Master of Science / Department of Entomology / Kun Yan Zhu / Cytochrome P450 monooxygenases (P450s) are important enzymes involved in the metabolism of a variety of xenobiotics, including insecticides and plant allelochemicals, and endogenous compounds, including juvenile hormones, ecdysteroids and fatty acids, in insects. Despite rapid advances in revealing various P450 genes in insects, our knowledge on the role of these genes in detoxification of insecticides is very limited. This research was to perform a genome-wide analysis of P450 genes and evaluate the role of selected P450 genes in detoxification of three commonly used pyrethroid insecticides in the yellow fever mosquito (Aedes aegypti). Our genome-wide analysis of revealed 159 P450 genes that can be classified into 18 families and 63 subfamilies. These genes are distributed in four clans, including 11 genes in the CYP2 clan, 80 in the CYP3 clan, 58 in the CYP4 clan and 10 in the mitochondrial CYP clan. The largest families are CYP6, CYP9, CYP4 and CYP325. The intron-exon organization of the genes is very diverse among the gene families, and the highest conservation of gene structures was observed in the CYP6 and CYP9 families predominantly containing single-intron genes. The phylogenetic analysis suggested that the CYP6 and CYP9 families might be derived from a common ancestor. The expression patterns of five transcripts including three individual genes (CYP6AA5, CYP6AL1 and CYP9J32) and two alternative splicing variants (CYP4J16A and CYP4J16B) of CYP4J16 were investigated in various tissues and at different developmental stages of the mosquito. Our results indicated differential expressions of these transcripts in different tissues and at different developmental stages examined. Furthermore, the exposure of the mosquitoes (larvae and adults) to each of three pyrethroid insecticides (permethrin, cypermethrin and deltamethrin) resulted in either down or up-regulation of these transcripts. Functional analyses of the selected P450 transcripts were conducted by using RNA interference (RNAi) followed by insecticide bioassay. RNAi was achieved by feeding mosquito larvae with chitosan/double stranded RNA (dsRNA) nanoparticles or injecting dsRNA to the adults. For the larvae, we obtained relatively low repressions of the P450 transcripts but the repressions were sufficient for carrying out our functional studies. Our study showed increased mortalities by 41.2% to cypermethrin when CYP6AA5 was silenced and 46.0% to permethrin when CYP9J32 was silenced. Similarly, the injection of dsRNAs in adults resulted in significant repressions of the P450 transcripts, and subsequent insecticide exposures led to a 29.3% increase in the adult mortality to cypermethrin when CYP6AA5 was silenced. Our further analysis of the nuclear receptor HR96 in the up-regulation of the P450 genes showed that when HR96 was silenced by RNAi, the up-regulation of CYP4J16B by cypermethrin was reduced by 10.1-fold but silencing HR96 did not affect the up-regulation of other P450 genes examined. These results suggest that HR96 is likely involved in regulating the expression of CYP4J16B in Ae. aegypti. However, different regulatory mechanism (s) may be involved in the up-regulation of other P450 genes examined. Model structure of CYP6AA5 was created by homology modeling and insecticides substrates were docked into the active site of this protein. Our results indicate that all three insecticides can fit into the catalytic pocket. The interaction distances between the heme iron and the putative aromatic hydroxylation site were 9.2, 9.4 and 7.2 Å for permethrin, cypermethrin and deltamethrin, respectively, whereas for aliphatic hydroxylation site these distances were 5.3, 2.8 and 2.9 Å. These results showed that CYP6AA5 may be able to metabolize cypermethrin and deltamethrin preferentially by aliphatic hydroxylation as indicated by the close interaction with the heme iron.

Doctor of Philosophy / Department of Entomology / David C. Margolies / James R. Nechols / Foraging traits such as prey consumption rate and the efficiency with which predators convert their prey into offspring are important determinants of local predator-prey dynamics. However, in environments with patchy prey distribution, predator dispersal and aggregation in response to prey-induced volatile cues becomes more critical. My dissertation addressed predator-prey population dynamics in response to variation in four foraging traits in the predatory mite, Phytoseiulus persimilis (Acari: Phytoseiidae): consumption rate, conversion efficiency, dispersal, and olfactory response related to prey. The dispersal response and olfactory sensitivity in predatory mites is modified by prey-related cues. For example, the dispersal response increased with decreasing prey density in a patch and increasing prey-related volatiles from outside the prey patch. The olfactory response of predatory mites also increased with increasing numbers of prey per plant or with the length of time a plant was infested by prey. These results formed the basis for development of bioassays used to examine genetic variation in dispersal and olfactory response of predatory mites. Through artificial selection I documented additive genetic variation in all four traits. After relaxation of selection, high-level phenotypes were stable compared to their low counterparts. There were significant genetic correlations between some of the foraging traits. However, there were no correlations between foraging traits and life-history traits. The existence of genetic variation and covariation among the foraging traits suggests that predatory mites must be able to adopt different foraging strategies in the evolution of prey-finding in a tritrophic system. High consumption, high conversion efficiency and high dispersal response phenotypes interacted differently with prey in a spatially complex landscape. All foraging traits were comparable in terms of predator-prey densities and plant damage; but they were lower than the unselected control. Spatial association and correlation analysis showed that all foraging traits were positively associated with prey; but the strongest association was observed for the high conversion efficiency and dispersal lines. The variability in foraging behaviors of the predatory mite affects its ability to locate patchily distributed prey, thereby influencing foraging efficiency and population dynamics. This research provides new information about the critical link between predator foraging and population dynamics relevant to biological control.

Master of Science / Department of Biochemistry / Michael R. Kanost / Laccases are multicopper oxidases that catalyze the oxidation of a broad range of substrates, typically phenols and anilines. Research on laccases in fungi, plants, and bacteria has indicated that they have roles in detoxification, pigmentation, wound healing, morphogenesis and lignin synthesis and degradation. However, there has been relatively little investigation on laccases that exist in insects or other invertebrates. Insects have multiple laccase genes, but the function of just one type is known; laccase-2 (Lac2) orthologs are required for tanning of newly synthesized exoskeleton. In the mosquito Anopheles gambiae and other insect species whose genomes have been sequenced, alternative exon splicing may generate two isoforms of Lac2. The objective of this study was to characterize the two isoforms of AgLac2. They are identical in their first 500 residues, but the carboxyl-terminal 262 residues derived from alternative exons are 81% identical. Recombinant Lac2A and Lac2B were expressed and purified. They are both glycoproteins of ~81 kDa, and both can oxidize the laccase substrate ABTS as well as the catechols, N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA). Lac2A and Lac2B with ABTS have pH optima of 5.0-5.5 and 4.5-5.0, respectively. The pH optima with NBAD and NADA are 5.5-6.5. The Km values (mM) for Lac2A and Lac2B with NBAD are 5.4 ± 2.1 and 5.0 ± 2.6, respectively. The Km values (mM) for Lac2A and Lac2B with NADA are 0.7 ± 0.2 and 1.4 ± 0.5, respectively. Thus, there is little difference between the isoforms in K[subscript]m for these two substrates. The K[subscript]m values do indicate that both isoforms have a greater affinity for the substrate NADA. The kcat values (s[superscript]-1) for Lac2A and Lac2B with NBAD are 14.2 ± 3.5 and 6.0 ± 1.8, respectively. The k[subscript]cat values (s[superscript]-1) for Lac2A and Lac2B with NADA are 2.4 ± 0.2 and 0.5 ± 0.04, respectively. The most apparent difference between the two isoforms detected in the study is that Lac2A was four-fold more active than Lac2B when NADA was used as a substrate. Although the two isoforms are very similar in their amino acid sequences, the differences in catalytic properties may indicate different roles in insect physiology.

Master of Science / Department of Entomology / Srinivas Kambhampati / Termites (Isoptera) are separated into morphologically and behaviorally specialized castes of sterile workers and soldiers, and the reproductive alates. Previous research on eusocial insects has indicated that caste differentiation has a genetic basis. Although much has been studied about the genetic basis of caste differentiation and behavior in the honey bee, Apis mellifera, termites remain comparatively understudied. Therefore, my objective was to compare the gene expression patterns of different castes of the termite Reticulitermes flavipes based on EST analyses and a microarray. Soldier, worker, and alate caste and two larval life stage cDNA libraries were constructed, and ~15,000 randomly chosen clones were sequenced to compile an EST database. Putative gene functions were assigned based on a BLASTX Swissprot search. Categorical expression patterns for each library were compared using the in silico methods of BLAST2GO and r-statistics. I chose 2,240 unique-ESTs based on their putative function and sequence quality, which I used to fabricate a Combimatrix microarray. I used the microarray to compare expression levels between workers and soldiers from Kansas and Florida populations. Seventy to ninety percent of the sequences from the ESTs of each caste and life stages had no significant similarity to those in existing databases. All libraries contained sequences with putative reproductive functions, which was unexpected in the non-reproductive soldier and worker castes. Sequences of interest that showed a putative bias among castes include a viral protein in soldiers and a possible chemosensory protein in alates, which may be involved in termite reproductive functionality or communication. The microarray showed increased expression in the soldier caste of a sequence that matched tropomyosin and an increased expression level of a sequence that matched a PDZ-domain containing protein in some worker samples. This study leads to several candidate genes of potential caste specific function, which can be further tested using functional analysis and between the different castes and life stages of R. flavipes. These genes include the sequences similar to pebIII and RGS-GAIP. I have also expanded upon the available sequences for this termite and utilized the r-statistic in silico method for the first time to putatively compare gene expression in the different castes of a eusocial insect. The in silico analysis allowed us to identify several genes which may show biased expression patterns in the different cDNA libraries and which may reveal caste-specific expression controlling important functions after further analysis. These candidates include: an alate-biased gene, which had a predicted function of neurotransmitter secretion and cholesterol absorption as well as a late larval-biased gene which was predicted to be involved in protein biosynthesis and ligase activity.

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.

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.