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.

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.

Universiẗat, Diss., 2001--Osnabrück.

Universiẗat, Diss., 2005--Osnabrück.

Universiẗat, Diss., 2004--Saarbrücken.

Doctor of Philosophy / Biochemistry / Michael R. Kanost / Insect innate immune responses include prophenoloxidase activation and antimicrobial peptide production. These responses involve extracellular serine proteinase cascades that are regulated by serpins. This work involved the study of serine proteinase 16 (HP16) and serpin-3 from hemolymph of the tobacco hornworm, Manduca sexta. HP16 has an amino-terminal domain with no similarity to any characterized protein and a carboxyl-terminal S1 family serine proteinase domain. HP16 levels in plasma were highest during the wandering, prepupal, and pupal stages. HP16 mRNA levels in fat body were highest at the wandering stage. Injection of bacteria into fifth instar larvae stimulated HP16 expression. To further characterize and investigate the biological function of HP16, recombinant proteins for proHP16, two HP16 mutants, the amino-terminal domain (NT16), and three NT16 mutants were purified. Recombinant HP16 was cleaved at the predicted activation site during expression, and its amino-terminal and catalytic domains remained connected by a disulfide bond. ProHP16 in plasma was apparently activated in the presence of the microbial elicitor, zymosan. Recombinant HP16 formed a complex with serpin-1Z, indicating that it was catalytically active, but no other natural or artificial substrates were identified. Analysis of NT16 and NT16 mutants led to the discovery that multiple disulfide bond arrangements were formed in the recombinant amino-terminal domain of HP16. This work furthered the understanding of HP16 and laid a foundation for subsequent experiments involving the proteolytic activity, regulation, and biological function of HP16. Active serine proteinases in insect hemolymph are often regulated by serpins. Immunoaffinity chromatography was used to identify plasma proteinases that are inhibited by serpin-3. Four serpin-3-proteinase complexes purified from plasma were identified by immunoblot analysis as serpin-3 complexes with HP8, PAP-1, PAP-2, and PAP-3. MALDI-TOF/TOF or ESI-MS/MS analysis after separation by 1D- or 2D-PAGE confirmed serpin-3 complex formation with HP8, PAP-1, and PAP-3. ProHP8 in plasma was activated by exposure to the β-1,3-glucan curdlan and inhibited by serpin-3. Purified recombinant serpin-3 and active HP8-Xa formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.

Natural selection acts on multiple traits simultaneously. How mechanisms underlying such traits enable or constrain their response to simultaneous selection is poorly understood. We show how antagonism and synergism among three traits at the developmental level enable or constrain evolutionary change in response to simultaneous selection on two focal traits at the phenotypic level. After 10 generations of 25% simultaneous directional selection on all four combinations of body size and development time in Manduca sexta (Sphingidae), the changes in the three developmental traits predict 93% of the response of development time and 100% of the response of body size. When the two focal traits were under synergistic selection, the response to simultaneous selection was enabled by juvenile hormone and ecdysteroids and constrained by growth rate. When the two focal traits were under antagonistic selection, the response to selection was due primarily to change in growth rate and constrained by the two hormonal traits. The approach used here reduces the complexity of the developmental and endocrine mechanisms to three proxy traits. This generates explicit predictions for the evolutionary response to selection that are based on biologically informed mechanisms. This approach has broad applicability to a diverse range of taxa, including algae, plants, amphibians, mammals, and insects.

Two proposed models will be used to help answer a long observed question in the dynamics of \textit{Manduca sexta} and its related parasitoid wasps-Why is there a large difference in diversity in hyperparasitoid species between tobacco and other related plants such as tomato? Two stage structured differential equation models are presented. The first is a single patch model to study the changes in dynamics that occur between hosts, parasitoids, and hyperparasitoids as the amount of nicotine in the plant increases. The second is a two patch model that allows hyperparasitoids to choose between patches that are nicotine negative (i.e. tomato plants) and nicotine positive (i.e. tobacco plants). Both models will be used to investigate how host nicotine sequestration may impact hyperparasitoid diversity.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Michael Kanost / Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC₅₀ of 12 μM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta. Biophysical analysis showed that diapausin-1 binds to the β-1,3 glucan component of the S. cerevisiae cell wall. A second insect peptide investigated in this project was M.sexta stress-response peptide 1(SRP1), an immune-related peptide upregulated under different stress conditions including immune-challenge. Preliminary results for NMR structure determination are presented. Most of the amino acid residue spin systems were assigned, and we determined the connectivities of many amino residues as a first step to solve the NMR structure. The circular dichroism spectrum of SRP1 indicates that the peptide lacks alpha-helical structure and may contain beta strands and turns.