As operárias de abelhas sem ferrão são peculiares com relação à divisão do trabalho reprodutivo para a manutenção da colônia, visto que podem produzir ovos reprodutivos que originam os machos, além dos ovos tróficos destinados a alimentar a rainha. Desta forma, os estudos de expressão de genes e de proteínas envolvidos na biossíntese de vitelo vitelogênese - e incorporação deste material aos ovócitos contribuem para evidenciar diferenças intra e inter-específicas entre as fêmeas, em relação à fertilidade e comportamento reprodutivo. Os perfis de expressão do gene codificador da vitelogenina e da própria proteína, precursora da principal constituinte do vitelo, foram determinados para várias etapas do desenvolvimento de três espécies de abelhas sem ferrão. Para este estudo foram selecionadas as espécies Frieseomelitta varia, cujas operárias nunca põem ovos, mesmo em condições de orfandade, Scaptotrigona postica e Melipona scutellaris, cujas operárias desenvolvem os ovários e participam ativamente da produção de machos. O RNA total de corpo gorduroso sítio de biossíntese de vitelogenina de operárias destas espécies foi extraído e o cDNA obtido por transcrição reversa semiquantitativa foi amplificado, clonado e seqüenciado utilizando-se primers específicos para a vitelogenina de Apis mellifera. Os resultados revelaram que os cDNA parciais obtidos são bastante conservados entre F. varia, S. postica e M. scutellaris e mostram alta identidade (93-100%) em relação à região 3-terminal do cDNA da vitelogenina de A. mellifera. Entretanto, o perfil de abundância do transcrito difere entre as espécies de meliponíneos e entre estas e as abelhas melíferas. Em F. varia e S. postica a expressão do transcrito mostrou-se constitutiva ao longo dos períodos pupal e adulto, mas M. scutellaris mostrou diminuição da abundância de transcritos nas fases pupais mais avançadas e nas operárias recém-emergidas. Estas espécies diferem de A. mellifera cujas pupas não expressam o gene da vitelogenina. A expressão constitutiva deste gene em F. varia e S. postica mostra que a atividade do gene em questão não é modificada pela variação dos títulos de ecdisteróides e hormônio juvenil descrita para A. mellifera e outros insetos, indicando, portanto, ausência de controle da transcrição de vitelogenina por hormônios nas espécies de meliponíneos estudadas. No entanto, os resultados indicam a existência de controle nutricional da atividade do gene da vitelogenina, dado o aumento de expressão verificado em operárias F. varia alimentadas com dieta rica em proteínas (contendo pólen - a fonte de proteínas para as abelhas - e açúcar) em comparação com aquelas que receberam dieta exclusiva de carboidrato (açúcar). A presença da proteína vitelogenina na hemolinfa de F. varia ocorre concomitantemente com a expressão constitutiva do transcrito. Neste aspecto, difere de S. postica e M. scutellaris que também expressam o transcrito da vitelogenina ao longo do estágio pupal e adulto, mas a proteína correspondente somente é detectada nas operárias destas espécies que estão exercendo a função de nutridoras de crias. Pode-se concluir que o gene da vitelogenina é conservado entre as espécies de abelhas até aqui estudadas, porém sua expressão e possivelmente sua regulação diferem entre meliponíneos e abelhas melíferas, refletindo as diferentes estratégias utilizadas na divisão do trabalho reprodutivo. / The stingless bee workers are peculiar with relation to reproductive division of labor to colony maintenance, since they are able to produce reproductive eggs that will develop in males, beyond trophic eggs to feed the queen. In this way, studies about gene and protein expression involved on the yolk biosynthesis vitellogenesis and the incorporation of this material to the oocytes contributes to evidence intra and inter-specific differences between females, in relation to fertility and reproductive behavior. The vitellogenin, the main yolk constituent precursor, gene expression and the protein itself profile were determined to several developmental stages of three stinglessbee species. To this work were selected the species Frieseomelitta varia, whose workers never lay eggs, even in a queenless condition, Scaptotrigona postica and Melipona scutellaris, whose workers develop their ovaries and participate actively to male production. The whole RNA of the fat body the vitellogenin biosynthesis site - of these worker species was extracted and the cDNA obtained by semiquantitative reverse transcription amplified, cloned and sequenced through Apis mellifera vitellogenin specific primers. The results reveal that the obtained partial cDNAs are very conserved among F. varia, S. postica and M. scutellaris and show high identity (93-100%), in relation to 3-end A. mellifera vitellogenin gene. However, transcript abundance profile is different among stinglessbee species and with honeybees. In F. varia and S. postica the transcript expression is constitutive during pupal and adult periods, but M. scutellaris showed transcript reduction in the advanced pupal phases and newly emerged workers. These species are different from A. mellifera pupae that do not express vitellogenin gene. This constitutive gene expression in F. varia and S. postica shows that the gene activity is not modified by the ecdysteroid and juvenile hormone titers as descript to A. mellifera and other insects, indicating absence of vitellogenin transcriptional control by hormones in the studied stingless bee species. However, the results indicate the existence of vitellogenin gene activity nutritional control, given the verified expression increase in F. varia workers and drones fed with a rich protein diet (with pólen the protein bee source and sugar) in comparison with which ones received exclusive sugar diet. The presence of vitellogenin protein on the F. varia hemolimph occurs concomitantly with the constitutive transcript expression. In this aspect, is different of S. postica and M. scutellaris which also express the vitellogenin transcript during pupal and adult stages, but the correspondent protein is only detected on the workers of these species that are exerting nurse tasks. Is possible conclude that vitellogenin gene is conserved among the studied bee species, but its expression and possibly regulation are different among stingless bees and honey bees, reflecting different strategies used on reproductive division of labor.

As abelhas e plantas apresentam diferentes graus de especialização em suas interações. Parceiros mais especialistas frequentemente apresentam uma história evolutiva mútua e sobreposição nas áreas de ocorrência. No entanto, a estrutura espacial dos ambientes nos quais esses grupos se distribuem é caracterizada por padrões complexos e dinâmicos. Para analisar a influência dos fatores que atuam na distribuição de espécies de abelhas e plantas associadas foram utilizadas a modelagem de distribuição de espécies, análise multivariada e ferramentas de sistemas de informações geográficas. Os resultados indicaram que a distribuição de gêneros estritamente associados, como é o caso de Peponapis e Cucúrbita, é influenciada pelo clima das áreas de ocupação, bem como provavelmente, por sua história evolutiva e pela domesticação das abóboras (Cucúrbita). Já os gêneros Krameria e Centris apresentam um padrão mais intrincado de distribuição, uma vez que a interação entre ambos é mais complexa. Centris é um grupo diverso que utiliza recursos florais de outras famílias botânicas além de Krameriaceae, o que provavelmente influencia seus padrões de distribuição. No entanto, os resultados obtidos para Krameria demonstraram de maneira geral, a influência de características climáticas na distribuição do grupo e uma provável dependência maior de Centris em alguns casos. Finalmente, foi também demonstrada a importância da inclusão de dados bióticos no processo da modelagem de distribuição, resultando no aumento da acurácia dos modelos e na alteração da projeção da distribuição para o futuro, considerando-se um cenário de mudança climática. Os resultados foram mais conspícuos quando foram consideradas interações mais estreitas entre espécies de abelhas parasitas e hospedeiras do gênero Bombus, do que entre Centris e Krameria. As técnicas utilizadas, em especial a modelagem de distribuição, representaram uma importante contribuição para a análise efetuada. No entanto, embora crescentemente utilizada, a modelagem de distribuição de espécies demanda técnicas e testes mais robustos para avaliar a acurácia dos modelos gerados. Além disso, um desafio adicional a ser vencido consiste no aumento e melhoria da qualidade dos pontos de ocorrência das espécies, principalmente no Brasil. Faz-se necessário um esforço adicional de coleta, especialmente em algumas áreas específicas, bem como, a conservação e digitalização dos dados das coleções biológicas. Porém, as técnicas utilizadas mostraram um grande potencial a ser explorado em outras análises, envolvendo questões biológicas diferentes, ou outros grupos taxonômicos e camadas de dados. / Bees and plants present different degrees of specialism in their interactions. More specialized partners generally present a mutual evolutionary history and overlap with their occurrence areas. Nevertheless, the spatial structure of environments occupied by them is characterized by complex and dynamic patterns. Species distribution modelling, multivariate analyses and geographical system information tools were used in order to analyze the influence of different factors that act in the geographical distribution of associated bees and plants. Results showed that the geographical distribution of close associated genera, such as Peponapis and Cucúrbita are influenced by the clime of occurrence areas, and also, by their evolutionary history and cucurbits domestication (squashes and pumpkins). On the other hand, Centris and Krameria genera presented a more intricate distribution pattern, since their interaction is more complex. Centris is a diverse group that uses other floral resources than those provided by the Krameriacea family, which has probably influenced its distribution, also. However, the results obtained for Krameria showed the influence of clime in its distribution and a stronger relationship with Centris in some cases. Finally, the importance of including biotic data in the species distribution modelling process was also demonstrated, resulting in a general increase in the models accuracy and also altering future scenarios projection, considering climate changes. Stronger interaction, such as the host-parasite bee species of Bombus showed more conspicuous results than those found for Krameria and Centris. The techniques, especially distribution modelling, made an important contribution to the analyses. However, in spite of being increasingly used, distribution modelling demands more robust tests and techniques to evaluate the accuracy of final models. Besides, an additional challenge to be achieved consists in the increase and improvement of species occurrence data, mainly in Brazil. An additional survey effort is necessary, especially in specific areas, as well as the conservation and data digitalization of biological collections. However, the techniques used here showed a great potential to be further explored in other analyses, involving different biological issues, other taxonomic groups and other data layers.

Orientador: Ricardo de Lima Zollner / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T07:59:14Z (GMT). No. of bitstreams: 1 Ferro_KarlaPriscilaVieira_D.pdf: 2593573 bytes, checksum: dbc61c553ff3b0a43d9d67bfd357abee (MD5) Previous issue date: 2011 / Resumo: As reações alérgicas à ferroada de inseto resultam de resposta exacerbada do sistema imune, com produção de elevados níveis de anticorpos IgE alérgeno-específicos e padrão de citocinas Th2, envolvidas na diferenciação de linfócitos B específicos para aquele antígeno em células produtoras de IgE e recrutamento de células efetoras da resposta alérgica. Neste contexto, granulocitos são células efetoras importantes na fase tardia da resposta alérgica e estão envolvidos na patogênese de diferentes doenças. Eosinófilos e neutrófilos, especificamente, modulam a resposta imune por meio de diferentes mecanismos, como a secreçao de citocinas, quimiocinas e mediadores lipídicos. A IgE desempenha papel central na patogênese das doenças alérgicas, interagindo com dois receptores de membranas: alta afinidade FcsRI e baixa afinidade FcsRII (CD23). A ligação da IgE ao seu receptor em mastocitos e basófilos promove a liberação de mediadores inflamatórios, dentre eles, a histamina. A histamina além de induzir os sintomas agudos da reação alérgica, sustenta a reação inflamatória até a fase crônica, sendo estes efeitos mediados através da ativação de diferentes receptores (H1, H2, H3 e H4). Os fatores liberadores de histamina (HRF), particularmente, HRF-dependentes de IgE, induzem a liberação de histamina na fase tardia da resposta alérgica, permitindo a perpetuação dos eventos inflamatórios crônicos. Muitos estudos demonstram a eficácia da imunoterapia específica na dessensibilização e no desenvolvimento de tolerância em indivíduos com quadros graves de hipersensibilidade à ferroada de insetos, sobretudo da classe Hymenoptera. Com base nestas informações, foram objetivos do presente trabalho avaliar os efeitos modulatórios da imunoterapia sobre a expressão gênica dos receptores de histamina (H1, H2 e H4), HRF- IgE dependente e de fatores apoptóticos (Bcl-2 e BID) por RT-PCR, além da expressão gênica, através da técnica de PCR em tempo real de fatores de transcrição envolvidos na diferenciação de granulocitos como PU.1, C/EBPa, C/EBPpe GATA-1, receptores de alta (FcsRla e FcsRly) e baixa afinidade de IgE (CD23), cuja detecção protéica foi realizada por imunofluorescência e citometria de fluxo, respectivamente. Além disso, foram avaliados os níveis séricos de IgE específica, secreçao de RANTES e IL-8 nos sobrenadantes das culturas celulares e quantificação de granulocitos apoptóticos através da técnica de TÚNEL. Os granulocitos foram isolados de pacientes submetidos à imunoterapia específica ao veneno de abelha, em diferentes períodos do tratamento (Pré, 1, 3, 6, 12, 18 e 24 meses), após injeção subcutânea, e submetidas à cultura por 72 horas, com estimulo de 1 ng/mL veneno de abelha. Indivíduos não alérgicos foram estudados como grupo controle. De maneira geral, a imunoterapia específica ao veneno de abelha foi capaz de modular os elementos analisados, reduzindo significativamente a expressão dos mesmos ao final de 24 meses de tratamento. Não verificamos, apenas, modulação no número de granulocitos apoptóticos ao longo da imunoterapia. Nossos resultados inéditos fornecem informações adicionais sobre os efeitos da imunoterapia sobre granulocitos, reforçando as propriedades supressoras e tolerogênicas desta forma de tratamento / Abstract: Allergic reactions to insect stings results from a exacerbated response of the immune system, resulting in the production of high levels of allergen-specific IgE antibodies and Th2 cytokine pattern, which are involved in the differentiation process of B lymphocytes, specific for that antigen, into IgE producing cells and the recruitment of effector cells of allergic response. Eosinophils and neutrophils, specifically, modulate the immune response through different mechanisms, such as the secretion of cytokines, chemokines and lipid mediators. IgE plays a central role on allergic diseases pathogenesis, interacting with two membrane receptors: high affinity FcsRI and low affinity FcsRII (CD23). Biding of IgE with receptors on mast cells and eosinophils promotes the release of inflammatory mediators, among them, histamine. Histamine, besides inducing acute symptoms of allergic reaction, supports inflammatory response until its chronic stage; these effects are mediated through the activation of distinct receptors (H1, H2, H3 and H4). Histamine releasing factors (HRF), particularly, IgE dependent HRF, induce histamine release during the late phase of allergic response, allowing the perpetuation of chronic inflammatory events. In this context, many studies have demonstrated the efficacy of specific immunotherapy on desensitization and tolerance development in subjects with severe hypersensivity to insect stings, especially Hymenoptera. Based on all these information, the aim of the present study were to evaluate the modulating effects of immunotherapy on gene expression of histamine receptors (H1, H2 and H4), IgE dependent HRF and apoptotic factors (Bcl-2 and BID), through RT-PCR; in addition to gene expression, through real time PCR, transcriptional factors involved at granulocytes differentiation as of PU.1, C/EBPa, C/EBPp and GATA-1, and protein expression of high (FcsRIa e FcsRly)and low affinity (CD23) IgE receptors, assessed by immunofluorescence and flow cytometry, respectively. Serum levels of specific IgE were also assessed, along with RANTES and IL-8 secretion in cell culture supernatant and quantification of apoptotic granulocytes through TUNEL technique. Granulocytes were isolated from patients undergoing bee venom specific immunotherapy in different periods of treatment (Pre, 1, 3, 6, 12, 18 and 24 months), after subcutaneous injection, and cultured for 72 hours, with bee venom 1ng/ml_. Non allergic subjects were studied as control group. Overall, bee venom specific immunotherapy was able to modulate the analyzed elements, significantly reducing their expression at the end of 24 months of treatment. Modulation on the number of apoptotic granulocytes were not observed during immunotherapy. Our results provide additional information about the effects of immunotherapy over granulocytes, reinforcing the suppressor and tolerogenic properties of this treatment / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

The role of the queen in wax secretion and comb building was studied in the the Cape honeybee, Apis mellifera capensis (Escholtz). The percentage of bees bearing wax and the amount of wax borne by these bees did not differ between the experiments. This meant that the queenless and queenright colonies had the potential to construct equal amounts of comb as the amounts of wax available for comb building was the same. Contrary to this prediction, queenright colonies constructed 8 times more comb than their queenless counterparts. Queenright Apis mellifera scutellata colonies constructed 4 times more comb than their queenless counterparts. The increased amount of 9-oxo-2-decanoic acid (90DA) in the A.m.capensis mandibular gland secretions could not alone account for this difference. In fact, A.m.capensis and A.m.scutellata colonies constructed similar amounts of comb when they were given their own queens or queens from the other race. Worker bees need to have direct contact with their queen for comb building to be enhanced. Even when the queen had her mandibular glands extirpated and tergite glands occluded large amounts of comb were constructed than when access to the queen was limited. Direct access to the head of a mated queen proved to be the stimulus enhancing comb building. No comb was constructed when the workers had access to the abdomen of the queen. Virgin queens did not stimulate comb building. The relatively large amounts of 90DA and 9HDA from the mandibular glands of Cape virgin queens had not influenced comb building. Worker sized cells were generally constructed. These cells were slightly smaller than those constructed by European honeybees, but were indicative of African bees. A few queen less colonies constructed cells that were of an intermediate drone and worker size. Four mandibular gland pneromones were measured by gas chromatography. No correlations between these pheromones and the comb construction measurements were found. It is unlikely that the mandibular gland pheromones are the only pheromones that stimulate comb building. Pheromones from other glands on the head may contribute towards the enhancement of comb building, and they are not present in virgin queens

In a honeybee colony, the loss of a queen is considered to be a serious occurrence and, if a new queen is not produced, ultimately doomed. However, in colonies of Apis mellifera capensis (Cape honeybees), numerous pathways are available for a colony which unexpectedly losses a queen. At the onset of this experiment, four colonies of A. m. capensis were dequeened. Following this photographs of all brood frames in each colony were taken and the contents of the cells analysed. Cells were chosen at random but once selected were repeatedly analysed for the duration of the experiment. The contents of a total of 44 888 individual cells were analysed. Any queen cells constructed during the sampling period were removed, maintaining a queenless state. In each colony, as predicted, the removal of the queen evoked a variety of responses in an attempt to rectify the sudden loss. However, ultimately three of the four colonies absconded, leaving little by way of stores. Three of the four colonies initially attempted to rear a new queen while one colony was immediately invaded by a presumed foreign queen and hence any attempt at queen cell construction ceased. An increased number of queen cells in the swarm position were recorded in all colonies. The invasion of a colony by a foreign queen was considered to be a new pathway available for queenless colonies of A.m capensis. Worker policing and suspected brood cannibalism was prevalent in all sampled colonies yet in addition, the transfer of eggs and larvae from cell to cell was also observed which may have increased the suspected cases of policing and cannibalism. It was unclear whether an egg or larva had been consumed or simply moved to another cell on the brood frame. All colonies contained eggs from laying workers to varying degrees, based on the length of each individual sampling period which varied between colonies due to differences in absconding dates. A steady breakdown in the effectiveness of the division of labour amongst the worker bees was observed in each of the colonies highlighting the vital role of a queen. This breakdown was clearly seen in the reduction in general housekeeping within a colony. An increase in stores indicated a possible increase in the number of forager bees, thereby reducing the number of worker bees available for other duties. Pathways available to broodright colonies and strategies used following sudden queen loss are discussed.

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.

Customer experience in retail banking has become a key source of differentiation. Increased competition in the financial services industry and the global financial crisis caused financial institutions to find themselves in a difficult operating environment. A sustainable competitive advantage in the current environment cannot be achieved only through excellent service delivery, service quality and prices, but through the creation of memorable customer experiences. The current study expands on the conceptual framework for the creation of customer experience proposed by Verhoef, Lemon, Parasuraman, Roggeveen, Leonard and Schlesinger (2009). In this paper the nature and extent to which the determinants discussed by Verhoef et al. impact on the customer experience is assessed. Using a quantitative method, a survey questionnaire measuring customers’ perceptions about their experiences with the retail banking firms, was distributed to 1043 employees of the air transportation company. The study finds that the past experiences, brand and prices impacts more on customer experience than other determinants identified. The study also evaluates the impact of transformation and Black Economic Empowerment on customer experience. Copyright / Dissertation (MBA)--University of Pretoria, 2010. / Gordon Institute of Business Science (GIBS) / unrestricted

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.

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.