Populations of honey bees have declined worldwide in recent years. One suspected cause is the widespread use of pesticides in agriculture. Experiments were conducted to examine potential exposure routes of pesticides to honey bees in the Midsouth. Neonicotinoid seed treatment compounds were studied to determine the rate at which they drift during planting and the rate at which they diminish in crop tissue during crop development. Honey bee foraging activity in Midsouth crops was observed to determine when and at what densities foraging honey bees could be active during pesticide applications. This project was designed to aid in understanding the risks that pesticides could potentially pose to honey bees in the Midsouth.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Apis mellifera (honey bees) are essential pollinators and thus considered a keystone species. Reproductive success is therefore important to the survival of the species, with drone sperm quality playing a vital role in fertilization success and colony health. Drone sperm quality is determined by multiple aspects including both structural and functional parameters. Particularly, motility, a functional parameter of sperm, plays an important role in reproductive success by determining the ability of sperm to move in the reproductive tract of the queen and to participate in fertilization. However, for the South African honey bee subspecies, the Cape honey bee (Apis mellifera capensis Escholtz), data on sperm quality parameters, are lacking. / 2023

The European honey bee, Apis mellifera, is a primarily beneficial insect for mankind. It has been utilized by humans for thousands of years for the products and services it provides. Crop pollination and honey production are two of the most economically beneficial activities of the honey bee. Though they have been important for many centuries and immeasurable amounts of effort have been expended investigating the methods and means to harness their natural abilities, a far lesser amount of attention has been directed towards exploring their molecular makeup. These experiments involve identifying modification of gene transcription as a result of exposure to a pheromone or a parasite. This data will provide information on the general types of transcripts involved in the biochemical response of the honey bee to the two stimuli and will also provide specific candidates for further investigation of their potential role in downstream behavioral events.

The European honey bee, Apis mellifera, is a primarily beneficial insect for mankind. It has been utilized by humans for thousands of years for the products and services it provides. Crop pollination and honey production are two of the most economically beneficial activities of the honey bee. Though they have been important for many centuries and immeasurable amounts of effort have been expended investigating the methods and means to harness their natural abilities, a far lesser amount of attention has been directed towards exploring their molecular makeup. These experiments involve identifying modification of gene transcription as a result of exposure to a pheromone or a parasite. This data will provide information on the general types of transcripts involved in the biochemical response of the honey bee to the two stimuli and will also provide specific candidates for further investigation of their potential role in downstream behavioral events.

The honey bee is one of the most familiar insects in the world, and plays an important role in the global economy providing essential pollination services to crops, fruit trees and vegetables. However, honey bee health is severely threatened by the ectoparasitic mite Varroa destructor, which feeds on the hemolymph of pupal and adult bees, resulting in loss of nutrients and circulatory fluids, decreased overall body weight and eventually the death of the bees. To investigate the molecular defense mechanisms of the honey bee against varroa mite infestation, we employed DNA microarray analysis to compare gene expression of two contrasting honey bee colony phenotypes selected from the Saskatraz breeding program. One designated as G4 is susceptible to the varroa mite, while the other designated as S88 is highly tolerant to the varroa. Total RNAs were isolated from bees at two different stages, dark-eyed pupa and adult worker, infected or non-infected with varroa mites, and used for DNA microarray analysis. The results showed that distinct sets of genes were differentially regulated in the varroa-tolerant and varroa-susceptible honey bee phenotypes, with and without varroa infestation. In both phenotypes, there were more differentially-expressed genes identified at the pupal stage than at the adult stage, indicating that at the pupal stage honey bees are more responsive to the varroa infestation than adult bees. In the phenotype comparisons, substantially more differentially-expressed genes were found in the tolerant than susceptible line, indicating that the tolerant phenotype has an increased capacity to mobilize the expression of the genes in response to varroa mite infestation. Based on function, the differentially-expressed genes could be classified into groups that are involved in olfactory signal transduction, detoxification, metabolism and exoskeleton formation, implying several possible mechanisms for the host-parasite interaction and resistance. Quantitative RT-PCR was used to confirm the data obtained from the DNA microarray hybridization. Eleven out of twelve genes selected based on the microarray data showed consistent expression patterns measured by both methods. Overall, comprehensive evaluation of the gene expression of honey bees in response to the mite infestation by DNA microarray has revealed several possible molecular mechanisms for the host defense against the pest. Identification of highly differentially expressed genes between the two phenotypes provides potential biomarkers that can be used for breeding honey bees resistant to the varroa mite.

The goal of this study was to examine the impact of Africanization on the genetic structure of the Welder Wildlife Refuge feral honey bee population by scoring mtDNA and microsatellite polymorphisms. Adult honey bee workers, collected between 1991 and 2001, were screened for mtDNA using the cytochrome b/BglII, ls rRNA/EcoRI, and COI/HinfI PCR-based assays. The procedure allowed identification of four mitotypes: eastern European, western European, A. m. lamarckii, and A. m. scutellata. The relative frequencies of the four mitotypes changed radically during the 11-year period. Prior to immigration of Africanized honey bees, the resident population was essentially of eastern European maternal ancestry. The first colony of A. m. scutellata mitotype was detected in 1993. Between 1995 and 1996 there was a mitotype turnover in the population from predominantly eastern European to predominantly A. m. scutellata. From 1997 onward, most colonies (69 %) were of A. m. scutellata mitotype. The temporal change in mtDNA was paralleled by nuclear DNA. The 12 microsatellite loci analyzed indicated (1) the mechanism of Africanization of the Welder population involved both maternal and paternal bi-directional gene flow (hybridization) between European and Africanized honey bees; and (2) the resident panmitic European population was replaced by panmitic asymmetrical admixtures of A. m. scutellata and European genes. The steepest increase in the proportion of introgressed A. m. scutellata nuclear alleles occurred between 1994 and 1997. The post-Africanization gene pool was composed of a diverse array of recombinant classes with a substantial European genetic contribution (mean proportion of European-derived alleles was 37 % as given by mR estimator or 25 % as given by mY estimator, for 1998-2001). If European genes continue to be retained at moderate frequencies, then the Africanized population is best viewed as a "hybrid swarm" instead of "pure African". The most radical change in the genetic structure of the Welder Wildlife Refuge feral honey bee population (observed between 1995 and 1997) coincided with arrival of the parasitic Varroa mite. We suggest that Varroa likely hastened the demise of European honey bees and had a major role in restructuring the Welder Wildlife Refuge feral honey bee population.

Red sorrel is a perennial weed in wild blueberry fields that decreases yield. Multiple experiments were conducted to evaluate its impact on blueberry pollination, Botrytis blight incidence, and berry yield. Kerb applications did not significantly impact blueberry stem or floral bud formation. Removal of red sorrel with Kerb increased blueberry yield at both sites. However a double application had no difference than one application. Blueberry and red sorrel flowering overlapped and red sorrel pollen grains were found on blueberry flowers in both years at all sites. Red sorrel pollen grains increased the incidence of germinating spores in Petri dishes and this relationship was adequately modeled with a three parameter, exponential rise to a maximum. Red sorrel pollen significantly increased disease incidence on immature blueberry flowers. Honey bees foraged from blueberry and red sorrel flowers, but there was no evidence to suggest that they favored red sorrel flowers over blueberry flowers.

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. / 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 exposures 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. One method for protecting these pollinators is use of chemical deterrents to reduce the interaction times of foraging bees with pesticide-treated crops. Insect repellents (IRs) primarily have been used for biting and blood-feeding arthropod pests. 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 goals of this study were to: 1) examine the toxicological effects of the pheromonal compounds on bees, 2) develop a laboratory testing protocol for evaluating the deterrent effects of pheromonal bee compounds and heterocyclic amines (HCAs) to bees, 3) evaluate the deterrent effects of HCAs to bees using high-tunnel and semi-field experimentations, and 4) characterize the olfactory responses of bees to the above compounds.

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 / The pollination service provided by insects, primarily honey bees, is estimated to contribute approximately one-third of the diet consumed by the average American. Honey bees are vitally important pollinators due to their broad range of foraging activities and ease of husbandry within a managed colony. In recent decades, colony numbers have decreased in the developed areas of the planet and pesticide usage has been implicated in these losses. Atrazine is the second most commonly used agricultural herbicide in the country and has been linked to oxidative stress in beneficial insects in the past. Oxidative stress is the result of an uncontrolled build-up of reactive oxygen species in an aerobic organism. These reactive oxygen species are dangerous because they are capable of damaging proteins, DNA, and cell walls. Every aerobic organism also possesses antioxidant function which serves to prevent or counteract damage caused by reactive oxygen species. This study examined antioxidant enzyme activities and antioxidant-encoding gene expression levels, which were used as indicators of oxidative stress biomarkers, in honey bees exposed to atrazine in the laboratory and in the hive environment. Honey bees were exposed to atrazine at increasing concentrations in the laboratory for 24 h and at one environmentally relevant dose for 28 d in the hive. After exposure to atrazine in the laboratory and the hive, four out of five antioxidant enzyme levels of honey bees decreased which implied an increase in oxidative stress and a decrease in antioxidant defenses. Activity of one enzyme, lipid peroxidase, increased in honey bees after exposure to atrazine. Lipid peroxidase is the most common measure of cellular injury during oxidative stress, once again signifying an increase in reactive oxygen species production and oxidative stress. Expression levels of seven antioxidantencoding genes were examined in honey bees after atrazine exposure and expression levels of some genes changed compared to the untreated control and expression levels in some genes remained the same compared to the untreated control. These changes in antioxidant-encoding gene expression levels may imply an increase in oxidative stress due to exposure of the honey bees to atrazine. This study aimed to examine biomarkers of oxidative stress in honey bees exposed to the commonly used herbicide atrazine with the hope of raising awareness of harmful effects caused by atrazine and protecting these important pollinators from unintended exposure to agricultural pesticides.

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 / Honey bees are the most important crop pollinator known to humans. The domestication and use of these insects constitutes a multi-billion dollar industry. Their pollination services alone are a necessary part of modern day agriculture. One of the concerns raised today with regard to honey bee health is their exposure to insecticides used widely in modern agriculture to manage crop pests and protect our food supply from devastating crop loss. One insecticide family that has gained much attention lately are the neonicotinoids. These insecticides are reported to elicit sublethal effects to honey bees that can affect colony health. Some of the more widely used neonicotinoids include, but are not limited to, imidacloprid, thiacloprid, and acetamiprid. The goal of this study was to examine the acute toxicity of imidacloprid to honey bees collected from different colonies and to compare the metabolic detoxification enzyme activities of the honey bees to understand the mechanism(s) of imidacloprid sensitivity in the honey bees. Here, I report a 36-fold difference in the acute toxicity of imidacloprid to the honey bees collected from different colonies. A comparison of glutathione <i>S</i>-transferases activities in imidacloprid-susceptible and -tolerant honey bees suggest that these metabolic detoxification enzymes may assist in the conjugation of imidacloprid, or associated metabolites, and thus facilitate the removal of the insecticide from the honey bees.