Neural representations of odors get associated with other stimuli through experience. Are action potentials the neural representation that directly gets associated with reinforcement during conditioning? In Manduca , I found that odor presentations elicited only one or two spikes at odor onset (and sometimes offset) in each of a small portion of Kenyon cells, a population of neurons known to be crucial for olfactory associative learning. By using a series of odor-taste associative conditioning paradigms with various sucrose presentation timings, I carefully controlled the temporal overlap between Kenyon cell spiking and sucrose reinforcement timing. I found that in paradigms that led to learning, spiking in Kenyon cells ended well before the reinforcement was given. Further, increasing the temporal overlap between Kenyon cell spiking and sucrose reinforcement actually reduced learning efficacy. Therefore, spikes in Kenyon cells are not the neural representation that gets directly reinforced, and Hebbian spike timing--dependent plasticity in Kenyon cells alone cannot underlie this learning. / Two important focuses in neuroscience are to study how animals process sensory stimuli, and how such stimuli get associated with other sensory modalities through experience. Often, sensory stimuli elicit the oscillatory synchronization of neurons in different parts of the brain, and thus may constitute an important stage in sensory processing. Odor-evoked oscillatory synchronization has been observed in a wide variety of animals, including mammals and insects. Despite differences in details of anatomical structure, animals from widely different phyla appear to use similar strategies to encode odors. Here, using the moth Manduca sexta, I examined the factors that cause odor-evoked oscillatory synchronization of olfactory neurons and that determine the frequency of these oscillations. I found that frequency of oscillations decreased from ∼40 Hz to ∼20 Hz during the course of a lengthy odor pulse. This decrease in oscillatory frequency appeared in parallel with a decrease in net olfactory receptor output, suggesting that the intensity of olfactory receptor neuron input to the antennal lobe, the first olfactory relay center, may determine oscillatory frequency. However, I found that changing odor concentration had little effect on oscillatory frequency. Combining the results of recordings made in vivo and computational models, I found that increasing odor concentration recruited additional, but less well-tuned olfactory receptor neurons to respond to the odor. Firing rates of these neurons were tightly constrained by adaptation and saturation. My work established that, in the periphery, odor concentration is mainly encoded by the size of the olfactory receptor neuron population that responded to the odor, whereas oscillatory frequency is determined by the adaptation and saturation of this response. / Ong, Chik Ying Rose. / Advisers: Siu Kai Kong; Mark Stopfer. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 132-147). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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.

In vivo protein turnover was measured during the last 4 days of flight muscle development in tobacco hornworm pupa. Linear synthesis rates were measured up to 2 hours after injection of 30 μmol (3H) phenylalanine. Since the results with this technique did not differ from another established method, the large bolus injection of phenylalanine did not affect protein synthesis. The former method is advantageous because only a single time point is required. Flight muscle growth and protein synthesis decreased in parallel between 100 and 24 hours. During this time free phenylalanine turnover decreased, and the total pool diminished, indicating that this pool could be a major sink for muscle protein synthesis. Proteolysis was rapid even in the growing muscle. 20-Hydroxyecdysone increased muscle growth at certain times by inhibiting proteolysis. Protein synthesis either decreased or was unchanged after injection of the hormone. Therefore ecdysteroids may play a role in controlling growth of the dorsolongitudinal flight muscle during adult development, especially by retarding proteolysis.

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.

<p>A key question in developmental biology is how organisms attain a final size. Deviations in growth patterns can produce different/new phenotypes and these changes can play fundamental roles in ecology and evolution. The size of an organism and of its constitutive organs is determined by the growth rate and the duration of the growing period. In insects, peptide hormones such as insulin-like growth factors have been shown to be involved in determining the growth rates by coordinating metabolism, cell proliferation and cell size. In contrast, steroid hormones, such as ecdysone, are involved in determining life stage transitions, and thus the termination of the growing period. Although it is clear that insulin and steroid hormones are both involved in the regulation of growth, the ways in which these two regulators interact is yet to be determined. Furthermore, it is not clear how organs and body growth are coordinated during development to arrive to their correct proportions. In this study, using the tobacco hornworm Manduca sexta and its wings as a model system, I examine the developmental mechanisms involved in the regulation of organ growth and how developmental processes can drive morphological evolution. First, I examine how the hormonal events that take place during the termination of the body growth period affect wing disk growth. Second, by using gene expression assays and in vitro cultures, I examine the interaction between bombyxin, the Lepidopteran insulin-like growth factor, and ecdysone, the molting hormone, and their contributions to wing imaginal disk growth. Finally, by using three different size strains of M. sexta, I examine the developmental basis of the allometric relationship between the wings and the body. My results show that during the final instar of M. sexta larval development, wing imaginal disks are sensitive to the hormonal events that terminate the growth period. Furthermore, I show that the bombyxin requirement for wing disk growth is restricted to the early days of the final instar unlike the constitutive effects seen in other species. After the larva has passed a particular critical weight, bombyxin is not necessary for wing disk growth, although its absence does decrease the growth rate. In contrast, ecdysone is required for promoting the growth of wing imaginal disks primarily through its stimulation of cell proliferation. Finally, I show how selection on body size has unpredictable consequence for the response of wing size. These results demonstrate how specific allometries have a developmental basis in the cross-talk of the various signals that regulate growth itself. Therefore, direct selection on allometric relationships may not need to be strong in order to hold scaling relationships constant, at least over short evolutionary periods.</p> / Dissertation

The gaseous messenger, nitric oxide (NO), has emerged as a key component of olfactory systems. Localization and imaging studies in the moth, Manduca sexta, suggest that NO may affect the excitability of olfactory neurons by modifying neuronal membrane properties through sGC-dependent mechanisms. This hypothesis was tested using a multidisciplinary approach, including two types of physiological recording techniques and immunocytochemical analysis of sGC antibody expression in the Manduca brain. The excitability of large populations or individual antennal lobe (AL) neurons was monitored with in vivo physiological recordings while various NO pharmacological agents were bath applied to the brain. To examine possible targets of NO, the binding site of sGC was blocked and the results were compared to NO blockade. Finally, sGC immunocytochemistry was used to also determine possible targets of NO.Two NO synthesis inhibitors and a sGC blocker were potent effectors of resting, baseline activity in the Manduca brain. Blocking NO synthesis caused significant decreases in AL neuron conductance. This conductance decrease led to changes in baseline activity, including the appearance of bursts in some neurons, and increased and decreased firing rates in other neurons. Further, the neurons had a decreased responsiveness and excitability to presynaptic input. Blocking the sGC binding site caused similar effects in most neurons, which indicates that NO likely acts through sGC-dependent signaling to exert its effects in at least a subset of neurons. However, some neurons had different responses to NO and sGC blockade, which indicates that NO may act through other signaling mechanisms in some neurons. Further examination using sGC immunocytochemistry revealed that only about 90% of projection neurons (PNs) and 30% of local neurons (LNs) contained sGC immunoreactivity.The results in this dissertation indicate that NO performs a global function in the antennal lobe to maintain the resting membrane conductance of AL neurons. NO likely exerts its effects through both sGC-dependent and sGC-independent mechanisms. Finally, these results have major implications for odor coding in all species, as NO has been found in the olfactory systems of every animal examined thus far.

The different reproductive strategies of males and females underlie differences in behavior that may also lead to differences in nutrient use between the two sexes. We studied sex differences in the utilization of two essential amino acids (EAAs) and one non-essential amino acid (NEAA) by the Carolina sphinx moth (Manduca sexta). On day one post-eclosion from the pupae, adult male moths oxidized greater amounts of larva-derived AAs than females, and more nectar-derived AAs after feeding. After 4 days of starvation, the opposite pattern was observed: adult females oxidized more larva- derived AAs than males. Adult males allocated comparatively small amounts of nectar-derived AAs to their first spermatophore, but this allocation increased substantially in the second and third spermatophores. Males allocated significantly more adult-derived AAs to their flight muscle than females. These outcomes indicate that adult male and female moths employ different strategies for allocation and oxidation of dietary AAs.

A variety of diseases lead to the atrophy and/or death of skeletal muscle. To better understand the molecular mechanisms that mediate these processes, I have taken advantage of the intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta, which undergo sequential programs of atrophy and programmed cell death at the end of metamorphosis. ISM death is mediated by changes in gene expression and numerous cell-death associated transcripts have been identified. MicroRNAs (miRs) are small (~22 nucleotide) non-coding RNAs that bind to sequences in messenger RNAs (mRNAs) and either cause translational arrest or mRNA degradation. To test the hypothesis that developmentally regulated miRs may control the stability and/or translatability of target mRNAs in the ISMs, putative mRNA targets for the test miRs have been identified and their 3’ untranslated region (UTR) have been cloned into a dual luciferase reporter plasmid. The microRNA mir-92b binds to the 3’ UTR of the Small Cytoplasmic Leucine Rich repeat Protein (SCLP) mRNA. Expression of miR-92b declines during development and SCLP expression increases with the commitment to die. I found that the miR-92b inhibits luciferase mRNA translation (spectrophotometric plate assays), but does not lead to transcript degradation (quantitative polymerase chain reaction; qPCR). miR-92 plays a survival role in several mammalian tissues and is repressed in two types of cardiomyopathy. Consequently, understanding how miRs regulate mRNA translation and stability may provide a better understanding of the regulation of muscle atrophy and death as well as provide novel tools for diagnostics or therapeutics.