Entomology
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Item Using CRISPR/Cas9 to functionally dissect Blimp1, a newly identified pair-rule gene in the hemipteran Oncopeltus fasciatus(2024) Reding, Katie; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genetic screens in the fruit fly Drosophila melanogaster identified a class of mutants displaying half as many segments as seen in wild type fly larvae (Nüsslein-Volhard and Wieschaus 1980). Careful examination of the larval cuticle revealed that one out of every two segments were deleted across the anterior-posterior (AP) axis, an unexpected phenotype suggesting that segmentation in Drosophila follows a ‘pair rule’. Thanks in part to the many genetic tools available for this model species, we now have a clear picture of how the AP axis of the Drosophila embryo is polarized and subsequently divided into distinct segments, and how the pair-rule genes (PRGs) define alternate segments during this process. Since all insects share a similar body plan, it is reasonable to expect that the processes regulating establishment of this body plan would be conserved. However, studies of the Drosophila segmentation gene orthologs in non-model insects suggest that this is not always the case. While the use of model organisms enables an unmatched depth of understanding of the mechanisms underlying development, it comes at the expense of understanding the diversity of these mechanisms across taxa. The milkweed bug Oncopeltus fasciatus (Ofas) (Hemiptera) is a particularly useful insect to study in this regard, as none of the orthologs of the Drosophila PRGs have clear PR-function in this species (Liu and Kaufman 2005b; Auman and Chipman 2018; Reding et al. 2019), while the gene E75A, which has no role in segmentation in Drosophila, is expressed in a PR pattern and its knockdown yields PR segmentation defects (Erezyilmaz et al. 2009). These results suggested that PR-regulation of segmentation in Oncopeltus might require a different set of factors than those discovered in Drosophila. To identify other non-canonical PRGs in this species, I conducted an expression pattern-based screen of transcription factor-encoding genes that are co-expressed with E75A during embryogenesis, and have identified the gene Blimp1 as an Oncopeltus PRG. Like the Drosophila PR mutants, Ofas-Blimp1 mutants display loss of alternate segments across the AP axis. No roles of Blimp1 in insect segmentation had been identified prior to this finding. This result suggests that while insect segmentation may be constrained to follow a pair rule, the genes responsible for regulating PR-segmentation are evolutionarily labile. Further, a major barrier to studying gene function in non-models is the lack of genetic tools such as visible markers and established methods for gene editing. Here I will describe deployment of CRISPR/Cas9 technology in Oncopeltus for targeted mutagenesis. While mutation of the ABC transporter-encoding gene white proved to be recessive lethal, I was able to generate a viable visible marker line by disrupting the X-linked gene Ofas-vermilion (v). Of-v is required for production of dark brown eye pigments, thus Ofas-v mutants have bright red eyes, easily discernible from the black eyes of wild type bugs. I show that a co-CRISPR approach using Of-v as a marker of germline mutation is a helpful strategy to identify mutations of interest at unlinked loci, enabling many future genetic manipulations in this species.Item COMPARATIVE GENOMICS OF CHEMOSENSORY GENE FAMILIES AMONG MEMBERS OF THE HELIOTHINAE(2023) Guo, Rong; Fritz, Megan L; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Insect chemosensory systems play crucial roles in the perception of chemical signals that regulate sexual behaviors and odors that mediate insect-host plant interactions. These processes, mate-finding and acceptance, as well as host plant identification and use, strongly contribute to diversification and speciation among plant-feeding insects, including the Lepidoptera. Chloridea virescens and Chloridea subflexa are an ideal pair species to study the evolution of insect chemosensory systems because they are closely related but show pheromone-based sexual isolation and divergent host plant preferences. My dissertation focuses on the development of genomic tools that enable investigation into genetic mechanisms of host plant and mate recognition, and applies these tools to examine inter- and intraspecific diversity of chemosensory genes among members of the Heliothinae.In chapter 2, I produced a novel Illumina short read C. subflexa genome assembly and an improved, highly contiguous C. virescens genome assembly. Due to quality limitations common to short read assemblies, I used our Heliothine genomes to examine the feasibility of reference-assisted assembly, an approach that leverages existing high quality genomic resources for genome improvement in closely related taxa. My work demonstrated that reference-assisted assembly has the potential to enhance contiguity and completeness of existing insect genomic resources with minimal additional laboratory costs. Both the potential and pitfalls of reference-assisted assembly are discussed in light of my results. In chapter 3, I manually curated two chemosensory gene families, the odorant receptors (ORs) and odorant binding proteins (OBPs), in C. virescens. In total, I identified 80 ORs, 1 Orco and 49 OBPs. Three types of OBPs were identified according to the number and positions of conserved cysteine residues: 34 classic OBPs, 8 Minus-C OBPs, and 7 Plus-C OBPs. In addition, I used phylogenetic analyses to study evolutionary divergence of OR and OBP gene families among Heliothine moths, which revealed both gene duplications and losses. In chapter 4, I studied the strength and nature of selection on the ORs of field-collected C. virescens and C. subflexa, with focus on the pheromone receptor genes. I characterized the host plant use of these species in central Maryland by comparing the larval densities and infestation rates in 2020 and 2021. Sequencing followed by analysis of selection on field-collected samples indicated that the pheromone receptor, OR6, was under very strong purifying selection in both C. virescens and C. subflexa. AMOVA tests suggested that in C. virescens, host plant-associated population differentiation existed in genes OR6, OR55, OR66 and OR78. Further analyses of genetic divergence analysis focused on OR6 showed that the most highly divergent sites were all in introns. The new genomic tools and analyses of chemosensory gene families described here will serve as a platform for future investigations into the genetic mechanisms underlying host plant specialization and sexual communication among lepidopteran insects.Item Quantifying the relative contribution and furthering qualitative understanding of ftz cis-regulatory elements in Drosophila melanogaster(2022) Fischer, Matthew Douglas; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Embryonic development is coordinated by interactions within gene regulatory networks. This process is orchestrated at the level of transcription through the regulatory properties of enhancers, which direct spatiotemporal expression patterns when bound by specific trans-acting factors. Though enhancers can act upon promoters located at great distances irrespective of orientation, the contributions from these cis-regulatory elements (CREs) are limited by insulators and/or tethering elements that organize chromatin architecture. Much research has been conducted towards understanding the coordination of the segmentation genes that pattern the basic body plan of the fruit fly, Drosophila melanogaster, during embryogenesis. The pair-rule genes (PRGs) of this pathway, such as fushi tarazu (ftz), are expressed in seven alternating stripes across the embryo. These PRGs are required for the development of body segments, and the mis-regulation of a single transcriptional domain can result in the loss of a segment. Here, I have investigated the ftz CREs to more precisely determine their sufficiency to direct expression within ftz stripe domains and their necessity for doing so in the native context of the gene. To investigate the sufficiency, I have generated 36 standardized reporter transgenes from 18 CREs, tested in both forward and reverse orientations. All CREs examined have been inserted into the same XbaI site of the reporter plasmid, and the transgenes have been inserted into the same genomic region. Through in situ hybridization experiments, I have determined that the qualitative patterns conferred by every CRE is orientation-dependent, and I have identified two putative insulators and/or tethering elements, proposed to explain this observation. To investigate their necessity, I targeted four genomic regulatory regions for precise deletion using the CRISPR/Cas9 system to generate seven deletion mutants. Though deletions were expected to cause lethality, most of the mutants are homozygous viable and fertile; only a mutant simultaneously removing two seven-stripe CREs was homozygous lethal. Quantitative gene expression analysis by fluorescent in situ hybridization chain reaction revealed that there is a critical threshold of ftz abundance required in each stripe for segmentation to proceed. In conclusion, I have determined that the ftz CREs are redundant and function together in a non-additive manner.Item SEQUENTIAL EXPRESSION OF NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNITS SUPPORTS DEVELOPMENT AND PLASTICITY OF A DROSOPHILA CENTRAL SYNAPSE(2021) Rosenthal, Justin Samuel; Pick, Leslie LP; Yuan, Quan QY; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The central nervous system (CNS) of animals is arguably one of the most sophisticated instruments designed by nature, and one its cardinal components is the postsynaptic specialization. Decades of studies on the cholinergic neuromuscular junction and the central glutamatergic synapses in vertebrate organisms has informed us of just how many factors are at play during postsynaptic development. However, despite its importance, the central cholinergic synapse is one system lacking the same knowledge base as the above models. The thesis work presented here was designed with the aim of understanding if and how nicotinic acetylcholine receptor (nAchR) activity at the postsynapse is used by a developing neuron to shape the structural and functional properties of the synapse and its dendrite arbor during normal periods of maturation. To this end, we employed the ventral lateral neuron (LNv) as a cellular model for Drosophila CNS development. This small group of cells are second-order projection neurons which convey visual activity to higher brain centers and are also critical mediators of adjusting the fly’s internal circadian clock. We report how nAchRs not only play a role in LNv neurophysiology by the end of larval development but show how in fact they actively participate during the formation and refinement of the LNv postsynapse. Our transcriptomic, morphological and physiological approaches reveal that two functionally distinct nAchR subunit genes, Da1 and Da6, are preferentially expressed during separate periods of larval development. Here, young and immature LNvs are characterized by high Da6 expression which facilitates synaptic formation. As the animal grows, Da6 is downregulated and Da1 is upregulated, which is necessary for synaptic stabilization and maturation. We also expand the scope of our primary investigation by identifying promising candidate genes, including transcription factors, molecular chaperones and membrane-associated proteins, that are key to orchestrating the successive stages of nAchR expression, maturation and postsynaptic activity. In summary, our findings will work to clarify in vivo subunit-specific functionalities for the insect nAchR and illustrate how individual nAchR subunits in the CNS are coordinately regulated within a single cell through time to actively regulate distinct properties of the synapse during development.Item COMBINING THE DROSOPHILA AND METARHIZIUM MODEL SYSTEMS TO INVESTIGATE HOST-PATHOGEN INTERACTIONS(2021) Wang, Jonathan; St. Leger, Raymond; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Even closely related individuals vary in their response to infection. In this dissertation, I combined the fruit fly model system with multiple pathogens, including 16 strains of the fungus Metarhizium, to dissect how pathogens with different virulence strategies interact with variable host resistance and tolerance mechanisms. I began by infecting 188 sequenced Drosophila melanogaster lines [the Genetic Reference Panel (DGRP)] with broad host range Metarhizium anisopliae (Ma549) or the bacterium Pseudomonas aeruginosa (Pa14, originally from a human host). Resistance to the two pathogens was correlated (suggestive of general multipurpose defense mechanisms) and associated with oxidative stress sensitivity, starvation resistance, and in particular sleep indices (flies that take a lot of naps are particularly resistant to disease). I followed up by showing that this nonspecific defense extends to the specialist co-evolved Drosophila pathogen, Entomophthora muscae. A genome wide association study implicated several metabolic pathways and physiological processes in individual variation to disease, but not the canonical antifungal Toll immune pathway.Indeed, Metarhizium strains that killed faster induced a stronger and earlier Toll immune response, indicating virulence does not depend on suppressing immunity. Disrupting the Toll pathway component Dif only increased susceptibility to the early diverged broad host range Metarhizium frigidum, whereas flies disrupted in Persephone (a sensor of pathogen proteases) succumbed quickly to all Metarhizium strains. Microarray analysis of mutants revealed a suppressed transcriptomic response to infection when either Persephone or Dif were disrupted, with overlap with GWAS-implicated pathways.