Call number: LD2668 .T4 1986 J86 / Master of Science / Biology

Fat body tissue was taken from female Manduca larvae throughout the fifth instar. The samples were prepared for histological study and electron microscopy. Hemolymph samples were collected and analyzed for protein profile and concentration. The data showed that the fat body underwent a series of changes with development. These include an increase in cell size, and accumulation of lipid and glycogen during the feeding phase of the instar, and the formation of protein and urate granules during the wandering stage. Also apparent was the cyclic development of a reticular system on the cell surfaces. Maximum development of the system coincided with the period of highest protein concentration in the hemolymph, while its disappearance is coincident with a drop in hemolymph protein concentration and formation of fat body granules. Thus the fat body plays a synthetic role early in the instar and becomes a storage tissue as pupation approaches.

Universiẗat, Diss., 2002--Osnabrück.

Master of Science / Graduate Biochemistry Group / Michael R. Kanost / Prophenoloxidase-activating proteinase (proPAP) activates prophenoloxidase when bacteria or fungi invade Manduca sexta. Upon activation, phenoloxidase initiates synthesis of melaninin, which can encapsulate the invaders and kill them. M. sexta contains three proteases that can activate prophenoloxidase, proPAP1, proPAP2, and proPAP3. The study of proPAP function has been slowed by the difficulty of expressing the proteins in recombinant systems. ProPAP1 contains one clip domain and one serine proteinase domain, a simpler structure than proPAP2 and proPAP3, which have two clip domains. For this reason, proPAP1 was selected for this investigation, to develop an improved system for expression of recombinant proPAP zymogens. In past experiments proPAP1 had a low expression level in insect cells using a baculovirus vector. In Escherichia coli, proPAP1 was expressed as an insoluble protein that could not be refolded successfully. The Bacillus subtilis expression system offers a potential improvement for expression of recombinant clip domain proteases because it can secrete recombinant proteins into the medium, it is a Biosafety Level 1 organism that is easy to handle, and it is less expensive to culture than insect cells. Four constructs for expression of proPAP1 and proPAP1 mutants were produced in the plasmid shuttle vector pHT43, which is compatible with both E. coli and B. subtilis. Experiments were carried out to test and optimize expression and purification of proPAP1 in B. subtilis. Conditions were optimized for IPTG (isopropyl β-D-1-thiogalactopyranoside) concentration, IPTG induction time, growth medium and induction temperature. Results showed that 0.5mM IPTG with 20 hours induction at 37°C in 2xYT medium was the optimum condition for proPAP1 production in the B. subtilis system. The recombinant proPAP1 was precipitated from the medium in 50% saturated ammonium sulfate and partially purified by nickel affinity chromatography. In addition to the full length proPAP1 protein, degradation of proPAP1 was also observed. Further experiments should be done to try to solve this problem. With purified protein, future work can be aimed at study of the structure and function of proPAP1.

Parasitoid insects face the fundamental problem of finding a suitable host in environments filled with competing stimuli. Many are deft sensors of olfactory cues emitted by other insects and the plants they live on, and use these cues to find hosts. Using olfactory cues from host-plants is effective because plants release volatile organic compounds (VOCs), in response to herbivory or oviposition, that contain information about the presence of hosts. However, plant-produced cues can also be misleading because they are influenced by a variety of stimuli (abiotic variation, infection and multiple sources of induction via herbivory or oviposition). Flexible behavior is one strategy that parasitoids may use to cope with variation in olfactory cues. We examine the innate and learned responses of a natural population of wasp egg parasitoids (Trichogramma deion and Trichogramma sathon) using a series of laboratory and field Y-olfactometer experiments. Wasps typically attack eggs of the hawkmoth Manduca sexta and Manduca quinquemaculata on native Datura wrightii plants in the southwestern United States. We show that Trichogramma wasps responded innately to VOCs produced by D. wrightii and could distinguish plants recently attacked by M. sexta from non-attacked plants. Furthermore, adult Trichogramma wasps were able to learn components of the VOC blend given off by D. wrightii, though they did not learn during exposure as pupae. By further exploring the behavioral ecology of a natural population of Trichogramma, we gain greater insight into how egg parasitoids function in tri-trophic systems.

The moth Manduca sexta has been a common model for the study of the insect olfactory systems. The neuronal architecture in the antennal lobes (ALs) of insects and in the olfactory lobes of vertebrates is similar in structure and development. In Manduca, as in other olfactory systems, sensory receptor neurons send axons into the AL where they form synapses with local interneurons (LNs) and projection neurons (PNs) within the structural units of glomeruli. Here, I present the morphological development of one type of interneuron, the uniglomerular projection neuron (uPN), in normal AL development and in AL development in the absence of olfactory receptor neurons (ORNs). Using fluorescent-dye labeling of uPNs and confocal microscopy, my results show that in the absence of ORNs, uPN dendritic arborization is uncharacteristic of that in normally developing ALs, reinforcing the concept that afferent input guides the development of architecture in sensory areas of the brain.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 88-93).

Universiẗat, Diss., 2001--Osnabrück.

Universiẗat, Diss., 2005--Osnabrück.

Universiẗat, Diss., 2004--Saarbrücken.