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.

Doctor of Philosophy / Department of Entomology / David C. Margolies / Yoonseong Park / The predatory mite Phytoseiulus persimilis (Acari, Phytoseiidae) is one of the most frequently released natural enemies for biological control of spider mites in greenhouse and outdoors crops. In this research, I utilized Expresses Sequence Tags (ESTs), the most cost effective approach for transcriptome exploration, to study three different aspects of this arachnid species for which there is little genomic information. I combined two EST datasets from different whole body cDNA libraries and analyzed by bioinformatics means. Approximately 54% of 10,256 uniESTs were annotated based on the homology to sequences in the National Center for Biotechnological Information (NCBI) database. A list of these uniESTs, sorted from most to least likelihood based on the expected value from the blast search in public databases, was used to create tools for each of the three studies. First, I described sixty-one genes encoding products known to be important in pesticide metabolism and in endocrinology, including cytochrome P450s, glutathione-S-transferases, acetylcholinesterase homologs, neuropetides and neurohormones. Findings on arachnid specific esterases and neuropetides, and possible benefits to pest management programs, were discussed. Next, I inferred divergence time for Acari and the point of divergence of two lineages within anactinotrichid mites, Ixodes scapularis and Phytoseiulus persimilis. I used expresses sequence tags from the predatory mite P. persimilis to pull out 74 orthologous amino acid sequences of invertebrates species: nine insect species, Daphnia pulex, Ixodes scapularis, and Caenorhabditis elegans. I estimated a similar origin for Chelicerata (578.1 ± 38.2 - 482.2 ± 7.2 Mya) as in other recent studies. However, divergence dating using amino acid sequences suggested a Devonian origin of anactinotrichid mites (487.6 ± 32.2 - 410.1 ± 6.1 Mya) based on four reference dates (two fossil records and two molecular clocks) and four amino acid substitution methods; this estimate is much earlier that those in the current literature. This discrepancy of divergence times may be due to the use of a global clock. Finally, I developed molecular markers from the EST dataset to examine inheritance in the haplodiploid system in P. persimilis. Biparental contribution of chromosomes is required among the predatory mites but the paternal chromosome set seems to be eliminated or loss (Paternal genome loss, PGL) in male offspring. However, genetic studies in other two phytoseiid species were suggested diploid males with PGL only in the germ cells. In the present study, haploid adult males of P. persimilis have been observed using five independent EST-derived markers. Single mites derived from inter-population crosses were genotyped after whole genome amplification. The parahaploid genetic system in P. persimilis is supported by this study, in which both sexes arise from fertilized eggs but the paternal chromosome set is subsequently lost in males.

Master of Science / Department of Entomology / Kun Yan Zhu / Raymond A. Cloyd / Protected environments, such as greenhouses and interior plantscapes provide optimal conditions for arthropod (insect and/or mite) pests to survive, develop, and reproduce. Two commonly encountered insect pests in protected environments include the citrus mealybug (CMB), Planococcus citri, and the western flower thrips (WFT), Frankliniella occidentalis. It is difficult to mitigate CMB and WFT populations due to the behavioral characteristics of the insects and few pesticides that are registered for use in protected environments. This research involved two distinctly different studies. The objectives of the first study were to determine the efficacy and residual activity of systemic insecticides registered for use against CMB and to quantify CMB feeding locations. The objectives of the second study were to determine the compatibility and efficacy of commonly used binary pesticide mixtures against the WFT under both laboratory and greenhouse conditions. To determine the efficacy of systemic insecticides against CMB, greenhouse experiments were conducted in which coleus, Solenstemon scutellarioides, plants were artificially infested with CMB. Drench applications of each designated treatment were applied to each plant. Results associated with drench applications of the systemic insecticides against CMB indicated minimal CMB mortality (<30%) for both preventative and curative drench applications of azadirachtin and spirotetramat. Thiamethoxam, a neonicotinoid-based insecticide, at the labeled and twice the labeled rate provided the highest CMB mortality; however, not until 21 days after treatment was this observed, and CMB mortality was <80%. In all cases, significantly more CMB were located on the stem of green coleus plants compared to the leaf top and bottom. Pesticide mixture compatibility was determined using jar tests. In addition, phytotoxicity and efficacy of pesticide mixtures against WFT was determined through a series of laboratory and greenhouse experiments for each individual pesticide, and the mixtures to determine synergism, antagonism, or no effect. Results associated with the jar tests indicated that all the mixtures were compatible. Furthermore, the mixtures were not phytotoxic to the horticultural plant species evaluated. Laboratory results indicated that mixtures containing spinosad + bifenazate were antagonistic against WFT. Greenhouse experiments demonstrated significantly reduced efficacy associated with the abamectin + azadirachtin mixtures; however, each binary mixture provided approximately 80% mortality of WFT.

Doctor of Philosophy / Department of Entomology / James F. Campbell / Kun Yan Zhu / Aerosol applications of reduced risk insecticides such as synergized pyrethrin and insect growth regulators (IGR) are part of food industry integrated pest management programs. Since aerosols cannot penetrate into hidden areas exploited by pests such as the red flour beetle, Tribolium castaneum, the potential for these insecticides to effect beetle populations was evaluated. Because IGRs do not cause immediate mortality, the potential of horizontal transfer for an IGR from treated to untreated individuals was also examined. Results showed that when untreated T. castaneum, larvae or pupae, were added to flour containing methoprene, IGR, treated larvae, pupae or adults, the untreated individuals exhibited evidence of methoprene exposure (external deformities and reduced survival). Evaluation of the different mechanisms of transfer indicated that contact with methoprene treated individuals or flour that had been in contact with treated individuals may be the primary method of methoprene transfer. Since aerosols are often applied as a combination of IGR and pyrethrin with a carrier, the effect of these components was evaluated. Applications of synergized pyrethrin caused knockdown of adults but affected adults recovered and progeny production was not effected. Exposure of eggs to these insecticides reduced egg hatch. Food material accumulations inside food facilities can potentially increase or reduce insecticide efficacy. Evaluation of different flour residue levels, representing different sanitation levels, revealed that sanitation alone reduced immature development. As flour residue depths increased more individuals developed into adults but very few developed in the insecticide treatments. Food facilities that use aerosol insecticides apply them at regular intervals, so the cumulative effects of these treatments were considered. Experiments evaluating repeated insecticide exposures indicated that the direct morality from synergized pyrethrin not the horizontal transfer of methoprene was the primary factor in population reduction. Overall findings suggested that methoprene is highly mobile between different surfaces. Exposure of untreated individual beetle larvae to treated larvae or pupae or to flour that has been in contact with exposed beetles can have detrimental effects on development or survival, but these effects may be highly variable and even in cumulative exposures the overall level of population suppression is limited.

Master of Science / Department of Grain Science and Industry / Bhadriraju Subramanyam / The insecticides, β-cyfluthrin and chlorpyrifos-methyl plus deltamethrin, are approved in the United States for treating empty bin surfaces. Chlorpyrifos-methyl plus deltamethrin and spinosad insecticides are approved for direct treatment of wheat. The efficacy of commercial formulations of β-cyfluthrin and chlorpyrifos-methyl plus deltamethrin at labeled rates was evaluated against adults of 16 field strains of the red flour beetle, Tribolium castaneum (Herbst); seven strains of sawtoothed grain beetle, Oryzaephilus surinamensis (L.); and two strains of the lesser grain borer, Rhyzopertha dominica (F.). Concrete arenas in plastic Petri dishes (9 cm diameter) were used to simulate the concrete floor of empty bins. The time for ~100% knockdown and mortality of adults of laboratory strains of the three species was first established by exposing them to insecticide-treated concrete surfaces for 1 to 24 h. Adults of field strains of each species were exposed to specific established insecticide-time combinations. Mortality of all species was lower than knockdown, suggesting recovery after seven days when placed on food. Chlorpyrifos-methyl plus deltamethrin did not control all R. dominica and most O. ]surinamensis field strains. β-cyfluthrin was extremely effective against R. dominica but ineffective against T. castaneum and O. surinamensis field strains, even at four times the high labeled rate. Field strains of R. dominica were highly susceptible to spinosad and chlorpyrifos-methyl plus deltamethrin at labeled rates on hard red winter wheat. Strains of T. castaneum and O. surinamensis were susceptible only to the latter insecticide. Dose-response tests with spinosad on the two least susceptible field strains of each species showed the lethal dose for 99% mortality (LD[subscript]99) for T. castaneum and R. dominica field strains were similar to that of the corresponding laboratory strains. Corresponding values for the two O. surinamensis field strains were significantly greater (~6 times) than the laboratory strain. The effective dose for progeny reduction (ED[subscript]99) of only one R. dominica field strain was significantly greater (~2 times) than that of the laboratory strain. The baseline susceptibility data of field strains of three insect species to spinosad will be useful for monitoring resistance development once this product is commercially released as a grain protectant.

Master of Science / Department of Entomology / James F. Campbell / Thomas W. Phillips / Knowing the factors that influence the distribution patterns, establishment and persistence of stored product insects aids in the development of a more effective pest management program in grain storage structures. This research focuses mainly on the insect communities of stored wheat and stored rice in two different geographical locations, their temporal relationships and the most important or abundant species within that community. Stored wheat was sampled for one season in Manhattan, KS and for rice stored in Beaumont, TX was sampled for two seasons. Hairy fungus beetle, Typhaea stercorea (Coleoptera: Mycetophagidae) was one of the most abundant species and was present in every bin of either wheat or rice and appeared to move into and out of the grain mass. In wheat bin, Indianmeal moth, Plodia interpunctella (Lepidoptera: Pyralidae) was a predominant species captured in the bin headspace, but was not frequently recovered in the grain mass. Headspace temperatures tended to be warmer than grain temperatures and outside temperatures. Other major insects recovered in wheat bin included the following groups or species: Anthicidae, Lathridiidae, Cryptolestes, foreign grain beetle Ahasverus advena (Coleoptera: Silvanidae), sawtoothed grain beetle Oryzaephilus surinamensis (Coleoptera: Silvanidae), red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae), smalleyed flour beetle (Coleoptera: Tenebrionoidea) and minute pirate bug Xylocoris favipes (Hemiptera: Anthocoridae). In rice bins, the predominate species were hairy fungus beetle, foreign grain beetle and Angoumois grain moth Sitotroga cerealella (Lepidoptera: Gelechiidae). Angoumois grain moth was one of the most abundant species in rice, and was captured in the headspace as well as below the grain surface. Rice bins varied considerably in the relative abundance of different species between bins within a season and between seasons. Foreign grain beetle and hairy fungus beetle were especially variable among bins. Two species of weevil (Coleoptera: Curculionidae) that are not grain pests, the sugar cane rootstock weevil, Apinocis deplanata and rice water weevil, Lissorhoptrus oryzophilus (Coleoptera: Curculionidae), were present in high numbers in rice bins from September-December 2009.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / C. Michael Smith / The Hessian fly, Mayetiola destructor (Say), is a major pest of wheat, and is controlled mainly through deploying fly-resistant wheat cultivars. This study investigated five M. destructor populations collected from Texas, Louisiana, and Oklahoma, where infestation by Hessian fly has been high in recent years. Eight resistance genes including H12, H13, H17, H18, H22, H25, H26, and Hdic, were found to be highly effective against all tested M. destructor populations in this region, conferring resistance to 80% or more of plants containing one of these resistant genes. The frequency of biotypes virulent to resistant genes ranged from 0 to 45%. A logistic regression model was established to predict biotype frequencies based on the correlation between the percentages of susceptible plants obtained in a virulence test. In addition to the virulence test, the log-odds of virulent biotype frequencies were determined by a traditional approach to predict the logistic regression model. Characterization of a bacterial artificial chromosome (BAC) clone identified a gene encoding a protein with sequence similarity to bacterial levanases. Blast searching with the levanase-like protein identified 14 levanase/inulase-like genes or gene fragments. In this study, we determined the expression levels of these genes in different developmental stages and different tissues of 3-d old larvae of M. destructor. Sequence analysis revealed that six genes encode full length proteins, three were truncated at the 5’ end, and five truncated at the 3’ end. Sequences of putative proteins showed approximately 42% similarities to bacterial levanases or inulases, and 36% similarity to fungal levanases or inulases. No sequence similarities were found with any known animal or plant proteins. Comparative analysis of sequences among 14 levanase/inulase-like genes revealed that positions for intron/exon boundaries are conserved among different genes even though the length of each intron and exon varied among different genes. The expression patterns of the levanase/inulase-like genes were different among developmental stages and larval tissues of M. destructor. Interestingly, three genes presented alternative splicing bands in different developmental stages, and two genes exhibited splicing bands in different tissues of 3 d old M. destructor. This study would be useful for future studies of the characterization and function of levanase/inulase-like genes of these enzymes in plant-insect interactions.

Master of Science / Department of Entomology / C. Michael Smith / The Dectes stem borer, Dectes texanus LeConte, is a pest of soybean, Glycine max (L.) Merrill, in North America. Larval feeding weakens plant stems, triggering lodging of the infested plants and causing significant yield losses. D. texanus infestations in soybean fields are increasing across Kansas and other states, necessitating the development of effective tactics to control this pest. The use of D. texanus -resistant soybean cultivars is a desirable strategy to control this pest since cultural and chemical control options are lacking. In previous studies, the soybean plant introduction PI165673 was identified to be resistant to D. texanus. The objective of this research was to determine the inheritance of resistance to D. texanus in PI165673. F[subscript]2 progeny plants from crosses between the D. texanus susceptible genotypes KS5004N and K07-1544, and the resistant genotype PI165673 were tested in the field for resistance to D. texanus in 2011. Seeds from the cross K07-1544/PI165673 were advanced to the F[subscript]3 generation, and F[subscript]2:3 families were tested in the field for resistance to D. texanus in 2012. At 20 d after infestation with adults, the numbers of oviposition punctures and larvae on each plant were counted to estimate the oviposition puncture per larvae resistance ratio. Segregation for resistance to D. texanus and heritability estimates in the F[subscript]2 and F[subscript]2:3 populations indicated that resistance is controlled by more than one gene. Thirteen F[subscript]2:3 families had a higher (more resistant) resistance ratio than the susceptible parent K07-1544. Mean head capsule widths of larvae collected from K07-1544 and PI165673 plants in 2012 were similar, as was the percentage of larvae per larval instar. According to heritability estimates for each phenotypic trait, progress in breeding for D. texanus resistance using PI165673 will benefit from marker assisted selection. Identification of additional sources of D. texanus resistance besides that in PI165673 is needed to slow larval growth in the stem.

Master of Science / Department of Entomology / Marcelo Ramalho-Ortigao / Sand flies (Diptera:Psychodidae) are vectors of parasites if the genus Leishmania, the causative agent of leishmaniasis, a neglected tropical disease in several countries around the world. Sand flies transmit Leishmania to suitable vertebrates during the blood meal and following a complex development parasites undergo within the fly. Many aspects of the Leishmania development within the sand fly vector are well known, however details about how sand fly molecules affect the parasite are still not yet known. Our group previously identified that RNAi knockdown of PpChit1, a midgut specific chitinase from the sand fly Phlebotomus papatasi, led to a significant decrease in the load of Le. major. In this study, we assessed potential fitness effects of antisera anti-PpChit1 on three laboratory-reared sand fly species (P. papatasi, Phlebotomus duboscqi, and Lutzomyia longipalpis). Our results suggest that feeding sand flies with anti-PpChit1 sera led to a one day delay in the onset of oviposition, and also suggested that anti-PpChit1-fed flies survived on average up to three days longer that control flies. Analyses of the peritrophic matrix (PM) indicated a significant increase in thickness 72 hours post anti-PpChit1 feeding compared to control sera. Altogether the results suggest that feeding sand flies with anti-PpChit1 likely affects the kinetics of sand PM, which in turn affects the flow of nutrients and certain aspects of sand fly fitness. In the course of this study, we also evaluated the ability of American Foxhounds naturally infected with Leishmania infantum to transmit these parasites via bites of phlebotomine sand flies to suitable vertebrates. Since 1999, an outbreak of canine visceral leishmaniasis (CVL) has been reported in the U.S especially among Foxhounds. The ability of sand flies to pick up and transmit this pathogen represents an important health risk for companion dogs and humans. Our results indicate that Foxhounds naturally infected with Le. infantum are highly infectious to sand flies and that the parasites are able to fully develop within these vectors and de successfully transmitted during blood feeding. Thus, the risk exists for these parasites to become endemic in North America where sand flies are also known to occur.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Each year, over 500 million people are infected with mosquito-borne diseases, including malaria, yellow fever and dengue fever, which cause several million deaths, and long-term disability and suffering. This dissertation focused on the mosquito Aedes aegypti, a vector for dengue virus and yellow fever virus. Since Sindbis virus (SINV) is an arthropod-borne virus (arbovirus) that is vectored by A. aegypti and is well characterized at the molecular level, the SINV - A. aegypti model was used to determine whether apoptosis plays a role in the control of vector competency. In Chapter 2, the effects of inducing or inhibiting apoptosis on SINV replication were tested in mosquito cells. It was observed that recombinant SINVs expressing pro-apoptotic genes caused extensive apoptosis in mosquito cells, with decreased virus production after the cells underwent apoptosis. Infection of mosquito cells with SINV expressing the caspase inhibitor P35 inhibited actinomycin D-induced apoptosis, but had no observable effects on virus replication. This study was the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells. Chapter 3 examined the effects of silencing apoptosis regulatory genes on SINV replication and dissemination in A. aegypti. Genes which either positively or negatively regulate apoptosis were silenced by RNA interference in mosquitoes, which were then infected with a recombinant SINV expressing green fluorescent protein (GFP). Reciprocal effects were observed on both the occurrence and intensity of expression of GFP in various tissues. These results suggest that systemic apoptosis positively influences SINV replication in A. aegypti. This was the first direct study to explore the role of apoptosis in determining mosquito vector competence for arboviruses. Finally, in Chapter 4, the mechanisms of apoptosis were explored in A. aegypti. Overexpression of IAP antagonists caused extensive cell death in mosquito cells, while silencing the expression of IAP antagonists attenuated apoptosis. The results showed that the IAP binding motif (IBM) of IAP antagonists was critical for their binding to AeIAP1. The IAP antagonists released initiator and effector caspases from AeIAP1 by competing for the binding sites and caused caspase-dependent apoptosis. These findings imply that the mechanisms of IAP antagonists regulating apoptosis are conserved between mosquitoes and the model insect where apoptosis has been mainly studied, Drosophila melanogaster.