No description available.

Typescript, etc. / Digitized by Kansas State University Libraries

Call number: LD2668 .T4 1986 E42 / Master of Science / Entomology

Doctor of Philosophy / Department of Entomology / R. Jeff Whitworth / The Hessian fly, Mayetiola destructor (Say), has been a historically significant pest of wheat in Kansas. However, it has been 60+ years since research has been conducted examining the flies’ activity throughout the year. Results of pheromone trapping in 4 counties in Kansas shows that Hessian fly (HF) males are actively flying in the fall, at least 1 month after the historical fly-free dates. Therefore, the Hessian Fly-Free Date is no longer valid and should be referred to as the Best Pest Management Date. Using pheromones for fall and spring trapping also indicated that HF is more active throughout the spring than previously thought, with almost continuous fly emergence and numerous emergence peaks in both spring and fall. The use of resistant wheat cultivars has been adapted to protect seedling plants from HF larval feeding in the fall. However, it is unknown if these cultivars are still providing protection after winter vernalization. Greenhouse trials indicated that ‘Armour’, a cultivar considered intermediately resistant, remains resistant under infestation levels of 1 fly/tiller but significant seed weight losses occured under infestations of 3 flies/tiller. In the field, Armour did not provide protection post-vernalization, with plants containing similar numbers of flaxseeds (pupae) as the susceptible cultivar, ‘Fuller’, and having significant losses of culm height (cm), number of spikelets/spike, number of seeds/spike, and seed weight (grams) when infested. ‘Duster’, a cultivar considered highly resistant, appeared to provide resistance to HF larval feeding in both the greenhouse and the field, and even produced significantly heavier seeds when infested with 3 flies/tiller in the greenhouse. These results suggest that post-vernalization screening should be conducted on all HF resistant cultivars to determine if each continues to provide protection. Little information is available showing if and how HF larval feeding on more mature wheat (Feekes 7-10), post-vernalization, impact plants, aside from lodging. Greenhouse and field infestations of a susceptible cultivar, Fuller, showed that significant losses of culm height (cm), number of seeds/spikelet, and seed weight will result from as few as 1 larva /culm. Yield losses averaged 0.13g/spike (65 kg/ha) compared to non-infested plants.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / John C. Reese / Keeping in view the important roles of bacteria in almost every aspect of insect’s life, the current study is the first systemic and intensive work on microbes associated with Hessian fly, a serious pest of wheat crop. A whole body analysis of Hessian fly larvae, pupae, or adults suggested that a remarkable diversity of bacteria is associated with different stages of the insect life cycle. The overriding detection of genera Acinetobacter and Enterobacter throughout the life cycle of Hessian fly suggested a stable and intimate relationship with the insect host. Adult Hessian flies have the most dissimilar bacterial composition from other stages with Bacillus as the most dominant genus. Analysis of 5778 high quality sequence reads obtained from larval gut estimated 187, 142, and 262 operational taxonomic units at 3% distance level from the 1st, 2nd, and 3rd instar respectively. Pseudomonas was the most dominant genus found in the gut of all three instars. The 3rd instar larval gut had the most diverse bacterial composition including genera Stenotrophomonas, Pantoea, Enterobacter, Ensifer, and Achromobacter. The transovarial transmission of major bacterial groups provided evidence of their intimate relationship with the Hessian fly. The Hessian fly is known to manipulate wheat plants to its own advantage. This study demonstrated that the combination of a decrease in carbon compounds and an increase in nitrogen compounds in the feeding tissues of Hessian fly-infested plants results in a C/N ratio of 17:1, nearly 2.5 times less than the C/N ratio (42:1) observed in control plants. We propose that bacteria associated with Hessian fly perform nitrogen fixation in the infested wheat, which was responsible for shifting the C/N ratio. The following findings made in the current study i.e. the presence of bacteria encoding nitrogenase (nifH) genes both in Hessian fly and infested wheat, exclusive expression of nifH in infested wheat, presence of diverse bacteria (including the nitrogen fixing genera) in the Hessian fly larvae, presence of similar bacterial microbiota in Hessian fly larvae and at the feeding site tissues in the infested wheat, and reduction in survival of Hessian fly larvae due to loss of bacteria are consistent with this hypothesis. The reduction in Hessian fly longevity after the loss of Alphaproteobacteria in first instar larvae, highest proportion of Alphaproteobacteria in insects surviving after the antibiotic treatments and the nitrogen fixation ability of associated Alphaproteobacteria strongly implies that Alphaproteobacteria are critical for the survival of Hessian fly larvae. This study provides a foundation for future studies to elucidate the role of associated microbes on Hessian fly virulence and biology. A better understanding of Hessian fly-microbe interactions may lead to new strategies to control this pest.

Doctor of Philosophy / Department of Plant Pathology / Bikram S. Gill / H13, a dominant resistance gene transferred from Aegilops tauschii into wheat (Triticum aestivum), confers a high level of antibiosis against a wide range of Hessian fly (HF, Mayetiola destructor) biotypes. Previously, H13 was mapped to the distal arm of chromosome 6DS, where it is flanked by markers Xcfd132 and Xgdm36. A mapping population of 1,368 F2 individuals derived from the cross: PI372129 (h13h13) / PI562619 (Molly, H13H13) was genotyped and H13 was flanked by Xcfd132 at 0.4cM and by Xgdm36 at 1.8cM. Screening of BAC-based physical maps of chromosome 6D of Chinese Spring wheat and Ae. tauschii coupled with high resolution genetic and Radiation Hybrid mapping identified nine candidate genes co-segregating with H13. Candidate gene validation was done on an EMS-mutagenized TILLING population of 2,296 M₃ lines in Molly. Twenty seeds per line were screened for susceptibility to the H13-virulent HF GP biotype. Sequencing of candidate genes from twenty-eight independent susceptible mutants identified three nonsense, and 24 missense mutants for CNL-1 whereas only silent and intronic mutations were found in other candidate genes. 5’ and 3’ RACE was performed to identify gene structure and CDS of CNL-1 from Molly (H13H13) and Newton (h13h13). Increased transcript levels were observed for H13 gene during incompatible interactions at larval feeding stages of GP biotype. The predicted coding sequence of H13 gene is 3,192 bp consisting of two exons with 618 bp 5’UTR and 2,260 bp 3’UTR. It translates into a protein of 1063 amino acids with an N-terminal Coiled-Coil (CC), a central Nucleotide-Binding adapter shared by APAF-1, plant R and CED-4 (NB-ARC) and a C-terminal Leucine-Rich Repeat (LRR) domain. Conserved domain analysis revealed shared domains in Molly and Newton, except for differences in sequence, organization and number of LRR repeat in Newton. Also, the presence of a transposable element towards the C terminal of h13 was indicative of interallelic recombination, recent tandem duplications and gene conversions in the CNL rich region near H13 locus. Comparative analysis of candidate genes in the H13 region indicated that gene duplications in CNL encoding genes during divergence of wheat and barley led to clustering and diversity. This diversity among CNL genes may have a role in defining differences in the recognition specificities of NB-LRR encoding genes. Allele mining for the H13 gene in the core collection of Ae. tauschii and hexaploid wheat cultivars identified different functional haplotypes. Screening of these haplotypes using different HF biotypes would help in the identification of the new sources of resistance to control evolving biotypes of HF. Cloning of H13 will provide perfect markers to breeders for HF resistance breeding programs. It will also provide an opportunity to study R-Avr interactions in the hitherto unexplored field of insect-host interaction.

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.

Doctor of Philosophy / Genetics Interdepartmental Program / Jesse A. Poland / Wheat is perhaps the most important component in human diet introduced since the conception of modern agriculture, which provides about 20% of the daily protein and calorie intake to billions of people. Adaptable to wide range of climates, wheat is grown worldwide, lending it the potential to mitigate the imminent risk of food security for future population of 9.5 billion people. For developing improved crop varieties in the future, genetic diversity is a key factor in plant breeding. Constraints in wheat evolution and artificial selection practices have resulted in erosion of this ingredient in elite germplasm. However, wheat wild relatives, such as Ae. tauschii, D-genome donor of wheat, are a storehouse for unexploited genetic diversity that can be used for improving wheat for disease and insect resistance, yield, quality, and tolerance to abiotic stresses. More than 1700 genebanks around the world hold over 7 million accessions of these wild relatives. These genebanks are expensive to maintain, therefore, efficient curation is necessary. We developed and implemented a protocol to identify duplicate accessions using genomic tools. Implementing this approach with three genebanks, we identified over 50% duplicated accessions across genebanks. There are over a million Triticeae accessions held collectively, and it is likely as more number of genebanks are tested, there will be decreasing number of unique accessions. Selecting and utilizing the wild genetic diversity is no easy task. Historically, breeders and geneticists have chosen the accessions primarily based on associated phenotypic data. Unless focusing on a targeted trait, this practice is imperfect in capturing the genetic diversity with some other limitations, such as confounding phenotypic data with the testing environment. Utilizing next-generation sequencing methods, we selected a MiniCore consisting of only 40 accessions out of 574 capturing more than 95% of the allelic diversity. This MiniCore will facilitate the use of genetic diversity present in Ae. tauschii for wheat improvement including resistance to leaf rust, stem rust, Hessian fly, and tolerance to abiotic stresses. Hessian fly is an important insect pest of wheat worldwide. Out of 34 known resistance genes, only six have been mapped on the D sub-genome. With swift HF evolution, we need to rapidly map and deploy the resistance genes. Some of the undefeated HF resistance genes, such as H26 and H32, were introgressed from Ae. tauschii. In this study, we mapped three previously known genes, and a new gene from Ae. tauschii accession KU2147. Genes were mapped on chromosomes 6B, 3D, and 6D. Further, identification and cloning of resistance genes will enhance our understanding about its function and mode of action. In conclusion, wild wheat relatives are genetically diverse species, and utilizing the novel genetic diversity in Ae. tauschii will be fruitful for wheat improvement in the wake of climate change to ensure future food security to expected 2 billion newcomers by 2050.

Doctor of Philosophy / Department of Entomology / Brian P. McCornack / Timely enactment of insect pest management and incursion mitigation protocols requires development of time-sensitive monitoring approaches. Numerous passive monitoring methods exist (e.g., insect traps), which offer an efficient solution to monitoring for pests across large geographic regions. However, given the number of different monitoring tools, from specific (e.g., pheromone lures) to general (e.g., sticky cards), there is a need to develop protocols for deploying methods to effectively and efficiently monitor for a multitude of potential pests. The non-random movement of the Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), toward several visual, chemical, and tactile cues, makes it a suitable study organism to examine new sensor technologies and deployment strategies that can be tailored for monitoring specific pests. Therefore, the objective was to understand Hessian fly behavior toward new sensor technologies (i.e., light emitting diodes (LEDs) and laser displays) to develop monitoring and deployment strategies. A series of laboratory experiments and trials were conducted to understand how the Hessian fly reacts to the technologies and how environmental factors may affect the insect’s response. Hessian fly pupae distribution within commercial wheat fields was also analyzed to determine deployment of monitoring strategies. Laboratory experiments demonstrated Hessian fly attraction to green spectrum (502 and 525 nm) light (LEDs), that response increased with light intensity (16 W/m2), and that they responded in the presence of wheat odor and the Hessian fly female sex-pheromone, but, response was reduced under ambient light. These laboratory experiments can be used to build a more targeted approach for Hessian fly monitoring by utilizing the appropriate light wavelength and intensity with pheromone and wheat odor to attract both sexes, and mitigating exposure to ambient light. Together this information suggested that light could be used with natural cues to increase attraction. Therefore, a light source (green laser display) was applied to a wheat microcosm, which resulted in greater oviposition in wheat covered by the laser display. Examination of Hessian fly pupal distribution within commercial wheat fields showed that proportion of wheat within a 1 km buffer of the field affected distribution between fields. This helps to inform deployment of monitoring strategies as it identified fields with a lower proportion of wheat within a 1 km buffer to be at higher risk Hessian fly infestation, and therefore monitoring efforts should be focused on those fields. Together this work demonstrates Hessian fly behavior toward new sensor technologies, how those technologies interact with environmental cues, and how environmental composition affects pupal distribution. Collectively this information will enable cheaper, more accurate and more efficient monitoring of this destructive pest.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / Gerald E. Wilde / Analysis of transcriptomes from salivary glands and midgut of the Hessian fly [Mayetiola destructor (Say)] identified a diverse set of cDNAs that were categorized into five groups, group I – V, based on their phylogenetic relationship. All five of these groups may encode putative protease inhibitors based on structural similarity with known proteins. The sequences of these putative proteins among different groups are highly diversified. However, sequence identity and structural analysis of the proteins revealed that all of them contained high cysteine residues that were completely conserved at their respective positions among these otherwise diversified proteins. Analysis of bacterial artificial chromosome (BAC) DNA for two groups, group I (11A6) and group II (14A4), indicated that group I might be a single copy gene or genes with low copy number whereas group II exists as multiple copies clustered within the Hessian fly genome. To test the inhibitory activity and specificity of these putative proteins, recombinant proteins were generated. Enzymatic analysis of the recombinant proteins against commercial and insect gut proteases demonstrated that recombinant proteins indeed are strong inhibitors of proteases with different specificities. Northern analysis of the representative members of five groups revealed that the group I-IV genes were expressed exclusively in the larval stage with variations among groups at different larval stages. The group V (11C4) genes were expressed in the late larval and pupal stage. Tissue specific gene expression analysis revealed that group I-IV genes were predominantly expressed in malpighian tubules whereas the group V genes were abundantly expressed in the salivary glands. Localization experiments with the antibody for representative members from group II (14A4) demonstrated that the protein was predominantly localized in the malpighian tubules and in low amounts in the midgut, suggesting that malpighian tubules are the primary tissue of 14A4 inhibitor synthesis. The overall results indicated that the Hessian fly contains a complex network of genes that code for protease inhibitors which regulate protease activities through different developmental stages of the insect.