To examine the interactions of disease and pollution on amphibian populations around the world, I investigated the effect of infection on contaminant susceptibility in pickerel frog, Rana palustris, larvae. I conducted standard 48-hr toxicity tests to examine effect of trematode parasite, Echinostoma trivolvis, infection (0, 10, or 30 cercaria) on the susceptibility of pickerel frog tadpoles to the widely used organophosphate insecticide malathion. LC50 values ranged from 16.5 – 17.4 mg/L, within the range reported for other amphibian species. I found no differences in susceptibility to malathion among parasite treatments. Nevertheless, this crucial question remains to be tested in other amphibian host-parasite systems. Second, I studied the reverse interaction, the effect of pesticide exposure on susceptibility to parasite infection. I exposed pickerel frog embryos to low doses of malathion, then subjected morphologically normal tadpoles to E. trivolvis later in development. Malathion significantly decreased hatching success and viability rates at concentrations lower than previously documented for anuran embryos. After 7 wk of development in water with no malathion, tadpoles previously exposed to malathion as embryos suffered increased parasite encystment rates compared to controls. My research identifies embryonic development as a sensitive window and the potential for increased susceptibility to infection long after pesticide exposure has ceased. With potential for increased parasite prevalence from eutrophication and climate change, my data underscore the importance of understanding the reciprocal influences of parasites and pesticides in amphibians. / Master of Science

Threshold and non-threshold linear models that govern toxicology are challenged by an alternative model, hormesis. It is defined as low-dose stimulation and high-dose inhibition from a stressor. Insecticide-induced hormesis has been studied in a plethora of insect-insecticide models at biochemical, individual and population levels. This research focuses on the effects of sublethal concentrations of insecticides on reproductive responses of green peach aphid, Myzus persicae (Sulzer), at individual and population level besides regulation of stress, dispersal and developmental genes during hormesis. In laboratory studies, irrespective of the duration and route of exposure, sublethal concentrations of imidacloprid induced stimulations in fecundity of M. persicae but the nature of response differed intra- and trans-generationally. Fitness tradeoffs could be rendered due to declined fecundity in successive generations. However, continuous exposure to sublethal concentrations does not compromise overall fitness trans-generationally, considering recovered levels of fecundity as controls in successive generations and the total reproduction after four generations. Greenhouse experiments affirmed uncompromised fitness where reproductive stimulations were noted in aphids exposed to imidacloprid treated potato plants. Up- and down- regulation of stress, dispersal and developmental genes was noted during imidacloprid-induced hormesis in M. persicae that mirrored the reproductive responses in few instances. Global DNA methylation results emphasized the heritability of adapted traits trans-generationally via hypermethylation. Dispersal related genes (OSD, TOL and ANT) that are predominantly expressed in alates (about 2- to 5-fold) were affected in apterous aphids continuously exposed to sublethal concentrations of imidacloprid. No direct relation with the previously noted fecundity was established implying adaptive cellular stress response pathways might be triggered rather than normal regulatory processes due to low-dose imidacloprid exposure. At a biochemical level, a study noted that imidacloprid-induced hormesis concurrently stimulated juvenile hormone III (JH) production and fecundity in M. persicae. Precocene, an anti-JH, at sublethal concentrations induced reproductive stimulations in M. persicae. Gene regulation during precocene-induced hormesis mirrored imidacloprid results for few genes including FPPS, a JH precursor gene, with a higher magnitude of regulation. Considering these stimulatory effects that insecticide-induced hormesis at various biological hierarchies, causes for pest resurgence, hormesis could have ramifications from declines in natural enemy population.

The near absence of bed bugs from human dwellings for fifty or more years has left us with limited knowledge of its biology and few answers to eliminate populations. I explored a diverse set of objectives to answer key questions concerning bed bug biology and control. Major objectives were studies of circadian rhythmicity, pyrethroid resistance, sublethal effects of insecticides, synergism of pyrethroids, and evaluation of a pyrrole insecticides, chlorfenapyr. Additional studies included persistence of Borrelia in bed bugs after ingestion, and aggregation factors from feces. In the absence of host stimuli, insects were much more active in the dark than in the light. Nocturnal activity was periodical under continuous light conditions, which indicates that locomotion is endogenously generated by a circadian clock. Circadian rhythm was entrained to reverse dark-light regimes. Short–term starved adults moved more frequently than long-starved adults. These results suggest that starved bugs reduce locomotor activity as a strategy to conserve metabolic reserves. Pyrethroid resistance in C. lectularius was documented for the first time. Extremely high levels of resistance to deltamethrin and λ-cyhalothrin, was detected in populations collected in Kentucky and Ohio. The resistance ratios reported are among the highest documented in any arthropod. Evaluations of more than 20 populations from across the United States indicate that resistance to pyrethroid insecticides is widespread. Bed bugs avoided resting on surfaces treated with deltamethrin but not with chlorfenapyr. Video recordings of bed bugs showed that insects increased their activity when they contacted sublethal doses of deltamethrin. However, harborages treated with a deltamethrin remained attractive. A nearby heat source overcame avoidance to deltamethrin. The P450 inhibitor piperonyl butoxide (PBO) enhanced toxicity of deltamethrin to resistant bed bugs. However, the residual resistance after PBO treatment indicated that other resistance mechanisms are involved. The effectiveness of combining PBO with pyrethroids varied among populations, which indicates that this synergist is not a comprehensive solution to pyrethroid resistance. Chlorfenapyr was effective against pyrethroid resistant strains. While it does not cause quick knockdown, long residual activity and no avoidance behavior to dry residues appears to make this insecticide a useful tool for bed bug control.

Examination of the swimming capabilities of fish is increasingly being considered as an effective method for determining sublethal toxicity. Acute toxicant exposure is known to cause decreases in swim performance in fish but less is known about how developmental exposure can cause persistent effects that hinder swimming. In addition, little is known about how triglyceride levels fluctuate during fish swimming upon both acute and developmental exposure to toxicant. In this thesis, two studies, one acute and one developmental, were carried out using two different toxicants in order to address these issues.<p> In order to examine acute effects, adult zebrafish (Danio rerio) were exposed to ethanol vehicle or increasing concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24 h period. Following exposure, fish were placed in a swim tunnel for critical swimming speed (Ucrit) determination and swim motion analysis. Whole body triglyceride levels were then determined. Ucrit was decreased in a concentration dependent manner in both the 6 mg/L and 12 mg/L DNP exposure groups, with 6 mg/L DNP being considered sublethal and 12 mg/L approaching the LC50. A decrease in tail beat frequency was observed and is likely the main cause for the decrease in Ucrit in the DNP exposure groups. Triglyceride levels were elevated in a concentration dependent manner in the DNP exposure groups. This increase in triglyceride stores may be due to a behavioral adaption limiting swimming capabilities or due to a direct toxic action of DNP on lipid catabolism.<p> The second study examined whether developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would cause persistent toxic effects. Zebrafish embryos were exposed to dimethyl sulfoxide control or increasing concentrations of TCDD between 2-4 days post fertilization (dpf). At 5 dpf, cytochrome P450 1A (CYP1A) activity was determined. Fish were raised to 90 dpf with mortalities and deformities being recorded at 5 dpf, 10 dpf, and 90 dpf. At 90 dpf, fish were placed in swim tunnel and Ucrit , swimming motion, and aerobic scope (oxygen consumption rate during exercise minus oxygen consumption rate during rest) were determined. Following swimming, some fish were used for whole body triglyceride analysis while others were used for histological examination. Ucrit was shown to be decreased in the two highest sublethal TCDD exposure groups (0.1 and 1 ng/L) but not in the lowest TCDD exposure group (0.01 ng/L). The exact cause of the decrease in Ucrit is not known, but may be linked to the observed decrease in dorsal aorta diameter, an inability to mobilize triglyceride stores, behavioral adaptations limiting swimming, decreased body length, or a combination of these factors. This TCDD related defect in swimming ability is not due to any increases in gross deformity or mortality rates, nor does it appear that CYP1A induction is required to mediate the toxic effects. Thus, it appears that examination of swim performance may serve as an effective measure of both sublethal acute and developmental toxicities.

Examination of the swimming capabilities of fish is increasingly being considered as an effective method for determining sublethal toxicity. Acute toxicant exposure is known to cause decreases in swim performance in fish but less is known about how developmental exposure can cause persistent effects that hinder swimming. In addition, little is known about how triglyceride levels fluctuate during fish swimming upon both acute and developmental exposure to toxicant. In this thesis, two studies, one acute and one developmental, were carried out using two different toxicants in order to address these issues.<p> In order to examine acute effects, adult zebrafish (Danio rerio) were exposed to ethanol vehicle or increasing concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24 h period. Following exposure, fish were placed in a swim tunnel for critical swimming speed (Ucrit) determination and swim motion analysis. Whole body triglyceride levels were then determined. Ucrit was decreased in a concentration dependent manner in both the 6 mg/L and 12 mg/L DNP exposure groups, with 6 mg/L DNP being considered sublethal and 12 mg/L approaching the LC50. A decrease in tail beat frequency was observed and is likely the main cause for the decrease in Ucrit in the DNP exposure groups. Triglyceride levels were elevated in a concentration dependent manner in the DNP exposure groups. This increase in triglyceride stores may be due to a behavioral adaption limiting swimming capabilities or due to a direct toxic action of DNP on lipid catabolism.<p> The second study examined whether developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would cause persistent toxic effects. Zebrafish embryos were exposed to dimethyl sulfoxide control or increasing concentrations of TCDD between 2-4 days post fertilization (dpf). At 5 dpf, cytochrome P450 1A (CYP1A) activity was determined. Fish were raised to 90 dpf with mortalities and deformities being recorded at 5 dpf, 10 dpf, and 90 dpf. At 90 dpf, fish were placed in swim tunnel and Ucrit , swimming motion, and aerobic scope (oxygen consumption rate during exercise minus oxygen consumption rate during rest) were determined. Following swimming, some fish were used for whole body triglyceride analysis while others were used for histological examination. Ucrit was shown to be decreased in the two highest sublethal TCDD exposure groups (0.1 and 1 ng/L) but not in the lowest TCDD exposure group (0.01 ng/L). The exact cause of the decrease in Ucrit is not known, but may be linked to the observed decrease in dorsal aorta diameter, an inability to mobilize triglyceride stores, behavioral adaptations limiting swimming, decreased body length, or a combination of these factors. This TCDD related defect in swimming ability is not due to any increases in gross deformity or mortality rates, nor does it appear that CYP1A induction is required to mediate the toxic effects. Thus, it appears that examination of swim performance may serve as an effective measure of both sublethal acute and developmental toxicities.

Agriculture ditch management (i.e. removal of vegetation and/or sediments) may disturb native wildlife, such as amphibian bioindicator species. The objective of this thesis was to determine whether ditch management affected northern leopard frogs (Lithobates pipiens). Twelve embryo and tadpole health endpoints were compared in vegetated and managed (treeless or dredged) ditches using in situ cages in 2018 and 2019, while water quality was monitored. The managed treatment did not negatively affect frog health compared to the vegetated treatment. The significantly faster development and larger body size due to warmer water temperatures at the managed treatment were likely advantageous. The water quality was typical of the region regardless of the ditch treatment and the pesticide mixture detected had limited toxicity. The main effects of the studied ditch managements on resident L. pipiens (and potentially other amphibians) appeared sub-lethal, likely beneficial, and mainly the result of temperature differences. Although poorer embryo health was observed at the vegetated treatment, it was likely unrelated to ditch management and possibly due to high specific conductivity causing premature hatching and mortality. Ditch management may be less important for frog health than the surrounding land use, and efforts should be directed at increasing suitable habitat for amphibians in agroecosystems. La gestion des fossés agricoles (plus spécifiquement l'élimination de la végétation des bandes riveraines et /ou le dragage des sédiments) peut perturber la faune indigène, comme les espèces bio-indicateurs d'amphibiens. L'objectif de cette thèse était de déterminer si la gestion des fossés affectait la grenouille léopard du Nord (Lithobates pipiens). Douze paramètres d'évaluation de la santé des embryons et des têtards ont été comparés dans des fossés végétalisés et gérés (sans arbres ou dragués) à l'aide de cages expérimentales in situ en 2018 et 2019, pendant que la qualité de l'eau a été suivie. Le traitement géré n'a pas eu d'effets négatifs sur la santé des grenouilles par rapport au traitement végétalisé. Le développement beaucoup plus rapide et la taille corporelle plus grande en raison des températures plus chaudes de l'eau lors du traitement géré étaient probablement avantageux. La qualité de l'eau était typique de la région quel que soit le traitement du fossé et le mélange de pesticides détecté avait une toxicité limitée. Les principaux effets de la gestion des fossés étudiés sur L. pipiens résidents (et potentiellement d'autres amphibiens) semblaient sublétaux, probablement bénéfiques, et résultaient principalement de différences de température. Bien que la santé des embryons ait été moins bonne lors du traitement végétalisé, elle n'était probablement pas liée à la gestion des fossés et pourrait être due à une conductivité spécifique élevée entraînant une éclosion prématurée et la mortalité. La gestion des fossés peut être moins importante pour la santé des grenouilles que l'utilisation des terres environnantes, et les efforts de protection devraient être dirigés vers une augmentation générale de l'habitat approprié pour les amphibiens dans les agroécosystèmes.

Neonicotinoids are primarily used in agriculture where they are applied as seed coatings, foliar sprays, and soil drenches or through drip irrigation. In urban areas neonicotinoids are used in home garden products and tree treatments. The maximum foraging ranges of honey bees are usually 10 – 15 km (median distances are 1 – 6 km) from the hive. Hence bee exposure to neonicotinoids is dependent upon the land use type within limited foraging distances from the hive. However, there are virtually no data showing levels of neonicotinoid use in urban areas and few studies have been done to compare urban and agricultural exposure. Several neonicotinoids have shown various toxic effects on pollinators and particularly honey bees. Honey bees have a limited arsenal of detoxification proteins to withstand neonicotinoid exposure, which makes them more sensitive and less able to develop tolerance to these insecticides compared to other insects. Sublethal exposure of honey bees to neonicotinoids can cause behavioral disturbances, orientation difficulties, impairment of social activities, and respiratory pattern changes. These behavioral changes can cause insufficient foraging behavior in honey bees due to the sublethal effect of neonicotinoids, thus putting the colony at risk of food shortage and eventually collapse. My objectives were to (1) develop a highly sensitive and selective, multi-residual analytical method for neonicotinoids in honey bee and pollen samples, (2) investigate the impacts of land use type (agriculture vs. urban) on the exposure of honey bees to neonicotinoid, (3) investigate the sublethal effect of imidacloprid on honey bees’ behavioral performance, and (4) investigate the sublethal effect of field-realistic concentrations of imidacloprid on honey bees’ metabolism at different ambient temperatures.To address my first objective (Chapter 2), I tested three sample cleanup methods (silica SPE, NH2-silica SPE, and Z-Sep SPE) based on solid phase extraction (SPE), which were investigated for determination of neonicotinoid insecticides and selected metabolites in honey bee and pollen samples by LC-MS/MS. Samples were extracted by hexane and ethyl acetate and then cleaned up with a SPE cartridge packed with silica gel, which showed a better cleanup efficiency compared to the aminopropyl silica SPE and zirconium-based sorbents method. Matrix effects of the three cleanup methods were evaluated and compared. Silica gel showed the highest analyte recoveries and method detection limit for this method were 2.0 to 9.1 μg/kg for honey bees and 2.4 to 4.7 μg/kg for pollen. Recovery studies were performed at three spiking levels (10, 60, and 120 μg/kg) and ranged from 78 to 140% with RSDs between 3 to 18% in honey bees and 83 to 124% with RSDs between 3 to 17% in pollen. The silica gel SPE cleanup method was then applied using honey bee and pollen samples that were collected from different apiaries. To address my second objective (Chapter 3), I analyzed honey bee and beebread (pollen) samples from apiaries in agricultural, developed, and undeveloped areas that were collected during two years in Virginia to assess if landscape type or county pesticide use were predictive of honey bee colony exposure to neonicotinoid insecticides. Trace concentrations of the neonicotinoid imidacloprid were detected in honey bees (3 out of 84 samples, 2.02 – 3.97 ng/g), while higher levels were detected in beebread (5 out of 84 samples, 4.68 – 11.5 ng/g) and pollen (3 out of 5 pollen trap samples, 7.86 – 12.6 ng/g). Imidacloprid was only detected in samples collected during July and August and were not detected in honey bees from hives where neonicotinoids were detected in pollen or beebread. Number of hives sampled at a site, county pesticide use, and landscape characteristics were not predictive of neonicotinoid detections in honey bees or beebread (all P>0.05). Because of the low detection rates, field surveys may underestimate honey bee exposure to field realistic levels of pesticides or the risk of exposure in different landscapes. Undetectably low levels of exposure or high levels of exposure that go undetected raise questions with regard to potential threats to honey bees and other pollinators. To address my third objective (Chapter 4), I investigated the effects of sub-lethal concentrations of imidacloprid on late fall forager honey bees’ behavior by accessing their activity levels and walking performance after being fed ad libitum with six different concentrations (2 – 125 μg/kg) of imidacloprid-dosed syrup for up to 48 hours in laboratory. Honey bee activity levels and motivation to move after being released into a UV light illuminated tunnel decreased significantly as dosages of neonicotinoid in their diet increased. However, their walking speeds were not significantly affected by imidacloprid. The behavioral changes I observed in honey bees chronically exposed to neonicotinoid via diet could negatively affect individual honey bee performance of their hive duties and consequently, colony survival during late fall and winter. To address my fourth objective (Chapter 5), I measured honey bee (Apis mellifera) foragers’ CO2 production rates at different temperatures (25, 30, or 35°C) after they consumed syrup dosed with a field realistic (5 μg/L) or high (20 μg/L) concentration of a neonicotinoid insecticide (i.e. imidacloprid) for 48h. We found that imidacloprid exposure significantly disrupted honey bees’ non-flight metabolic rates and there was a significant interaction between imidacloprid dosage and ambient temperature. Honey bee foragers dosed with 5 μg/L imidacloprid displayed higher average metabolic rates and those dosed with 20 μg/L imidacloprid displayed similar average metabolic rates compared to the corresponding control group across all temperatures. Exposure to field realistic concentrations of neonicotinoid may have a higher energetic cost for honey bees at 25℃ than at higher ambient temperatures. Disrupted energy costs in honey bees fed imidacloprid might be due to the thermoregulation, nerve excitation, or detoxification processes. Metabolic rate changes caused by pesticide exposure could result in less available energy for honey bees to perform hive duties and forage, which could negatively affect colony health.

Previous findings showed that Listeria monocytogenes exhibits higher heat tolerance to thermal exposure at 60°C when pre-exposed to sublethal heat stress at 48°C for 30/60 min. The objective of this study was to investigate the morphological changes that occurred in L. monocytogenes serotype 1/2a cells as visualized by transmission electron microscopy (TEM) after exposure to sublethal heat stress at 48°C for 60 min and in combination with lethal concentration of carvacrol for 30 min. The TEM micrographs revealed thickening of cell wall and cell membrane, and clumping of cytoplasm when subjected to sublethal heat stress followed by carvacrol treatment. These studies indicate that L. monocytogenes cells when adapted to heat stress may alter its morphology to protect themselves against carvacrol. Further studies will investigate the effect of increased contact time with carvacrol and with other essential oils on the ultrastructural changes in L. monocytogenes cells adapted to heat stress.

The objective of this study was to investigate the differences in survival of L. monocytogenes EGD (Bug600) (serotype 1/2a) in various disinfectants and essential oils after sublethal heat stress at 48°C for 60 min. The survival of heat stressed L. monocytogenes cells was decreased in lethal acid (HCl or H3PO4 pH 2.5), lethal oxidative stress (H2O2 1000 ppm) and lethal quaternary ammonium compounds (QAC 2.5-3.5 ppm) compared to non-stressed control cells. By contrast, the survival of heat stressed L. monocytogenes cells was higher in lethal alkali (NaOH or KOH pH 12) than the control cells. Also, the survival of heat stressed L. monocytogenes cells was higher in lethal carvacrol (428 ppm) and bay oil (1100 ppm) but was decreased in lethal red thyme oil (300 pm) compared to control. These findings indicate that the heat stressed cells of L. monocytogenes are not easily killed by alkali-based disinfectants and essential oils containing carvacrol and bay oil. Therefore, disinfectants and essential treatments should be carefully considered when heat stressed cells of L. monocytogenes may be present.

Listeria monocytogenes is an important foodborne pathogen that can adapt to stress conditions to persist in food processing environments. Our findings show that there was a development of low-level tolerance to quaternary ammonium compound (QAC) and antibiotics ciprofloxacin and trimethoprim in L. monocytogenes after sublethal adaptation to QAC. Using eight L. monocytogenes strains, we determined the changes in MIC, growth rate, and surviving CFU for homologous and heterologous stress-response after sublethal exposure to daily cycles of fixed or gradually increasing concentration of QAC. Three main findings were observed: (1) Short-range MIC of QAC, ciprofloxacin, and trimethoprim increased by 1.6 to 2.3, 1.5 to 2.9, and 1.7 to 2.5 fold against QAC-adapted phenotypes of L. monocytogenes as compared to the non-adapted cells; (2) QAC-adapted phenotypes of L. monocytogenes exhibited a significant increase in growth rate by 2.5 to 7.1, 2.1 to 6.8, or 1.4 to 4.8 fold in the broth model containing QAC, ciprofloxacin, or trimethoprim respectively, as compared to non-adapted cells; and (3) QAC-adapted phenotypes of L. monocytogenes exhibited a significant increase in survival by 1.5 to 4, 2.2 to 4.3, or 1.3 to 3.2 log CFU/ml in the agar model containing QAC, ciprofloxacin, or trimethoprim respectively, as compared to non-adapted cells (P < 0.05). There were strain differences in QAC-adapted phenotypes of L. monocytogenes for both homologous and heterologous stress-response with some strains exhibiting a significant increase in short-range MIC, growth rate, and survival while others exhibiting no changes as compared to non-adapted cells. These findings suggest the potential formation of low-level QAC-tolerant and antibiotic-tolerant phenotypes in some L. monocytogenes strains under residual QAC concentrations (where QAC may be used widely) and such cells if not inactivated may survive longer to increase food safety risk.