Search

The productivity and survival of managed honey bee colonies is negatively impacted by a diverse array of interacting factors, including exposure to agrochemicals, such as pesticides. This study investigated the use of volatile heterocyclic amine (HCA) compounds as potential short-term repellents that could be employed as feeding deterrents to reduce the exposure of bees to pesticide-treated plants. Parent and substituted HCAs were screened for efficacy relative to the repellent N,N-diethyl-meta-toluamide (DEET) in laboratory and field experiments. Additionally, electroantennogram (EAG) recordings were conducted to determine the level of antennal response in bees. In video-tracking recordings, bees were observed to spend significantly less time with an HCA-treated food source than an untreated source. In a high-tunnel experiment, the HCA piperidine was incorporated in a feeding station and found to significantly reduce bee visitations relative to an untreated feeder. In field experiments, bee visitations were significantly reduced on melon flowers (Cucumis melo L.) and flowering knapweed (Centaurea stoebe L.) that were sprayed with a piperidine solution, relative to untreated plants. In EAG recordings, the HCAs elicited antennal responses that were significantly different from control or vehicle responses. Overall, this study provides evidence that HCAs can deter individual bees from food sources and suggests that this deterrence is the result of antennal olfactory detection. These findings warrant further study into structure–activity relationships that could lead to the development of short-term repellent compounds that are effective deterrents to reduce the contact of bees to pesticide-treated plants. Published version

Multiple interacting stressors negatively affect the survival and productivity of managed honey bee colonies. Pesticides remain a primary concern for beekeepers, as even sublethal exposures can reduce bee immunocompetence, impair navigation, and reduce social communication. Pollinator protection focuses on pesticide application guidelines; however, a more active protection strategy is needed. One possible approach is the use of feeding deterrents that can be delivered as an additive during pesticide application. The goal of this study was to validate a laboratory assay designed to rapidly screen compounds for behavioral changes related to feeding or feeding deterrence. The results of this investigation demonstrated that the synthetic Nasonov pheromone and its terpenoid constituents citral, nerol, and geraniol could alter feeding behavior in a laboratory assay. Additionally, electroantennogram assays revealed that these terpenoids elicited some response in the antennae; however, only a synthetic Nasonov pheromone, citral, and geraniol elicited responses that differed significantly from control and vehicle detections. Published version

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, 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. In the work described here, we have demonstrated the use of flock house virus (FHV) as a model system for virus infection in bees and revealed an important role for the regulation of the bee antiviral immune response by ATP-sensitive inwardly rectifying potassium (KATP) channels. We have shown that treatment with the KATP channel agonist pinacidil increases survival of bees while decreasing viral replication following infection with FHV, whereas treatment with the KATP channel antagonist tolbutamide decreases survival and increases viral replication. Our results suggest that KATP channels provide a significant link between cellular metabolism and the antiviral immune response in bees. This material is based upon work that is supported by the National Institute of Food and Agriculture, United States Department of Agriculture, under award number 2017-67011- 26048.

Honey bees (Apis mellifera) are the primary pollinators of major horticultural crops. Over the last few decades, a substantial decline in honey bees and their colonies have been reported. While a plethora of factors could contribute to the putative decline, pathogens, and pesticides are common concerns that draw attention. In addition to potential direct effects on honey bees, indirect pesticide effects could include alteration of essential gut microbial communities and symbionts that are important to honey bee health (e.g., immune system). The primary objective of this study was to determine the microbiome associated with honey bees exposed to commonly used in-hive pesticides: coumaphos, tau-fluvalinate, and chlorothalonil. Treatments were replicated at three independent locations near Blacksburg Virginia, and included a no-pesticide amended control at each location. The microbiome was characterized through pyrosequencing of V2–V3 regions of the bacterial 16S rRNA gene and fungal ITS region. Pesticide exposure significantly affected the structure of bacterial but not fungal communities. The bee bacteriome, similar to other studies, was dominated by sequences derived from Bacilli, Actinobacteria, α-, β-, γ-proteobacteria. The fungal community sequences were dominated by Ascomycetes and Basidiomycetes. The Multi-response permutation procedures (MRPP) and subsequent Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis indicated that chlorothalonil caused significant change to the structure and functional potential of the honey bee gut bacterial community relative to control. Putative genes for oxidative phosphorylation, for example, increased while sugar metabolism and peptidase potential declined in the microbiome of chlorothalonil exposed bees. The results of this field-based study suggest the potential for pesticide induced changes to the honey bee gut microbiome that warrant further investigation.

Honey bee colony loss continues to be an issue and no factor has been singled out as to the cause. In this study, we sought to determine whether two beekeeper-applied pesticide products, tau-fluvalinate and Fumagilin-B®, and one agrochemical, chlorothalonil, impact the nutrient levels in honey bee workers in a natural colony environment. Treatments were performed in-hive and at three different periods (fall, spring, and summer) over the course of one year. Bees were sampled both at pre-treatment and two and four weeks post-treatment, weighed, and their protein and carbohydrate levels were determined using BCA and anthrone based biochemical assays, respectively. We report that, based on the pesticide concentrations tested, no significant negative impact of the pesticide products was observed on wet weight, protein levels, or carbohydrate levels of bees from treated colonies compared with bees from untreated control colonies. Published version

A series of bis(n)-tacrines were used as pharmacological probes of the acetylcholinesterase (AChE) catalytic and peripheral sites of Blattella germanica and Drosophila melanogaster, which express AChE-1 and AChE-2 isoforms, respectively. In general, the potency of bis(n)-tacrines was greater in D. melanogaster AChE (DmAChE) than in B. germanica AChE (BgAChE). The change in potency with tether length was high in DmAChE and low in BgAChE, associated with 90-fold and 5.2-fold maximal potency gain, respectively, compared to the tacrine monomer. The optimal tether length for Blattella was 8 carbons and for Drosophila was 10 carbons. The two species differed by only about twofold in their sensitivity to tacrine monomer, indicating that differential potency occurred among dimeric bis(n)-tacrines due to structural differences in the peripheral site. Multiple sequence alignment and in silico homology modeling suggests that aromatic residues of DmAChE confer higher affinity binding, and the lack of same at the BgAChE peripheral site may account, at least in part, to the greater overall sensitivity of DmAChE to bis(n)-tacrines, as reflected by in vitro assay data. Topical and injection assays in cockroaches found minimal toxicity of bis(n)-tacrines. Electrophysiological studies on D. melanogaster central nervous system showed that dimeric tacrines do not readily cross the blood brain barrier, explaining the observed nonlethality to insects. Although the bis(n)-tacrines were not good insecticide candidates, the information obtained in this study should aid in the design of selective bivalent ligands targeting insect, pests, and disease vectors.

Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (kcat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC50>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC50 = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito. Published version

A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD50 bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance. Published version

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.

My dissertation and research focused on the effects of insecticides on squash bugs, its egg parasitoid, and pollinators in the production of cucurbits in Virginia. Plants in the cucumber family are dependent on insect pollination for successful fruit set, and are also susceptible to plant eating insects. Squash bugs are capable of transmitting cucurbit yellow vine decline, and their feeding can cause significant wilt and death in many varieties. To control for squash bug and other pests, growers commonly combine the application of broad-spectrum insecticides with the frequently applied prophylactic fungicides. Broad-spectrum insecticide applications are known to have negative effects on natural enemy populations, are capable of promoting insecticide resistance, and can have negative effects on pollinators if care in their use is not taken. Squash bugs have several natural enemies, but their predominant egg parasitoid is most effective at reducing damaging populations. The scelionid wasp Gryon pennsylvanicum Ashmead, is a prevalent egg parasitoid in Virginia and can be negatively affected by the application of broad-spectrum insecticides. Through survey efforts I found that G. pennsylvanicum is widely distributed throughout Virginia and is capable of high rates of egg parasitism (>90%). This is contrary to the 20% level previously assumed for the East Coast. I explored the effects of narrow-spectrum insecticides on the fate of the egg parasitoids, those developing in the host egg and emerged adults of G. pennsylvanicum. Contact assays showed that the insecticides λ-cyhalothrin and sulfoxaflor had caused high adult parasitoid mortality. As new insecticides get registered for use there is often concern about their effect on pollinators, specifically the European honey bee Apis mellifera L. I evaluated the use of large flight cages as a method to measure the sub-lethal effects of narrow-spectrum insecticides to honey bees, as a means to qualify risk. The method utilizes small colonies of honey bees (with stores of nectar and pollen) and their feeding at a treated sucrose solution after being trained to a feeder in an enclosed arena. This choice-test style behavioral experiment shows promise in qualifying the risks associated with insecticide exposure in the field. In the case of pyrifluquinazon, colonies repeatedly choose to avoid feeding at tainted feeders even after training with no other outside sources of food present. Further researching the sub-lethal behavioral effects that insecticides have on bees in a colony can help us better qualify their risk. Ph. D.

La Crosse virus (LACV) encephalitis is the most common and important endemic mosquito-borne disease of children in the U.S. with an estimated 300,000 annual infections. The disease is maintained in a zoonotic cycle involving the eastern treehole mosquito, Aedes triseriatus and small woodland mammals such as chipmunks and squirrels. The objectives of this study were 1) to conduct surveillance of LACV and other mosquito-borne viruses; 2) to evaluate the effect of virus infection on mosquito host-seeking and neurotransmitter levels, and 3) to determine the effectiveness of barrier sprays to control infected mosquito vectors. Our surveillance study demonstrated the involvement of an invasive species, Aedes japonicus, in the transmission cycle of Cache Valley virus (CVV). CVV is a mosquito-borne virus that is closely related to LACV. Thus, surveillance is a critical step in public health, providing pathogen distribution and frequency data as well as identifying and incriminating new vectors. LACV infection did not affect the host-seeking behavior of Ae. triseriatus females. Using high performance liquid chromatography with electrochemical detection (HPLC-ED), the levels of serotonin and dopamine were measured in infected and uninfected mosquitoes. Serotonin is known to affect blood-feeding and dopamine affects host-seeking. Serotonin levels were significantly lower in LACV-infected mosquitoes but dopamine levels were unaffected by virus. A previous study found that LACV infection caused an alteration in mosquito blood-feeding in a way that could enhance virus transmission. This work showed that LACV infection can reduce the level of serotonin in the mosquito, promoting virus transmission through altered blood-feeding without impairing the vector's ability to locate a host. Standard CDC bottle assays were used to evaluate the efficacy of two pyrethroids and two essential oil sprays on LACV infected and uninfected mosquitoes. LACV-infected Ae. triseriatus females were more susceptible to both pyrethroids than uninfected ones. Infection status did not affect the susceptibility of Ae. albopictus to either pyrethroid. The essential oils were inconsistent in their effects. These results demonstrate that barrier sprays may be a viable part of a mosquito control program, not just to reduce the biting rate but to potentially reduce the virus-infected portion of the vector population. 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 decline of honey bee (Apis mellifera) colony numbers in recent years presents an economic and ecological threat to agriculture. One outstanding threat to honey bees is the unintended exposure to agricultural pesticides. Previous studies report that acute exposures to the common-use herbicide atrazine elicit oxidative stress in non-target insects; however, little information is currently available on the exposure risk of atrazine to honey bees. This project examined biochemical and molecular oxidative stress response markers of honey bees following laboratory and field treatments of atrazine. Laboratory experiments were conducted with honey bees exposed to increasing concentrations of atrazine for 24 h whereas hive experiments were conducted with bees exposed to one sub-lethal concentration of atrazine for 28 d. The overall antioxidant enzyme activities of atrazine-treated honey bees were decreased compared to the untreated honey bees in both the laboratory and hive experiments. After exposure to atrazine in the laboratory and field, semi-quantitative RT-PCR analysis of antioxidant-encoding genes reveals the differential expression of genes in atrazine-treated bees that are important for oxidative stress tolerance in the laboratory and field experiments. Here, we provide evidence that the laboratory and hive exposure of honey bees to the common-use herbicide atrazine results in oxidative stress responses that can compromise the health of bee colonies. The data will be discussed with regard to the protection of these pollinators against the untended exposure of agricultural pesticides. Master of Science in Life Sciences

Mosquitoes affect human health worldwide as a result of their ability to vector multiple diseases. Mosquitocide resistance is a serious public health challenge that warrants the development of improved chemical control strategies for mosquitoes. Previous studies demonstrate the mosquito blood-brain barrier (BBB) to interfere with the target-site delivery and action of anticholinesterase chemistries. The ATP-binding cassette (ABC) transporters are efflux proteins that assist in maintaining the BBB interface and serve as a first line of defense to mosquitocide exposures. To date, there are three subfamilies (ABC -B, -C, -G) of ABC transporters; however, knowledge of these chemistries interacting with mosquito ABC transporter(s) is limited. Here, I report that tacrine and bis(7)-tacrine are relative non-toxic anticholinesterases at solubility limits; however, the addition of verapamil enhances toxicity of both tacrine and bis(7)-tacrine to mosquitoes. Verapamil significantly increases the mortality of mosquitoes exposed to tacrine and bis(7)-tacrine compared to the tacrine- and bis(7)- tacrine-only treatments. Tacrine and bis(7)-tacrine reduce acetylcholinesterase activity in mosquito head preparations compared to the untreated mosquitoes; however, the addition of verapamil significantly increases the anticholinesterase activity of tacrine and bis(7)-tacrine compared to the tacrine-and bis(7)-tacrine-only treatments. Tacrine and bis(7)-tacrine increase ATPase activity in Aedes aegypti at lower concentrations compared to that of verapamil (Fig. 3). The differential increase in ATPase activity suggests that tacrine and bis(7)-tacrine are more suitable substrates for ABC transporter(s) compared to verapamil and, thus, provides putative evidence that ABC transporter(s) is a pharmacological obstacle to the delivery of these anticholinesterases to their intended target site. 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 continue to be a big threat to human health worldwide. Despite using various vector control methods, we lose a great number of lives to this malicious disease in tropical and subtropical countries each year. Not surprisingly, mosquito is considered as the deadliest animal on the earth, because mortality rates from mosquito-vectored infections only lag behind other major diseases such as HIV and tuberculosis. Current approaches of vector control are mostly limited to using insecticidal bed nets, thus novel techniques are required to prevent a staggering loss to human health and quality of life. Advances in the genome sequencing in the past decade have helped to uncover numerous secrets of diverse genomes. The genome of malaria mosquito Anopheles gambiae was first sequenced in 2002 and since then has been updated to include additional scaffolds, their orientations and correction of mis-assemblies. Yet, the greatest challenge remains in assembling the heterochromatin regions, that are repeat rich part and contain relatively low-gene density. Although previously neglected by scientific studies due to its characteristic paucity of genes, heterochromatin is now recognized to be crucial for several processes such as cell viability, chromosome pairing, meiosis, longevity etc. It is therefore not surprising that heterochromatin comprises of a significant portion of the genome in many species. The efforts to analyze the genome of malaria mosquito in order to identify potential new leads for vector control warrant a better understanding of the heterochromatin. Mosquitoes diploid chromosome number equal 6. While autosomes 2 and 3 are submetacentric and present in both sexes, females are homogametic with XX and males are heterogametic with XY sex chromosomes. To achieve a better understanding of the Anopheles heterochromatin, we investigated heterochromatic region of the X chromosome. Despite one arm of the X chromosome being completely heterochromatic, few studies have investigated the molecular content of this region. Protocols were developed for performing fluorescent in situ hybridization (FISH) on mitotic X chromosomes in An. gambiae. Using cytogenetics and molecular biology techniques, we characterized the X chromosome heterochromatin in members of the An. gambiae complex. Specific satellite DNA and 18S ribosomal DNA probes (major components of heterochromatin) were mapped to X chromosomes enabling their differentiation and characterization in the An. gambiae complex. Microarray studies have highlighted the importance of X chromosome during investigation of nascent species An. gambiae and An. coluzzii. Here for the first time qualitative differences in heterochromatin in between nascent species are described. Cytogenetic idiograms are developed as to include the molecular and qualitative differences between the species of the An. gambiae complex. These idiograms are expected to provide a better resolution of the X chromosome heterochromatin for comparison in major malaria vectors, closing some of the gaps present due to poor sequencing of unassembled repeat rich regions in An. gambiae complex. The current understanding of Y chromosome for transgenic manipulation is poor and limited to very few genes. Due to its near total heterochromatic composition, it is the hardest part of the genome to assemble. In collaboration with other researchers, the Y chromosome content was characterized among sibling species of the An. gambiae complex. Our data revealed the swift changes the Y chromosome has undergone in a relatively short evolutionary time period. These include a rapid rate of turnover not only in heterochromatin but also in euchromatin. In addition to previously described repeats, a novel highly repetitive element called Zanzibar was discovered and mapped to the males of various Anopheles sibling species. Our data can form the basis for evolutionary studies in heterochromatin for male mosquitoes within the An. gambiae complex while also help identify novel targets to create successful transgenic male populations. Along with the X chromosome heterochromatin, to our knowledge this is the most extensive contribution to improve the understanding of mitotic chromosome heterochromatin in malaria mosquitoes. This study also investigated if epigenetics play role in mosquito development, fecundity and heterochromatin formation. DNA methylation, histone modifications and small noncoding RNAs are among the epigenetic mechanisms scrutinized in mammals. However, knowledge about epigenetic mechanisms and their effects is sparse in mosquitoes. A protocol for testing the various effects of epigenetics on different stages of malaria mosquito was developed. An epigenetic drug was utilized to probe the effects on immature and adult malaria mosquitoes. Different concentrations of DZNep, a histone methyltransferase inhibitor, were administered to An. coluzzii larvae. Total survivorship and pupation were compared for treated and untreated groups. The drug was also administered to adult blood feeding females to determine any effects on fecundity and egg morphology, revealing a negative association with an increase in drug concentration. A dose dependent decrease in SAH hydrolase concentration in An. coluzzii was also noticed. These results suggest epigenetics plays a critical role in mosquito pupation and ovarian development. Our work lays the groundwork for future investigations into the field of epigenetics in mosquitoes by revealing its effect on several important developmental stages in malaria mosquitoes. Although genomics and next-gen sequencing technology have come a long way in the last decade since the first Anopheles genome was sequenced, considerable gaps still exist in case characterization of heterochromatin function in an organism. Through our work, we have endeavored to elucidate a few of the major roles that heterochromatin may play in organization, evolution and adaptation of the malaria mosquitoes. Ph. D.

The insecticide imidacloprid, has been found to be highly effective in suppressing hemlock woolly adelgid, Adelges tsugae. Laricobius nigrinus is a predatory beetle released as a biological control of adelgids in the eastern U.S. This project was designed to develop a pest management strategy that utilizes both tactics concurrently within the same site. It will assess the efficiency of this strategy in reducing HWA populations and improving the health of hemlock forests. The project was started in 2010 and data were collected annually through 2016. The project spanned three sites in three different states (KY, WV, and TN). Results show that tree health has generally declined across all sites for each year. HWA population index values are highly variable and are more strongly influenced by the occurrence of low winter temperatures than by treatment effect. Cross-correlation analysis of tree health and HWA population, revealed characteristics of their temporal relationship. In two of the three sites, tree health lagged up to three years behind changes in HWA population, and HWA populations lagged approximately one year behind changes in tree health. L. nigrinus did not establish at any site as of 2016. The lack of sustained recovery of the beetle may be attributable to the occurrence of extremely cold temperatures during the winters of 2014 and 2015 which produced subsequent crashes in the HWA population at two of the three sites. In TN, the L. nigrinus population may have never established due to a decline in the HWA population shortly after release. Master of Science in Life Sciences

The incidence of mosquito-borne human diseases is increasing worldwide, with effective chemical control limited due to widespread insecticide resistance in the insect. Recent evidence also suggests that bacterial symbionts of mosquitoes, known to be essential in nutritional homeostasis and pathogen defense, may play a significant role in facilitating mosquitocide resistance. Here, I examined the metabolic detoxification and toxicity of two mosquitocides, propoxur and naled, and the capacity of bacterial symbionts to modify the detoxification of the mosquitocides and, thus, alter their toxic action in the yellow fever mosquito, Aedes aegypti. The insecticide synergists piperonyl butoxide (PBO), triphenyl phosphate (TPP), and S,S,S-tributyl phosphorotrithioate (DEF) were used to examine the metabolic detoxification and toxic action of the two mosquitocides in mosquito larvae. A significant increase in the toxicity of propoxur was observed when applied in combination with PBO; however, there was no corresponding decrease in AChE activity. Naled applied in combination with PBO resulted in a decrease in anticholinesterase activity (higher residual AChE activity) and a subsequent decrease in toxicity of the insecticide. This suggests that esterases play a major role in the metabolic detoxification of both insecticides in mosquito larvae. The acute toxicities of naled and propoxur to Ae. aegypti larvae were also studied following a reduction of bacterial symbionts with the broad-spectrum antibiotics gentamycin, penicillin, and streptomycin. Antibiotic-treated mosquito larvae showed increased susceptibility and a reduction in cytochrome P450 monooxygenase and general esterase activities when treated with naled and propoxur. A reduction of bacteria in mosquito larvae treated with broad-spectrum antibiotics, therefore, appears to affect the metabolic detoxification of standard-use mosquitocides, such as propoxur and naled. The results also suggest that the bacteria themselves may contain metabolic detoxification enzymes that are functionally similar to those in the mosquito larvae. Additional experiments, however, are needed to fully elucidate the contribution of bacterial symbionts in Ae. aegypti larvae in the metabolic detoxification of mosquitocides. Master of Science in Life Sciences

The Common bed bug, Cimex lectularius L., suddenly reappeared in developed countries in the past 15 years. The factor contributing to the sudden resurgence of the bed bugs is insecticide resistance. In this study, we investigated the mechanisms of reduced cuticular penetration type insecticide resistance in bed bugs. First, we determined and compared the lethal dosage (LD50) of a pyrethroid insecticide using topical and injection application. The resistant strain not only had significantly greater resistance ratios, but also demonstrated significantly greater penetration resistance ratios. This provided the evidence of the reduced cuticular penetration in bed bugs. Second, we determined the levels of gene transcription (CPR-type cuticle protein genes) using real-time quantitative polymerase chain reaction (qRT-PCR). We identified 62 putative bed bug cuticle protein-encoding contigs based on the presence of the Chitin-binding 4 (CB4) domain. Based on the qRT-PCR analysis of the mRNAs, we found many of the genes were up-regulated in the resistant strain suggesting thickening of the cuticle or increasing the cuticular proteins might be involved in the reduced cuticular penetration. Third, we identified and described the cuticular proteins using the matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometry (MALDI-TOF/TOF). The total of 265 peptides were identified, among which 206 belonged to one of 50 confidently identified proteins. We identified the CPRL, CPF, CPFL, TWDL, and CPAP1 family proteins. The profile of the cuticular proteins between the resistant and the susceptible strains bed bugs were almost identical. Fourth, we determined and compared the cuticular thickness using Scanning Electron Microscopy (SEM). We found statistical differences of the cuticular thickness among different strains (populations), however, correlation between the levels of insecticide resistance and cuticular thickness were not found. Finally, we identified and described bed bug cuticular hydrocarbon profiles using Gas-Chromatography and Mass-Spectrometry (GC-MS). The total of 87 compounds in addition to n-alkanes were extracted and identified. There were no correlation found with the concentration and the levels of insecticide resistance. However, several additional compounds exhibited the correlation between the concentration of the compounds and the levels of insecticide resistance. Overall, we found three lines of evidence to support reduced cuticular penetration as a mechanism of insecticide resistance in some bed bug populations. This study provides additional evidence of the reduced cuticular penetration type resistance in bed bugs. Ph. D.

The honey bee is a widely managed crop pollinator that provides the agricultural industry with the sustainability and economic viability needed to satisfy the food and fiber needs of our society. Excessive use of agrochemicals such as the acaricides coumaphos and tau-fluvalinate, and the fungicide, chlorothalonil is implicated in the reduced number of managed bee colonies available for crop pollination services. Here, I report the effects of pesticide exposures on the nutritional and immune health of the honey bee. Total protein concentration was significantly reduced in the coumaphos- and chlorothalonil-treated individuals compared to the pesticide-untreated bees. Total carbohydrate concentration was significantly reduced in the tau-fluvalinate-, coumaphos-, and chlorothalonil-treated individuals compared to the pesticide-untreated bees. Total lipid concentration was significantly decreased in the chlorothalonil-treated individuals compared to the pesticide-untreated bees. Body weight was significantly reduced for the tau-fluvalinate-, coumaphos-, and chlorothalonil-treated individuals, respectively, compared to the pesticide-untreated bees. Head width was significantly reduced for the chlorothalonil-treated individuals whereas the wing length was significantly reduced for the coumaphos and chlorothalonil-treated individuals, respectively, compared to the pesticide-untreated bees. Phenoloxidase activity was significantly increased in the coumaphos-treated individuals compared to the pesticide-untreated bees. Glucose oxidase activity was significantly increased in the chlorothalonil-treated individuals compared to the pesticide-untreated bees. While more research is needed to verify the observed effects of the pesticides on the nutritional and immunity health of the honey bee, it is important for beekeepers to consider alternative methods for control of varroa mites and the use of fungicides near their colonies. Master of Science in Life Sciences

Aedes aegypti (Ae. aegypti) is one of the most important vectors of dengue, chikungunya and yellow fever viruses. The use of chemical control strategies such as insecticides is associated with problems including the development of insecticide resistance, side effects on animal and human health, and environmental concerns. Because current methods have not proven sufficient to control these diseases, developing novel, genetics-based, control strategies to limit the transmission of disease is urgently needed. Increased knowledge about mosquito-pathogen relationships and the molecular biology of mosquitoes now makes it possible to generate transgenic mosquito strains that are unable to transmit various parasites or viruses. Ae. aegypti genetic experiments are enabled, and limited by, the catalog of promoter elements available to drive transgene expression. To find a promoter able to drive robust expression of firefly (FF) luciferase in Ae. aegypti embryos, an experiment was designed to compare Ae. aegypti endogenous and exogenous promoters. The PUb promoter was found to be extremely robust in expression of FF luciferase in different stages of embryonic development from 2-72 hours after injection. In subsequent experiments, transformation frequency was calculated using four different promoters (IE1, UbL40, hsp82 and PUb) to express the Mos1 transposase open reading frame in Mos1-mediated transgenesis. Germline transformation efficiency and size of transgenic cluster were not significantly different when using endogenous Ae. aegypti PUb or the commonly used exogenous Drosophila hsp82 promoter to express Mos1 transposase. This study also describes the development of new tools for gene editing in the Ae. aegypti mosquito genome and the use of these tools to design an efficient gene drive system in this mosquito. Homing endonucleases (HEs) are selfish elements which catalyze double-stranded DNA (dsDNA) breaks in a sequence-specific manner. The activities of four HEs (Y2-I-AniI, I-CreI, I-PpoI, and I-SceI) were investigated for their ability to catalyze the excision of genomic segments from the Ae. aegypti genome. All four enzymes were found to be active in Ae. aegypti; however, the activity of Y2-I-AniI was higher compared to the other three enzymes. Single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways were identified as mechanisms to repair HE-induced dsDNA breaks. TALE nucleases (TALENs) are a group of artificial enzymes capable of generating site-specific DNA lesions. To examine the ability of TALENs for gene editing in Ae. aegypti, a pair of TALENs targeted to the kmo gene were expressed from a plasmid following embryonic injection. Twenty to forty percent of fertile G0 produced white-eyed progeny which resulted from disruption of the kmo gene. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. A small deletion of one to seven bp occurred at the TALEN recognition site. These results show that TALEN and HEs are highly active in the Ae. aegypti germline and can be used for gene editing and gene drive strategies in Ae. aegypti. Ph. D.

Leptinotarsa decemlineata (Say) is the most important defoliating pest of potato Solanum tuberosum L., in North America and Europe. Management of this pest relies heavily on chemical control and insecticide resistance is a persistent problem. This phenomenon has increased the need for developing novel insecticides, resistance evaluation, and the development of alternative control strategies regarding this insect pest. From 2010 to 2013, field and lab experiments were conducted to evaluate the efficacy of a novel insecticide tolfenpyrad on L. decemlineata. In leaf-dip assays, tolfenpyrad was highly toxic to L. decemlineata with LC50 values of 0.013 and 0.164 g ai/L for larvae and adults, respectively. Tolfenpyrad was also toxic to eggs with 0% hatching after being dipped in a field rate concentration. In field efficacy trials, potato plots treated with tolfenpyrad at rates as low as 153 g ai/ha effectively controlled L. decemlineata. In 2012, populations of L. decemlineata were collected from the Eastern Shore of VA and subjected to toxicity assays to determine current susceptibility to permethrin and oxamyl. The toxicity assays indicated an increase in toxicity to permethrin in L. decemlineata larvae (LC50 = 3.931 g ai/L) and an increase in toxicity to oxamyl in adult beetles (LC50 = 9.695 g ai/L) compared with LC50 values previously reported in 1990. In 2012, populations of L. decemlineata from Cheriton, VA, New Church, VA, Painter, VA, and Plymouth, NC were also evaluated for enzyme activity after exposure to sub-lethal concentrations of permethrin, oxamyl, and tolfenpyrad. Adult beetles were subjected to enzyme assays to measure the activity of cytochrome P450 mono-oxygenase (P450), glutathione-S-transferase (GST), general esterases, and protein content. Results from the enzyme assays indicated significantly greater esterase activity in beetles from Painter, VA exposed to permethrin [±-naphthol (F= 11.66, df= 4, 20, P