Doctor of Philosophy / Department of Entomology / Larry L. Buschman / Kun Yan Zhu / Genomic information for lepidopteran insects, particularly agricultural pest species, is very limited but urgently needed due to their economic importance and biodiversity. The huge economic losses ($ 1-2 billons / year) caused by the European corn borer (Ostrinia nubilalis, Hübner, ECB) makes this insect species one of the major pests of corn in the United States and western world. Management of ECB by conventional methods is limited but has had a great success by transgenic Bt (Bacillus thuringiensis) corn, which targets insect gut. However, the widespread use of Bt corn may lead to the development of Bt resistance in ECB. Knowledge of genes expressed in the insect gut is considered crucial for understanding basic physiology of food digestion, their interactions with Bt toxins and pathogens, and for discovering new targets for pest management. A large database of 15,000 expressed sequence tags (ESTs) was established from the ECB larval gut. To our knowledge, this database represents the largest gut-specific EST database from a lepidopteran pest. Analysis of 10 aminopeptidase-like genes between Cry1Ab–resistant and –susceptible ECB larvae revealed that aminopeptidase P-like (OnAPP) gene is a strong candidate for its role in Bt toxicity and resistance. The RNA interference mediated reduction in the transcript level of OnAPP gene in ECB larvae resulted in their reduced susceptibily to Cry1Ab. Analysis of the chitinase-like gene (OnCht) revealed its essential role in regulating chitin content of peritrophic membrane (PM). Our results suggest that OnCht may influence food digestion, nutrient absorption or movement of digestive enzymes through the PM and can be an important target for insect management. We also identified and characterized six genes involved in the innate immune defense response in ECB and showed that the expression of these genes were induced when challenged with bacteria. In addition to these results, this research generated significant genomic information for the development of microarray from the larval gut of ECB. The establishment of the feeding-based RNA interference technique could potentially help in delivering dsRNA orally to ECB for high throughput screening of effective genes to be targeted for insect pest management.

Doctor of Philosophy / Department of Entomology / Ludek Zurek / Stable flies (Stomoxys calcitrans L.) are blood-feeding insects with a great negative impact on livestock resulting in annual losses around $2 billion in the USA alone. One of the main reasons for such an impact is because stable fly management is very difficult. Stable fly larvae develop primarily in animal manure and live bacteria were shown to be essential for stable fly development. I hypothesized that the microbial community of horse manure changes over time and plays an important role in stable fly fitness. Two-choice bioassays were conducted using 2 week old horse manure (control) and aging horse manure (fresh to 5 week old) to evaluate the effect of manure age on stable fly oviposition. The results showed that fresh manure did not stimulate oviposition and that the attractiveness increased as manure aged but started to decline after 3 weeks. Stable fly eggs artificially placed on 1, 2, and 3 week old manure resulted in significantly higher survival and heavier adults comparing to those developing in fresh, 4, and 5 week old manure. Analysis of the bacterial community of aging horse manure by 454-pyrosequencing of 16S rDNA revealed a major shift from strict anaerobes (e.g. Clostridium, Eubacterium, Prevotella, Bacteroidales) in fresh manure to facultative anaerobes and strict aerobes (e.g. Rhizobium, Devosia, Brevundimonas, Sphingopyxis, Comamonas, Pseudomonas) in 1-5 week old manure. Identified volatile compounds emitted from 2 and 3 week old horse manure included phenol, indole, p-cresol, and m-cresol. However, none of them stimulated stable fly oviposition in two-choice assays. In conclusion, the microbial community of 2 and 3 week old horse manure stimulates stable fly oviposition and provides a suitable habitat for stable fly development. Manure at this stage should be the main target for disrupting the stable fly life cycle. Volatile compounds acting as oviposition stimulants/attractants and their specific bacterial origin remain to be determined. Better understanding of stable fly microbial ecology is critical for development of novel management strategies based on alteration of the microbial community of stable fly habitat to generate a substrate that is non-conducive to fly oviposition and/or larval development.

Doctor of Philosophy / Department of Entomology / Raymond A. Cloyd / The life history of the rove beetle, Atheta coriaria (Kraatz) (Coleoptera:Staphylinidae), predation against the fungus gnat Bradysia sp. nr. coprophila (Lintner) and compatibility with pesticides and plant growth regulators was investigated under laboratory conditions using Sunshine LC1 Professional Growing Mix as a substrate. Duration of life stages was 2.2, 7.1, and 7.8 days for egg, larva and pupa respectively, at 26°C, whereas total development time from egg to adult was 17.0 days. In addition, A. coriaria male and female adult longevity was 60.3 and 47.8 days. Average fecundity was 90.2 eggs per female and the number of adults produced per female was 69.1. There were no significant differences in prey consumption when using second and third instar fungus gnat larvae as prey and starved and non-starved rove beetles. Overall, predation efficacy in Petri dishes was high (70 to 80%) as fungus gnat larval density increased with 3.9, 7.0, 11.1, and 15.3 larvae consumed in 24 hours after exposure of 5, 10, 15 and 20 fungus gnat larvae to one rove beetle adult. However, lower predation rates were found at different predator:prey ratios when using 1 to 5 rove beetles and growing medium as a substrate. The direct and indirect effects of pesticides and plant growth regulators on A. coriaria were investigated under laboratory conditions. Rove beetle survival was consistently higher when adults were released 24 hours after rather than before applying pesticides. Acetamiprid, lambda-cyhalothrin, and cyfluthrin were directly harmful to rove beetle adults, whereas Beauveria bassiana, azadirachtin and organic oils were compatible with A. coriaria. Similarly, the plant growth regulators acymidol, paclobutrazol and uniconazole were not harmful to rove beetle adults. In addition, Beauveria bassiana, azadirachtin, kinoprene, organic oils, and the plant growth regulators did not negatively affect A. coriaria development. However, Beauveria bassiana did negatively affect rove beetle prey consumption. This study demonstrated that A. coriaria is not compatible with the pesticides acetamiprid, lambda-cyhalothrin and cyfluthrin, whereas there is compatibility with organic oils, Beauveria bassiana, azadirachtin, and the plant growth regulators. As such, these compounds may be used in combination with A. coriaria in greenhouse production systems.

Doctor of Philosophy / Department of Entomology / James F. Campbell / Phillip E. Sloderbeck / Robert "Jeff" J. Whitworth / Knowledge of factors influencing the establishment, persistence and distribution of stored-product pests aids the development of effective Integrated Pest Management (IPM) programs in food storage and processing facilities. This research focused primarily on Tribolium castaneum, which is one of the most important pests of mills. Populations of T. castaneum from different food facilities can potentially be interconnected by either their own dispersal behavior or by human transportation. Population genetic structure analyses based on microsatellites and other insertion-deletion polymorphisms (“indels”) showed that populations from different mills around the US are genetically distinct from each other, but the level of differentiation was not correlated with the geographic distance. A potential source of insect infestation within a food facility is spillage that accumulates outside or movement from bulk storage facilities on site. Results from three facilities showed that most stored-product species were captured both inside and outside buildings, but T. castaneum was rarely captured outside of the facilities. Spatial distribution of all species outside was associated with the proximity of buildings, not necessarily with areas with accumulated spillage. T. castaneum populations inside facilities are potentially exposed to frequent genetic bottlenecks resulting from structural fumigations. Changes in allele frequencies through time, based on the analysis of microsatellites and other indels in individuals collected in a mill, confirmed bottleneck effects. To understand how spatial distribution of T. castaneum within a mill could be influenced by environmental and physical factors, a range of variables were measured at each trap location. There was significant variation among trap locations regarding beetle captures and the variables measured, but increase in beetle captures correlated only with increase in temperature and spillage production. Tribolium castaneum response to visual cues could influence attraction to pheromone and kairomone olfactory cues used in traps. Results of laboratory experiments showed that adults respond to tall narrow black shapes and placing traps in front of these shapes can increase captures. This research provides new insights into factors influencing the spatial distribution of T. castaneum and could help in improving monitoring programs for this important pest of the food industry.

Master of Science / Department of Entomology / David C. Margolies / James R. Nechols / Many crops grown in greenhouses are damaged by the twospotted spider mite, Tetranychus urticae. The predatory mite, Phytoseiulus persimilis, is a commercially-available predator that is commonly used to control twospotted spider mites on greenhouse crops; but its efficacy varies among crops, and it is generally ineffective at low prey densities. In general, predator foraging efficiency depends on how well predators find prey patches, the length of stay in prey patches, and consumption of prey while in prey patches. With respect to P. persimilis, I asked how this predator responds to different prey distributions, as might be encountered at different stages of spider mite infestations. I also asked how components of foraging, namely consumption rate and dispersal tendency, affected predator efficiency. To examine the former, I established T. urticae eggs on 6-leafed cucumber plants in two distributions. To examine the latter, I imposed artificial selection on a population of P. persimilis to create a line that exhibited extremely high consumption and one that demonstrated a greater tendency for dispersal. Subsequently, foraging efficiency was assessed by observing predator oviposition and consumption of twospotted mite eggs on individual leaves of 6-leafed cucumber plants. The number of eggs laid by predators corresponded to the number of prey consumed regardless of predator line. In addition, predators from both lines distributed their eggs proportional to where they fed. However, prey consumption differed between selected lines in response to prey distribution. Predators selected for high consumption fed more on the basal leaf where they were released; whereas prey consumption by the high dispersal and control lines were more evenly distributed throughout the plant. These results contribute to a better understanding of how foraging behavior is modified in plant landscapes under different levels of expression of foraging traits. They also indicate that predator release strategies likely would need to modified in accordance with the kind of foraging trait(s) used in artificial selection programs. In general, my research, when combined with future studies at a broader landscape level, will facilitate decisions by biological control practitioners about whether changes in foraging efficiency resulting from artificial selection justify the cost investment of producing selected lines of P. persimilis

Master of Science / Department of Biology / Rollie Clem / Apoptosis, or programmed cell death, is a crucial conserved process among organisms for deleting damaged unwanted cells, as well as for development and viral defense, and plays an important role in multiple diseases. Too much apoptosis may lead to Alzheimer’s disease, and too little may result in cancer. Therefore, the ability to understand this process is essential for improved medical knowledge today. Apoptosis has been explored in a number of species and pathways seem relatively conserved among most, with unique aspects contained in each, but little is known about apoptosis in mosquitoes. Improved knowledge and growing interest concerning apoptosis in mosquitoes is necessary considering the vast health effects seen across the globe as a result of diseases transferred by the mosquito vector. The Dengue virus mosquito vector Aedes aegypti was the focus here. A new player named defense repressor 1 was discovered in Drosophila melanogaster (DmDnr1), shown to play a role in apoptosis, and the homolog discovered in A. aegypti (AeDnr1). Silencing Dmdnr1 resulted in cells sensitized to apoptosis but was not enough to induce spontaneous apoptosis. In contrast, silencing Aednr1 in the A. aegypti cell line, Aag2, led to spontaneously induced apoptosis. This showed the importance of AeDnr1 as a member of the apoptotic pathway in this species. Epistasis experiments showed that apoptosis induced by silencing Aednr1 requires the initiator caspase Dronc and the effector caspase CASPS8, whereas apoptosis induced by silencing the inhibitor of apoptosis, Aeiap1, also requires Dronc but acts through the effector caspase CASPS7. Further epistasis experiments showed that apoptosis induced by silencing Aednr1 requires the IAP antagonist Mx, but not IMP. This showed for the first time a gene regulating upstream of an IAP antagonist. Biochemical studies showed that AeDnr1 regulates active CASPS8 but not CASPS7, and interacts with Mx and CASPS8 but not AeDronc, CASPS7 nor AeIAP1. Studies also showed Mx competes effectively with CASPS8 but not CASPS7 for AeIAP1 binding, and IMP competes effectively with CASPS7 but not CASPS8 for AeIAP1 binding. An improved apoptosis pathway for the mosquito A. aegypti emerged involving a potential feedback loop with explanations for the upstream IAP antagonist preference as well as the downstream effector caspase preference resulting from apoptosis induced by Aednr1 silencing. Through the discussed research, multiple unique findings resulted. Studying the mosquito model will allow us to find certain gene relations that are more difficult to uncover in the Drosophila model. Because Dnr1 is found in most systems, this improved pathway may shed light not only on a potential role of Dnr1 in apoptosis in insects but higher organisms as well.

Master of Science / Department of Entomology / John C. Reese / The soybean aphid, Aphis glycines is documented to have arrived in North America in mid 2000 and has ever since established itself as a formidable pest of soybeans, with the capacity to cause immense crop losses. This formidable pest with its complex life cycle and habits represents a current threat to soybean production. Host plant resistance is a promising avenue that can offer considerable control over the soybean aphid problem. Antibiosis being the most effective host plant resistance category, this study was aimed at attempting to understand the effects of induction on the antibiosis levels of soybeans. In the first set of experiments, different soybean genotypes and two soybean aphid biotypes were tested to comprehend if and how the genotypes and biotypes affected the survival and reproduction of the aphid. The experiments revealed mixed results that can be attributed to the genotypes tested and the biotypes used. While some genotypes showed no significant changes due to previous infestation, K1621 suggested signs of induced resistance to biotype 1 and PI567301B showed induced resistance to biotype 2, while K1639 pointed towards induced susceptibility to biotype 2. A follow up feeding behavior study with Electrical Penetration Graph (EPG) technique was carried out on PI567301B to elucidate if the induced resistance was tissue-specific, which could affect the feeding behavior of the aphid (biotype 2); but the results showed no appreciable differences in the feeding behavior of the aphids on clean vs. infested plants. Induced response studies shed light on how plants respond to herbivory and help us identify how changes in plant physiology affect the various herbivores that visit it for food and shelter. This knowledge can thus be applied to the development of superior varieties of crops that can defend themselves better against recurring infestations.

Doctor of Philosophy / Department of Entomology / Marcelo Ramalho-Ortigao / The emergence and reemergence of vector-borne diseases pose significant threats to humans and other animals worldwide. Although vector control relies mostly on insecticides, the emergence of insecticide resistance urges for the development of new strategies to control the spread of such diseases. For sand fly-transmitted leishmaniasis, Transmission Blocking Vaccines (TBV) may constitute a feasible strategy to impair Leishmania transmission from infected to uninfected vertebrate hosts. Moreover, sand fly saliva-based vaccines represent an alternative or complementary approach as these vaccines protect different mammalian hosts against Leishmania. Based on the potential use of sand fly molecules as vaccines against leishmaniasis, we assessed the potential of Phlebotomus papatasi midgut secreted proteins as TBV candidates and the expression variability of sand fly salivary gland genes. Regarding the TBV approach, we took advantage of the RNA interference (RNAi) technique to evaluate the effects of knocking down P. papatasi midgut-specific genes on Leishmania major development within the sand fly midgut. Whereas peritrophin 1 (PpPer1) knock down led to increased Le. major load by 39%, knocking down chitinase 1 (PpChit1) reduced Le. major load in P. papatasi midguts by 63%. Thus, our data strongly suggest that PpChit1 constitutes a potential target for TBV approaches against Leishmania transmission in endemic areas. Concerning protective vaccines based on salivary gland secreted proteins, we searched for expression polymorphism in selected salivary gland genes in natural and colonized populations of P. papatasi. Significant differences in salivary gland gene expression were not only exhibited in P. papatasi specimens collected in different geographic habitats but also seasonal difference in gene expression was displayed by specimens belonging to the same population. As antigen dose is an important component of immune responses, different doses of salivary protein inoculated into host skin may interfere with vaccine protection. Thus, the efficacy of sand fly saliva-based vaccine upon exposure to different salivary protein doses must be evaluated before deployment in endemic areas. Our data also ruled out some biotic factors as responsible for fine-tuning the expression of such genes. Overall, this dissertation makes significant contribution to the development of sand fly-based vaccines against leishmaniasis.

Doctor of Philosophy / Department of Entomology / R. Jeff Whitworth / The Hessian fly, Mayetiola destructor (Say), has been a historically significant pest of wheat in Kansas. However, it has been 60+ years since research has been conducted examining the flies’ activity throughout the year. Results of pheromone trapping in 4 counties in Kansas shows that Hessian fly (HF) males are actively flying in the fall, at least 1 month after the historical fly-free dates. Therefore, the Hessian Fly-Free Date is no longer valid and should be referred to as the Best Pest Management Date. Using pheromones for fall and spring trapping also indicated that HF is more active throughout the spring than previously thought, with almost continuous fly emergence and numerous emergence peaks in both spring and fall. The use of resistant wheat cultivars has been adapted to protect seedling plants from HF larval feeding in the fall. However, it is unknown if these cultivars are still providing protection after winter vernalization. Greenhouse trials indicated that ‘Armour’, a cultivar considered intermediately resistant, remains resistant under infestation levels of 1 fly/tiller but significant seed weight losses occured under infestations of 3 flies/tiller. In the field, Armour did not provide protection post-vernalization, with plants containing similar numbers of flaxseeds (pupae) as the susceptible cultivar, ‘Fuller’, and having significant losses of culm height (cm), number of spikelets/spike, number of seeds/spike, and seed weight (grams) when infested. ‘Duster’, a cultivar considered highly resistant, appeared to provide resistance to HF larval feeding in both the greenhouse and the field, and even produced significantly heavier seeds when infested with 3 flies/tiller in the greenhouse. These results suggest that post-vernalization screening should be conducted on all HF resistant cultivars to determine if each continues to provide protection. Little information is available showing if and how HF larval feeding on more mature wheat (Feekes 7-10), post-vernalization, impact plants, aside from lodging. Greenhouse and field infestations of a susceptible cultivar, Fuller, showed that significant losses of culm height (cm), number of seeds/spikelet, and seed weight will result from as few as 1 larva /culm. Yield losses averaged 0.13g/spike (65 kg/ha) compared to non-infested plants.

Doctor of Philosophy / Department of Entomology / David C. Margolies and Yoonseong Park / Organisms are capable of sensing environmental conditions through diverse mechanisms. Transient receptor potential channels (TRPs) are a cation channel family that has been found to function in diverse sensing mechanisms. In this dissertation, I identified the function of several TRPs in thermosensing and mechanosensing in the red flour beetle, Tribolium castaneum. Candidate TRPs were chosen based on homology to TRPs found and studied in Drosophila melanogaster. To identify the function of candidate TRPs in T. castaneum, I suppressed the expression of target genes by RNA interference technique and investigated the phenotype of each treated beetle. Temperature is a major limiting environmental factor for organisms. I tested the function of candidate TRPs in thermotaxis (behavior) and thermal acclimation (physiology). Using bioinformatics approaches, I identified three candidate TRPs – painless, pyrexia, and trpA1 – involved in high temperature sensing. To test thermotactic behavior, I investigated beetle movement on a temperature arena with two separate temperature zones. Thermal acclimation was tested by pre-exposing beetles to either 42 °C for 10 min. When treated with double stranded RNA of TRPA1 (dstrpA1), the thermotactic response of beetles at 39 and 42 °C was reduced when compared to control groups. With pre-exposure at 42 °C, survivorship of dstrpA1-treated beetles significantly increased after one minute exposure at 52 °C compared to beetles that were not pre-exposed. With dspainless treatment, beetles showed lower response to thermal acclimation and lower long-term survivorship. Beetles treated with dspyrexia showed lower recovery after heat treatment without pre-exposure at 42 °C. To identify the function of candidate TRPs in mechanosensing, I evaluated dsRNA treated beetles for survival, walking behavior, and tonic immobility. Treatment with dsnompC and dstrpA5 resulted in failure in eclosion, causing 93 % mortality in both treatments. Survivors in dsnompC showed defects in elytra sclerotization. In dsnanchung and dsinactive treatments, adults showed abnormal walking behavior and reduced walking speed that were likely caused by defects of mechanosensing in folding of the joint between the femur and tibia. For tonic immobility, beetles with dsnanchung, dsinactive, dswaterwitch and dsick2 (insect cytokine 2) treatments showed increased sensitivity to mechanical stimulation leading to tonic immobility.