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.

Master of Science / Department of Entomology / Marcelo Ramalho-Ortigao / Sand flies (Diptera:Psychodidae) are vectors of parasites of the genus Leishmania transmitted to suitable vertebrate host during blood feeding. For blood feeding arthropods, including sand flies, blood meal digestion requires the secretion of inhibitory molecules, such as Kazal-type serine proteinase inhibitors that are involved in preventing the blood from coagulating within the mouthparts and the midgut. Previous studies have identified such molecules in mosquitoes, ticks, and triatomine bugs. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). We are interested in the role of these proteins as inhibitors of coagulation cascades, in addition to their potential effects on blood digestion in P. papatasi. Ppkzl1 is similar to thrombin and trypsin inhibitors in triatomines and mosquitoes and Ppkzl2 is similar to Kazal-type inhibitors in mosquitoes with unknown function. Analyses of expression profiles indicated that although both transcripts are expressed prior to blood feeding in the midgut of P. papatasi they are tightly regulated by the blood meal. Reverse genetics studies using RNAi-targeted knockdown of PpKzl1 and PpKzl2 by dsRNA injection did not result in a detectable effect on mRNA expression levels. Thus, we expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess activity against various serine proteinases. Recombinant PpKzl2 inhibited chymotrypsin at nanomolar levels and also inhibited thrombin and trypsin at micromolar levels, suggesting that native PpKzl2 is an active serine proteinase inhibitor and may regulate digestive enzymes and thrombin in the midgut. Leishmania development within the sand fly midgut is faced with several barriers that can severely impact the parasites. For transmission to occur, parasites must be able to overcome these barriers including digestive proteinases, escape from the peritrophic matrix, and midgut attachment. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sand fly midgut. Thus, targeting serine proteinase inhibitors may provide a new strategy to prevent transmission of Leishmania.

Master of Science / Department of Entomology / Thomas W. Phillips / Resistance of stored-grain insects to the fumigant gas phosphine is becoming common, due to genetic-based resistance. Using proper resistance management, phosphine can continue to be effective with efficient monitoring methods. This thesis focuses on assessing the levels of phosphine resistance across North America in a common stored-product pest, the red flour beetle, Tribolium castaneum (Herbst), and on refining a rapid bioassay method so that resistance can be easily and accurately detected. Previous work found that phosphine resistance was present in two regions of the USA, the Southeast and Midwest. In this study a discriminating-dose bioassay was used with adult beetles to determine the number of resistant and susceptible individuals in a test population. Adult T. castaneum from 25 locations across the United States and Canada were collected, and 25-60 adults from each population were assessed for the percentage of resistant individuals. Thirteen populations were deemed susceptible with no resistant insects in the samples, while eight populations had 52% or fewer individuals scored as resistant, and four populations had between 88 and 100% resistant individuals. Dose-mortality experiments were conducted to characterize the “strength” of resistance. One population with 41% resistant beetles in the discriminating dose assay was 4.5-fold resistant relative to the susceptible laboratory strain, compared to 127-fold resistant for a population with all 100% scored as resistant. Adult beetles from twelve populations were used to determine if a “knockdown” test of insects exposed to a high concentration of phosphine (3000 ppm) could assess resistance as well as the discriminating-dose mortality assay. The time required for five out of ten beetles to be knocked down, called the KT[subscript]50 for the time to knockdown 50% of the sample, was useful to characterize resistance. Phosphine susceptible beetles had KT[subscript]50 values less than 15 minutes, while samples from resistant populations had KT[subscript]50’s between 15 and 52 minutes. The refined quick test and knowledge of current levels of phosphine resistance in the United States and Canada reported in this thesis point to the importance of such information in developing phosphine resistance management programs for grain insects.

Master of Science / Entomology / Ming-Shun Chen / Robert "Jeff" J. Whitworth / MicroRNA (miRNA) plays a role in nearly all the biological pathways and therefore may provide opportunities to develop new means to combat the Hessian fly, Mayetiola destructor, a destructive pest of wheat. This study presents a comprehensive analysis of miRNA species via deep-sequencing samples from Hessian fly second instar larvae, pupae and adults. A total of 921 unique miRNA species were identified from approximately 30 million sequence reads. Among the 921 miRNA species, only 22 were conserved among Hessian fly and other insect species, and 242 miRNA species were unique to Hessian fly, the remaining 657 share certain sequence similarities with pre-miRNA genes identified from various insect species. The abundance of the 921 miRNA species based on sequence reads varies greatly among the three analyzed stages, with 20 exclusively expressed in adults, two exclusively expressed in pupae and two exclusively expressed in second instar larvae. For miRNA species expressed in all stages, 722 were with reads lower than 10. The abundance of the remaining 199 miRNA species varied from zero to more than eight-fold differences among different stages. Putative miRNA-encoding genes were analyzed for each miRNA species. A single putative gene was identified for 594 miRNA species. Two putative genes were identified for 138 miRNA species. Three or more putative genes were identified for 86 miRNA species. The three largest families had 14, 23 and 34 putative coding genes, respectively. No gene was identified for the remaining 103 miRNA species. In addition, 1516 putative target genes were identified for 490 miRNA species based on known criteria for miRNA targets. The putative target genes are involved in a wide range of processes from nutrient metabolism to encoding effector proteins. Analysis of the expression patterns of miRNA and pre-miRNA for the miRNA family PC-5p-67443, which contains 91 genes, revealed that miRNA and pre-miRNA were expressed differently in different developmental stages, suggesting that different isogenes are regulated by different mechanisms, or pre-miRNAs had other functions in addition to as an intermediate for miRNA biogenesis. The large set of miRNA species identified here provides a foundation for future research on miRNA functions in Hessian fly and for comparative studies in other species. The differential expression patterns between a pre-miRNA and its encoded mature miRNA in a multigene family is an initial step toward understanding the functional significance of isogenes in dramatically expanded miRNA families.

Master of Science / Entomology / C. Michael Smith / The bird cherry oat aphid, Rhopalosiphum padi, and one of the viruses it vectors, barley yellow dwarf virus (BYDV), form a very damaging pest unit on cereals. Understanding how people can better manage crops to prevent damage or recognize environmental or geographic factors that put their crops at risk for BYDV could lead to improved virus aphid management strategies. One of the most successful methods for mitigating pest damage is using pest-resistant varieties of crops. Seven candidate wheat varieties were screened for resistance to R. padi, by testing aphid population densities and aphid host choice. Results of this research identified six varieties of wheat that show resistance to R. padi. One of the major knowledge gaps in BYDV management is forecasting potential damage. Our objective was to create viral presence maps, to start building the foundation of correlations between persistent and changing frequency of viruliferous R. padi. The results of BYDV assays in R. padi in wheat fields across Kansas indicated that the amount of BYDV infection in viruliferous aphids changes rapidly from year to year, and differs considerably between geographic regions and field landscape characteristics in Kansas. Neonicotinoid wheat seed treatment is a management technique that reduces R. padi populations. However, a common question among producers is whether or not seed treatments stop viral transmission. Results of greenhouse seed treatment experiments with plants from neonicotinoid treated and untreated seed infested with viruliferous R. padi indicated no significant difference in viral transmission due to seed treatment. This new information about R. padi wheat varietal resistance, geographic distribution of BYDV in Kansas, and neonicotinoid seed treatment creates a better understanding of aphid-virus-wheat interactions. Results from this thesis directly enhance producer ability to forecast risk from BYDV, select wheat varieties that effectively reduce R. padi as a BYDV vector, and to make better decisions about the use of insecticide to reduce BYDV infection.

Master of Science / Entomology / Ludek Zurek / House flies (Musca domestica L.) and stable flies (Stomoxys calcitrans L.) are insects of medical and veterinary importance. House flies are recognized as mechanical vectors of human foodborne pathogens and stable files are known for their painful bites resulting in reduction of body weight gain and milk production in cattle. The larval development of both fly species takes place in decaying organic materials (primarily animal manure), resulting in large fly populations in confined cattle environments. Shiga toxin-producing Escherichia coli (STEC) are a major foodborne pathogen. Cattle are the asymptomatic reservoir of STEC with bacteria being released to the environment via their feces. STEC O157 is the main serogroup causing human illness. However, infections with non-O157 STEC are increasing: more than 70% of non-O157 infections are caused by six serogroups of non-O157, referred as "Big six" (O26, O45, O103, O111, O121, and O145). In addition, there was a large 2011 outbreak in Europe caused by STEC O104. The objectives of my thesis were: 1) To assess the prevalence of seven serogroups of non-O157 STEC (O26, O45, O103, O104, O111, O121, and O145) (STEC-7) in house flies and stable flies collected from confined cattle environments; 2) To investigate the vector competence of house flies for non-O157 STEC-7. A total of 463 house flies from feedlots and dairies from six states, and 180 stable flies collected from a feedlot in Nebraska were processed for the isolation and identification of STEC-7 using a culture-based approach followed by PCR for the confirmation of serogroups, and virulence genes. A total of 34.3% of house flies and 1.1% of stable flies tested positive for at least one serogroup of E. coli of interest, and 1.5% of house flies harbored STEC with the Shiga-toxin gene (stx1). No STEC were detected in stable flies. Vector competence bioassays for non-O157 STEC revealed that house flies can carry non-O157 STEC for at least six days with the exception STEC O145. Overall, the findings of this research demonstrate that house flies, but not stable flies, likely play an important role in the ecology and transmission of non-O157 STEC in confined cattle environments.