectividade de abobrinha de moita mostraram que apenas o isolado de pimentão não infectou plantas de abobrinha de moita quando transmitido por meio dos afídeos A. gossypii e M. persicae. Também não houve infecção quando inoculado mecanicamente. Os demais isolados infectaram abobrinha de moita através da transmissão por ambas espécies de afídeos. Análise da proteína capsidial dos diferentes isolados do CMV indicaram que todas apresentaram a mesma mobilidade em gel de SDS-PAGE. A origem do isolado o CMV, a eficiência da espécie de afídeo na sua transmissão e a interferência dos potyvirus PRSV-W e ZYMV podem explicar em parte a menor incidência desse cucumovirus em cucurbitáceas no país. / The cucurbits in Brazil can be infected by different viruses, such as Papaya ringspot virus - type W (PRSV-W); Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV). The first two belong to the genus Potyvirus and in general they occur more frequently than CMV, which is a species of the genus Cucumovirus. The two potyviruses and the cucumovirus are transmitted by means of aphids in a non persistent way. The main objective of this work was to obtain subsidies that can explain the lower incidence of CMV in cucurbit species, studying: (a) the interference of the potyviruses PRSV-W and ZYMV in the transmission of CMV by means of Aphis gossypii and Myzus persicae to zucchini squash plants (Cucurbita pepo 'Caserta') and (b) the effect of isolates of CMV from passion flower (Passiflora edulis f. flavicarpa), bell pepper (Capsicum annuum), cucumber (Cucumis sativus), melon (Cucumis melo) and Commelina virginica in the infectividade of zucchini squash plants through the transmission by aphids. To evaluate the possible interference of the potyvirus in the transmission of CMV, zucchini squash plants were inoculated with aphids that acquired each one of the viruses separately; CMV simultaneously with each one of the potyvirus; one of the potyvirus follow by CMV and vice-versa. The results showed that the transmission of PRSV-W, ZYMV and CMV separately was more efficient than in mixture. The potyviruses in general were more efficiently transmitted by both species of aphids than CMV. When in mixture (simultaneous or sequential acquisition), there was a reduction in the rate of transmission of CMV as well as that of the potyvirus present in the mixture. The evaluation on the effect of the origin of the isolate of CMV in the infectivity of zucchini squash showed that only the isolate from bell pepper did not infected the plants when inoculated by means of A. gossypii and M. persicae. This isolate also did not infecte zucchini squash when inoculated mechanically. The others isolate infected zucchini squash when transmitted by both species of aphids. Analysis of the capsidial protein of the different isolates of CMV indicated that all presented the same mobility in SDS-PAGE. The origin of the isolate of CMV, the efficiency of the species of aphid and the interference of the potyviruses PRSV-W and ZYMV on its transmission can partly explain the lower incidence of this cucumovirus in cucurbits species in Brazil.

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No. of bitstreams: 2 Dissertação_Sheila Avila.pdf: 1444685 bytes, checksum: 5af742f17f8020ce9558feb7331065cc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Pesquisar adubações orgânicas que almejam o equilíbrio trofobiótico na cultura de interesse é de extrema importância na busca de sistemas de produção de alimentos mais sustentáveis. Com esta premissa objetivou-se no trabalho analisar a influência de diferentes adubações orgânicas sobre a preferência alimentar de Brevicoryne brassicae em Brassica oleracea var. acephala, estabelecendo inter-relações entre o metabolismo vegetal da couve, as fontes de adubação testadas e a resistência e/ou suscetibilidade aos afídeos. Para tanto, foi feito o cultivo da hospedeira em casa de vegetação, onde a mesma foi cultivada em vasos com adubação orgânica acrescentada ou não de fitoprotetores, obedecendo aos seguintes tratamentos: T1) substrato orgânico comercial a 70% + 30% de húmus (SO+H) como controle; T2) (SO+H) + biofertilizante Vairo via solo; T3) (SO+H) + biofertilizante Supermagro via solo T4) (SO+H) + urina de vaca via solo; T5) (SO+H) + Húmus líquido pulverizado; T6) (SO+H) + soro de leite pulverizado e, T7) (SO+H) + biodinâmico pulverizado. O delineamento experimental foi inteiramente casualizado com 10 repetições, sendo cada repetição (parcela) uma planta. Para os bioensaios de preferência alimentar de múltipla escolha em laboratório foi feita a criação do afídeo, sendo as avaliações realizadas as 24 e 48 horas através da contagem de indivíduos em todas as folhas (halos) representando todos os tratamentos. Também foi feito análise química dos substratos e das couves, além dos fitoconstituintes das mesmas após receber os diferentes tratamentos. Os dados foram submetidos ao teste de hipótese de KruskalWallis (p<0,05), com comparação de grupos pelo método de Simes-Hochberg, sendo após, submetidos à análise de componentes principais. A aplicação dos tratamentos influenciou na preferência alimentar de B. brassicae em B. oleracea var. acephala. Estes forneceram diferentes fontes de nutrientes ao substrato e à planta hospedeira, afetando assim, seu metabolismo vegetal e conseqüentemente a resistência e/ou suscetibilidade aos afídeos. As plantas tratadas com húmus líquido e Vairo apresentaram menor preferência pelos pulgões. Estes induziram a resistência, envolvendo o acúmulo de ácido ascórbico que se correlacionou ao manganês. Os tratamentos urina de vaca, Supermagro e em menor grau o soro de leite foram os que apresentaram folhas com maior preferência pelos afídeos, sendo estes ricos em nitratos e açúcares solúveis, estando estes compostos relacionados respectivamente aos nutrientes nitrogênio, ferro e cálcio. Já os tratamentos controle e biodinâmico apresentaram resultados intermediários de preferência e de correlação aos nutrientes e fitoquímicos. / To search for organic fertilizers that target the trophobiotic balance in the culture of interest and of extreme importance in the search for more sustainable food production systems. The objective of this work was to analyze the influence of different organic fertilizers on the Brevicoryne brassicae food preference in Brassica oleracea var. acephala, establishing interrelations between the vegetable metabolism of the cabbage, the sources of fertilization tested and the resistance and / or susceptibility to the aphids. For this, the host was cultivated in a greenhouse, where it was cultivated in pots with organic fertilization added or not of phytoprotectants, following the following treatments: T1) commercial organic substrate at 70% + 30% humus (SO + H) as control; T2) (SO + H) + biofertilizer Vairo via soil; T3) (SO + H) + biofertilizer Supermagro via soil T4) (SO + H) + cow urine via soil; T5) (SO + H) + pulverized liquid humus; T6) (SO + H) + powdered whey and, T7) (SO + H) + pulverized biodynamic. The experimental design was completely randomized with 10 replicates, each replicate (plot) being one plant. For the bioassays of multiple choice food preference in the laboratory was created the aphid, and the evaluations were performed at 24 and 48 hours by counting individuals on all leaves (halos) representing all treatments. Chemical analysis of the substrates and cabbage was carried out, besides the phyto-constituents of the same ones after receiving the different treatments. The data were submitted to the Kruskal-Wallis hypothesis test (p <0.05), with a comparison of groups by the Simes-Hochberg method, after which they were submitted to principal components analysis. The application of the treatments influenced the food preference of B. brassicae in B. oleracea var. acephala. These provided different nutrient sources to the substrate and host plant, thus affecting its plant metabolism and consequently the resistance and/or susceptibility to aphids. Plants treated with liquid humus and Vairo showed less preference for aphids. These induced resistance, involving the accumulation of ascorbic acid that correlated with manganese. The treatments of cow urine, Supermagro and, to a lesser extent, whey were the ones that presented leaves with a higher preference for aphids, being these rich in nitrates and soluble sugars, these compounds being related respectively to nutrients nitrogen, iron and calcium. The control and biodynamic treatments presented intermediate results of preference and of correlation to nutrients and phytochemicals.

The thesis studied the biological control of woolly aphid (Eriosoma lanigerum Hausm.) using European earwig (Forficula auricularia L.) in an Australian apple orchard IPM program. Woolly aphid populations were monitored over the 1995/96 and 1996/97 growing seasons, completing a four year study of the pest's status and management under IPM programs at Bathurst in the Central Tablelands of NSW. Woolly aphid infestation in 2 IPM treatments, based on alternative control techniques for codling moth (Cydia pomonella L.), namely mating disruption and fenoxycarb, were compared with a conventional insecticide azinphos-methyl program. Further, the thesis studied the toxicity, to adult European earwigs, of chemicals commonly used in Australian apple orchards. The pesticide alpha-cypermethrin is highly toxic to weevil spp. and is used by apple growers in Western Australia for their control, but it is also toxic to earwigs. This trial investigated the potential of alpha-cypermethrin to disrupt the biological control of woolly aphid. Following a single application as a butt spray, alpha-cypermethrin suppressed the number of earwigs in apple trees for 14 weeks. The single application did not reduce earwig numbers to the extent that the biological control of woolly aphid was lost, however a full season program with applications every 14 to 21 days to all trees, as recommended to control weevils, within an orchard is likely to be very disruptive. The research also studied the control of woolly aphid using insecticide root-soil drenches. Imidacloprid provided excellent control of woolly aphid for 3 seasons. Pirimcarb provided some suppression of woolly aphid during the first season, but not in subsequent seasons. Chlorpyrifos and vamidothion failed to control woolly aphid in any season. / Doctor of Philosophy (PhD)

Mounted ill. Bibliography: leaves 263-265.

Most Aphidinae species produce and use (E)-ß-farnesene (Eßf) as an alarm pheromone. This sesquiterpene is released by individuals under attack by a predator, and nearby aphids exhibit a variety of alarm behaviours. This PhD thesis aims to better understand how aphids manage their production and emission of alarm pheromone (Chapter IV). We also wanted, in a second step, to improve our knowledge on the roles that Eßf could play in the relationships that aphids have with their predators (Chapter V) and tending ants (Chapter VI), in order to better pinpoint the problem in this very tough context. The aphid predators have indeed a real advantage to be able to use the odorant cues emitted by their prey, to locate them and to select an adequate oviposition site. Ants establish with certain aphid species mutualistic relationships, which occurrence could be facilitated by the use of aphids odours. In Chapter IV, we have highlighted that aphid colonies non subjected to attack by predators release constantly small quantities of Eßf in their headspace, which means that this molecule could have additional roles than just acting as an alarm pheromone. In a second study, we demonstrated that the release of Eßf was not contagious, and therefore that a non stressed aphid receiving the alarm signal does not release additional Eßf. Since the production of alarm pheromone is likely to entail physiological cost, we tested and validated the hypothesis that aphids regulate their Eßf production according to their social environment. In Chapter V, we studied the ability of the hoverfly predator Episyrphus balteatus (Diptera, Syrphidae) to be used as biological control agent against aphids infesting tomato plants (Lycopersicon esculentum). After identifying the odours emitted by aphid infested plants, we have demonstrated that although this Diptera is able to perceive all the odours released by the system, it mainly uses Eßf to select its oviposition site. However, the E. balteatus larvae are not adapted to the architecture of tomato plants. We also showed that the Asian ladybeetle Harmonia axyridis (Coleoptera, Coccinellidae) olfaction was adapted to the perception of Eßf and that this beetle is also attracted by this sesquiterpene. Finally, in Chapter VI, we characterized the benefits accruing to aphid populations that have established mutualistic relationships with Lasius niger (Hymenoptera, Formicidae), and have demonstrated the role of Eßf and honeydew, respectively in locating aphid colonies and in the persistence of the mutualism. La plupart des espèces de pucerons appartenant à la sous-famille des Aphidinae produisent et utilisent le (E)-ß-farnésène (Eßf) comme phéromone dalarme. Ce sesquiterpène est relargué par les individus stressés par lattaque dun prédateur et cause chez les individus qui le perçoivent un comportement dalerte. La présente thèse de doctorat a pour objectif de comprendre comment les pucerons gèrent la production et émission de phéromones dalarme (Chapitre IV). Nous voulions aussi, dans une seconde étape, améliorer les connaissances sur les rôles potentiels que cet Eßf peut jouer au sein des relations que les pucerons entretiennent avec leurs prédateurs (Chapitre V) et avec les fourmis (Chapitre VI) Les prédateurs de pucerons retirent en effet un réel avantage à pouvoir saider des odeurs émises par leurs proies pour les localiser et pour sélectionner un site doviposition adéquat. Les fourmis, quant à elles, établissent des relations de mutualisme avec certaines espèces de pucerons. Les rencontres entre fourmis et pucerons pourraient être facilitées par lutilisation des odeurs de pucerons. Les résultats obtenus peuvent être résumés de la manière suivante: Dans le chapitre IV, nous avons mis en évidence que les colonies de pucerons non soumises à lattaque de prédateurs relarguent constamment de faibles quantités dEßf, ce qui permet dassumer que cette molécule puisse avoir dautres fonctions que celle de phéromone dalarme. Dans une deuxième étude, nous avons démontré que lémission du Eßf nétait pas contagieuse, et donc quun puceron non stressé percevant le signal dalarme német pas à son tour de le Eßf. Puisque la production de phéromone dalarme a inévitablement un coût physiologique, nous avons testé et validé lhypothèse selon laquelle les pucerons régulent leur production de Eßf en fonction de leur environnement social. Dans le chapitre V, nous avons étudié la possibilité dutiliser le syrphe prédateur Episyrphus balteatus (Diptera, Syrphidae) en lutte biologique contre les pucerons infestant les plants de tomate (Lycopersicon esculentum). Après avoir identifié les odeurs émises par les plants infestés, nous avons démontré que, si ce Diptère est capable de percevoir lensemble des odeurs émises par ce système tritrophique, il utilise principalement lEßf pour sélectionner son site doviposition. Cependant, les larves dE. balteatus ne sont pas adaptées à larchitecture des plants de tomate. Nous avons également montré que la coccinelle asiatique Harmonia axyridis (Coleoptera, Coccinellidae) possède le matériel olfactif nécessaire à la perception du Eßf et quelle est aussi attirée par ce sesquiterpène. Enfin, dans le chapitre VI, nous avons caractérisé les bénéfices retirés par les populations de pucerons ayant établi des relations de mutualisme avec Lasius niger (Hymenoptera, Formicidae), et avons démontré le rôle du Eßf et du miellat, respectivement dans la localisation des colonies de pucerons et dans la persistance du mutualisme.