This thesis analyzes mixtures of neem oil and amitraz as alternative control for Varroa destructor, a major pest of Apis mellifera, under different agricultural settings. In organic and conventional farms, the different treatments were applied in colonies to determine impacts on mite loss, colony strength, and honey yield. The results demonstrated neem to have the least effective control on mite mortality, while the neem and amitraz mixture had the most. Furthermore, no long term impacts on queen fecundity and colony strength were noticed between treatments. However, queen fecundity and honey yield was significantly higher in sites with higher flower abundance and diversity, demonstrating higher colony strength in these sites. Further understanding of the relationship between apiculture and agricultural management is vital for conservation of pollinator health and associated habitats.

Urease of Helicobacter pylori is an important virulence factor implicated in the pathogenesis of many clinical conditions, such as chronic gastritis, peptic ulceration, and gastric cancer. Many urease inhibitors have been discovered, like phosphorodiamidates, hydroxamic acid derivatives, and imidazoles. Despite good activities at the enzyme level and excellent kinetic properties most of them have not been used as therapeutic agents in vivo because of their side effects, toxicity and instability. This has led to much attention to focus on exploring the novel urease inhibitory activities of natural products because of their low toxicity and good bioavailability. Honey, a natural product has been used in folk medicine due to its antitumor, antioxidant, antimicrobial and anti-inflammatory properties. The aims of this study were to isolate, characterise, purify urease produced by H. pylori and investigate the inhibitory effects of solvent honey extracts on its enzymatic activity. Urease was found to be both surface-associated and cytoplasmic. Maximum cytoplasmic urease activity was found to occur after 72 hr whereas maximum extracellular urease activities were found to occur after 96 hr. Characterization of the crude cytoplasmic urease revealed optimal activity at a pH of 7.5 and temperature of 40°C. The kinetic parameters Vmax and Km were 45.32 U ml-1 and 61.11 mM respectively.The honey extracts inhibited the activity of the crude urease in a concentration dependent manner. The Lineweaver-Burk plots indicated a non-competitive type of inhibition against H. pylori urease. The two honey extracts gave promising inhibitory activities against urease of H. pylori. Thus the results of this study delineates that inhibition of urease can ease development in therapeutic and preventative approaches based on the enzymatic activity of this Helicobacter protein.

This paper deals with the distributional outgrowths of Prosopis chilensis (Molina) Stuntz, the common mesquite, in Denton County, Texas.

abstract: Vitellogenin (Vg) is an ancient and highly conserved multifunctional protein. It is primarily known for its role in egg-yolk formation but also serves functions pertaining to immunity, longevity, nutrient storage, and oxidative stress relief. In the honey bee (Apis mellifera), Vg has evolved still further to include important social functions that are critical to the maintenance and proliferation of colonies. Here, Vg is used to synthesize royal jelly, a glandular secretion produced by a subset of the worker caste that is fed to the queen and young larvae and which is essential for caste development and social immunity. Moreover, Vg in the worker caste sets the pace of their behavioral development as they transition between different tasks throughout their life. In this dissertation, I make several new discoveries about Vg functionality. First, I uncover a colony-level immune pathway in bees that uses royal jelly as a vehicle to transfer pathogen fragments between nestmates. Second, I show that Vg is localized and expressed in the honey bee digestive tract and suggest possible immunological functions it may be performing there. Finally, I show that Vg enters to nucleus and binds to deoxyribonucleic acid (DNA), acting as a potential transcription factor to regulate expression of many genes pertaining to behavior, metabolism, and signal transduction pathways. These findings represent a significant advance in the understanding of Vg functionality and honey bee biology, and set the stage for many future avenues of research. / Dissertation/Thesis / Doctoral Dissertation Evolutionary Biology 2019

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy / In this dissertation the physical and chemical alterations induced by mastication and manipulation of wax by the worker bee in honeycomb construction, and the subsequent contribution afforded the structural integrity of the nest, are elucidated. In comb building, the freshly secreted wax scales are mandibulated together with a frothy salivary emulsion, and added piece-meal to form honeycomb. Textural modifications were revealed using X-ray crystallography. While virgin scale wax is highly structured, with the crystallites aligned approximately perpendicular to the planar surface, comb wax has a random crystallographic arrangement. This reflects a disruption of the crystallite structure following the mechanical insult of mastication. Chemical analyses included investigation of both lipid and proteinaceous elements. Lipid composition was evaluated by enzyme-catalyzed as well as thin-layer and gas-liquid chromatographic methods. The results indicate a reduction in scale diacylglycerols with a corresponding increase in comb saturated monoaeylglycerols. Such modifications are highly suggestive of lipase activity within the salivary addition. The proteins of comb and scale wax were analyzed electrophoretically, under reduced conditions. Each wax possesses unique polypeptide fractions, in addition to sharing common protein species, It is speculated that those in common represent integral proteins, such as transport molecules, while the disparities noted may be due to salivary enzymatic degradation, or even glycosylation. The effects of these textural and chemical alterations on the mechanical behaviour of the waxes was assessed. Tensile tests were performed on a variety of scale and comb wax preparations over the range of temperatures likely to impinge on the honeybee nest. These investigations reveal the specific structural contributions made by each of the physico-chemical alterations described. Further, they demonstrate that while scales are ideal moulding materials due to their high distensibility and low stiffness, the greater resistance to deformation and lower potential for extension makes comb wax a superior structural material. The mechanical advantage for including propolis and cocoon silk within the comb structure was also investigated. Tensile testing indicates that the resultant composite material is structurally superior, largely due to the presence of silk reinforcement. / Andrew Chakane 2018

<p>    </p> <p>Domestication is the process by which a previously wild population is managed by humans, thereby being subjected to a different set of selective pressures than experienced in its natural setting. Its opposite, feralization, is therefore when a domesticate escapes or is released from a captive setting, reasserting natural selective pressures. The genomics underpinning both domesti- cation and feralization have not been studied in insects; the Western honey bee (<em>Apis mellifera </em>L.) is a good model for this system, as honey bees exist in both a managed and feral state, and have extensive historic and genomic resources to document population changes. My goal in this thesis was to 1) improve upon our understanding of honey bee importation and genetics to the United States to support demographic assertions, and 2) to sequence managed and feral stocks of honey bees to identify the population structure and 3) genetic differences underpinning domestication. Ultimately, I reconstructed 400 years of honey bee importation and management history, creating the most comprehensive understanding to date of importation dates and locations, historical man- agement practices, and genetic bottlenecks. Additionally, I summarized thirty years of honey bee genome sequencing to provide a road map for future studies. Then, I conducted whole genome pooled sequencing on six managed and three feral stocks of honey bees from the United States. The mitochondrial and whole genome ancestry of feral colonies holds relics from their importation history, while managed colonies show evidence of more recent importation events. The managed stocks in my sample set have higher overall genetic diversity, but exhibit little differentiation, but feral stocks exhibit varying levels of differentiation, indicating different levels of ferality likely dictated by the level of reproductive isolation from managed colonies. </p>

Honey bees can form distinct spatiotemporal memories that allow them to return repeatedly to different food sources at different times of day. Although it is becoming increasingly clear that different behavioral states are associated with different profiles of brain gene expression, it is not known whether this relationship extends to states that are as dynamic and specific as those associated with foraging-related spatiotemporal memories. We tested this hypothesis by training different groups of foragers from the same colony to collect sucrose solution from one of two artificial feeders; each feeder was in a different location and had sucrose available at a different time, either in the morning or afternoon. Bees from both training groups were collected at both the morning and afternoon training times to result in one set of bees that was undergoing stereotypical food anticipatory behavior and another that was inactive for each time of day. Between the two groups with the different spatiotemporal memories, microarray analysis revealed that 1329 genes were differentially expressed in the brains of honey bees. Many of these genes also varied with time of day, time of training or state of food anticipation. Some of these genes are known to be involved in a variety of biological processes, including metabolism and behavior. These results indicate that distinct spatiotemporal foraging memories in honey bees are associated with distinct neurogenomic signatures, and the decomposition of these signatures into sets of genes that are also influenced by time or activity state hints at the modular composition of this complex neurogenomic phenotype.

In the field of insect circadian rhythms, the honey bee is best known for its foraging time-sense, or Zeitgedächtnis, which permits the forager bee to make precise associations between the presence of food and the time of day. A number of studies, now considered classics, established that bees could be trained to collect food at virtually any time of the circadian cycle and that this timekeeping ability was controlled by an endogenous circadian clock. Recently, behavioral rhythms in bees have been examined using a variety of approaches, in both laboratory and field studies. The following areas of new research are reviewed: (a) the ontogeny of behavioral rhythmicity in newly emerged worker bees; (b) the integration of behavioral rhythmicity with the colony's division of labor; (c) the evidence for social entrainment of behavioral rhythms and for a 'clock of the colony'; (d) the potential linkage between circadian rhythms of general locomotor activity and the foraging time-sense; (e) learning and entrainment hypotheses proposed to explain the mechanism underlying the time-sense; (f) the interplay between extinction and persistence of the time-memory as revealed from the differential behavior of individuals within the foraging group; and (g) comparisons of the Zeitgedächtnis with food-anticipatory rhythms in other animals.

Honey bees utilize their circadian rhythms to accurately predict the time of day. This ability allows foragers to remember the specific timing of food availability and its location for several days. Previous studies have provided strong evidence toward light/dark cycles being the primary Zeitgeber for honey bees. Work in our laboratory described large individual variation in the endogenous period length of honey bee foragers from the same colony and differences in the endogenous rhythms under different constant temperatures. In this study, we further this work by examining the temperature inside the honey bee colony. By placing temperature and light data loggers at different locations inside the colony we measured temperature at various locations within the colony. We observed significant oscillations of the temperature inside the hive, that show seasonal patterns. We then simulated the observed temperature oscillations in the laboratory and found that using the temperature cycle as a Zeitgeber, foragers present large individual differences in the phase of locomotor rhythms for temperature. Moreover, foragers successfully synchronize their locomotor rhythms to these simulated temperature cycles. Advancing the cycle by six hours, resulting in changes in the phase of activity in some foragers in the assay. The results are shown in this study highlight the importance of temperature as a potential Zeitgeber in the field. Future studies will examine the possible functional and evolutionary role of the observed phase differences of circadian rhythms.

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.