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Doctor of Philosophy / Department of Biology / Susan J. Brown / How animals polarize and establish the main axis during embryogenesis has been one of the most attractive questions in Biology. Increasing body of work in various model organisms implicates that most metazoans utilize the canonical Wnt signaling pathway to pattern the anterior-posterior (AP) axis, despite the limited evidence from arthropods. In Drosophila, a highly derived insect, canonical Wnt activity is not required for global AP patterning, but in typical insects including Tribolium castaneum, loss of canonical Wnt activity results in posterior truncation. To determine the eff ects of increased canonical Wnt levels, I analyzed the function of axin, encoding a highly conserved negative regulator of the pathway. Tc-axin transcripts are maternally localized to the anterior pole in freshly laid eggs. Parental RNAi for Tc-axin produced progeny phenotypes that ranged from mildly a ffected embryos with cuticles displaying a graded loss of anterior structures, to severely a ffected embryos lacking cuticles and condensing to the posterior pole of the egg without any de finable structures. Altered expression patterns of several blastodermal markers indicated anterior expansion of posterior fates. Epistasis analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the eff ects of Tc-axin depletion are mediated through this pathway and that canonical Wnt activity must be repressed for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt activity must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt signaling in de fining AP polarity and patterning in metazoan development. Additionally, as an anterior structure, the extraembryonic serosa is reduced in Tc-axin RNAi progeny. However, in Tc-pangolin (Tc-pan, a homolog of Wnt downstream component) RNAi progeny, an interesting phenotype was produced that serosa was not only reduced but also separated into distinct anterior and dorsal domains. I carefully recorded this phenomenon with live imaging using a Tribolium transgenic line that expresses GFP in each nucleus. Through careful examination with embryonic fate-map markers, I found that the tissue between separated serosa domains is dorsally extended head lobe. And I also found that in severe phenotype, dorsal serosa was completely gone while anterior serosa not, suggesting independent regulation mechanisms for anterior and dorsal serosa formation. This descriptive data will complement future study in the genetic mechanism underlying serosa formation by providing more details in morphogenesis.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Hulya Dogan / Larvae of the Indian meal moth, Plodia interpunctella (Hübner), have the ability to invade or penetrate packaging materials and infest the food within. Energy bars with three polypropylene packaging types were challenged with eggs (for first instars), third, and fifth instars of P. interpunctella to determine package resistance to larvae at 28°C and 65% r.h. to determine which provided the greatest protection against P. interpunctella larval penetration. Third and fifth instars showed a higher propensity to infest all packaging variations. First instars showed a reduction in package pentration abiltiy compared to third and fifth instars. Methoprene is an insect growth regulator labeled in the USA for use as an aerosol spray, a residual surface treatment, and as a grain protectant, and recently has been impregnated into a polymer-based packing film to prevent insect infestations of packaged products. The objectives of these studies were 1) determine the effect of short term exposure time and temperature on four week old larvae, continual exposure on egg-to-adult emergence of beetles, and sub-lethal effect on adults of the red flour beetle, Tribolium castaneum (Herbst) and warehouse beetle, Trogoderma variabile Ballion, on the inside and outside surfaces of methoprene-treated woven packaging material at 27 and 32°C at 60% r.h.; 2) evaluate fecundity, egg hatch, and egg-to-adult emergence of T. castaneum and T. variabile, when exposed to two methoprene-impregnated polymer packaging on the inside and outside surfaces at 27 and 32°C at 60% r.h.; and 3) determine the effect of methoprene-treated foil packaging on larval emergence, penetration, and invasion ability of T. variabile and P. interpunctella at 27°C and 60% r.h. Short term exposure results indicated that adult emergence from larvae of T. castaneum and T. variabile decreased with increasing exposure time. The number of eggs laid per female of T. castaneum and T. variabile did not vary from their controls. Continual exposure demonstrated 100% supression of T. castaneum adult emergence, irrespective of exposure to outside or inside surfaces. T. variabile exposed to inside surfaces were unaffected and normal adult emergence was reduced in those exposed to outside surfaces.. The number of T. variabile eggs laid per female was not significantly different among polymer packaging types. The methoprene-treated polyethylene terephthalate to polyethylene packaging, PET-PE reduced the number of T. castaneum eggs laid per female. Both polymer packaging reduced the percent hatch of both species. No T. castaneum adults emerged on the inside surface of PET-PE and both sides of the polyethlyene to polyethylene (PE-PE). Egg-to-adult emergence of T. variabile was arrested at the pupal stage on the outside surface of PE-PE packaging. The PET-PE packaging greatly reduced the number of normal adults by 87 to 97% when exposed to inside surfaces at both temperatures. The foil packaging had no significant effect on hatch of either species. T. variabile were unable to penetrate/invade any foil packages. P. interpunctella invaded all packaging containing pinholes. Therefore, continual exposure of T. castaneum and T. variabile to methoprene impregnated packaging could be a vaiable tool to protect food packages.

<p>The initiation of metamorphosis causes the cessation of the larval growth period which determines the final body size of adult insects. Because larval growth is roughly exponential, differences in timing the initiation of metamorphosis can cause large differences body size. Although many of the processes involved in metamorphosis have been well characterized, little is known about how the timing of the initiation of metamorphosis is determined. </p> <p>Using different strains from <italic>Tribolium castaneum<italic>, <italic>Tribolium freemani<italic>, and <italic>Manduca sexta<italic> and varied nutritional conditions, I was able to document the existence of a threshold size, which determines when the larva becomes competent to metamorphose. Threshold size, however, does not dictate the exact timing of initiation. The exact timing for the initiation of metamorphosis is determined by a pulse of the molting hormone, ecdysone, but only after threshold size has been reached. Ecdysone pulses before the larva attains threshold size only cause the larva to molt to another larval instar. These results indicate the timing of metamorphosis initiation is controlled by two factors: (1) attainment of threshold size, at which the larva becomes competent to initiate metamorphosis and (2) the timing of an ecdysone pulse after attaining threshold size. </p> <p>I hypothesize the attainment of threshold size, and therefore competence to metamorphose, is mediated by the effect of changing juvenile hormone concentrations caused by the increase in size of the larva. While the larval body grows nearly exponentially, the corpora allata, which secretes juvenile hormone, grows very little if at all. The difference in relative growth causes juvenile hormone concentrations to gradually become diluted. When juvenile hormone concentrations fall below a threshold, changes in protein-protein binding occur that can cause changes in signaling networks and ultimately gene expression. These changes make the larva competent for metamorphosis. </p> <p>I have demonstrated that only threshold size is consistently correlated with body size; other growth parameters such as growth rate, duration of instars, or number of instars do not consistently correlate with variation in body size. Using the black mutant strain of <italic>M. sexta<italic> I have shown that lower juvenile hormone titers correlate with lower threshold sizes. My hypothesis is consistent with the large body of literature indicating the involvement of juvenile hormone. I also hypothesize that the diversity of metamorphosis types in holometabolous insects can be explained by heterochronic shifts in the timing of threshold size and other developmental events related to metamorphosis. The heterochronic shifts affect not only the morphology of organs, but can also affect the overall phenotypic response of the larva to changes in the environment. The different phenotypic responses among species may make the more or less suited for certain types of niches.</p> / Dissertation

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Intraspecific genetic diversity is an important attribute of natural populations and is deemed critical for their adaptive potential and persistence. However, we have limited empirical understanding of the impact of genetic diversity on population performance under different conditions. For my dissertation, I conducted long-term laboratory experiments with populations of the flour beetle Tribolium castaneum to test the consequences of genetic variation for population dynamic stability and niche evolution. In Chapter 1, I show that genetic variation prevented population extinction in a novel habitat. In addition, genetically diverse populations were more stable, both in a novel heterogeneous habitat and in their ancestral habitat. In the ancestral habitat, alleles from a single founding lineage dominated the dynamics, leading to increased stability of genetically diverse populations. However, such as selective effect was not observed in the novel heterogeneous habitat. Therefore, while genetic variation within populations increased their stability and persistence, the magnitude of the impact and its mechanism depended on the selective habitat. In Chapter 2, I ask whether genetic variation also facilitates resource niche expansion, i.e., use of a novel resource. Using stable carbon isotopes, I analyzed diets of beetles sampled from the above experiment and quantified the rate of change in resource use. Contrary to theoretical predictions, I found that genetic variation for resource use had no effect on the rate of niche evolution. Furthermore, behavioral niche expansion accounted for most of the adaptation to the novel resource, and the behavioral change hindered subsequent evolutionary change in resource use. It is thus apparent that in the short term, behavioral plasticity in niche use may impose far greater constraints on niche evolution than the amount of standing genetic variation. Mathematical models predict that intraspecific competition generates selection for niche evolution, and that genetic variation increases the response to selection. Therefore, I hypothesized that the impact of genetic variation on resource niche evolution may depend on the degree of intraspecific competition. In the final chapter of this thesis, I describe results of an experiment to test this hypothesis. I found that genetic variation and competition indeed interacted to increase the rate of niche expansion in T. castaneum, but that their impacts were temporally variable. Furthermore, the two factors acted on different components of niche evolution: while competition only affected the degree of niche expansion, genetic variation also promoted maintenance of individual variation in resource use. In summary, my thesis describes experiments to test for the ecological and evolutionary impacts of intraspecific genetic variation; and its interaction with behavioral plasticity, intraspecific competition, and resource availability. Genetic diversity and behavioral plasticity are common features of living organisms, and therefore it is vital to understand their combined consequences for population ecological and evolutionary dynamics. In addition, natural populations often face intense competition for limited resources. Hence the experimental results presented here can help us to better understand how populations overcome these resource constraints, given their specific genetic composition. Biologists are increasingly aware that the intricate connection between ecological and evolutionary dynamics is important to gain a more complete understanding of population biology. The work described here represents one of the few experiments providing such detailed mechanistic understanding of the interactions between- and consequences of - key ecological and evolutionary parameters. Finally, the results have important implications for conservation biology, because they show that the effects of genetic diversity can vary greatly depending on a number of population and environmental parameters. / text

Spatial segregation among life cycle stages has been observed in many stage-structured species, including species of the flour beetle Tribolium. Patterns have been observed both in homogeneous and heterogeneous environments. We investigate density dependent dispersal of life cycle stages as a mechanism responsible for this separation. By means of mathematical analysis and numerical simulations, we explore this hypothesis using stage-structured, integrodifference equation (IDE) models that incorporate density dependent dispersal kernels.In Chapter 2 we develop a bifurcation theory approach to the existence and stability of (non-extinction) equilibria for a general class of structured integrodifference equation models on finite spatial domains with density dependent kernels. We show that a continuum of such equilibria bifurcates from the extinction equilibrium when it loses stability as the net reproductive number n increases through 1. We give several examples to illustrate the theory.In Chapter 3 we investigate mechanisms that can lead to spatial patterns in two dimensional Juvenile-Adult IDE models. The bifurcation theory shows that such patterns do not arise for n near 1. For larger values of n we show, via numerical simulation, that density dependent dispersal can lead to the segregation of life cycle stages in the sense that each stage peaks in a different spatial location.Finally, in Chapter 4, we construct spatial models to describe the population dynamics of T. castaneum, T. confusum and T. brevicornis and use them to assess density dependent dispersal mechanisms that are able to explain spatial patterns that have been observed in these species.

Due to concerns with synthetic neurotoxic insecticides used in insect pest management, alternative control methods are sought. Methoprene is a juvenile hormone analogue, which is well-known for its lethal effects on immature insects, and is registered as a stored-grain insecticide in the U.S.A. and Australia. However, less is known about its sub-lethal effects. Experiments were conducted to investigate the effects of methoprene (Diacon II) on heat tolerance, cold tolerance and progeny production of Tribolium castaneum (Herbst) when late-instar larvae or adults were exposed to wheat treated with methoprene. Methoprene at 3.33 ppm or higher, reduced heat tolerance of adults at 46ºC. In contrast, methoprene did not affect the heat tolerance of larvae. Cold tolerance and cold acclimation of both adults and larvae was not affected by methoprene. Exposure of larvae to 0.001 or 0.0165 ppm of methoprene on wheat had 37 and 72% reduction in adult emergence, respectively. The surviving adults had normal movement, but their progeny production was reduced by 71%. Males were affected more than females. Exposure of adults to methoprene (66.6 ppm) did not reduce progeny production. Methoprene is used as a surface treatment to control insects in empty grain bins, processing facilities and warehouses. Experiments were conducted to assess the effect of surface material, temperature and cleaning practices on methoprene residual efficacy. A bioassay with late-instar larvae at 30ºC until adult emergence was used to measure methoprene residual efficacy. Methoprene applied at the label rate (0.0003 mg/cm2) on varnished wood, held at 20, 30 or 35ºC for 24 weeks, prevented all development of larvae into adults. Conversely, concrete surfaces allowed 22% adult emergence after 8 weeks and 69% after 24 weeks. Temperatures, 20, 30 or 35ºC, that the concrete surfaces were held before the bioassay, did not affect this decline in efficacy. Presence of flour or repeated removal of flour (cleaning) reduced the efficacy of methoprene on concrete surfaces, but not on varnished wood. Maintaining concrete surfaces at 65ºC for 48 hours did not reduce efficacy. The implications of these results are discussed in the context of controlling stored-product insects, as well as insect physiology.

No description available.