Entomology, Department of


Date of this Version

Fall 11-25-2009


A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Entomology. Under the Supervision of Professor David W. Stanley
Lincoln, Nebraska: December, 2009
Copyright (c) 2009 Sean M. Putnam


I report on the chemical identification of lipid signaling molecules in three species of insects and describe the chemical isolation of a bacterial product that inhibits eicosanoid biosynthesis. Chapter 2 reports that eicosanoids mediate nodulation reactions to bacterial infection in newly-emerged, but not forager honeybees. Cyclooxygenase and lipoxygenase inhibitors attenuate nodulation, which is reversed in the presence of arachidonic acid. Older adult honeybees do not produce bacterial-induced nodules, and they have fewer circulating hemocytes, from which I infer that foraging honeybees express a physiological trade-off between maintaining a biologically expensive hemocytic immune system and flight activity associated with foraging. Chapters 3 and 4 report on eicosanoid production in two insect tissues, Manduca sexta midgut and Zophobus atrata fat body. Optimal reaction conditions for prostaglandin biosynthesis were developed. Microsomal-enriched fractions of the tissues produced four PGs, PGA/B2, PGD2, PGE2 and PGF2&#;. Chemical structures of each PG was confirmed by gas chromatography-mass spectrometry. PG biosynthesis was reduced by two cyclooxygenase inhibitors, indomethacin and naproxen. In contrast to the mammalian model, PG biosynthesis was more prevalent in cytosolic fractions compared to microsomal fractions. The chemical confirmation of PG structures provides strong evidence that PGs are definitely produced by insect tissue and that these lipid mediators facilitate important roles in biological actions. Chapter 5 describes the chemical properties of a factor produced from an insect pathogenic bacterium, Xenorhabdus nematophilus, which attenuates immune responses. The bacterium suppresses nodulation responses to bacterial infections by inhibiting eicosanoid biosynthesis. The immunity-suppressing factor from living X. nematophilus was present in the organic, and not aqueous, fraction of the bacterial culture medium. This chemical work was the first step in identification of a new class of eicosanoid biosynthesis inhibitor. This work advances the eicosanoid hypothesis by chemically confirming that PGs are synthesized in insect tissues and that they represent significant mediators of biological actions in insects. Beyond that, the work illustrates the power of multidisciplinary research to advance our understanding of insect biology generally.

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