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Movement and spatial costs of resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say), Coleoptera: Chrysomelidae
Kathleen Schnaars / Uvino
Year of Dissertation:
Dr. Robert Rockwell
Abstract Movement and Spatial Costs of Resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say), Coleoptera: Chrysomelidae by Kathleen Schnaars Uvino Advisers: Professor Robert F. Rockwell Professor Mitchell Baker The Colorado potato beetle, Leptinotarsa decemlineata is infamous for its' ability to develop resistance to insecticides and remains the most important insect defoliator of potatoes today. Long Island populations of the Colorado potato beetle have been at the forefront of developing resistance to every newly developed insecticide. Managing the evolution of resistance requires cultural as well as chemical means. Cultural efforts include field rotation, crop rotation, chemical rotation as well as refugia. Movement plays an integral part of both, the cultural schemes intended to thwart resistance evolution and the life history traits of the Colorado potato beetle. The use of refuges and crop rotation are often promoted to supplement the use of chemical pesticides in an effort to control crop pests. Refuges are untreated areas adjacent to treated crops, where susceptible genes can survive. The efficacy of refuges depends on movement between treated and untreated areas. Differences in movement between resistant and susceptible beetles can play a big role in the success of the refuge or rotation plan. Crop rotation can reduce the amount of insecticide used through dosage levels or frequency of application and slows insects' resistance evolution. Resistance to insecticides often has fitness costs associated with that resistance. I hypothesized that resistance to the insecticide Imidacloprid is correlated with reduced movement capability in Colorado potato beetles, Leptinotarsa decemlineata (Say) (henceforth potato beetles), the primary insect defoliator of potato plants. I examined whether migratory ability or flight propensity have a cost of resistance to imidacloprid in Colorado potato beetles, Leptinotarsa decemlineata (Say) by examining LD50's of flying emergers and walking emergers in the spring. Imidacloprid is the most widely used and in some cases the only effective insecticide for Colorado potato beetle control and there is currently a wide range of variation in resistance. In the spring overwintering potato beetle adults halt diapause and emerge from overwintering sites. For the purposes of this work I will use the definition of diapause presented by Tauber et al (1986): "a neurohormonally mediated, dynamic state of minimal activity that occurs during a genetically determined stage(s) of metamorphosis, usually in response to environmental stimuli that precede unfavorable conditions." Diapause in the Colorado potato beetle begins before the harsh conditions set in (loss of host and cold temperatures). It is an important strategy employed by many temperate zone insects for overwintering. Upon emergence from the overwintering site they emigrate to colonize local and distant fields. Emergence from diapause therefore offers an opportunity to sample genetically diverse groups of beetles. My results indicate that emerging flyers have a higher level of resistance than emerging walkers from overwintering sites. I also examined populations that were under intense selection pressure from one chemical, Spinosad, and largely isolated from other fields or populations. Spinosad is produced by a soil dwelling bacterium called Saccharopolyspora spinosa and it kills by ingestion. Spinosad is currently the only approved chemical available to Organic farmers on Long Island. These results indicate complete failure of Spinosad on that population but less resistance on distant populations and less resistance on populations from conventionally managed fields, all in Suffolk County, Long Island. Additionally early spring colonists of rotated and `non-rotated' fields were evaluated for resistance levels for 3 years. For two of the three years, colonists on long distance rotated fields had high LD50. Assuming long distance colonization is more likely dependent on flight, this is consistent with my results that emerging flyers have a higher LD50 than emerging walkers.
RELATIONSHIPS BETWEEN POLLUTANT-INDUCED DIGESTIVE TOXICITY AND THE ASSIMILATION AND SUBCELLULAR PARTITIONING OF ELEMENTS BY GRASS SHRIMP PALAEMONETES PUGIO
Year of Dissertation:
Aquatic invertebrates inhabiting urbanized estuaries are typically exposed to pollutants through multiple pathways, including the diet. Biochemical and physical processes within invertebrate guts can be impacted by ingested pollutants, which may influence the assimilation of essential nutrients as well as pollutants. Pre-assimilatory digestive toxicity may result from pollutants circulating in gut fluid. Post-assimilatory toxicity could be due to incorporation of a pollutant into consumer tissues as a result of chronic exposure. This series of studies investigated the influence of chronic exposure to impacted field conditions or pre-exposure to dietary metal (Cd and Hg) in the laboratory on the assimilation of elements (organic carbon, Cd and Hg), subcellular partitioning of assimilated metal and digestive physiology (gut residence time [GRT], feces elimination rates [FER], gut pH and digestive protease activities) in the grass shrimp Palaemonetes pugio. Carbon and Cd assimilation and endpoints related to digestion were also assessed for naïve shrimp following ingestion of a pulse of Cd-contaminated food. Based on these studies, it appears that grass shrimp may be able to maintain carbon assimilation in the laboratory under different forms of pollutant-induced dietary stress. For field-collected shrimp, this phenomenon may be attributable to digestive plasticity (e.g., increased GRT to compensate for reduced digestive enzyme activities). Increased assimilation of Cd and Hg was observed for shrimp collected along an impact gradient. Enhanced non-essential metal assimilation may have implications for accumulation and toxicity in impacted shrimp. In the case of Cd assimilation, a positive correlation with GRT and negative relationship with protease activities suggests that digestive plasticity may also influence assimilation of non-essential elements in the field. Variability in Cd assimilation by shrimp pre-exposed to dietary metal in the laboratory was not dose-dependent, which may be related to interactions between post-assimilatory impacts on gut physiology. Increased assimilation of Hg by Hg pre-exposed shrimp may have been related to a corresponding increase in gut pH (i.e., decreased concentrations of H+ ions in circulating gut fluid).
In vitro effects of Insulin and VEGF on the Choroidal and Scleral Components of Eye Growth
Ka Lee Caren Sheng
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Emmetropization occurs in most vertebrates to regulate the axial length of the growing eyes, so that the focal plane of the eye can match its ocular length, and a clear image of a distant object can fall on the retina. It has been well established that vision can control eye growth (change in the ocular length). When the image of the distant object is not on the retina, change in eye growth occurs to correct this visual error, so that the position of the retina can be moved and the image of a distant object can fall on the retina again. Visual error can be induced by using optical lenses, or by depriving the eye of form vision (form deprivation), or by allowing the eye to recover from form deprivation. The eye will compensate the induced error by changing the choroidal thickness and rate of ocular elongation. This compensation can occur without any connection to the brain, which suggests that eye growth can be regulated by local retinal signals. A signal cascade is presumed to be present at the posterior part of the eye, where retina produces the signal to act on the retinal pigment epithelium (RPE), and RPE produces another signal to affect choroid and/or sclera. It is also possible that the signal that regulates the choroid is different from the signal that regulates the sclera. Many molecules have been suggested to be involved in eye growth. Among them, insulin and vascular endothelial growth factor (VEGF) are potential regulatory signals. Insulin injected into the eye can decrease choroidal thickening caused by positive lenses and increase ocular elongation as well as scleral glycosaminoglycan (GAG) synthesis, an indicator of ocular elongation in vitro. VEGF mRNA expression in the RPE increases when eye growth is enhanced. In this thesis, we used a new experimental system to study the in vitro effect of insulin and VEGF on the choroidal and scleral components of eye growth. Eye-cups with vitreous and retina removed were prepared. The RPE and choroid of the eye-cups can be removed separately. Therefore, the effect of how the RPE and choroid mediate the effect of insulin and VEGF to affect the choroidal and scleral components (indicated by scleral GAG synthesis in vitro) of eye growth can be studied. We found that in in vitro as in in vivo, insulin can reduce choroidal thickening and increase scleral GAG synthesis. Our findings also suggest that insulin can cause the RPE to produce secondary signaling molecules that thin the choroid. Furthermore, we found that VEGF can reduce choroidal thickening transiently and increase scleral GAG synthesis in the eye-cups with choroid and sclera. We suggest that both insulin and VEGF act on the choroid to affect scleral GAG synthesis. We also suggest that insulin might cause the RPE to produce VEGF to thin the choroid, and VEGF might be one of the initial signals that cause choroidal thinning in eye growth. We suggest future experiments to explore further this relationship between insulin and VEGF in guiding eye growth.
LIM Domain Proteins TRIP6 and LPP Associate with Shelterin to Mediate Telomere Protection
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POT1 is the single stranded telomeric overhang binding protein of the shelterin complex, a group of six proteins essential for proper telomere function. The abrogation of POT1 DNA binding activity results in telomere elongation, or activation of the ATR DNA damage response at telomeres. Therefore, overhang binding represents the functionally relevant activity of POT1. Novel protein associations with the POT1 DNA binding domain are of great interest to explore and these possible interacting factors were sought using the yeast two-hybrid system. Bait containing the POT1 DNA binding domain was used leading to the isolation of LIM domain protein TRIP6 as a novel POT1 interacting factor. TRIP6 could co-immunoprecipitate with other shelterin components, arguing for association with the whole complex. Additionally, TRIP6 was detected at telomeres by Chromatin Immunoprecipitation and Immunofluorescence in Hela and HTC75 cells, which suggests association with telomeric DNA. TRIP6 depletion by siRNA led to the induction of telomere dysfunction induced foci, indicating a role in telomere protection. A closely related LIM protein, LPP, was also found at telomeres and was important for repressing the DNA damage response. A related LIM protein Zyxin was found not to associate with telomeres. We propose that TRIP6 and LPP represent a novel class of molecules at human telomeres involved in the repression of inappropriate DNA damage response at chromosome ends. All assays incorporate human cancer cell lines HTC75 and Hela 1.2.11. These results could advance our understanding on the repression of telomere-based senescence, an important tumor suppressor mechanism.
Regulation of Microtubule Stability in Saccharomyces Cerevisiae
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The integrity of chromosome segregation during mitosis is essential for the propagation of genetic information to daughter cells during cell division. In yeast, it is achieved in four microtubule-dependent steps: first is spindle assembly, which involves the migration of duplicated microtubule organizing centers to form a bipolar spindle by prophase; second is orientation of the mitotic spindle at the site of cytokinesis; third- chromosome movement along kinetochore microtubules (anaphase A); and fourth, complete chromosome segregation through spindle elongation through interpolar microtubules (anaphase B). Signaling pathways have been implicated in the regulation of microtubule dynamics and stability, which is required for these processes. This work identifies additional protein regulators of microtubule stability using mutants of key mitotic motor proteins, specifically, Cin8p, Kip1p, and Dyn1p. Loss of Cin8p function in the absence of either Kip1p or Dyn1p is lethal. Haploid cells that carry the cin8-3 temperature sensitive allele in a deletion background of either KIP1 or DYN1 cannot grow above 35◦C. Our studies suggest that suppressors of these mutant genotypes act by stabilizing microtubules. We propose that the mechanism of suppression involves enhancing signal-transduction cascades that regulate microtubule stability and dynamics. We found that FCP1 overexpression supressess the microtubule defect in our background and that this suppression requires the following genes to manifest: SWI6, SWI4, CLB2, ELM1, HSL1 and MRS6. These proteins, with a previously uncharacterized role in microtubule stability, may be candidate microtubule-associated proteins (MAPs) or novel regulators of MAPs (direct or indirect). In addition, a putative pathway to MT stability was drawn based on genetic interactions we established, epistatic experiments that were done and physical data we produced, combined with existing knowledge.
The Systematics and Evolution of the Nightjars and their allies (Aves: Caprimulgiformes)
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Recent studies have shown that the avian order Caprimulgiformes includes eight families: the owlet-nightjars (Aegothelidae), the nightjars (Caprimulgidae), the potoos (Nyctibiidae), the frogmouths (Podargidae), the monotypic oilbird (Steatornithidae) and the three families traditionally placed in the order Apodiformes: the swifts (Apodidae), the tree-swifts (Hemiprocnidae) and the hummingbirds (Trochilidae). In this study, a total-evidence approach was utilized to address the relationships of these families. A phylogenetic analysis of a combined dataset of 134 skeletal morphological characters, 14 nuclear loci (exons and introns) and a presence/absence indel matrix, with a taxon sampling of all eight families as well as 16 outgroup taxa provided a better resolved phylogeny for the group than obtained by previous studies. New relationships include the placement of the frogmouths (Podargidae) as the sistergroup to a clade containing the owlet-nightjars (Aegothelidae) and the three "apodiform" families. Both morphological and molecular data supported the sister-relationship of the nightjars (Caprimulgidae) and the potoos (Nyctibiidae). A phylogeny for the three New World radiations of nightjars (Caprimulgidae) was produced from a four-loci molecular dataset. The taxon sampling was the densest of any phylogenetic study of the group, not only were all but three New World nightjar species sampled, but also 78 of 101 recognized subspecies. This provided an opportunity to address species- and intraspecific-level relationships. The taxonomic modifications resulting from the phylogeny included a reduction in the number of genera for the three radiations, from 14 to 10, and nine subspecies were elevated to full phylogenetic species status resulting in an increase in total species numbers from 89 to 98 for the family. The modified phylogeny of the New World nightjars was utilized to investigate temporal patterns of diversification, historical biogeography and evolution of habitat choice and migratory behavior. The three New World radiations are for the most part temporally congruent but they show highly independent histories of spatial and ecological diversification that have resulted in divergent patterns of extant species distributions as well as ecology, impacted by multiple independent vicariant events, long-distance dispersal and habitat shifts.
Phylogeny and Population Genetics of the Endangered Dwarf Bear-poppy, Arctomecon humilis Coville (Papaveraceae) Using Microsatellite Markers
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The genus Arctomecon (Papaveraceae) is comprised of three narrowly endemic rare species that are largely restricted to gypsum soils of the eastern Mojave Desert. The small, remaining populations of these species have become increasingly isolated by urban development and habitat fragmentation. Arctomecon humilis is federally listed as endangered due to its limited distribution within a ~15 km radius of an actively expanding city. Organizations involved with land management and conservation have called for greater insight into the genetic variation and population structure of the remaining subpopulations as they make important decisions regarding where to focus their efforts and resources. The goal of this study was to provide answers to some of the remaining research gaps involving Arcotmecon species particularly conservation genetics by developing microsatellite markers and comparing community dynamics. First, a phylogenetic study using six gene regions (nrITS, cpDNA (matK, rbcL, trnH-psbA, rpl32-trnL, ndh-rpl32)) was conducted for members of the genus and three outgroup species. Thorough sampling throughout the geographic range of Arctomecon was conducted in order to obtain a complete representation of the genetic variability present across multiple populations. A total of 1176 plants were sampled from 35 locations with DNA extraction being performed on 949 of those, to be included in different stages of research. Outgroup taxa included a member of the sister genus Argemone, a Meconopsis species from within the subfamily, and an Eschscholzia from a separate subfamily. It was hypothesized that this increased sampling and number of gene regions would provide a more robust species tree, as compared to previous studies. Additionally, I hypothesized that new genetic markers could identify isolated populations that would be more informative to conservation management. The phylogenetic analysis did result in a well-supported species tree in addition to exhibiting broad structure among populations within each species. Notably, the population sampled in the Grand Canyon is genetically and morphologically divergent from all the other populations of A. californica that were sampled. Polymorphic microsatellite markers revealed the micro-evolutionary structure from within and between populations of A. humilis. This was the first time that genetic markers of this type have been developed for any Arctomecon species. Sixteen markers with 2 to 31 alleles (mean=12) per marker were used to determine the level of variation and admixture among 341 individual plants from thirteen sampling localities. The number of individuals per locality ranged from 26 at Price Hills to 49 at Boomer Hill. Each marker was tested for amplification and variability within the sister species A. californica and A. merriamii where cross-amplification occurred with less success and fewer alleles than in A. humilis. Population genetic analyses identified localities with greater amounts of admixture, as well as those more isolated and at risk of inbreeding depression. Through Bayesian analysis and genetic cluster assignment the overall trend suggests that populations are becoming more isolated. Analysis of Molecular Variance found 30% of the genetic variability between populations, and the FST analogues indicated substantial genetic differentiation (G'ST=0.427). A concern among land managers and conservation organizations concerned the effectiveness of the reserve system. An analysis of the allele frequencies located within the protected areas does indicate that the reserve system is effectively capturing genetic diversity. However, allele frequency data also suggest that a small number of new annual recruits represent only a subset of potential alleles. Due to the small effective population sizes and the already rare habitat supporting Arctomecon humilis the conservation efforts should continue to monitor and protect this unique species in all locations.
Functions of Notch and Neuralized in Drosophila hematopoiesis
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In vertebrates, hematopoiesis is commonly divided into two temporal phases, primitive (embryonic) and definitive (adult). Genetic studies in zebrafish and mice have implicated signaling pathways and molecular networks of transcription factors in the control of primitive and definitive hematopoietic programs. Notch signaling is essential for the proper execution of a wide array of cell fate decisions and developmental processes, including hematopoiesis. Many of these same signaling and transcriptional mechanisms also control hematopoiesis in simpler animals, such as the fruit fly Drosophila melanogaster. Because of its simple organization and genetic accessibility, Drosophila hematopoiesis has recently gained attention. Drosophila larvae produce three cell types: plasmatocytes, crystal cells and lamellocytes. While plasmatocytes and crystal cells arise in embryonic stages, lamellocytes do not. In fact very few lamellocytes are typically found in healthy third instar larvae. However, their differentiation is induced in large numbers upon oviposition by parasitic wasps. Circulating blood cells divide continuously as the animal grows in size. In addition, a small hematopoietic organ flanking the dorsal vessel supports the growth and development of blood cells. In larval stages, the Notch pathway regulates the differentiation of crystal cells: loss or reduction of Notch signaling results in the reduction of crystal cells while an increase in Notch signaling leads to the expansion of the crystal cells population in circulation and in the lymph gland (Duvic et al., 2002; Lebestky et al., 2003). Notch encodes the receptor/transcription factor that mediates short range cell-cell signaling. Notch ligand Serrate is expressed in the niche of the lymph gland. Serrate activates Notch in the pro-crystal cells and promotes commitment of the crystal cell fate. This step requires the functions of transcription factor Suppressor of Hairless (Lebestky et al., 2003). Duvic et al. (2002) also reported that Notch function is essential for lamellocyte differentiation, although how this occurs was not explored in the Duvic study. The goal of this work was to analyze the contributions of Notch and Neuralized in lamellocyte differentiation. Neuralized encodes an E3 ligase for ubiquitination of Notch ligands, and its role in hematopoiesis remains unexplored. The thesis contains three chapters. In Chapter 1, I report the expression of Notch in the lymph gland and circulating blood cells. Using RNA interference and clonal analysis, I show that Notch maintains lamellocytes in their progenitor state. This requirement is non cell-autonomous. Lamellocytes induced by loss of Notch appear mostly in the peripheral cortical zone of the anterior lobes, that houses both mature cells and progenitors. Notch target genes are expressed in most anterior lobe cells. In Chapters 2 and 3, the functions of Neuralized are explored in three ways: RNA interference, clonal analysis (Chapter 2), and using putative alleles of neuralized, l(3)hem1 and l(3)hem2 (Chapter 3). These studies show that Neuralized function is essential in maintaining hematopoietic stem-like progenitors in their undifferentiated state. In the medullary zone, where these undifferentiated progenitors reside, Neuralized plays an essential role in cell division and differentiation. Further, like Notch, it provides an inhibitory non cell-autonomous influence on pro-lamellocytes in the cortical zone, and keeps them from differentiating in the absence of infection. In chapter 3, I characterize a classical hematopoietic mutation, l(3)hem1, a putative weak allele of neur. The lymph glands and blood cells of this homozygous mutant are severely affected, with multiple defects in cell division and differentiation. These studies support the three functions of Neuralized uncovered in Chapter 2. Our studies provide novel insights into hematopoieitic stem/progenitor division and differentiation. Notch signaling plays an essential role in mammalian hematopoiesis. Misregulation of the Notch pathway leads to hematopoietic malignancies in humans. Because of the high molecular conservation between flies and mammals, understanding the regulation of Notch signaling in Drosophila hematopoiesis will yield insights into its role in mammalian hematopoiesis and potentially in developing therapies for treatment of human malignancies.
ECOLOGICAL NICHES, SPECIES DISTRIBUTIONS, AND BIOGEOGRAPHIC PROCESSES IN RODENTS ON NEOTROPICAL SKY ISLANDS
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This dissertation focused on the methodological and theoretical improvement of correlative ecological niche models (ENMs) to better understand the processes governing species distributions and associated evolutionary divergence in rodents inhabiting mesic conditions in the Neotropics. Focusing on a widespread rodent from northern South America (Heteromys anomalus), in the first chapter I proposed and tested a methodological approach to surmount the challenge of incorporating environmental information from the margins of species geographic ranges into ENMs. In so doing, I argue how populations that exist on the borders of species' local ranges (spatial margins) can lead to exaggerated estimates of their niches and potential geographic distributions due to issues of variable choice and resolution. In the second chapter, I demonstrated how the approach developed in Chapter 1 improved the ability of ENMs to detect an obvious environmental barrier fostering isolation and potential divergence between continental and peninsular populations in three rodent lineages in northern South America: Proechimys guairae, Rhipidomys venezuelae, and the Heteromys anomalus/H. oasicus species pair. In the third chapter, I integrated ENMs with molecular data to test the effect of the climatic oscillations of the late Quaternary Period in two species of rodents restricted to the sky islands of Costa Rica and western Panama: Reithrodontomys creper and Nephelomys devius. Overall, results revealed how, despite sharing similar distribution patterns, niche differences in these species resulted in idiosyncratic responses to past climate change that match currently observed patterns of genetic diversity. Finally, in the fourth chapter, I developed a perspective of the ecological niche concept that takes into account the responsiveness of phenotypes and the variability of ecological strategies that a species can perform. Integrating these aspects into niche theory leads to a more holistic perspective that reduces conflict between niche definitions, dissolves existing paradoxes, and has multiple implications for the study of niches, their evolution, and their effect on lineage divergence. Overall, this dissertation contributes to the conceptual and methodological development of correlative approaches for modeling species Grinnellian niches and their associated potential geographic distributions; the understanding of how these relate to the evolutionary history of Neotropical montane taxa with regards to past climate change; and finally, to a more holistic perspective of the niche concept that has multiple implications for the study of niches in general, as well as our understanding of how they evolve and affect lineage divergence.
Nuclear Pore Protein Nup211 Is Essential for Cell Cycle Progression and Cell Shape Maintenance in Fission Yeast
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The nuclear pore complexes (NPCs) are large multi-protein channels that traverse the nuclear envelope and mediate nucleo-cytoplasmic transport. Proteomic studies have revealed that NPCs are composed of about thirty different proteins called nucleoporins. I explored a particular nucleoporin, Nup211, in fission yeast. nup211 is an essential gene; its deletion is lethal and causes morphological defects. In this study, I characterized the cell morphological defects caused by the down-regulation of Nup211 and found that restoring the N-terminal domain of Nup211 was sufficient to rescue the lethal phenotype and partially suppress the morphological defects. Additionally, I investigated the role Nup211 plays in cytokinesis. RNA-Sequencing analysis revealed that Nup211 down-regulation and overexpression elicited a global change in gene expression. Specifically in Nup211-down-regulated cells, there was a decrease in ace2 gene expression and its downstream targets- adg1, adg2, adg3, mid2, cfh4, eng1, agn1 and rgf3. ace2 and its target genes play a dominant role in normal septum formation, dissolution, and cell separation (cytokinesis). I show that directly restoring ace2 expression significantly suppressed the morphological defects caused by Nup211 down-regulation. These results suggest that Nup211 partially controls cytokinesis through control the expression of ace2. Furthermore, we identified proteins important for cytokinesis, such as actin, were affected by Nup211 down-regulation. Taken together, our data indicate that Nup211 is fundamental for fission yeast cell shape maintenance, cytokinesis and global gene expression.