Master of Science / Department of Entomology / Franklin Arthur / Kun Yan Zhu / Tribolium spp. are major pests in structures used for the processing and storage of grain-based products (e.g., flourmills, warehouses, retail stores). Consumers and regulators have little tolerance for insect-damaged or contaminated food products. The direction and breadth of pest-control strategies in the food industry have changed significantly over the past few years, creating the need to optimize insecticides through improved integrated pest management (IPM) techniques, specifically through the identification of new control agents that are low in mammalian toxicity, as well as any factors that might affect susceptibility to these agents. There is currently renewed interest in developing reduced-risk, low toxicity chemicals that can be effectively utilized in a setting in which grain and other food commodities are vulnerable to insect infestation, as a means of replacing outdated, and at times, less effective methods of insect control. Over the past decade, developed countries have made significant progress toward alternative insect control strategies by employing a variety of applied insecticides. Two classes of insecticide include natural pyrethrum and insect growth regulators (IGRs), which are substances that mimic insect hormones essential to normal development and reproduction. Pyrethrin is a highly efficient, broad spectrum, botanical insecticide that causes a rapid knockdown in exposed insects. Synergists are used to extend the economic usage of natural pyrethrins and because pyrethrum is rapidly metabolized, it is often mixed with a synergist. Methoprene, a juvenile hormone analog, is labeled as an aerosol and surface treatment inside mills, warehouses and other food storage facilities. There is little recent research with large-scale aerosol applications in stored-food facilities; furthermore, there are few published references regarding the efficacy of using methoprene in combination with synergized pyrethrin, in aerosol form. Therefore, the purpose of this research was to evaluate the use of aerosol applications of two aerosol concentrations on flour and finished stored-product packaging materials for the control of Tribolium spp. Results of this research show that T. castaneum are effectively controlled with 1% aerosol application, while the 3% formulation is required to effectively control T. confusum. With regards to the various packaging material surfaces, few differences between the surfaces emerged.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / John C. Reese / Keeping in view the important roles of bacteria in almost every aspect of insect’s life, the current study is the first systemic and intensive work on microbes associated with Hessian fly, a serious pest of wheat crop. A whole body analysis of Hessian fly larvae, pupae, or adults suggested that a remarkable diversity of bacteria is associated with different stages of the insect life cycle. The overriding detection of genera Acinetobacter and Enterobacter throughout the life cycle of Hessian fly suggested a stable and intimate relationship with the insect host. Adult Hessian flies have the most dissimilar bacterial composition from other stages with Bacillus as the most dominant genus. Analysis of 5778 high quality sequence reads obtained from larval gut estimated 187, 142, and 262 operational taxonomic units at 3% distance level from the 1st, 2nd, and 3rd instar respectively. Pseudomonas was the most dominant genus found in the gut of all three instars. The 3rd instar larval gut had the most diverse bacterial composition including genera Stenotrophomonas, Pantoea, Enterobacter, Ensifer, and Achromobacter. The transovarial transmission of major bacterial groups provided evidence of their intimate relationship with the Hessian fly. The Hessian fly is known to manipulate wheat plants to its own advantage. This study demonstrated that the combination of a decrease in carbon compounds and an increase in nitrogen compounds in the feeding tissues of Hessian fly-infested plants results in a C/N ratio of 17:1, nearly 2.5 times less than the C/N ratio (42:1) observed in control plants. We propose that bacteria associated with Hessian fly perform nitrogen fixation in the infested wheat, which was responsible for shifting the C/N ratio. The following findings made in the current study i.e. the presence of bacteria encoding nitrogenase (nifH) genes both in Hessian fly and infested wheat, exclusive expression of nifH in infested wheat, presence of diverse bacteria (including the nitrogen fixing genera) in the Hessian fly larvae, presence of similar bacterial microbiota in Hessian fly larvae and at the feeding site tissues in the infested wheat, and reduction in survival of Hessian fly larvae due to loss of bacteria are consistent with this hypothesis. The reduction in Hessian fly longevity after the loss of Alphaproteobacteria in first instar larvae, highest proportion of Alphaproteobacteria in insects surviving after the antibiotic treatments and the nitrogen fixation ability of associated Alphaproteobacteria strongly implies that Alphaproteobacteria are critical for the survival of Hessian fly larvae. This study provides a foundation for future studies to elucidate the role of associated microbes on Hessian fly virulence and biology. A better understanding of Hessian fly-microbe interactions may lead to new strategies to control this pest.

Master of Science / Department of Entomology / Frank Arthur / The control of Indianmeal moth [Plodia interpunctella (Hübner)], a commonly found serious stored product pest around the world, relies mainly upon chemical control methods. Because of recent changes in the laws and regulations governing pesticide usage in the United States, there is an increasing need for finding safer chemicals to control insect pests. Hydroprene, an insect growth regulator, is considered to be a safe alternative. In this study, I quantified the effects of hydroprene on two critical life stages of Indianmeal moth, the eggs and 5th instar wandering phase larvae. Maximum development time in the untreated controls was 13.6 ± 0.6 d at 16°C and minimum development time was 2.3 ± 0.4 d at 32°C. At 20°C and 24°C, the effect of hydroprene on egg development became more evident; development time generally increased with exposure interval, with some variability in the data. The mean egg mortality among all temperatures was 7.3 ± 4.6%. Among the treatments, mortality of eggs increased as the exposure periods increased within any given temperature, with a dramatic increase in mortality with increase in temperature. Egg mortality was lowest at 16°C when exposed for 1 h (0 ± 3%), but mortality gradually increased up to 32 ± 3% when exposed for 18 h. Within each exposure interval, there was a direct increase in mortality as the temperatures increased. For the 5th instar wandering phase larvae, the longest development time among the treatments of 47.2 ± 1.3 d occurred at 16ºC when the larvae were exposed for 30 h, whereas the shortest development time of 7.0 ± 0.5 d occurred when the larvae were exposed for 1 h at 32ºC. Among treatments, the greatest larval mortality (82.0 ± 0.1%) occurred when larvae were exposed for 30 h at 28ºC, while the minimum mortality of 0.0 ± 0.5% occurred at 16ºC when larvae were exposed for 1 h. Response-surface models derived from this study can be used in simulation models to estimate the potential consequences of hydroprene on Indianmeal moth population dynamics.

Doctor of Philosophy / Department of Entomology / Gerald R. Reeck / John C. Reese / Salivary secretions are a key component of aphid-plant interactions. Aphids’ salivary proteins interact with plant tissues, gaining access to phloem sap and eliciting responses which may benefit the insect. In an effort to isolate and identify key components in salivary secretions, we created a salivary gland cDNA library. Several thousand randomly selected cDNA clones were sequenced. We grouped these sequences into 1769 sets of essentially identical sequences, or clusters. About 22% of the clusters matched clearly to (non-aphid) proteins of known function. Among our cDNAs, we have identified putative oxido-reductases and hydrolases that may be involved in the insect's attack on plant tissue. C002 represents an abundant transcript among the genes expressed in the salivary glands. This cDNA encodes a novel protein that fails to match to proteins outside of aphids and is of unknown function. In situ hybridization and immunohistochemistry localized C002 in the same sub-set of cells within the principal salivary gland. C002 protein was detected in fava beans that were exposed to aphids, verifying that C002 protein is a secreted protein. Injection of siC002-RNA caused depletion of C002 transcript levels dramatically over a 3 day period after injection. With a lag of 1 – 2 days, the siC002-RNA injected insects died, on average 8 days before the death of control insects injected with siRNA for green fluorescent protein. It appears, therefore, that siRNA injections of adults will be a useful tool in studying the roles of individual transcripts in aphid salivary glands.

Doctor of Philosophy / Department of Entomology / C. M. Smith / Brenda Oppert / Coleopteran insects compensate for dietary protease inhibitors by a number of mechanisms. To study this compensation response at the molecular level, the digestive proteases of Tenebrio molitor were studied. Biochemical studies of the pH optima and inhibitor sensitivity of proteases indicated the cysteine proteases were mostly in the anterior and serine proteases were in the posterior midgut of T. molitor larvae. Expressed Sequence Tags (ESTs) from T. molitor larval midgut cDNA libraries contained sequences encoding putative digestive proteases. Of a total of 1,528 cDNA sequences, 92 cDNAs encoded proteases, and 50 full-length cDNAs were grouped into serine, cysteine and metallo protease classes. Sequences tmt1a, tmt1b and tmt1c were identified as genes encoding isoforms of T. molitor trypsin, and tmc1a encoded T. molitor chymotrypsin. The general distribution cysteine protease transcripts in the anterior and serine protease transcripts in the posterior midgut, of T. molitor larvae, was in agreement with the biochemically-characterized compartmentalization of proteases. Expression analyses of selected transcripts demonstrated varied expression patterns across five developmental stages of T. molitor, with maximal expression of most protease transcripts in first instar larvae. Dietary serine and cysteine protease inhibitors fed in combination to early-instar T. molitor larvae caused a significant delay in larval growth in 21-day-old larvae. Real-time quantitative PCR analysis of RNA isolated from larvae fed different protease inhibitor treatments indicated that dietary inhibitors affected the expression of serine and cysteine proteases. Larvae fed soybean trypsin inhibitor, a serine protease inhibitor, compensated by the hyperproduction of proteases from the same class, as well as the upregulation of cysteine proteases. A cysteine protease inhibitor, E-64, caused a reduction in the hyperproduction of all proteases, and, in combination with the soybean trypsin inhibitor, lowered the compensation response of T. molitor larvae to negligible levels. These data suggest that T. molitor larvae are more sensitive to the effects of cysteine protease inhibitors, perhaps because these proteases are the first line of defense for larvae against plant protease inhibitor. The bioassay and molecular studies suggested that combinations of inhibitors that target both serine and cysteine proteases are needed to effectively control larval infestations of T. molitor.

Doctor of Philosophy / Department of Entomology / James R. Nechols / Tetranychus kanzawai Kishida and Panonychus citri (McGregor) are the two major arachnid pests of screenhouse-cultivated papayas in Taiwan. Control of these mites has become more difficult because both pests have become resistant to most registered miticides. This laboratory study investigated the feeding behaviors, predatory potential, and prey preference of a domesticated line of Mallada basalis Walker, a commonly-occurring chrysopid in Taiwan, to both of these pest mites. A laboratory assessment on control efficacies of different predator:prey release ratios to single and mixed-pest species was also conducted. Behavioral study showed that all larval stages of M. basalis exhibited a high rate of acceptance of all life stages of both T. kanzawai and P. citri. Second and third instar predators foraged actively during most of the 2-h tests. Numbers and rates of prey consumption were measured for each instar of predator and prey. Results showed that consumption increased and prey handling time decreased as predator life stage advanced, and prey stage decreased. Mallada basalis exhibited both a shorter handling time and corresponding higher consumption rate on P. citri compared with T. kanzawai. Handling time and consumption rate also were positively affected by increasing prey density. Mallada basalis did not exhibit notable species or life stage preferences, and prior feeding experience on one mite species did not affect subsequent prey choice between the two mites. Lacewings significantly reduced T. kanzawai and P. citri populations at a predator:prey ratio of 1:30 and this improved at ratios of 1:15 and 1:10. Control of T. kanzawai was slightly better than P. citri when the mites occurred singly and together. Consumption by M. basalis increased with temperature up to 30C. I conclude that M. basalis has high potential for augmentative biological control of papaya mites. Further field investigations are needed for making final recommendations.

Doctor of Philosophy / Department of Entomology / James F. Campbell / James R. Nechols / Theoretical and empirical ecological research has emphasized the need for understanding how animals perceive and respond to landscape structure and the importance of integrating both behavioral and landscape approaches when studying movement behavior. Knowledge of insect movement behavior is essential for understanding and modeling dispersal and population structure and developing biologically-based integrated pest management programs. My dissertation research addresses questions concerning how insects respond to landscape structure by examining movement behavior of an important stored-product pest, red flour beetle (Tribolium castaneum), in experimental landscapes. Results show that beetles modify movement behavior depending on landscape structure. Edge effects and interpatch distances may influence landscape viscosity, or the degree to which landscape structure facilitates or impedes movement, resulting in significant differences in velocity and tortuosity (amount of turning) of movement pathways, as well as retention time in landscapes with different levels of habitat abundance and aggregation. Perceptual range, or the distance from which habitat is detected, appears to be limited while beetles are moving in a landscape as they did not respond to a flour resource before physical encounter. Beetles showed differential responses to patches with various characteristics, entering covered patches more quickly than uncovered patches with more resource or the same amount of resource. Permeability of patches changed with subsequent encounters suggesting that full evaluation of patch quality may only occur after entering a patch. Beetles responded to landscape structure differently depending on the activity in which they were engaged. Distribution of movement pathways was similar to that of the habitat, but distribution of oviposition sites were significantly more aggregated than pathways and habitat. Oviposition site choice may be influenced by a complex set of factors which include previous visitation, amount of resource, travel costs, and edge effects. Insights were gained concerning how red flour beetle perceives resources, modifies search strategies, responds to boundaries, and chooses reproductive sites in patchy landscapes. This research provides new information regarding how red flour beetle interacts with landscape structure that has implications in the areas of behavioral and landscape ecology and applications in stored-product insect ecology.

Doctor of Philosophy / Department of Entomology / C. Michael Smith / The Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae) (RWA), is an important pest in the U.S. Western Plains, causing hundreds of millions of dollars of losses to wheat and barley production through reduced yields and insecticide application costs. The objectives of this research were to evaluate the performance of two RWA-resistant barley varieties planted approximately one month earlier than normal in experimental fields at Fort Collins, Colorado; Tribune, Kansas; and Sidney, Nebraska during 2007, 2008, and 2009. The experimental design was a split-plot design with two main plot treatments (early and normal planting dates), and four split plot treatments (barley varieties) that were randomized within each main treatment plot. The varieties included two RWA-barley resistant varieties, Sidney and Stoneham, and the susceptible variety, Otis, under thiamethoxam-protected and unprotected regimes. Sampling of RWA, other cereal aphids, and natural enemy populations was conducted on four dates from mid May through early July. RWA populations collected from early-planted plots (first week of March) were significantly lower than normal-planted plots in 2007-2009 at the Fort Collins, Colorado and Tribune, Kansas sites. In samples collected from early planting date plots, RWA-resistant varieties yielded RWA populations similar to those found on the insecticide-treated susceptible variety at both Fort Collins and Tribune. At the Sidney, Nebraska site, very low RWA populations were present and there were no differences between either planting date or varietal treatments. The combined effect of early planting and RWA-resistant varieties reduced RWA populations at the Fort Collins, Colorado site in all three years. Results were similar at the Tribune, Kansas site in 2007, but differences due to planting date or variety were not observed in 2008 or 2009. The lowest RWA populations occurred at the Sidney, Nebraska site, were independent of planting date and varietal treatments. The RWA-resistant barley varieties had no negative impact on populations of other cereal aphids compared to those found on the susceptible variety, Otis at any of the three research sites. The only treatment effective in reducing other cereal aphids was the insecticide, thiamethoxam. There was also no clear response of populations of other cereal aphids to different planting date. Neither the RWA-resistant barley varieties nor the systemic, short residual action insecticide treatment had adverse affects on the abundance of natural enemies.

Master of Science / Department of Entomology / David C. Margolies / James R. Nechols / A two-year greenhouse experiment was conducted to compare the relative effectiveness of biological control versus chemical control for western flower thrips, Frankliniella occidentalis, as a means of reducing the spread of Tomato spotted wilt virus (TSWV) on tomatoes. To compare efficacy of different thrips management tactics for reducing TSWV incidence, tomatoes were subjected to one of three treatments: 1) biological control based on weekly releases of the predatory mite, Amblyseius cucumeris, at a commercially-recommended rate, 2) a single chemical treatment with Conserve®, a spinosad formulation, or 3) no treatment. TSWV was introduced into the greenhouse either by starting with 20% of the crop already infected and releasing non-viruliferous thrips, or by making a single release of viruliferous thrips. Analyses were done among thrips management tactics for each virus introduction method to examine the cumulative number of weeks plants were infected, the weekly proportion of infected plants, and total marketable yield. The effects of different virus introduction methods were also compared. A comparison of virus introduction methods showed that, among all plants, the average number of weeks they were infected by TSWV was significantly lower when virus was introduced through infected plants than by infected thrips. In addition, when virus was introduced by infected thrips, a significantly greater proportion of plants were infected in any given week than when virus was introduced on infected plants. Finally, crop yields were significantly lower when virus was introduced via infected thrips than on infected plants. Among thrips management methods, plants were infected for significantly less time, and the proportion infected was lower in any given week, when biological or chemical control was applied compared to no thrips management. Tomato yields were not affected by thrips management tactic. There was no significant difference between biological and chemical control in the length of time that plants showed symptoms. However, the proportion of infected plants was marginally greater with biological control in weeks 4 and 5 than with chemical control; differences were not significant thereafter. My findings suggest that inundative releases of biological control may provide as adequate a level of protection from TSWV as chemical control in commercial greenhouse tomato crops.

Master of Science / Department of Entomology / Yoonseong Park / Neuropeptides are diffusible signal molecules mediating vital physiological processes. We have been interested in a group of neuropeptides and their receptors involved in osmoregulatory neuroendocrine system which has been suggested as a possible target for development of new biopesticides. Since the genome sequence of the T. castaneum has recently been completed, we were able to identify the respective genes encoding three peptide hormones from T. castaneum that were characterized for their diuretic activities in other insects: one calcitonin-like (CT-like DH31) and two corticotropin releasing factor-like (CRF-like DH37 and DH47, the numbers indicates the number of amino acid residues). This peptide is expressed at all developmental stages and in the central nervous system (CNS), Malpighian tubules (MT) and gut. The synthetic peptide TricaDH31 also has been show to be biologically active, inducing significant excretions in adults beetles. When Tcdh31 was silenced using RNAi, adults had deformed wings and abnormal body shape. Mortality in adults was high, the number of eggs laid was reduced as well as the hatchability of the eggs. The two biologically active CRF-like peptides in T. castaneum, are encoded by one gene which undergoes alternative splicing. When Tcdh47 was knocked down, high mortality occurred as well as low oviposition and egg hatchability. Similar effects were observed with silencing of both CRF-like genes. However, RNAi of Tcdh37 transcripts had similar, but less severe effects. Adults also had deformed wings when both CRF-like genes were silenced, but not when just one of them was knocked down. These results indicate that CRF-like genes could have additional biological functions to their roles in dieresis. We tested the in vivo activity of these peptides. TenmoDH47 induced high excretions in adults, whereas TenmoDH37 induces smaller excretions. We identified the respective genes encoding two putative receptors for TricaDH31 as Glean_13321 and Glean_02694 (Trica-ctr1 and Trica-ctr2, respectively) and two receptors for CRF-like peptide as Glean_12799 and Glean_07104 (Trica-crfr1 and Trica-crfr2, respectively). The CT-like receptors are expressed at all developmental stages, in the CNS and MT. RNAi of the receptors revealed that only Trica-ctr2 silencing caused significant mortality and reduction in the number of eggs laid. The CRF-like receptors are expressed at all developmental stages. Adults also had deformed wings and laid fewer eggs after RNAi of Trica-crfr1. RNAi of Trica-crf2 also caused significant mortality. These peptides and receptors seem to fine tune the beetle physiology and may have functions not yet known.