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The present study was conducted to investigate the composition of the oil of Cyperus rotundus L. rhizomes collected in Saudi Arabia, and to evaluate in vitro cytotoxic, antimicrobial and mosquito mortality activities of both the oil and the alcoholic extract of the aforementioned plant. Nineteen compounds were identified by GC-FID and GC-MS, of which α-cyperone (21.1%) and 4-oxo-α-ylangene (12.8%) were the major components. The essential oil recovered with diethyl ether demonstrated potent cytotoxic activities against colon (HCT-116), hepatocellular (Hep G-2) and breast (MCF-7) human cancer cell lines with IC50s of 1.06, 1.17 and 2.22 μg/mL, respectively. This oil also showed a comparable zone of inhibition against the gram positive bacteria Staphylococcus epidermidis and the gram negative bacteria, Klebsiella pneumoniae, Shigella flexneri and Salmonella enteritidis when compared to the standard drugs ampicillin and gentamycin. The ethanolic extract showed moderate activity against human cancer cells and pathogens. The essential oil was significantly more toxic to 1st instar Aedes aegypti larvae than the ethanol extract; however, the ethanol extract was more toxic to adult Ae. aegypti than the essential oil. Based on the results obtained, C. rotundus collected from Riyadh city could provide a potential and cheap source of biologically active compounds.

The first asymmetric total synthesis of both enantiomers of the natural products colletorin A and colletochlorin A is presented. The proposed methodology is based on the coupling reaction between highly substituted aromatic Gilman cuprates and optically active allyl bromides, in turn obtained by Sharpless asymmetric dihydroxylation. The latter ensured a high degree of regio- and stereocontrol in the enantioselective step of the synthesis. The same synthetic strategy has been also applied for the preparation of differently halogenated synthetic analogues of colletochlorin A in high enantiomeric purity. The enantioselective synthesis of colletorin A and colletochlorin A allows to reliably assign their absolute configuration. Preliminary assessment of their herbicidal and insecticidal properties evidence the possibility to modulate the bioactivity of these compounds, highlighting its dependence on both the absolute stereochemistry and the halogen nature.

Honey bees are economically important pollinators of a wide variety of crops that have attracted the attention of both researchers and the public alike due to unusual declines in the numbers of managed colonies in some parts of the world. Viral infections are thought to be a significant factor contributing to these declines, along with exposure to agricultural and apicultural pesticides, but viruses have proven a challenging pathogen to study in a bee model and interactions between viruses and the bee antiviral immune response remain poorly understood. Recent studies have demonstrated an important role for inwardly-rectifying ATP-sensitive potassium (KATP) channels in the cardiac regulation of the fruit fly antiviral immune response, but no information is available on their role in the heart-specific regulation of bee immunity. The results of this work demonstrate that KATP channel modulators have an observable effect on honey bee heart rate that supports their expected physiological role in bee cardiac function. Here, it is also reported that the entomopathogenic flock house virus (FHV) infects adult bees, causing rapid onset of mortality and accumulation of viral RNA. Furthermore, infection-mediated mortality can be altered by pre-exposure to KATP channel modulators. Finally, this work shows that exposure to environmental stressors such as commonly used in-hive acaricides can impact bee cardiac physiology and tolerance to viral infection. These results suggest that KATP channels provide a significant link between cellular metabolism and the antiviral immune response in bees and highlight the significant impact of environmental stressors on pollinator health. Ph. D.

The loss of managed honey bee colonies, resulting from their unintentional exposure to pesticides, is a topic of concern for the agricultural and apicultural industry. Current methods for reducing pesticide exposure to bees involve the application of pesticides before crop bloom or in the evening when foraging bees are less likely to be exposed to these applications. There is an urgent need for additional protection procedures to reduce the annual losses of managed bee colonies. Another method for protecting these pollinators is the use of chemical deterrents to reduce the interaction times of foraging bees with pesticide-treated crops. Historically, insect repellents (IRs) have been used to prevent the spread of deadly human diseases by arthropod vectors. However, it has been shown that bees can be repelled from pesticide-treated crops using DEET and bee pheromonal compounds. Here, I report the toxicological and deterrent effects of bee pheromonal compounds, as well as the deterrent effects of heterocyclic amines (HCAs) on bees. The results of this study indicate that the bee pheromonal compounds, at 8, 20, 60 and 100% concentrations, are toxic to bees and inhibit the feeding of bees within a confined space. Additionally, the pheromonal compounds and the HCAs are as efficacious as DEET in deterring bees from treated food sources. The HCA piperidine was observed to effectively deter bee foragers from a sugar feeder in a high-tunnel experiment as well as from melon flowers and knapweed in field experiments. Electroantennogram recordings were conducted to verify an olfactory response of the bees to the tested compounds. Pheromonal compounds were readily detected by bee antennae; whereas, the HCAs did not elicit significant responses in the bee antennae. These data suggest that bee pheromonal compounds, as well as HCAs, may serve as candidates for the further investigation as repellents to protect bees from unintentional pesticide exposures. Ph. D.

Dry cured meat production is a costly and long term investment for producers. Ham mites (Tyrophagus putrescentiae) are a common pest of dry cured products and cause devastating effects, potentially nullifying producers' investments due to loss of salable product, as well as regulatory concerns. Methyl bromide, a chemical fumigant used to control mite populations, is damaging to stratospheric ozone and will no longer be available. Presently, no alternative control measure has been approved to combat the ham mite; therefore, it is essential to identify potential alternatives. Interest in safe alternatives to control arthropod pest populations is gaining momentum, and garlic (Allium sativum) has been used to control other arthropod species including the northern fowl mite, mosquitos, and aphids. We chose to explore the efficacy of garlic juice in controlling T. putrescentiae. Using a choice test design, approximately 65% of the inoculated mites colonized on the control ham cubes, while no mites remained on garlic juice-dipped cubes. Garlic was ineffective when examined for volatile efficacy, but was effective in direct contact assays. However, as garlic juice was aged and diluted, efficacy was reduced even after treatments with antioxidants, metal chelators, and pH neutralization. In total, garlic juice acted as a short term repellent and showed efficacy in contact models, but application is time sensitive due to variable enzymatic degradation. Master of Science

The honey bee, Apis mellifera, provides about $15 billion USD in crop value each year in the U.S. alone in the form of pollination services. Since 2006, commercial beekeepers have reported an average annual overwintering loss of about 28.6% of all managed colonies. There are many factors that are thought to contribute to colony loss including bee-specific pests (e.g. the Varroa destructor mite), bee-specific pathogens (e.g. Nosema fungus), modern beekeeping practices, diminished genetic variability, poor queens, climate change, and exposure to agricultural pesticides. While not the single cause of colony loss, the neonicotinoid insecticides elicit sublethal effects to honey bees that could increase their sensitivities to other stressors that affect colony health. Previous studies found that honey bees have differential sensitivities to the neonicotinoid insecticide imidacloprid, which suggest a mechanism of tolerance to the insecticide in certain colonies. In this study, I examined the imidacloprid sensitivity of honey bees collected from different colonies. After determining a range of LC50 values in the tested colonies, I examined the metabolic detoxification activities of honey bees collected from two colonies that represented the highest and lowest LC50 values, between which there was a 36-fold difference in their LC50 values. I discovered that of the three main families of metabolic detoxification enzymes, general esterases, cytochrome P450 monooxygenases, and glutathione S-transferases (GSTs), a reduction of GST activity with diethyl maleate (DEM) significantly increased imidacloprid-mediated mortality to the honey bees. A comparative analysis of GST kinetic activity from imidacloprid-susceptible and -insensitive honey bees revealed a lower bimolecular inhibition rate constant (ki) for imidacloprid-insensitive individuals (5.07 ± 0.098 nmol/min/mg protein) compared to the imidacloprid-sensitive honey bees (17.23 ± 1.235 nmol/min/mg protein). The IC50 of DEM estimated for bees from each colony showed that the imidacloprid-susceptible honey bees possess a higher IC50 (10 μM) than that of the tolerant honey bees (3 μM). These data suggest that the GSTs in the imidacloprid-tolerant honey bees might be a more efficient detoxification mechanism for the conjugation and elimination of imidacloprid, or imidacloprid metabolites, compared to that of imidacloprid-susceptible honey bees. Therefore, I hypothesize that the differences in metabolic detoxification enzyme activities of honey bees collected from different colonies can result in the differential toxicities of honey bees exposed to neonicotinoid insecticides, such as imidacloprid. However, a thorough examination of imidacloprid detoxification in honey bees is warranted to confirm this hypothesis. Master of Science in Life Sciences

The varroa mite is a primary driver behind periodical losses of honey bee colonies. The mite requires bees for food and reproduction and, in turn, elicits physiological deficiencies and diseases that compromise bee colony health. The mite nervous system is a target site for existing acaricides. These acaricides not only have adverse health effects on bees, but resistance limits their use to reduce mites and diseases in bee colonies. Voltage-gated chloride channels are involved in the maintenance of nerve and muscle excitability in arthropod pests, which suggests that these channels might be exploited as targets for acaricides. Apistan® (the pyrethroid tau-fluvalinate), Checkmite+® (the organophosphate coumaphos), and Apivar® (the formamidine amitraz) are control products for mite management. The effectiveness of these chemistries has diminished as a result of the increasing incidence resistance in mite populations. I report a toxicological analysis of stilbene products against acaricide-susceptible and -resistant mites. My results find a significant increase in metabolic detoxification enzyme activities in acaricide-resistant mites compared to susceptible mites. Acetylcholinesterase of coumaphos-resistant mites was significantly less sensitive to the toxic coumaphos metabolite compared to susceptible mites, which suggests target-site insensitivity as a mechanism of acaricide resistance. The stilbene product DIDS had significantly higher field efficacy to acaricide-resistant mites compared to Apistan®- and CheckMite+®. These data suggest that DIDS, and other stilbene products, might serve as candidate chemistries to continue field efficacy testing of alternative acaricides for Apistan® and CheckMite+® resistant mites. Master of Science in Life Sciences

Mosquito-borne diseases remain a significant burden on global public health. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although infection of the vertebrate host is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. It is well known that invertebrates rely on small RNA pathways as an adaptive antiviral defense. The canonical antiviral response in these organisms involves dicer enzymes that cleave viral double-stranded RNA replicative intermediates (RIs) into small interfering RNAs (siRNAs; ~21-24 nucleotides). One strand of the siRNA duplex guides the targeting and destruction of complementary viral RNAs when loaded and retained in a multi-protein complex called the RNA-induced silencing complex. Here, we show that mosquito vectors mount a redundant double defense against virus infection mediated by two different small RNA pathways. Specifically, we demonstrate that in addition to a canonical antiviral response mediated by siRNAs, virus infection of the mosquito soma also triggers an antiviral immune pathway directed by ping-pong-dependent PIWI-interacting RNAs (piRNAs; ~24-30 nucleotides). The complexity of mosquito antiviral immunity has important implications for understanding how viruses both induce and modulate RNA-silencing responses in mosquito vectors. In mammals, viral RIs induce a range of relatively nonspecific antiviral responses. However, it remains unclear if viral RIs also trigger RNA silencing in mammals. Mosquito-borne viruses represent an ideal model for addressing this question as their transmission cycles involve alternating replication in mammalian and invertebrate hosts. Although we report identifying a subset of virus-derived small RNAs that appear to be products of RNA silencing in two mammalian cell lines infected with the mosquito-borne chikungunya virus (CHIKV), our studies suggest these small RNAs have little biological relevance in combating virus infections. Thus, while the accumulation of virus-derived siRNAs is essential to the survival of mosquitoes infected with CHIKV, they appear to have little functional significance in mammalian antiviral immunity. Ph. D.

Malaria (vector: Anopheles gambiae) is a major infectious disease that kills about 1 million people each year. For the improvement of its treatment and vector control during the past decades, several issues such as high medicine cost, insecticide resistance, and lack of an effective vaccine have prevented adequate control of malaria. Additionally, the low selectivity of malaria vector insecticides also presents a public health problem. The purpose of developing novel carbamate insecticides in our laboratory is to offer effective and selective insecticide options to achieve the ultimate goal of malaria control. First, 50% inhibition concentration (IC50) data was collected from three mammalian AChEs with eight commercial carbamate insecticides by using the Ellman assay. The IC50 values varied from 57 nM to 7358 nM. The AChE sensitivity pattern and level were shown to be similar between the recombinant mouse and ICR male mouse brain cortex homogenate (slope = 0.99, R2 = 0.96). Then eight novel carbamate insecticides that are possible malaria vector control agents were selected for further neurotoxicity testing in non-target organisms. For commercial carbamate insecticides, the IC50 varied from 9.1 nM to 2,094 nM. For the novel carbamate insecticides, it varied from 58 nM to 388,800 nM. Based on IC50 data from previous work on A. gambiae, the selectivity index (IC50 of non-target species / IC50 A. gambiae) ranged from 0.17 to 5.64 and from 0.47 to 19,587 for commercial and novel carbamate insecticides, respectively. Subsequently, the AChE protein sequence alignment comparison and cladogram were used to compare the genetic and evolutionary relationship among five different organisms. The alignment score ranged from 88 for mouse vs. human to 54 for hen vs. T. californica. The evolutionary relationships among species was obtained from the cladogram. Recombinant mouse vs. recombinant human was shown to have the most similar inhibitor potency profiles (alignment score = 88, closest taxa position on cladogram, similar AChE sensitivity pattern [R2 = 0.81] and level [P > 0.05] to the novel carbamates). Neurotoxic esterase (NTE) assay showed that the novel carbamates did not significantly inhibit NTE, inhibition of which underlies a significant hazard for anticholinesterases, especially organophosphates, in several nontarget vertebrate organisms. The NTE activity in the presence of novel carbamate insecticides ranged from 93% to 116% of the control, while in the commercial group, bendiocarb significantly inhibited NTE, leaving only 76.5% of the initial reactivity at 1 mM inhibitor concentration. Further in vivo bioassay using Daphnia magna was conducted to compare the aquatic toxicity of commercial and novel carbamates. The data showed that except for PRC331 (3-tert-butylphenylmethylcarbamate), all novel carbamates were of similar potency as bendiocarb (LC50 = 611 nM) for aquatic toxicity, and their LC50 values ranged from 172 nM (PRC331) to 1109 nM. In conclusion, the novel carbamate insecticides would appear to be an improvement over commercial carbamate insecticides because of greater selectivity, negligible NTE inhibition capacity, but in some cases with potent in vivo toxicity to Daphnia magna. However, since the envisioned usage of these compounds is in bednets or as indoor residual sprays (IRS), any environmental exposures to nontarget aquatic organisms are expected to be minimal. Master of Science

To better understand the evolutionary dynamics of chromosomal inversions, a physical map for an Asian malaria vector, Anopheles stephensi, was created and compared with the maps of the major African malaria vectors A. gambiae and A. funestus No interchromosomal transposition was observed between A. gambiae and A. stephensi. Several cases of euchromatin and heterochromatin transitions weridentified between A. gambiae and A. stephensi. The study of paracentric inversions between lineages in Anopheles mosquitoes demonstrated that X chromosome has the fastest rate of inversion fixations and highest density of repetitive elements. Among the autosomes, 2R evolved faster than other autosomes. The slowly evolved autosomes have more M/SARs than rapidly evolving arms. Breakpoint regions are enriched with repetitive elements. The study revealed that fixed inversions are distributed nonrandomly and breakpoint clustering is common in lineages of A. gambiae and A. stephensi. The parallel association between the density of inversion fixations and polymorphisms suggests that polymorphic inversions can be fixed during evolution. To understand the direction of evolution in A. gambiae complex, the ancestral status of fixed inversions for this complex was identified. The presence of the 2La inversion in outgroups, A. stephensi and A. nili, confirmed the ancestral status of the 2La inversion. The presences of breakpoint structure of the 2Ro inversion in outgroup species, A. stephensi, indicated that the 2Ro is ancestral arrangement. The presence of SINE elements at the breakpoints of the 2R+p in A. gambiae PEST strain suggested that the 2R+p is a derived arrangement. Therefore, the carrier of 2Rop inversions, A. merus, was considered closest to the ancestral species. We have developed a new protocol for laser microdissection and whole genome amplification of polytene chromosomal fragments to obtain DNA for sequencing and assembly. The chromosomal regions spanning both breakpoints of the 2La in A. arabiensis and A. merus were laser microdissected from the polytene chromosomes. Subsequently, DNA samples were amplified using Illustra GenomePhi V2 DNA and Whole-pool amplification methods for obtaining amplicons. Successful amplification of our target DNA was confirmed by PCR with specific primers followed by Sanger sequencing. Ph. D.

Due to the increasing resistance demonstrated by insects to conventional insecticides, the need for compounds with novel modes of action is becoming more urgent. Also, the discovery and production of new insecticides is vital as regulations and restrictions on conventional insecticides become increasingly stringent (Casida and Quistad 1998). Research in this area requires screening of many candidate compounds which is costly and time-consuming. The goal of this research was to produce in vitro insect neurons from Sf21 insect ovarian cell lines, which could lead to new high throughput screening methods and a way to mass produce insect material for basic research. This study used a culture of Sf21 cells and a mixture of differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, Sf21 cells began to express neuronal morphology, or the production of elongated, axon-like processes within 2-3 days. Maximal differentiation occurred when in the presence of 42 μM 20-HE or 10 μM insulin. Effects were maximal on day 2 for 20-E and day 3 for insulin. Insulin was more potent at day 2 for inducing differentiation (EC₅₀ = 247 nM) than 20-HE (EC₅₀ = 13 μM). In combination, 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system (CNS) stimulant, inhibited induction of elongated processes by 20-HE and/or insulin. Caffeine was a potent inhibitor of 42 μM 20-HE, with an IC50 of 9 nM, and the inhibition was incomplete, resulting in about one quarter of the differentiated cells remaining, even at high concentrations (up to 1 mM). The ability to induce a neural phenotype simplifies studies with of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined. If they are present, high throughput screening for new insecticides will be accelerated and made more economical by the utility of this method. Master of Science

Parkinson's disease (PD) is a neurodegenerative Lewy body disorder characterized by severe motor deficits, followed by cognitive dysfunction with progression of the disease. Environmental exposure has been suggested as a possible contributor to the development of PD and this view is linked to the discovery of the nigrostriatal neurotoxin MPTP. MPTP can induce dopamine specific degeneration within the basal ganglia often resulting in motor deficits similar to PD. MPTP used in the C57BL/6 mouse is a widely used animal model of PD. The pyrethroid permethrin (PM), and the organophosphate chlorpyrifos (CPF), can produce changes in dopaminergic nigrostriatal neurons, the primary target of PD and MPTP-induced neurotoxicity. Such insecticide induced changes in the basal ganglia could exacerbate the onset or severity of PD. Chronic exposure to the metal manganese (Mn) can damage the globus pallidus (GP) of the BG, and produce motor deficits similar to PD. Since the GP is part of the BG circuitry essential for motor control, and is synaptically integrated with the nigrostriatal pathway, Mn may exacerbate MPTP-induced neurotoxicity. Because the BG is disynaptically linked to the mesocortical pathway, a dopaminergic pathway that is important for cognition, Mn induced damage in the BG could indirectly affect the mesocortical pathway as well. This study investigated the pesticides, permethrin and chlorpyrifos, and the heavy metal, manganese as possible environmental compounds that could exacerbate PD in the MPTP treated C57BL/6 mouse. The first part of this dissertation used immunohistochemistry to examine insecticide induced effets on MPTP-induced neurotoxicity in the dorsolateral striatum of the C57BL/6 mouse, the principal target of the nigrostriatal pathway. Tyrosine hydroxylase (TH) was used as a marker for loss of dopaminergic neuropil and glial fibrillary acidic protein (GFAP) was used as a marker of glial activation in the striatum. Three experiments assessed effects of 1) PM (200 mg/kg), 2) CPF (50 mg/kg) & 3) PM + CPF, on MPTP (30 mg/kg) neurotoxicity. Immunohistochemistry revealed a decrease in TH staining and an increase in GFAP staining with MPTP (30 mg/kg). A main effect increase in GFAP was observed for PM (200 mg/kg), but not for CPF (50 mg/kg) or PM+CPF. Insecticides, alone or combined, did not alter MPTP-induced toxicity. . However, the absence of the PM-induced increase in GFAP staining following combined insecticide treatment suggests a neuroprotective effect. The next set of experiments in this dissertation looked at the effect of Mn on MPTP-induced neurotoxicity in the nigrostriatal and mesocortical dopaminergic pathways of the C57BL/6 mouse. Inductively Coupled Plasma atomic emission spectrometry revealed striatal Mn levels were significantly increased with multiple dose 100, 50, and 25 mg/kg MnCl2. Administration of Mn (MnCl2 s.c., Days 1, 4, & 7) in the MPTP (20 mg/kg i.p., Day 8) treated C57BL/6 mouse revealed Mn and MPTP interactions for locomotor activity, grip strength, and repeated measures of learning. Mn attenuated the effect of MPTP on striatal DOPAC, and facilitated the effect of MPTP on cortical DA and DOPAC. Mn also attenuated the MPTP induced decrease in cortical DAT. While these data support the notion that insecticides can produce tissue damage in the nigrostriatal pathway, in this case, these insecticide induced changes were not found to be strong enough to facilitate PD-like tissue damage. While Mn did not always facilitate MPTP neurotoxicity in the mesocortical and nigrostriatal dopaminergic pathways, these results demonstrate Mn and MPTP can interact in a complex way to alter dopaminergic function as well as motor and cognitive behavior. Differences in brain uptake mechanisms and metabolism of Mn and MPTP, could explain why combined administration of Mn and MPTP differentially affect dopaminergic activity in the nigrostriatal and mesocortical pathways. Ph. D.

Aedes aegypti transmits the viruses which cause yellow fever, dengue fever, and dengue hemorrhagic fever. Homing endonucleases are selfish genetic elements which introduce double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this study, we aimed to validate a somatic assay to detect recombinant homing endonuclease (rHE)-induced dsDNA breaks in both cultured cells and adult female Ae. aegypti. While the cell culture-based two plasmid assay used to test rHE ability to induce dsDNA breaks was inconclusive, assays used to test rHEs in Ae. aegypti were successful. Recognition sequences for various rHEs were introduced into Ae. aegypti through germline transformation, and imperfect repair at each of these exogenous sites was evaluated. In mosquitoes containing a single exogenous HE site, imperfect gap repair was detected in 40% and 21% of clones sequenced from mosquitoes exposed to I-PpoI and Iâ SceI, respectively. In mosquitoes containing two exogenous HE sites flanking a marker gene (EGFP), 100% of clones sequenced from mosquitoes exposed to I-PpoI, I-CreI, and I-AniI demonstrated excision of EGFP. No evidence of EGFP excision or imperfect repair at any HE recognition site was detected in mosquitoes not exposed to a rHE. In summary, a somatic genomic footprint assay was developed and validated to detect rHE or other meganuclease-induced site-specific dsDNA breaks in chromosomal DNA in Ae. aegypti. Master of Science in Life Sciences

Prey-mediated effects of imidacloprid were evaluated for Laricobius nigrinus Fender and Sasajiscymnus tsugae Sasaji and McClure after feeding on hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae). Two methods were evaluated for detecting imidacloprid in hemlock tissues: a commercially available enzyme linked immunoassay (ELISA) kit and a high performance thin-layer chromatography technique for detecting and quantifying imidacloprid residues in hemlock wood and needle tissues. ELISA is advantageous because of its cost, sensitivity, and ease of use. However, matrix effects in the form of false positives and overestimated imidacloprid concentrations were evident in hemlock wood and needle tissue extracts. Matrix effects could be reduced by dilution with water, effectively raising the lower detection range of the kit from 0.2 to 200 ppb. High performance thin-layer chromatography was accurate, quick, easy to use, and matrix effects were not evident. However, the technique is sensitive in the lower ppm range and tissue samples from field-treated hemlocks are often in the ppb range, making this technique less desirable than more sensitive analytical methods. Lethal and sublethal effects on both predators were evident after eastern hemlock branches infested with HWA were spiked with imidacloprid in the laboratory. HWA mortality increased with dosage and time, and its 30 d LC50 was determined to be 242 ppb. Both predator species exhibited reduced survivorship and fitness parameters after feeding on HWA from the treated branches. In a topical application bio-assay, 6 d imidacloprid LD50 values for L. nigrinus and S. tsugae were 2.43 and 1.82 µg/g, respectively. Imidacloprid and its major metabolites in hemlock tissues were analyzed by liquid chromatography-tandem mass spectrometry. Imidacloprid recovery from beetle cadavers was correlated with beetle mortality from feeding on treated hemlock branches. Olefin was the primary imidacloprid metabolite recovered from hemlock wood tissues. When predators fed on HWA from field-treated trees, impacts on survivorship and fitness were variable. In 2005, significantly higher proportions of both species of beetles were affected by feeding on control branches compared with treated branches. In 2006, beetles feeding on HWA from some of the trees treated in the field exhibited longer fliptimes compared with beetles feeding on controls, although beetle mortality was not significant among treatements. In the field, imidacloprid controlled HWA populations 1-3 years post-treatment. Hemlock health improved in the highest dosage group, with significantly greater lengths of new shoots compared with shoots from control trees. Eastern hemlock trees primarily metabolized imidacloprid into the olefin metabolite, which can have increased insecticidal toxicity compared with imidacloprid. Imidacloprid was detected in beetle cadavers after feeding on HWA from treated branches, suggesting that prey-mediated impacts of systemic imidacloprid are possible on nontarget predators. However, because of HWA's sensitivity to imidacloprid, in field situations predators are more likely to be affected by reduced adelgid density and quality. Ph. D.

The tobacco-adapted form of the green peach aphid, Myzus persicae (Sulzer), is one of the most important insect pests of tobacco in the United States and around the world. Insecticides play a major role in controlling the aphid on tobacco because natural enemies usually fail to maintain its populations below damaging levels. The aphid has a history of developing resistance to many insecticides. Therefore, baseline information on the aphid's susceptibility to imidacloprid and other insecticides is critical for developing future resistant management programs to minimize losses attributed to the aphid. Studies were conducted on colonies of the tobacco-adapted form of the green peach aphid collected from nine states in the eastern United States in 2004-2007. The susceptibility of 151 colonies to imidacloprid was determined in serial leaf-dip bioassays. When combined over the four years, 18, 14, and 4% of the colonies had 10- to 20-fold, 20- to 30-fold, and 30- to 90-fold resistance ratios, respectively, suggesting that high levels of resistance to imidacloprid are present in field populations of the aphid. A colony collected near Clayton, NC had the highest LC50 value (31 ppm) combined over six tests and three years, but the average resistance ratios for the first three runs was over 130-fold (48 ppm). Geographic location had little effect on susceptibility to imidacloprid. Aphid colonies (136) including red, green, and orange color morphs were screened for total esterase activity using microplate assay with 1-Naphthyl acetate as the substrate. The green morphs generally had lower esterase levels than the red and orange morphs. All orange morphs had among the highest esterase activities. Esterase activities of red and green morphs were positively correlated with LC50 values as determined by leaf-dip bioassays for acephate and methomyl. All 25 colonies tested for esterase gene amplification had either E4 or FE4 gene amplification. The amplification of both E4 and FE4 seen as an 865-bp band characteristic of the FE4 gene and an additional 381-bp band characteristic of a deleted upstream region of the E4 gene occurred in all (4) orange morphs and one (1 of 9) green morph. Target-site insensitivity of acetylcholinesterase (AChE), as modified AChE resistance (MACE) was assessed in 65 colonies of field-collected tobacco-adapted forms of M. persicae. Eight colonies over a range of AChE activity were selected to study inhibition of AChE in the presence of two carbamate insecticides, methomyl and pirimicarb. IC50 values for methomyl ranged from 0.35 to 2.4 μM while six of eight colonies had lower values with a range of 0.16 to 0.30 μM for pirimicarb. Two colonies that were inhibited by methomyl had very high IC50 values of 40.4 and 98.6 μM for pirimicarb. Such insensitivity may be due to mutations in the ace2 gene, but this needs to be confirmed by genetic and molecular analysis. Glutathione S- transferases (GSTs), isoenzymes that are involved in the metabolism and detoxification of many xenobiotic compounds were quantified for 100 colonies by CDNB conjugation. There was a wide range of GST activity for the red (8 to 343 pmol/min/mg protein) and green (15.3 to 330 pmol/min mg protein) morphs, but all six orange morphs collected in 2007 had a narrower range (160 to 211 pmol/min/mg protein). About 45% of the red morphs had GST activity from 200-300 pmol/min/mg of protein, while 53% of the green morphs had a range of 100-200 pmol/min/mg protein. The influence of temperature-mediated synergisms on the toxicity of insecticides in red and green color morphs of the tobacco-adapted from of M. persicae were evaluated using leaf-dip bioassay procedures in laboratory incubators. Post-exposure temperatures of 15, 20, and 25°C were evaluated for four classes of insecticides, acephate, imidacloprid, lambda-cyhalothrin, and methomyl. The temperature change from 15 to 20°C caused almost a 3-fold increase in toxicity to the red and green color morphs for methomyl, acephate, and imidacloprid. In contrast, the toxicity of lambda-cyhalothrin decreased as the temperature increased, showing a negative temperature coefficient. Bioassay experiments conducted with the red morph for indirect estimates of imidacloprid concentrations in flue-cured tobacco showed that leaf position, imidacloprid rate and time after application affected the concentration of the toxicant in the leaf. The differences in aphid mortality between the lower and upper leaf positions indicate that the concentration of imidacloprid and its metabolites were unevenly distributed with the lowest mortality for aphids feeding on the younger, upper leaves and the highest for those feeding on the older, lower leaves. In field experiments, higher aphid populations occurred on tobacco treated with imidacloprid less than the field recommended rate of 41.4 ml/1,000 plants. The development of aphid populations in the field was consistent with the laboratory bioassays. Field trials were conducted to evaluate the performance of various insecticides currently registered for aphid control on tobacco. Imidacloprid applied as a tray drench treatment and acephate as foliar sprays were the most effective treatments. Moderate declines in control with imidacloprid were observed at 75-87 d after transplanting in 2006 and 2007. Aldicarb gave good to excellent control in one of three years, but only fair to poor control in the other two years. Methomyl and lambda-cyhalothrin gave good control in all three years except the residual was shorter. The poor performance of aldicarb in the two years may have been related to the presence of E4 or FE4 resistance in the naturally occurring TGPA in the experimental plots. Ph. D.

In this study, four anion transporter (AT) blockers, DIDS (4, 4′-diisothiocyanatostilbene-2, 2′-disulfonic acid), 9-AC (anthracene-9-carboxylic acid), NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid), and IAA-94 (indanyloxy acetic acid) were selected to evaluate ATs as potential target sites for insect and nematode control. All the AT blockers showed slowly developing toxicity against second-stage larvae of Meloidogyne incognita (Kofoid and White 1919) Chitwood 1949 and adults of Caenorhabditis elegans Maupas 1900 but not against third-stage larvae of Heterorhabditis bacteriophora Poinar 1975 even at 200 ppm. Symptoms of AT blocker toxicity observed in C. elegans adults were increased pharyngeal muscle contractions and decreased locomotion. Exposure of C. elegans as fourth-stage larvae to double-stranded RNA (dsRNA) of ceclc-1 and ceclc-2 (VGCC genes coding for CeClC-1 and CeClC-2, respectively) either alone or together for 24 h decreased their expression in F1 progeny in a time-dependent manner. Reduction in expression of ceclc-2 alone or together with ceclc-1 significantly increased pharyngeal contractions and decreased locomotion in significantly higher percentage of F1 progeny. The above findings suggested AT blockers nematicidal activity primarily comes from inhibition of CeClC-2 channels, while inhibition of CeClC-1 channels may enhance this activity. All the AT blockers showed slowly developing toxicity against adults of a susceptible strain (Oregon-R) of Drosophila melanogaster Meigen 1830, while DIDS, was equally toxic to dieldrin-resistant rdl flies. All AT blockers, except 9-AC, at 100 µM showed significant excitatory effect on desheathed central nervous system (CNS) of third-instar larvae of Drosophila, while DIDS showed a modest excitatory effect on ascending peripheral nerves. Feeding adult flies on 10% sugar solution mixed with 100 ppm of DIDS for 6 h decreased the midgut pH by 2 units approximately. All the AT blockers inhibited the growth of larvae (in weight), increased the developmental time, and decreased survival when Ostrinia nubilalis (Hübner 1796) second-instar larvae were fed for seven days. All the AT blockers decreased the midgut alkalinity and inhibited chloride ion transport from midgut lumen into epithelia in fifth-instar larvae when fed for 3 h on treated diet. Positive correlations observed among growth, midgut alkalinity, and midgut chloride transport in AT blocker-fed larvae suggested that inhibition of chloride/bicarbonate exchangers by AT blockers may have contributed to midgut alkalinity decrease affecting the digestion and resulting in observed lethal and sublethal effects. Ph. D.

Diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) is a serious pest of crucifer vegetables (Brassica sp.) worldwide because of a lack of effective natural enemies in certain regions and because of insecticide resistance. In 2003, laboratory and field studies were initiated in Virginia to better understand P. xylostella, its primary natural enemies, and their susceptibilities to insecticides in order to develop an economically and environmentally sound integrated pest management program for collards in the state. Ecological life table studies of P. xylostella immature stages on collards located on the Eastern Shore and on Kentland Farm, near Blacksburg at the New River Valley, VA revealed that most (98 to 99%) of P. xylostella died from natural causes. Mortality factors varied between the two regions. Neonates, small larvae, and large larvae disappearing were major mortality factors. Rainfall, predation, and dispersal probably contributed the most to this mortality. Egg mortality played a bigger role at the New River Valley compared with the Eastern Shore. Three parasitoid species were found that contributed to the mortality of P. xylostella: Diadegma insulare (Cresson) (Hymenoptera: Ichneumonidae); Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae); and Microplitis plutellae (Muesebeck) (Hymenoptera: Braconidae). Additional studies conducted in the laboratory using leaf-dip bioassays revealed that P. xylostella collected from the Eastern Shore of Virginia, showed significant tolerance levels to esfenvalerate, acetamiprid, methomyl, methoxyfenozide, indoxacarb, and acephate compared with a susceptible strain of P. xylostella. The highest tolerance ratio (1,876 fold) was to esfenvalerate, a commonly-used pyrethroid. All of the insecticides tested in this study were quite toxic to the adult stage of the parasitoids, D. insulare and O. sokolowskii. The insect growth regulator, methoxyfenozide was considerably less toxic than other insecticides such as esfenvalerate, methomyl, acephate, spinosad, indoxacarb, and emamectin benzoate at field-rate and 1% of field-rate concentrations. The aforementioned insecticides as well as some other insecticides were evaluated several times in the field for efficacy against P. xylostella as well as other pests of collards. The most efficacious insecticides over five field experiments included acephate, emamectin benzoate, esfenvalerate, methomyl, methoxyfenozide, novaluron, indoxacarb, and spinosad. These insecticides were followed in relative efficacy by Bt kurstaki, acetamiprid, and azadirachtin, which provided relatively inconsistent control of lepidopteran larvae over the experiments. Effective insecticide options in collards that are less toxic to natural enemies and that can fit well into integrated pest management programs include indoxacarb, spinosad, novaluron, emamectin benzoate, methoxyfenozide, and Bacillus thuringiensis subsp. kurstaki. Ph. D.

Organophosphorus (OP) compounds are found in household pest control products, plastics, and petroleum. Due to the neurotoxic nature of OP compounds, exposure can cause both acute and delayed symptoms, including organophosphate-induced delayed neuropathy (OPIDN). This syndrome is characterized by Wallerian-like degeneration of nerves in the central and peripheral nervous system after exposure to neuropathic OP compounds. There are many questions surrounding the mechanisms of the onset of OPIDN, including possible alterations in proteins associated with neuronal maintenance and repair. This dissertation investigated the changes in levels of neurotrophins in vivo and how in vitro levels of neurotrophin receptors and their downstream signaling cascades are affected after exposure to OP compounds. We also characterized the molecular weight of a soluble factor responsible for inducing neurite outgrowth in vitro after in vivo exposure to a neuropathic OP compound. We evaluated in vivo endpoints using enzyme-linked immunosorbant assays. Results indicated that nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are found in chicken spinal cord but do not increase as a result of exposure to neuropathic OP compounds. This study also noted that NGF, BDNF, and NT-3 concentrations were not altered after exposure to a non-neuropathic OP compound. We evaluated in vitro endpoints using Western blots, ultrafiltration, and digital morphometry. These studies revealed that activated forms of high-affinity and low-affinity neurotrophin receptors are present after OP compound exposure, that the ratio of these two receptors to each other is stable after OP compound exposure, and that the activated form of the low-affinity receptor, which can lead to apoptosis, was present in greater levels than the activated form of the high-affinity receptor. Furthermore, OP compound exposure resulted in time-dependent changes of protein levels central to the mitogen-activated kinase and phosopholipase C-gamma intracellular pathways. Changes in a third pathway, the protein kinase C pathway, were dependent on the concentration and type of OP compound. Finally, in vitro neurite length was not affected by the type of OP compound administered in vivo or when a whole protein fraction was separated by molecular weight. This research has revealed in vivo consequences and early effects on intracellular protein and activated neurotrophin receptor levels after OP compound exposure. These early effects may contribute to the delayed development of neurotoxic effects associated with OP compound exposure. Ph. D.

Numerous previous studies of GABAAR ligands have suggested that GABAAR agonists must be zwitterionic and feature an intercharge separation similar to that of GABA (approx. 4.7-6.0 Ã ). We have demonstrated that monomeric, homodimeric and heterodimeric non-zwitterionic GABA amides are partial, full, or superagonists at the murine GABAA receptor (GABAAR). The agonism of these GABA amides is comparable to that of THIP, as shown by in vitro assay results. The assay data indicate that the agonism of GABA amides is tether length-dependent. Optimum agonism is achieved with a tether length of four methylenes in GABA amide dimers and in GABA amides bearing pendant amide or amino groups. We have further investigated the structure-activity relationship for GABA amides on the GABAAR by performing structural modifications to both the superagonist 2c and the agonist 6c. Synergism and [3H]muscimol binding experiments show that 2c binds to the same sites as GABA. Structural modification of 2c demonstrated that partial rigidification of the tether eliminated agonism and caused ligands to behave as weak competitive antagonists. We have also investigated the agonism of four ZAPA derivatives in 36Cl- uptake functional assay. Two of them are found to be as potent as GABA. In our studies of 1,4-benzodiazepines, our goal was to synthesize three different subtypes of quaternary 1,4-benzodiazepines by use of the memory of chirality (MOC) strategy. Disappointingly, most of the deprotonation/alkylations failed, due to various reasons. The failure of the reactions of (S)-alanine-derived tetrahydro-1,4-benzodiazepin-3-ones was probably due to either the unexpected side reactions or the steric hindrance of enolate alkylation. In the case of tetrahydro-1,4-benzodiazepin-2-ones, computational studies suggested that steric hindrance by both the benzo ring and N4-allyl group might retard deprotonation at C3 by bulky bases like KHMDS or LDA. Finally, (S)-serine-derived 1,4-benzodiazepin-2-ones and their elimination products (ï ¡-methylene benzodiazepines) were prepared. These proved unreactive towards deprotonation/alkylations and conjugate additions, respectively. The low reactivity of the ï ¡-methylene benzodiazepines towards nucleophiles was attributed to highly delocalized LUMOs that failed to direct nucleophiles to the ï ¢-carbons. Ph. D.

Otiorhynchus sulcatus (Fabricius) is a serious pest of various horticultural crops in the United States. Despite its economic importance, this pest remains very challenging to manage. Integrated Pest Management Program for O. sulcatus has not yet been implemented due to lack of accurate information on its ecology. Investigation of temperature-dependent development and reproduction of O. sulcatus was conducted to enable the prediction of stage emergence more accurately. Immature-development study at constant temperatures 11-30 oC showed that there was the difference in optimal temperature regime among immature stages, which suggests that temperature can play a role to adapt each stage to the different temperature condition of the season. By applying mathematical models, optimum temperature for the fastest development was estimated to be 27.5, 25.1, and 24.1 oC for eggs, larvae, and pupae, respectively. Temperature also significantly influenced the adult reproductive life history traits, including preovipositional period, ovipositional period, longevity, per capita egg production, and egg viability. At 27 oC or above, reproductive success of O. sulcatus was substantially impaired due to shortened longevity, delayed reproduction, and lowered egg viability. Endosymbiont bacteria Wolbachia infect many arthropod species and they may alter the reproduction of their hosts. I first documented that Wolbachia is highly prevalent in O. sulcatus populations in the United States. Given that two antibiotics (tetracycline and gentamicin) differing only in their efficacy against Wolbachia, results strongly support the hypothesis that Wolbachia may be required for normal egg development of O. sulcatus. Despite intensive insecticide application, O. sulcatus populations often remain at unacceptable levels. I ask whether sublethal dosages of newer insecticides, imidacloprid and thiamethoxam, influence offspring production of O. sulcatus adults and its progeny under laboratory conditions. Weevils that survived short-term exposure to sublethal dosages of imidacloprid and thiamethoxam produce viable offspring when they have access to insecticide-free leaves after the exposure. Toxicity test showed the potential of imidacloprid and thiamethoxam for the control of first-instars but not for the control of eggs. Ph. D.

Field studies were conducted in southwestern Virginia to determine the bionomics and ovipositional preferences of Culex restuans Theobald and Culex pipiens Linnaeus using ovitraps and gravid traps. Both species have been implicated as enzootic and epizootic vectors of West Nile virus (WNV) and these studies provide information on the relative abundance of gravid mosquitoes. Ovitraps were used in the summers of 2002 and 2003 to measure the oviposition activity of Culex mosquitoes, mainly Cx. restuans and Cx. pipiens. In 2002, 1,345 egg rafts were collected from 5 traps set at different locations in the New River Valley (NRV). Cx. restuans constituted 93.2% of the catch; the remainder was Cx. pipiens (6.7%) and Cx. salinarius (

Pneumonic pasteurellosis is a significant disease in beef production medicine. The most recent information suggests that this disease is a $700 million dollar per year economic burden in bovine food animal production The medical and pathological characteristics of this disease are well documented. Many pathological findings associated with pneumonic pasteurellosis may be explained by disruption of the pulmonary vascular adrenergic system. However, only a limited amount of research has addressed the adrenergic system and its relationship to the etiology and pathophysiology of this disease. In an attempt to further investigate the contributions of the vascular adrenergic receptor mechanism to the development of pneumonic pasteurellosis a series of six experiments have been completed. It is to be noted, that in 1999 the organism Pasteurella haemolytica was renamed Mannheimia haemolytica. The name change was based on the taxonomic features of the organism from other closely related organisms, in particular Pasteurella multocida.. The differences noted were identified and described by Dr. Mannheim in 1974. The familiarity of the past nomenclature and the lack of familiarity for the new nomenclature suggests that the more commonly recognized name of Pasteurella haemolytica should be used throughout this document. Scientific evidence suggests that the disruption of the normal homeostatic mechanisms of the pulmonary vasculature to beta adrenergic agents may be part of the etiology of pneumonic pasteurellosis. The dynamics and kinetics of the involvement of the beta receptors, following prophylactic vaccination and in the disease state, has yet to be fully investigated with respect to the events associated with pneumonic pasteurellosis. Evaluation of the time frame of the onset and duration of the events associated with the disruption of pulmonary vascular beta adrenergic receptor mechanisms revealed that an escalating level of dysfunction occurs over the first 24-48 hour period after exposure to parenteral Pasteurella haemolytica and lasts for at least 21 days. A component of P.haemolytica organism or contained in the vaccine using the organism is likely associated with the disruption of vascular beta adrenergic mechanism. This factor is, as yet, not specifically identified, however the likely culprit is the lipid A moiety of the endotoxin. Using the well defined and purified Escherichia coli endotoxin, trials were run to examine the effect of endotoxin on the pharmacological response of vascular associated beta adrenergic receptor mechanisms. The effects of Escherichia coli endotoxin, administered parenterally, on beta adrenergic receptor mechanisms were pharmacologically indistinguishable from those effects following parenterally administered Pasteurella haemolytica. The nature of the disruption in the beta adrenergic receptor remains a mystery. The receptor mechanism involves at least two second messengers to initiate vascular relaxation. Initial activation of the beta adrenergic receptor with a beta selective drug starts a cascade of events involving adenylylate cyclase and cyclic adenylylate monophosphate (cAMP) and nitric oxide. A disruption in the receptor mechanism, as a result of the parenteral administration of Pasteurella haemolytica, which is "upstream" of adenylyl cyclase, would result in a diminished amount of cAMP when compared to the unvaccinated negative controls. An investigation of cAMP accumulation, at the receptor level was inconclusive. The assessment of some previously used vaccines has demonstrated that there is an, as yet unidentified virulence factor, associated with these vaccines that results in the pharmacological disruption of beta adrenergic receptor mechanisms. Two newer vaccines, Once PMH® and One Shot® have been evaluated and there is evidence to suggest that these currently used vaccines also have the ability to disrupt beta adrenergic receptor mechanisms in rats. The effects of parenteral P. haemolytica on the alpha-2 adrenergic receptor mechanism, is described. The alpha-2 receptor mechanism, unlike the beta receptor mechanism appears to increase the amount of vasoconstriction. The possibility that the alpha-2 adrenergic receptor could also mediate vasorelaxation under certain conditions was investigated. The evidence suggests that in the presence of high alpha-1 mediated vascular tone, the alpha -2 receptor can cause vasorelaxation. Evidence, from other scientists active in this area of investigation, suggests that a vasorelaxation response may be mediated by nitric oxide. Elimination of the nitric oxide mediated relaxation may offer an explanation for the increased vasoconstriction noted with alpha-2 selective drugs after exposure to parenteral P. haemolytica. Finally, the importance of the beta adrenergic receptor to the disease process is addressed by elucidation of one of the mechanisms by which Micotil 300® (tilmicosin phosphate) acts to improve cattle with symptomatic pneumonic pasteurellosis. The rapid improvement of animals on Micotil 300®, with-in 24 hours suggests that there is a mechanism beyond the antimicrobial effect of the drug that mediates the clinical improvement. Evaluation of the effect of Micotil 300® demonstrates a pharmacologically measurable amount of beta adrenergic activity with respect to the bovine pulmonary artery and vein. Based on the conclusions drawn as a result of these experiments, the adrenergic system in general, and the beta adrenergic system in particular are important to the development of pneumonic pasteurellosis in cattle. The beta adrenergic system is affected by endotoxin. Further, these receptors maybe responsible for the mediation of the pathological and clinical signs associated with pneumonic pasteurellosis. In conclusion, these investigations have suggested, that it is likely that a disruption in the homeostatic mechanisms mediated by the beta and alpha-2 adrenergic receptors are intimately involved in the development of post vaccination receptor failure as well as the pathophysiology associated with pneumonic pasteurellosis in cattle. Ph. D.

A cDNA clone encoding a turkey intestinal peptide transporter, tPepT1, was isolated from a turkey small intestinal cDNA library by screening with our chicken PepT1 (cPepT1) cDNA probe. The tPepT1 cDNA is 2,921-bp long and encodes a 79.4 kDa protein of 714 amino acids (AA) with 12 predicted transmembrane domains. The isoelectric point (pI) of tPepT1 is 5.9, which is much lower than that of PepT1 cloned from chicken (pI = 7.5) and other species. The AA sequence of tPepT1 is 94.3% identical to cPepT1 and ~ 60% identical to PepT1 from rat, sheep, rabbit, and human. Using a two-electrode voltage-clamp technique in Xenopus oocytes expressing tPepT1, Gly-Sar transport was pH dependent, but independent of Na+ and K+. For the dipeptides Gly-Sar and Met-Met, the evoked inward currents indicated that the transporter was saturable and had a high affinity for these substrates. However, transport of the tetrapeptide, Met-Gly-Met-Met, exhibited a possible substrate inhibition. To study developmental regulation of PepT1 in broiler and turkey embryos, 12 Nicholas turkey or Cobb x Cobb broiler embryos (six males and six females) were sampled daily from 5 d before hatch to the day of hatch (d 0). The abundance of PepT1 mRNA in the small intestine was quantified densitometrically from northern blots after hybridization with full-length cPepT1 and tPepT1 cDNA as probes. There was a quadratic increase (P < 0.001) in PepT1 mRNA abundance with age in turkey and broiler embryos. The relative increase in abundance of PepT1 mRNA in intestinal tissue from 5 d before hatch to d 0 was much less in the turkey than in the broiler (3.2-fold vs 14-fold). The dramatic increase in PepT1 mRNA abundance indicates a developmental regulation of the PepT1 gene and that there may be a crucial role for PepT1 in the neonatal chick and poult. Master of Science

Parkinson's disease (PD) is a debilitating motor disorder that primarily afflicts older individuals (> 50yrs). Although its cause is unknown, many factors are thought to contribute to the disease. There is growing epidemiological evidence supporting a link between pesticide exposure and PD. The present immunohistochemical study was undertaken to characterize the role of insecticide exposure in the etiology of idiopathic PD. The insecticides selected for study were the pyrethroid permethrin (PE) and the organophosphate chlorpyrifos (CP), both of which possess properties that could damage or disrupt the nigrostriatal pathway, which is the principal neurodegenerative target in PD. The present study examined possible alteration of the amount of dopamine re-uptake transporter protein (DAT), within the striatum of the C57BL/6 mouse, using DAT antibodies, following low (0.8, 1.5 & 3.0 mg/kg) and high (200 mg/kg) doses of PE, respectively. Possible nigrostriatal terminal degeneration was examined using antibodies to tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, following treatment with 50 mg/kg of CP alone or in combination with the high dose of PE. For both the high dose of PE alone and for the combined PE/CP treatment, glial fibrillary acidic protein (GFAP) antibodies were used to examine the possibility of non-degenerative tissue injury. Groups of matched treated/vehicle-control mice received three IP injections of the insecticide/dose of interest over a 2-week period. Counts of immunoreactive (IR) neuropil in the dorsolateral striatum were made from four pre-selected fields per striatal tissue section. Counts were compared between matched sections, processed on the same slide, from a treated mouse and its vehicle control. A mean difference score, across slides, for each treated/vehicle control pair was determined. All low dose PE groups showed a trend of decreased DAT IR neuropil, but only the 3.0mg/kg group showed a statistically significant reduction (p

To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken cDNA library. The cDNA was 2,914-bp and encoded a protein of 714 amino acid residues. Twenty-three di-, tri-, and tetra-peptides were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary (CHO) cells. For most di- and tripeptides tested, the Kt was in the micromolar range, except Lys-Lys and Lys-Trp-Lys. Northern analysis demonstrated that cPepT1 is expressed strongly in the small intestine, and at lower levels in kidney and cecum. These results demonstrated the presence and functions of a peptide transporter in chickens. cPepT1 mRNA abundance was evaluated in response to developmental and dietary regulations. In Experiment 1, eggs at incubation day 18 (E18) and Cobb chicks after hatch (d 0) were sampled before treatments. Three groups of chicks were fed diets containing 12, 18, or 24% crude protein (CP). Feed intake of chicks fed the 18 or 24% CP diets was restricted to that of chicks fed the 12% CP diet. In Experiment 2, a fourth group with free access to the 24% CP diet was added. cPepT1 mRNA abundance was quantified from northern blots. By d 0, there was a 50-fold increase in cPepT1 mRNA abundance compared with E 18. In chicks fed the 12% CP diet, cPepT1 mRNA abundance decreased throughout the 35 d. Chicks fed 18 or 24% CP diets showed an increase in cPepT1 mRNA abundance with time. In chicks with free access to the 24% CP diet, cPepT1 mRNA decreased until d 14 but returned to an intermediate level at d 35. Our results indicate that cPepT1 mRNA is regulated by both dietary protein and developmental stage. To investigate the kinetics of an ovine peptide transporter (oPepT1), CHO cells were transfected with oPepT1 cDNA. Uptake of Gly-Sar by transfected cells was pH-dependent, concentration-dependent, and saturable. Competition studies showed that all di-, tri-, and tetra-peptides inhibited uptake of Gly-Sar. Pretreatment of the cells with staurosporine resulted in an increase in peptide transport. This increase was blocked by pretreatment with PMA. The results indicate that protein kinase plays a role in oPepT1 function. Ph. D.

Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. Ph. D.

Certain organophosphorus compounds have the ability to induce a delayed neuropathic condition in sensitive species termed organophosphorus compound-induced delayed neurotoxicity (OPIDN). Esteratic changes associated with OPIDN have been successfully modeled in vitro. The physical characteristics of lesion development in OPIDN including the mode of nerve cell death, cytotoxic initiator and effector molecules, and cytoskeletal involvement have received little in vitro investigation. This dissertation evaluated the mode of cell death (apoptosis versus oncotic-necrosis), and cell cycle, cytoskeletal, nuclear, and mitochondrial alterations induced by OP compounds in SH-SY5Y cultures, an in vitro human neuroblastoma model. The distribution of in vivo neural degeneration in white Leghorn hen models was also assessed as a prelude to validating the mode of OP compound-induced in vivo neural cell death. These endpoints were evaluated by utilizing flow cytometry, spectrophotometry, gel electrophoresis, immunohistochemistry, light, and electron microscopy. Initial data gathered on culture parameters revealed that the mitotic status, basal rates of cell death, and total culture density were dependent on the condition of the media and the initial seeding density. Subsequent in vitro investigations used standardized culture conditions with OP compounds (diisopropylphosphorofluoridate (DFP), paraoxon, parathion, phenyl saligenin phosphate (PSP), tri-ortho-tolyl phosphate (TOTP), and triphenyl phosphite (TPPi); 1uM - 1mM). These studies revealed that OP compounds altered the cell cycle phase, decreased the amount of intracellular f-actin, altered the mitochondrial membrane potential, and induced caspase-3 activation and nuclear partitioning characteristic of apoptosis. The amount of change in these parameters was strongly dependent on the OP compound, the length of incubation time, and the presence of modulators of cytotoxicity such as phenylmethylsulfonyl fluoride (PMSF), carbachol, Ac-DEVD-CHO, Ac-IETD-CHO, and cyclosporin A. Preliminary in vivo experiments designed to validate in vitro results revealed neural degeneration involving fibers, terminals, and cell soma in spinal cord and brain tissue of PSP- and TPPi- exposed hens. The distribution and magnitude of these changes were contingent on the OP compound and length of time post-exposure. Subsequent experiments designed to evaluate the mode of cell death in these tissues revealed little evidence of either necrosis or apoptosis. These results, therefore, did not support or refute in vitro observations. Many OP compound-induced subcellular alterations have been demonstrated in our in vitro SH-SY5Y neuroblastoma model. Even though the mode of cell death observed in SH-SY5Y cells was not validated in in vivo experiments, in vitro observations nonetheless provide stimulating areas to further research the mechanisms of OPIDN and OP compound-induced cell death. Ph. D.

PCBs are known to cause thyroid disruption in laboratory rats and are thought to be the causal agent in thyroid gland alterations in herring gulls in the Great Lakes. This study examined the regulation of thyroid hormone supply during development in (1) domestic chicken embryos (Gallus domesticus) exposed to a specific dioxin-like PCB congener (PCB-126) and (2) herring gull (Larus argentatus) embryos and pre-fledglings from Great Lakes sites with different chemical pollutant exposures. Specifically, PCB effects on thyroid status were evaluated by measuring plasma thyroid hormone concentrations and brain type II 5'D activity (to determine if PCB exposure was associated with alteration in brain 5'D type II activity that could maintain local T3 supply to the brain). If PCB-126 and PCB mixtures altered thyroid function, we expected to see decreased plasma thyroid hormone concentrations and subsequent increases in 5'D-II activity. Chicken eggs were injected (into the air cell) before incubation with five dose levels (0.0512, 0.128, 0.32, 0.64, 0.8 ng/g) of PCB-126 (3,3, 4,4',5-pentachlorobiphenyl), or vehicle (sunflower oil); sampling was on day 20 of the 21-day incubation period. Studies on PCB-treated embryos included a preliminary study and a larger study encompassing a serious of smaller studies. Herring gull embryos (at pipping, on day 25 of the 26 day incubation), and 28-day pre-fledgling chicks were sampled (for two field seasons) at several Great Lakes sites with different contaminant exposures (with Kent Island being the reference site). In PCB-treated chicken embryos, there were no statistically significant decreases in plasma T4 or T3 concentrations and no significant increases in brain 5'D-II activity in either the preliminary or the larger study. We found no clear pattern of altered thyroid function in herring gulls from polluted Great Lakes' sites. Plasma TH concentrations were not significantly decreased and 5'D-II activity did not significantly increase in birds from more contaminated sites in comparison to birds from Kent Island or sites with less contamination. Although pipped embryos from Strachnan Island had a significant increase in 5'D-II activity when compared to Kent Island, there were no differences in plasma TH concentrations, and brain 5'D-II activity was not significantly increased in birds from sites with greater PCB loads than Strachnan Island. Plasma T4 and T3 concentrations were significantly decreased in prefledglings from West Sister Island and Detroit Edison in comparison to Kent Island, but there was no subsequent increase in brain 5'D-II activity. The present study is the first to evaluate the potential effects of PCBs, alone and in a mixed environmental exposure, on circulating THs and brain 5'D-II activity in developing birds. Although thyroid function was not altered by the specific PCB congener used in my study or by exposure to environmental pollutants, more complete evaluations are needed before determining whether PCBs alter thyroid function in birds. Master of Science

We reported the primary structure, tissue distribution, and in vitro functional characterization of a peptide transporter, oPepT1, from ovine intestine. The ovine intestinal oPepT1 cDNA was 2,829 bp long encoding a protein of 707 amino acid residues with an estimated molecular size of 79 kDa, and a pI of 6.57. The cDNA contained a 79-bp 5' untranslated sequence and a 630-bp 3' untranslated sequence. The proposed oPepT1 protein was 77.9, 81.3, and 82.6 percent identical to PepT1 from rabbit, rat, and human, respectively. High stringency northern blot analysis demonstrated that oPepT1 is expressed strongly in the small intestine, at lower levels in the omasum, and at much lower levels in the rumen, but is not expressed in liver and kidney. The presence of the peptide transporter in the forestomach at such levels could provide amino acid nitrogen for the ruminant in a nutritionally significant manner. Transport function of oPepT1 was assessed by expressing oPepT1 in Xenopus oocytes using a two-electrode voltage-clamp technique. Overall, the in vitro transport characteristics of oPepT1 expressed in oocytes were similar to those of PepT1 from other species. The transport process is electrogenic and pH-dependent, but independent of Na+, Cl-, and Ca2+. It displayed a broad substrate specificity that transported neutral and charged dipeptides and tripeptides. All dipeptides and tripeptides examined evoked inward currents in a saturable manner, with an affinity constant (Kt) ranging from 20 mM to .6 mM for dipeptides and .15 to 3.0 mM for tripeptides. No responses were detected from tetrapeptides or free amino acids. Although many of the properties displayed by oPepT1 were similar to those of PepT1 from other species, some differences were noted. First, the isoelectric point of oPepT1 was lower than that of others, but the oPepT1 protein appeared to have the same biological activity as that of others at a physiological pH. Second, more potential phosphorylation sites for protein kinases were present in oPepT1. Third, compared with PepT1 from other species, oPepT1 has more negatively charged amino acids at its C-terminus. Ph. D.

Epidemiological and analytical studies have suggested that environmental exposure to neurotoxic insecticides may exist as a factor in the etiology of Parkinson's Disease (PD). This study has focused on two insecticides, dieldrin and heptachlor epoxide, members of the cyclodiene class of insecticides. The cyclodienes are environmentally persistent, and brain residues of these compounds are correlated with the occurrence of PD. Cyclodiene mode of action has been attributed to two mechanisms: 1) facilitation of neurotransmitter release (with specificity for release of dopamine) and 2) antagonism of the inhibitory neurotransmitter, GABA. In order to assess the relative contributions of these two mechanisms leading to toxicity, eletrophysiological studies were undertaken in murine striatal slices. Extracellular recordings of spontaneous nerve discharge were used to compare the effects of the cyclodienes and the prototypical GABA antagonist, picrotoxinin, upon striatal neurons. At low micromolar concentrations of cyclodiene, depression of firing, consistent with dopamine release and not GABA antagonism, was seen. Alternatively, application of the prototypical GABA antagonist, picrotoxinin, produced excitation in slices. Additionally, the inhibitory action of dieldrin was blocked by a dopamine receptor (D1) antagonist, fluphenazine, verifying that cyclodiene-released dopamine was responsible for the observed depression of striatal neurons. These results suggest that the ability of these cyclodienes to evoke neurotransmitter release may significantly contribute to the neurotoxicity of these cyclodienes in vivo. In light of this data, the neurotoxic potential of the cyclodiene insecticides must be reassessed, particularly within the scope of PD. Master of Science

A process for disposing of pesticide rinsewater generated from the rinsing of application equipment is being developed at Virginia Polytechnic Institute and State University. This process involves the sorption of pesticides onto an organic matrix followed by degradation in a composting environment. We are now evaluating the hazards that might be associated with land-applying composted pesticide waste. Diazinon was the first pesticide selected for evaluation, which consisted of two studies. The first used the earthworm species Eisenia foetida to evaluate the toxicity of soil amended with composted diazinon waste. The second study determined the bioavailability of delta-2-14C-diazinon and its degradation products to E. foetida in soil amended with composted delta-2-14C-diazinon. Results from the first study indicate that uncomposted diazinon sorbent and 30-day composted diazinon sorbent were toxic to E. foetida at sublethal and lethal levels. However, E. foetida exposed 60-day composted diazinon sorbent did not experience mortality or demostrate sublethal effects commonly associated with acetylcholinesterase inhibition. Earthworms exposed to diazinon that was uncomposted or composted for 30 days in the radiolabelled study experienced higher mortality than in the field study. After 30 and 60 days of composting 14C-diazinon became unextractably incorporated into organic matter and very little was mineralized. Earthworms were shown to accumulate radioactivity when exposed to soil amended with 60- day composted delta-2-14C-diazinon. The majority of this radioactivity was unextractably bound to earthworm tissue and that which was extractable contained only trace levels of delta-2-14C-diazinon. Based on the absence of toxicity in the field study and the low levels of 14C-diazinon present in earthworm tissues, 60 days of composting appears to greatly reduce the hazard that diazinon rinsate poses to E. foetida. Master of Science

The nutritional ecology of the black carpenter ant, Camponotus pennsylvanicus (De Geer) was investigated by examining macronutrient preference and particle consumption in foraging workers. The crops of foragers collected in the field were analyzed for macronutrient content at two-week intervals through the active season. Choice tests were conducted at similar intervals during the active season to determine preference within and between macronutrient groups. Isolated individuals and small social groups were fed fluorescent microspheres in the laboratory to establish the fate of particles ingested by workers of both castes. Under natural conditions, foragers chiefly collected carbohydrate and nitrogenous material. Carbohydrate predominated in the crop and consisted largely of simple sugars. A small amount of glycogen was present. Carbohydrate levels did not vary with time. Lipid levels in the crop were quite low. The level of nitrogen compounds in the crop was approximately half that of carbohydrate, and exhibited seasonal dependence. Peaks in nitrogen foraging occurred in June and September, months associated with the completion of brood rearing in Camponotus. In choice tests, foragers demonstrated a preference for sucrose, fructose, and glucose, the most common honeydew sugars. Sucrose was preferred over other sugars in laboratory and field tests. Consumption rates peaked at a concentration of 20%. Casein hydrolysate and processed fish products stimulated the most feeding in choice tests of protein foods. Though a variety of lipids of plant and animal origin were offered in both field and laboratory tests, they were generally ignored. No effect of time was observed during choice tests of macronutrient preference. Overall, nitrogenous food was collected four-fold more intensively than carbohydrate, in contrast to the results obtained from examinations of the crop contents. These data suggest that accessible nitrogen is limited in the environment. Workers readily consumed fluorescent microspheres 0.5 - 45 μm diameter. Forty-five μm microspheres were excluded from the crop. Particles 3 - 10 μm reached the crop, but were never seen in the mid- or hindguts of either major or minor workers. They also filled the infrabuccal pocket, where they were compacted into pellets. It is thought that the proventriculus contains such particles in the foregut, where they are eventually filtered from the ingluvium. Microspheres 1 μm or less were difficult to observe in the infrabuccal pocket, suggesting that they are not as effectively sequestered as larger particles. Microspheres smaller than 1 μm were seen in the mid- and hindgut of both worker castes, indicating that particles of this size are immune to the proventricular filter. Caste exerted an effect at one μm diameter, the threshold of filtering efficiency. One μm microspheres consumed by minor workers were detected in the mid- and hindgut, whereas one μm microspheres were never detected beyond the proventriculus in major workers. Ph. D.

Epidemiological studies implicate pesticide exposure as a possible etiologic factor in idiopathic Parkinson's Disease, which results from degeneration of nigrostriatal neurons, along with reduced levels of the neurotransmitter, dopamine. Behavioral and neurochemical analyses in C57BL6 mice were performed following a subchronic dosing regime with the organochlorine insecticide heptachlor or the pyrethroid deltamethrin. Results were compared to those induced by the established parkinsonian neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). At the end of the treatment period, mice were assessed for effects on behavior, as well as levels of striatal dopamine, nerve terminal respiration, and synaptosomal dopamine transport. The primary behavioral effect of deltamethrin was incoordination, while heptachlor caused hyperexcitability and increased locomotion. The major neurochemical effect observed for both compounds was upregulation of the presynaptic dopamine transporter (DAT) by 70% and 100% for deltamethrin and heptachlor, respectively. The insecticides exerted only modest effects on striatal levels of dopamine and its metabolite, dihydroxyphenylacetic acid. However, doses of heptachlor higher than those which caused induction of DAT (e.g. greater than or equal to 25 mg/kg), when administered subchronically, were found to cause convulsions in some animals and caused marked, dose-dependent depression of basal striatal tissue respiration rates. No synergism was observed between the effects of insecticides and MPTP. Enhanced transport was thought to be a compensatory effect from increased release of transmitters by the insecticides, in vivo. Striatal dopamine, GABA and glutamate nerve terminals were differentially sensitive to the releasing effects of heptachlor compared to cortical serotonin terminals, and responded in the following rank order of sensitivity: dopamine > GABA > glutamate > serotonin. Additional experiments to characterize the mechanism(s) by which cyclodienes facilitate release of neurotransmitters in synaptosomes demonstrated a lack of distinct Ca²⁺ component and no involvement of retrograde DAT activity, suggesting that released label was of vesicular origin, but did not require Ca²⁺. Insecticidal toxicants, such as organochlorines and pyrethroids, which augment dopamine release and increase the maximal rate of dopamine uptake, may inundate the cytosol of nigrostriatal neurons with high concentrations of free dopamine, which has been shown by other researchers to induce apoptosis and may thereby contribute to the development of Parkinson's disease. Funding for this work was provided under grant number HHHREP 94-01 by the Hawaii Heptachlor Foundation, a non-profit organization. The Hawaii Heptachlor Foundation may be contacted at the following address: Ocean View Center PH#3, 707 Richards St., Honolulu, HI 96813. Ph. D.

Bioremediation was studied in soils containing high concentrations of formulated chlorpyrifos (5 mg kg-1 Dursban® 4E) and atrazine (5 mg kg-1 AAtrex® 4L) using amendments including lignocellulosic sorbents, microbial nutrients (vegetable oil, corn meal and fertilizers), and microbial extracts from organic media previously exposed to these pesticides (chlorpyrifos and atrazine, respectively). Radiolabeled atrazine was used to examine the various dissipation routes in contaminated soil, also amended with lignocellulosic sorbents and microbial nutrients. Both chlorpyrifos and atrazine dissipation from contaminated soils was enhanced by organic-based material amendments. The half-lives of chlorpyrifos based on extractability for soils unamended and amended with vegetable oil and peat moss were 87 and 52 days, respectively. The half-lives of atrazine in unamended and amended soil (vegetable oil, peat moss and fertilizers) were 175 and 40 days, respectively. The leachability of chlorpyrifos from contaminated soil was dramatically reduced by 82% during the first 30 days of incubation in treatments amended with vegetable oil and peat moss while only a 28% of reduction in leachability occurred in the corresponding unamended controls. Only a slight reduction of atrazine leachability was detected in amended treatments after 120 days of incubation. Differences were found in the leachability of chlorpyrifos and atrazine when they were applied to soil either as technical grade or formulated material. The presence of surfactants and other adjuvants in formulated chlorpyrifos (Dursban® 4E) reduced chlorpyrifos leachability in contaminated soil. Chlorpyrifos leachability was reduced by 43% in the formulated chlorpyrifos treatments, whereas there was a negligible decrease in technical chlorpyrifos treated soil during the first 3 days after contamination. Atrazine extractability and leachability was not affected by its formulation (AAtrex® 4L). Amendments with lignocellulosic sorbents and nutrients decreased atrazine®s volatility from contaminated soils. After 16 weeks of incubation, less than 1% of 14C-atrazine was volatilized from incubated soils. Overall, after 16 weeks of incubation less than 4% of 14C-atrazine was mineralized and more radioactivity was recovered from amended treatments than unamended treatments as 14CO2. The major portion of radioactivity (62%) was associated with physisorbed atrazine represented by the ethylacetate extract I from unamended treatments while only 28% of initial applied radioactivity was recovered in the corresponding amended treatments. Based on the sum of radioactivity in humic and fulvic acids, approximately 14% of radioactivity was incorporated or chemisorbed atrazine and its metabolites in both unamended and amended treatments. Forty-five percent of the initially applied radioactivity was associated with alkali insoluble fraction in amended treatments but only 17% of the initially applied radioactivity was detected in the corresponding unamended treatments. Less than 2 % of initial activity associated with physisorbed portions of fulvic acids and alkaline insoluble fraction indicated as the radioactivity in methylene chloride and ethylacetate extract II . Over time, more radioactivity was associated with polar atrazine hydroxylated degradation products. Ph. D.

The populations of aphidophagous insects in low-spray and conventional programs were surveyed in Virginia apple orchards. Two sampling methods were utilized;aphid colony collection and beating tray collection. The low-spray blocks employed the use of mating disruption for control of the key direct pests, codling moth, Cydia pomonella (Linnaeus), and leafrollers. The abundance and diversity of aphid predators in low-spray mating disruption programs and conventionally controlled apple orchards was compared. Both sampling methods suggested that aphid predators were more plentiful in the mating disruption blocks. Several mating disruption blocks were found to accumulate significantly more predator-days and diversity than the matched control blocks. The reduction of insecticide input into the low-spray blocks may have allowed higher populations of aphidophagous insects to occur. Aphid populations in mating disruption blocks sometimes declined faster and had less resurgence than those in the conventionally controlled blocks. The tufted apple bud moth (TABM), Platynota idaeusalis (Walker), populations in four apple orchards in Winchester, Virginia, were compared for resistance to azinphosmethyl. These orchards were suspected by the growers to have resistant populations of TABM. Pheromone traps with insecticide incorporated into the adhesive were used to collect and test the moths. The results were not analyzed due to the low number of moths collected and high variability in mortality. Master of Science

In order to verify the research from this laboratory that sheep omasal epithelium contains mRNA encoding for a peptide transporter (s) and to determine di- to octapeptide transport capability, poly(A)+ RNA isolated from sheep omasal epithelium was injected into Xenopus laevis oocytes. Poly(A)+ RNA was functionally expressed in Xenopus oocytes 4 to 7 d post-injection. Peptide (5 di-, 10 tri-, 6 tetra-, 2 penta-, 1 hepta-, 1 septa-, 1 octapeptide) transport capability was measured by impaling oocytes with a microelectrode to monitor membrane potential (Vm). Oocytes were maintained in pH 5.5 buffer. Peptide transport was identified as being expressed when, in the presence of a buffered peptide substrate (1 mM), the oocyte membrane showed persistent depolarization (a more positive Vm). In the absence of peptide transport, the membrane became depolarized with the addition of buffered substrate, but rapidly repolarized to the resting potential. Peptide transport was expressed for some di-, tri-, and tetrapeptides. Measured depolarization ranged from 9.6 mV to 42.1 mV. Larger peptides were not transported by the oocytes. When transport expression was measured with the substrates in a pH 7.5 buffer, no transport occurred indicating that transport was dependent on a proton gradient. The data indicate that sheep omasal epithelium contains mRNA that code for a protein(s) capable of proton-dependent di-, tri-, and tetrapeptide transport. This provides further evidence that absorption of peptides from the ruminant stomach is possible. Master of Science