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 / Larry L. Buschman / Kun Yan Zhu / European corn borer, Ostrinia nubilalis, is one of the most destructive insect pests of corn in the Midwest corn belt of the United States. The crystal protein toxin (Cry1Ab) expressed by the bacterium, Bacillus thuriginesis (Bt), specifically targets O. nubilalis gut and functions as “stomach poison”. Transgenic corn expressing Cry1Ab can effectively control O. nubilalis larval infestation. However, O. nubilalis has the potential to develop resistance to Bt toxins which prompts concerns that transgenic corn will lose its control efficacy. Previous studies found that O. nubilalis gut serine proteases and membrane proteins were involved in Bt toxicity and resistance. Therefore, this study was to identify and characterize gut transcripts potentially involved in Bt toxicity and resistance, and to compare their transcriptional responses to the ingestion of Cry1Ab protoxin and transgenic corn leaves expressing Cry1Ab toxin. We identified and characterized 34 cDNAs encoding putative trypsins, chymotrypsins, and trypsin- and chymotrypsin-like protease homologs from O. nubilalis gut-specific expressed sequence tags (ESTs). Blast and phylogenetic analysis of their deduced amino acid sequences indicated that 15 were putative trypsins belonging to Try-G2 and Try-G3 groups (none of them was grouped in Try-G1), another 15 were putative chymotrypsins in one large group (CTP-G1), and the remaining four were serine protease homologs in Try-G4 and CTP-G2 groups, respectively. The existence of diverse trypsins, chymotrypsins and serine protease homologs in O. nubilalis could be an adaptation to different food sources and also a defense mechanism against plant-specific protease inhibitors and Cry toxins from transgenic corn. The expressions of four putative trypsins (OnTry4, OnTry5, OnTry6 and OnTry14) were up-regulated in O. nubilalis larvae after the ingestion of Cry1Ab protoxin. The differential expressions of these protease transcripts may implicate a link to Cry1Ab intoxication. To better understand the basic physiology of insect gut and Bt toxin interactions, we developed a high-resolution 8×15K cDNA microarray chips based on the larval gut specific ESTs. Each microarray contains 12,797 probes representing 2,895 unique larval gut transcripts. The expressions of 174 transcripts were differentially regulated at least 2-fold (P-value ≤0.05) after the larvae fed Cry1Ab protoxin for 6 hours. Among them, 13 transcripts, putatively encoding eight serine protease, three aminopeptidase, one alkaline phosphatase, and one cadherin-like protein, were identified and further validated their expression ratios by quantitative PCR (qPCR). Three trypsin transcripts were up-regulated by more than 5-fold in larvae fed Cry1Ab protoxin. Sequence analysis suggests that they may have role in protoxin activation and toxin degradation. The transcriptional responses of laboratory-selected Cry1Ab resistant (R) and susceptible (S) strains of O. nubilalis to the ingestion of transgenic corn (MON811) leaves expressing Cry1Ab toxin were also examined. Even though R-strain larvae showed 200-fold resistance to Cry1Ab protoxin as compared with S strain, the larvae from both strains eventually died after fed transgenic corn leaves. However, the survival time of R-strain larvae was significantly different from that of S-strain larvae. The median lethal time (LT50) for the early third-instar larvae of R- and S-strains were 5.4 and 3.6 days, respectively. Furthermore, we identified 398 and 264 transcripts from the larvae of the S and R strains, respectively, with a significantly increased or decreased expression (expression ratio cut off ≥2.0 fold with p-value ≤0.05) as compared with those in the larvae fed on non-transgenic corn leaves. The number of transcripts and their expression ratios of S-strain larvae are larger than these of R-strain larvae. These significantly differentially expressed transcripts may play important roles in influencing Cry1Ab toxicity from toxin degradation, toxin binding, to intracellular defense. Seventeen transcripts including serine protease and aminopeptdiase in S strain and nine in R strain were further analyzed by qPCR to validate their expression ratios. This study not only revealed information about the difference in the transcriptional responses of these genes to Cry1Ab between Bt-resistant and susceptible strains of O. nubilalis, but also provided new insights into potential interactions of the protoxin, toxin from transgenic corn with important proteins in the gut of O. nubilalis larvae.

Master of Science / Department of Entomology / Thomas W. Phillips / The rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae), is a very common worldwide pest of stored grains. The orientation of C. ferrugineus beetles to various semiochemicals was studied in both the laboratory and field. In laboratory experiments glass two-choice pitfall bioassay dishes were used. Mixed-sex populations of beetles responded positively and significantly to cucujolide I and II separately and in combination compared to controls. Bioassays using the two aggregation pheromones of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). Dominicalure 1 and 2 together elicited unexpected significant attractive responses by C. ferrugineus as did assays with the synthetic pheromones from other species. Bioassays showed that C. ferrugineus were not attracted to either corn meal or rolled oats, but they were attracted to commercial grain-based lures and also to ethanol, a natural fermentation product from grains. Several bioassays were conducted to test the attractiveness of naturally produced beetle volatiles to C. ferrugineus, but these showed no orientation to volatiles from either the beetles or their food only. Field tests were conducted in Kansas to test attractiveness of the synthetic pheromones cucujolide I and II in lures together with wheat versus wheat only as a control using Lindgren funnel traps. Field tests in 2014 with cucujolide II pheromone lures showed a significant response by feral C. ferrugineus when tested against wheat. Other field tests to examine dispersal of C. ferrugineus relative to grain storages showed that more C. ferrugineus were caught on the western side of grain bins compared to other cardinal directions at two different locations. Results from field and laboratory studies suggest that semiochemical-based tools can be developed to study dispersal behavior in field populations of C. ferrugineus.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Subbaratnam Muthukrishnan / The exoskeleton of insects, the cuticle, functions as a support structure and a physical barrier that protects insects from mechanical damage and dehydration. The exoskeleton is mainly made of chitin and proteins, some cross-linked to one another into certain patterns to form the rigid and resistant cuticle. In previous studies from our laboratory, cuticular proteins analogous to peritrophins (CPAPs) and Knickkopf (Knk) were identified and characterized mainly at the pharate adult stage during insect development. However, the dynamic assembly of both CPAP and Knk into the cuticle and the functions of the CPAPs are still not fully understood. Our study is to investigate how these cuticular proteins are assembled into the cuticle during different developmental stages and carry out their functional characterizations in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) experiments that resulted in down-regulation of transcripts for CPAP 1-C, CPAP1-H, CPAP 1-J, CPAP 3-C and Knk genes resulted in molting defects. Confocal and transmission electron microscopic analysis examined protein expression at twelve stages of development, as well as the span from young larva through adult day 3 stages. The results suggested that the CPAP 3-C protein is present in the lower part of endocuticle in the so-called assembly zone and it was not distributed thoughout the procuticle with chitin. Down-regulation of CPAP 3-C transcripts revealed a disorganized assembly zone; however, no loss of chitin content or the laminar architecture of the procuticle was found. Knk protein was present throughout the procuticle and some of the protein was found inside of the epithelial cells.

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 / Biochemistry / Michael R. Kanost / Insect innate immune responses include prophenoloxidase activation and antimicrobial peptide production. These responses involve extracellular serine proteinase cascades that are regulated by serpins. This work involved the study of serine proteinase 16 (HP16) and serpin-3 from hemolymph of the tobacco hornworm, Manduca sexta. HP16 has an amino-terminal domain with no similarity to any characterized protein and a carboxyl-terminal S1 family serine proteinase domain. HP16 levels in plasma were highest during the wandering, prepupal, and pupal stages. HP16 mRNA levels in fat body were highest at the wandering stage. Injection of bacteria into fifth instar larvae stimulated HP16 expression. To further characterize and investigate the biological function of HP16, recombinant proteins for proHP16, two HP16 mutants, the amino-terminal domain (NT16), and three NT16 mutants were purified. Recombinant HP16 was cleaved at the predicted activation site during expression, and its amino-terminal and catalytic domains remained connected by a disulfide bond. ProHP16 in plasma was apparently activated in the presence of the microbial elicitor, zymosan. Recombinant HP16 formed a complex with serpin-1Z, indicating that it was catalytically active, but no other natural or artificial substrates were identified. Analysis of NT16 and NT16 mutants led to the discovery that multiple disulfide bond arrangements were formed in the recombinant amino-terminal domain of HP16. This work furthered the understanding of HP16 and laid a foundation for subsequent experiments involving the proteolytic activity, regulation, and biological function of HP16. Active serine proteinases in insect hemolymph are often regulated by serpins. Immunoaffinity chromatography was used to identify plasma proteinases that are inhibited by serpin-3. Four serpin-3-proteinase complexes purified from plasma were identified by immunoblot analysis as serpin-3 complexes with HP8, PAP-1, PAP-2, and PAP-3. MALDI-TOF/TOF or ESI-MS/MS analysis after separation by 1D- or 2D-PAGE confirmed serpin-3 complex formation with HP8, PAP-1, and PAP-3. ProHP8 in plasma was activated by exposure to the β-1,3-glucan curdlan and inhibited by serpin-3. Purified recombinant serpin-3 and active HP8-Xa formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.

Master of Science / Department of Entomology / Raymond A. Cloyd / Western flower thrips (WFT), Frankliniella occidentalis, is one of the most destructive insect pests of greenhouse production systems because of the direct and indirect damage they cause and their ability to develop resistance to a wide variety of insecticides. A common method of avoiding resistance development is by rotating insecticides that have different modes of action. Entomopathogenic organisms (EPO), such as fungi and bacteria, have modes of action that are very different from standard insecticides. Thus, by incorporating EPO into rotation programs, greenhouse producers may preserve the efficacy of insecticides used for suppression of WFT populations. Therefore, the objectives of this study were to 1) determine the efficacy of entomopathogenic fungi on WFT adults and nymphs, and to assess product effectiveness when used beyond the expiration date; 2) evaluate the efficacy of entomopathogenic fungi against WFT nymphs when combined with the insect growth regulator, azadirachtin; and 3) evaluate different rotation programs that include EPO and standard insecticides commonly used to suppress WFT populations. To satisfy objective one and two, a series of laboratory bioassays were conducted in which WFT nymphs and adults were exposed to three entomopathogenic fungi (Beauveria bassiana, Isaria fumosoroseus, and Metarhizium anisopliae) at two label rates (maximum and minimum), and two product conditions (fresh and expired). Furthermore, a bioassay in which each entomopathogenic fungi was tested with and without azadirachtin was conducted to determine if there was any synergistic effect on WFT nymphal mortality. Results indicated that adults are generally more susceptible to infection by entomopathogenic fungi than nymphs, fresh products resulted in higher mortality than expired products, and azadirachtin, when mixed with the entomopathogenic fungi, did not increase mortality of WFT nymphs except when combined with M. ansiopliae. Insecticide rotation programs that included EPO were evaluated by conducting a series of greenhouse experiments in which chrysanthemum, Dendranthema x morifolium plants were artificially infested with WFT adults. Eight-week rotation programs were applied to each plant and weekly counts of adults captured on yellow sticky cards were recorded. A final quality assessment of damage due to WFT feeding on foliage and flowers (1 to 5 in which 1 = no damage, and 5 = greater than 75% damage) was also recorded. In addition, a cost comparison of each rotation program was determined. Generally, insecticide rotations programs which incorporated EPO resulted in no significant difference in WFT populations compared to standard insecticide rotation programs without EPO. Furthermore, there were no significant differences between any of the rotation programs in regards to foliage and flower quality. Based on the results of the cost comparison, there may be a cost savings associated with using EPO. Therefore, by incorporating EPO into insecticide rotation programs, greenhouse producers may reduce costs as well as reduce selection pressure on WFT populations, which may avoid or delay resistance development.

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.