Master of Science / Department of Entomology / Yoonseong Park / Neuropeptides are diffusible signal molecules mediating vital physiological processes. We have been interested in a group of neuropeptides and their receptors involved in osmoregulatory neuroendocrine system which has been suggested as a possible target for development of new biopesticides. Since the genome sequence of the T. castaneum has recently been completed, we were able to identify the respective genes encoding three peptide hormones from T. castaneum that were characterized for their diuretic activities in other insects: one calcitonin-like (CT-like DH31) and two corticotropin releasing factor-like (CRF-like DH37 and DH47, the numbers indicates the number of amino acid residues). This peptide is expressed at all developmental stages and in the central nervous system (CNS), Malpighian tubules (MT) and gut. The synthetic peptide TricaDH31 also has been show to be biologically active, inducing significant excretions in adults beetles. When Tcdh31 was silenced using RNAi, adults had deformed wings and abnormal body shape. Mortality in adults was high, the number of eggs laid was reduced as well as the hatchability of the eggs. The two biologically active CRF-like peptides in T. castaneum, are encoded by one gene which undergoes alternative splicing. When Tcdh47 was knocked down, high mortality occurred as well as low oviposition and egg hatchability. Similar effects were observed with silencing of both CRF-like genes. However, RNAi of Tcdh37 transcripts had similar, but less severe effects. Adults also had deformed wings when both CRF-like genes were silenced, but not when just one of them was knocked down. These results indicate that CRF-like genes could have additional biological functions to their roles in dieresis. We tested the in vivo activity of these peptides. TenmoDH47 induced high excretions in adults, whereas TenmoDH37 induces smaller excretions. We identified the respective genes encoding two putative receptors for TricaDH31 as Glean_13321 and Glean_02694 (Trica-ctr1 and Trica-ctr2, respectively) and two receptors for CRF-like peptide as Glean_12799 and Glean_07104 (Trica-crfr1 and Trica-crfr2, respectively). The CT-like receptors are expressed at all developmental stages, in the CNS and MT. RNAi of the receptors revealed that only Trica-ctr2 silencing caused significant mortality and reduction in the number of eggs laid. The CRF-like receptors are expressed at all developmental stages. Adults also had deformed wings and laid fewer eggs after RNAi of Trica-crfr1. RNAi of Trica-crf2 also caused significant mortality. These peptides and receptors seem to fine tune the beetle physiology and may have functions not yet known.

Doctor of Philosophy / Genetics Interdepartmental Program / James C. Nelson / QTL (quantitative-trait locus) analysis aims to locate and estimate the effects of genes that are responsible for quantitative traits, by means of statistical methods that evaluate the association of genetic variation with trait (phenotypic) variation. Quantitative traits are typically controlled by multiple genes with varying degrees of influence on the phenotype. I describe a new QTL analysis method based on shrinkage and a unifying framework based on the generalized linear model for non-normal data. I develop their extensions to multiple-trait QTL analysis. Expression QTL, or eQTL, analysis is QTL analysis applied to gene expression data to reveal the eQTLs controlling transcript-abundance variation, with the goal of elucidating gene regulatory networks. For exploiting eQTL data, I develop a novel extension of the graphical Gaussian model that produces an undirected graph of a gene regulatory network. To reduce the dimensionality, the extension constructs networks one cluster at a time. However, because Fuzzy-K, the clustering method of choice, relies on subjective visual cutoffs for cluster membership, I develop a bootstrap method to overcome this disadvantage. Finally, I describe QGene, an extensible QTL- and eQTL-analysis software platform written in Java and used for implementation of all analyses.

Master of Science / Genetics Interdepartmental Program / Bikram S. Gill / TILLING (Targeting Induced Local Lesions IN Genomes) is a high-throughput, reverse genetics strategy for scanning mutagenized populations for point mutations in loci of interest. Originally, TILLING was used to investigate gene function in Arabidopsis and has since been similarly applied for gene functional analysis in other organisms. TILLING also allows the generation of novel genetic variation in specific genotypes and, thus, has been implemented as a tool for crop improvement. Ethyl methanesulfonate (EMS) is a widely used mutagen to induce point mutations in most TILLING protocols. M1 plants are then self-pollinated and M2 seed harvested. A single seed is grown from each M2 progeny and tissue taken for DNA isolation. M3 seed is cataloged. DNA is pooled to increase the efficiency and aid in mutation detection. Polymerase chain reaction (PCR) is used to amplify a locus of interest using the M2 DNA pools as a template. The PCR products are digested with an endonuclease that cleaves mismatched, mutant DNA, and the digested products are visualized. The pools for which PCR products are positive for a mutation are deconvoluted to determine which individual plant of the pool was responsible for the mutation. DNA from the positive individual is sequenced to determine the type of mutation (missense, nonsense, synonymous). Individuals with mutations that are more likely to disrupt gene function (nonsense and certain missense) are studied further by growing the corresponding M3 generation. In bread wheat, Triticum aestivum, TILLING is complicated by polyploidy: genes that have homoeologs require that the functionality of each be studied. If functional homoeologs are present for all three genomes, mutants must be identified for each homoeolog, followed by successive intercrossing to produce a triple mutant plant. As a model for wheat genetics, we propose TILLING in diploid wheat. EMS mutant populations were created in diploid wheat (Triticum monococcum ssp. monococcum) and the hexaploid bread wheat cultivar ‘Jagger’. The diploid and hexaploid wheat populations were screened for mutations at the waxy locus, GBSS1, as a validation of our population and for comparative analysis of mutation rates in 2x and 6x wheat. For diploid wheat, GBSSI was screened in 716 M2 plants, and one mutant was found for 1.9 Mb screened. 3 For hexaploid wheat, GBSSI was screened in 518 M2 plants, and 30 mutants were identified within a total of 657 Kb screened, giving a mutation frequency of one mutation per 22 Kb. The reasons for this vast difference in mutation frequency between diploid and hexaploid wheat are discussed. The diploid wheat population was further examined by screening for mutations within four lignin biosynthesis candidate genes, for a total of 2 Mb screened. A single mutant was discovered for both of the lignin genes PAL6 and HCT, giving a mutation frequency of one mutation per 1 Mb screened.

Doctor of Philosophy / Department of Biology / Michael A. Herman / Determining the genetic mechanisms involved in organismal response to environmental change is essential for understanding the effects of anthropogenic disturbance. The composition of the bacterial-feeding nematode community is an excellent biological indicator of disturbance, particularly in grassland ecosystems. We have previously shown that grassland soil nematodes are responsive to perturbations in the field including the addition of nitrogen fertilizer. We are interested in how this perturbation affects the microbial community and downstream effects on the next trophic level, the bacterial-feeding nematodes. To determine the effects of disturbance on soil bacterial communities we used massively parallel sequencing and found that chronic nitrogen addition on tallgrass prairie significantly impacts overall bacterial community diversity and the abundance of specific bacterial taxa. Because native soil nematodes lack well developed genomic tools, we employed Caenorhabditis elegans as a model for native soil nematode taxa and used transcriptional profiling to identify 204 candidate genes regulated in response to altered bacterial diets isolated from grassland soils. To biologically validate our results we used mutations that inactivate 21 of the identified genes and showed that most contribute to fitness or lifespan in a given bacterial environment. Although these bacteria may not be natural C. elegans food sources, this study aimed to show how changes in food source, as can occur in environmental disturbance, has large effects on gene expression and those genes whose expression are affected, contribute to fitness. Furthermore, we identified new functions for genes of unknown function as well as previously well-characterized genes, demonstrating the utility of this approach to further describe C. elegans genome. We also investigated the function of previously well-characterized C. elegans defense pathways in our grassland soil bacterial environments and found that some are environment specific. Additionally, we found that cuticular collagen genes are important for lifespan, and appear to function downstream of known defense pathways. Overall, our results suggest that anthropogenic disturbance in grasslands alters the most basal components of the soil food web, bacteria and bacterial-feeding nematodes through the genes they possess and how they are expressed, and resultant bottom-up effects could have profound consequences on ecosystem health and function.

Doctor of Philosophy / Department of Biology / Susan J. Brown / RNA interference (RNAi) is a natural gene-silencing phenomenon triggered by dsRNA (dsRNA). While RNAi is an endogenous process that plays essential roles in regulating gene expression it can also be harnessed as a tool for the study of gene function. Introducing dsRNA that is homologous to target mRNA into a cell triggers the RNAi response causing the destruction of the homologous mRNA and a loss of function phenotype. In some organisms, such as the nematode Caenorhabditis elegans, once dsRNA is introduced into the body cavity, the RNAi effect is seen throughout the organism because the dsRNA is taken up by individual cells and is then spread from cell to cell. This process has been termed the systemic RNAi response. For other organisms, such as the fruit fly Drosophila melanogaster, introduction of dsRNA into the body cavity does not result in a systemic RNAi response. This may be due to the cell’s inability to take up dsRNA or spread that dsRNA from cell to cell. For other organisms, including mammals, introduction of dsRNA into the body cavity does not result in a systemic RNAi response because the immune response causes dsRNA destruction before it can be utilized in the RNAi pathway. For organisms that do not exhibit a systemic RNAi response, complex genetic methods are needed to introduce dsRNA into cells to induce the RNAi response. Therefore, one of the challenges in utilizing RNAi as a genetic tool is introducing the dsRNA into individual cells. In recent years, systemic RNAi responses have been documented in both model and non-model organisms, making RNAi an accessible genetic tool. The red flour beetle, Tribolium castaneum is an emerging model organism that has a robust systemic RNAi response. However, the mechanism of systemic RNAi and the specific parameters required to obtain a strong systemic RNAi response in this organism have not been thoroughly investigated. The aim of this work is to provide data that can allow RNAi to be better utilized as a genetic tool in Tribolium and to use this information as a basis for the use of RNAi in other insects in which it can be performed. Specifically we provide data on the essential parameters necessary to achieve an effective systemic response in Tribolium, we describe differences in the systemic RNAi response between Drosophila and Tribolium, we analyze the conservation and function of RNAi machinery genes in Tribolium and we provide information on the genes critical for a systemic RNAi response in Tribolium.

Master of Science / Department of Entomology / David C. Margolies / Yoonseong Park / Phytoseiulus persimilis (Acari: Phytoseiidae) is a specialist predator on tetranychid mites, especially on the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). The foraging environment of the predatory mites consists of prey colonies distributed in patches within and among plants. Quantitative genetic studies have shown genetic variation in, and phenotypic correlations among, several foraging behaviors within populations of the predatory mite, P. persimilis. The correlations between patch location, patch residence, consumption and oviposition imply possible fitness trade-offs. We used molecular techniques to investigate genetic variation underlying the foraging behaviors. However, these genetic studies require a sufficiently large amount of DNA which was a limiting factor in our studies. Therefore, we developed a method for obtaining DNA from a single mite by using a chelex extraction followed by whole genome amplification. Whole genome amplification from a single mite provided us with a large quantity of high-quality DNA. We obtained more than a ten thousand-fold amplified DNA from a single mite using 0.01ng as template DNA. Sequence polymorphisms of P. persimilis were analyzed for nuclear DNA Inter Transcribed Spacers (ITS1 & ITS2) and for a mitochondrial 12S rRNA. The sequence comparisons among individuals identified a number of polymorphisms in the 12S sequences. The foraging gene (for) associated with rover-sitter behavioral strategies of Drosophila is known to have role in feeding behaviors of honeybee and other arthropods. We surmised that the same or a similar gene may be present in P. persimilis. Among the foraging behavior(s) exhibited by this predatory mite, we were particularly interested in resource/prey-dependent dispersal behavior. We isolated a partial sequence that is presumed to be the orthologue of the foraging (for) gene. We named the putative foraging gene as Ppfor (for Phytoseiulus persimilis foraging gene). We used a fragment of Ppfor gene as a molecular marker between populations and among individuals and, further, to help understand behavioral phenotypes.

Doctor of Philosophy / Department of Biology / Susan J. Brown / All arthropods share a segmented body plan. Detailed studies on segmentation mechanisms in the long-germ insect Drosophila melanogaster identified a segmentation hierarchy composed of maternal, gap, pair-rule, and segment polarity genes. In this hierarchy, pair-rule genes play an important role to translate gradients of regional information from maternal and gap genes into segmental expression of segment polarity genes. However, our understanding of the role of pair-rule genes in other short-germ insects and basally branching arthropods is still limited. To gain insights into the role of pair-rule genes in short-germ segmentation, I analyzed genetic interactions as well as expression patterns and functions of homologs of Drosophila pair-rule genes in the short-germ insect Tribolium castaneum. Interestingly, despite the pair-rule like expression patterns of Tribolium homologs of almost all eight canonical Drosophila pair-rule genes, only five have a segmentation function. Knock-down of primary pair-rule genes caused asegmental and truncated phenotypes while knock-down of secondary pair-rule genes caused typical pair-rule phenotypes. Epistatic analysis between the genes revealed that primary pair-rule genes form a gene circuit to prepattern a two-segmental unit, and secondary pair-rule genes are downstream targets of the gene circuit. The typical pair-rule phenotypes observed in secondary pair-rule gene RNAi embryos led to a detailed comparative analysis of the role of paired (prd) and sloppy-paired (slp) between Drosophila and Tribolium. This study revealed that prd is functionally conserved while the functional parasegmental register for Tribolium slp is opposite that of Drosophila slp. The fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning. Despite this flexibility in pair-rule patterning between Drosophila and Tribolium, segmental expression of engrailed (en) and wingless (wg) at parasegmental boundaries is conserved in both insects. Analysis of double and triple RNAi for pair-rule genes in Tribolium revealed that the primary pair-rule genes even-skipped and runt are redeployed to directly regulate en and wg with prd or slp at parasegmental boundaries. This redeployment of primary pair-rule genes seem to compensate for the apparently fewer number of functional secondary pair-rule genes in Tribolium segmentation.

Doctor of Philosophy / Department of Plant Pathology / Bikram S. Gill / The wild relatives of crop plants are sources of useful genes, but such genes when transferred to agricultural crops are often associated with deleterious traits. Because most of the recombination and the disease resistance genes are localized towards the ends of wheat chromosomes, cryptic terminal alien segments, carrying rust resistance genes, were transferred from Aegilops geniculata (UgMg) and Ae. triuncialis (UtCt) into common wheat without the usual linkage drag. The alien segment with the leaf rust and stripe rust resistance genes Lr57 and Yr40 in translocation T5DL•5DS-5MgS(0.95) was found to be less than 3.3 cM in genetic length and spans less than four overlapping BAC/PAC clones of the syntenic rice chromosome arm 12L. The alien segment with leaf rust resistance gene Lr58, transferred from Ae. triuncialis, was found to be less than 5% of the chromosome arm 2BL of wheat in T2BS•2BL-2tL(0.95), further suggesting that it is feasible to transfer small alien segments with disease resistance genes. Resistance genes Lr57, Yr40 and Lr58 were transferred to Kansas hard red winter wheat cultivars by backcrossing and marker assisted selection. Tillering, a key component of grain yield, and seed color which influences seed dormancy and pre-harvest sprouting in wheat, are agronomically important domestication traits in wheat. A tiller inhibition mutant with monoculm phenotype was isolated and the mutated gene (tin3) was mapped on the distal region of chromosome arm 3AmL of T. monococcum. As a first step towards isolating candidate gene(s), the tin3 and the seed color gene (R-A1) of chromosome 3A were mapped in relation to physically mapped ESTs and STS markers developed based on synteny with rice. Physically mapped wheat ESTs provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 and R-A1 region. Comparative genomic analysis of the tin3 and R-A1 genomic regions with the corresponding region in rice localized the tin3 gene to a 324 kb region spanned by two overlapping BACs and the R-A1 gene was mapped to a single BAC of the colinear rice chromosome arm 1L.

Master of Science / Department of Entomology / Srinivas Kambhampati / Termites (Isoptera) are eusocial insects and exhibit highly complex eusocial behavior. Eusociality is characterized by the presence of castes (workers, soldiers, reproductives), polyphenisms (same genotype exhibiting multiple phenotypes), flexible developmental pathways, complex communication, cooperative brood care, construction and maintenance of complex nests, and division of labor. Previous studies on honey bees implicated several genes in caste-specific behavior; here, we investigate if orthologs of such genes are present in termites and if so, whether they are expressed differentially among the castes. A candidate gene approach using degenerate primers was used to amplify three candidate genes in the termite Reticulitermes flavipes. Quantitative real time PCR analysis revealed differential expression among termite workers, soldiers, and alates, with a general pattern of higher expression in alates. These results provide information on three novel genes in the termite R. flavipes.

Doctor of Philosophy / Department of Biology / Kimberly A. With / The loss, fragmentation, and degradation of grasslands have resulted in widespread declines in grassland birds. Nest predation is the leading cause of avian reproductive failure; therefore minimizing nest predation can lessen the severity of bird declines. Snakes are important predators of bird nests, but little is known about how snakes may enhance predation risk. To address this issue, I studied the habitat use, movement behavior, population genetic structure, and connectivity of snakes in the grasslands of northeastern Kansas. I addressed the connectivity of eastern yellowbelly racer (Coluber constrictor flaviventris) populations by using a landscape genetics approach at a broad scale (13,500 km2). I also radio-tracked the yellowbelly racer and Great Plains ratsnake (Pantherophis emoryi) at Konza Prairie Biological Station to understand their spatial ecology while simultaneously evaluating nest survival in grassland birds. Individual racers had limited dispersal (<3 km), but substantial admixture occurred within 30 km and populations were in migration-drift equilibrium and had high allelic diversity; therefore, racers must be abundant and continuously distributed for gene flow to be fluid throughout the region. Racers may be more likely to encounter bird nests, as they had more frequent movements and traversed greater distances on average than ratsnakes, which exhibited long periods of inactivity between directed movements. As for grassland birds, nest survival rates decreased with increasing shrubs and decreasing vegetation height. Discriminant function analysis revealed that successful nests were likely to occur in tall vegetation but reduced shrub cover, whereas higher shrub cover characterized snake habitats. Because snakes often use shrubs, nests in areas of increased shrubs may be at higher risk of predation by snakes. Targeted removal of shrubs may increase nest success by minimizing the activity of predators attracted to shrubs. Although predator removal is often a strategy for protecting bird populations, it may not be feasible in this instance, especially since snakes are a native component of the grassland community. Efforts to reduce snake predation on grassland bird nests should therefore focus on managing habitat within grasslands (i.e., shrubs) that influence snake activity, as no natural or anthropogenic habitat barriers currently limit snake movement across the landscape.