Master of Science / Department of Grain Science and Industry / Subramanyam Bhadriraju / The development, relative retention, and oviposition of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), on six different types of starches, wheat flour, and wheat flour plus yeast were investigated in the laboratory. The particle size of starch and flours were different; the mean size of 90% of particles for starches ranged from 15 μm for high amylose corn starch to 58 μm for potato, whereas that of the flour was ≤ 133 μm. Larval length, head capsule width, and weight gain of T. castaneum larvae were measured every 3 d on starches, flour, and flour plus 5% (by wt) yeast diet for 30 d at 28oC, 65% r.h., and 14:10 (L:D) photoperiod. Larvae reared on flour and flour plus yeast developed normally and showed better survival compared to those reared on starches. Larvae on the starches failed to develop beyond second, and rarely, third instars. Adults of T. castaneum did not show any preference to flour over starches in dual-choice tests in circular arenas. On average, T. castaneum laid less than 3 eggs/female over a 15-d period on starches compared to 97 and 109 eggs/female on flour and flour plus yeast diet, respectively. These studies suggest that starches are poor substrates for larval survival and development. Starches were as attractive as flour to adults; however, starches do not appear to be a suitable medium for egg-laying. Both aggregation pheromone and volatiles did not trigger oviposition behavior. Experiments by moving adults between wheat starch and wheat flour and vice versa showed that feeding on wheat flour was necessary for egg-laying, indicating the absence of essential nutrients in wheat starch. On wheat flour, feeding for 0.5 d was necessary to lay eggs. Females that were starved failed to lay eggs, reinforcing that the nutrional status of females and not males was essential for egg-laying. A minimum of 4% of wheat gluten (wheat protein) elicited egg-laying on starches, although 4-5 times fewer eggs were laid in starch gluten compared with wheat flour alone. Supplementing wheat starch with 1% cholesterol, in addition, to gluten, did not result in an increase in egg-laying by T. castaneum females. These findings suggest that starches may have potential in managing development and reproduction of T. castaneum—a pest that is common and severe in food-processing facilities. Furthermore, starches can be used as a suitable substrate for studying the nutritional ecology of T. castaneum.

Doctor of Philosophy / Department of Entomology / Alberto B. Broce / Ludek Zurek / Stable fly, Stomoxys calcitrans (L.), and house fly, Musca domestica L., are two major pests affecting both confined and pastured livestock in the United States. It costs livestock producers millions of dollars annually to reduce populations of these two pests. Control of stable flies and house flies based on chemical insecticides is only marginally effective and unsustainable in the long term due to the development of insecticide resistance. This has created a demand for alternative methods which are environmentally friendly and cost effective for the management of these pests. Information on stable fly and house fly oviposition behavior and the aggregation and segregation of their immatures may help in an integrated pest management control program for these pests. This research identified specific bacterial species from the surface of stable fly eggs which are suspected of releasing chemical cues used to induce gravid females to oviposit at sites where eggs have been deposited and inhibit additional deposition of eggs in the same habitat when it is already colonized. My research also showed that stable fly and house fly larvae tend to be aggregated in distribution, even in apparently homogenous habitats, and to be spatially segregated from each other. Finally, I evaluated the vector competence of stable flies for an emerging food-borne pathogen, Enterobacter sakazakii, showing that this fly species is potentially a good vector for this pathogen.

Master of Science / Department of Entomology / Jeremy L. Marshall / Sexual conflict is a process that occurs when the evolutionary interests of the sexes is not aligned. The theory began with Darwin in the 1800’s. His observations of sexual dimorphism and traits which did not seem to follow the rules of natural selection led him to the theory of sexual selection. Sexual selection resolved some of the issues which were noted in the evolutionary processes he observed but not all of them. For example, it did not explain traits which increased the male optimum fitness while decreasing the female optimum fitness. It was decades before the concept of sexual conflict was formulated, and even longer before the underlying mechanisms were understood. Sexual conflict is different than sexual selection and there are ways to differentiate which of these processes have occurred in a population. The main forms of sexual conflict are intralocus and interlocus conflict. These involve the interactions between alleles in the genome of the sexes. Intralocus conflict involves conflict at a single locus in the genome while interlocus conflict involves conflict between different loci. Interlocus conflict has been more extensively studied due to its association with sexually antagonistic coevolution (SAC). SAC draws the attention of scientists due to the possibility it is related to the fundamental biological process of speciation. Sexual conflict targets certain traits at defined periods in the mating process. These periods include first, the precopulatory stage which is before the act of copulation begins. Second, there is the stage during copulation but before fertilization of the embryo. Finally, there is the postcopulatory postzygotic stage which is after copulation has ended and fertilization has occurred. Each of these points in the process of mating has traits or behaviors which sexual conflict may target. This review concludes with a proposed experiment to determine if sexual conflict is occurring in a group of four genera of mosquitoes. The experiment utilizes the attributes of sexual conflict to differentiate between other processes. A major component is the consequence of mating systems on selective processes to determine if sexual conflict is involved in the evolution of male accessory gland proteins.

Doctor of Philosophy / Department of Entomology / Alberto B. Broce / Ludek Zurek / Stable flies, Stomoxys calcitrans (L.), are a serious pest to beef cattle in confined animal feeding operations (CAFOs) by causing economic losses in the form of reduced feed intake and feed efficiency, resulting in reduced weight gain. Integration of sanitation, parasitoids, and residual insecticides offers a much-needed reduction of this pest's impact on CAFOs. In the past two decades, stable flies have become the most important pest of pastured cattle. Further impact that stable flies have on cattle is when cattle seek protection from stable flies by standing in water, which results in water pollution with fecal matter, in addition to reduced foraging time. Sites of winter feeding of round hay bales have demonstrated to be important habitats for stable fly development during spring/summer. Cattle feeding on round bales can waste as much as 40% of the total amount of hay when fed in conventional ring feeders. Hay wastage is largely a function of the type of feeding method and the amount of agonistic behavior of the cattle. Feeding methods range from rolling hay directly onto the ground to the use of various types of feeders. Since traditional control methods utilized in CAFOs against stable flies have not been evaluated in pastures, producers rely heavily on organic insecticides in efforts to control this pest. At this time, there are no effective control methods available for stable fly management on pastured cattle. This research examined different management strategies that could minimize or eliminate stable fly larval habitats by reducing the amount of hay wasted being mixed with manure. In addition, different hay and manure mixtures were compared to characterize the larval habitat at these hay-feeding sites. Finally, the efficacy of boric acid, Metarhizium anisopliae, and tetrachlorvinphos in controlling the development of stable flies in hay substrates was evaluated.

Doctor of Philosophy / Department of Entomology / Franklin Arthur / James R. Nechols / The Indianmeal moth, Plodia interpunctella Hubner, is capable of infesting a number of different commodities including a wide variety of grains, nuts and finished stored products. Therefore, control of the Indianmeal moth is especially needed in areas where food is being stored for human consumption. Increased concerns of consumers and producers regarding the impact of conventional insecticides on the environment and on human health has prompted scientists and the agricultural chemical industry to search for insecticides that do not affect mammalian nervous systems and have limited effects on non-target organisms. One group of insecticides with reduced risks is insect growth regulators (IGRs), which are substances that mimic insect hormones essential to normal development and reproduction. Currently methoprene, a juvenile hormone analog, is labeled for direct application to stored grains, as well as a contact insecticide and as an aerosol application inside mills, warehouses, and indoor food storage facilities. Surface treatments and aerosol space applications can be effective ways to treat the interior surfaces and storage areas of warehouses and food processing facilities. There is little recent research with large-scale aerosol applications in storage sites; furthermore, there are no published references in the scientific literature regarding efficacy of using methoprene alone in aerosol form. Therefore, the purpose of this research was to evaluate the use of surface and aerosol applications of methoprene on finished stored-product packaging materials and facilities for the control of P. interpunctella. Results of this research showed that while methoprene has good residual activity, and efficacy is unaffected by temperature, surface applications of methoprene on packaging materials is not as effective for control of P. interpunctella as aerosol applications of methoprene. Aerosol methoprene is highly effective alone and in combination with conventional chemicals for control of eggs and wandering-phase larvae. Simulations with a population growth model make it possible to estimate impact of insecticide treatments at different temperatures and application times on populations of P. interpunctella. Aerosol treatments are also economically viable as part of an overall integrated pest management program.

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 / 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.

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.