Thesis (Ph.D. (Biochemistry, Microbiology & Biotechnology)) - Rhodes University, 2005.

Neonicotinoid exposure has been recognised as potentially impacting upon bee health, but whether realistic exposure scenarios are driving declines in bee health is not known. This thesis contributes new insights and perspectives to this research field investigating the use of molecular, physiological and behavioural endpoints as potential ecotoxicological markers for pesticide risk assessment. The thesis presents experimental data for dietary exposures of the European honey bee, Apis mellifera, and the buff-tailed bumble bee, Bombus terrestris, to one of two neonicotinoid pesticides, imidacloprid and thiamethoxam. The first part of this thesis explores impacts of chronic dietary exposures to neonicotinoid pesticides on bee immunocompetence- the ability to mount an immune response- using an artificial challenge to invoke an immune response in adult workers. Levels of phenoloxidase, an enzyme involved in melanisation and part of the bee’s defence system, were largely constitutive and resilient to exposure in honey bees and bumble bees. In honey bees, transient transcriptional changes in antimicrobial effector genes were observed following neonicotinoid exposure, but the physiological antimicrobial response was unaffected. In bumble bees, the induced antimicrobial response was impaired following neonicotinoid exposure, but only when exposed to concentrations likely higher than realistic environmental exposure scenarios. The next phase of this thesis investigates whether transcriptional, physiological and behavioural endpoints associated with the functioning of the honey bee hypopharyngeal gland were altered by imidacloprid exposure. Imidacloprid exposure led to transcriptional changes in foraging genes (associated with the control of temporal polyethism) and major royal jelly proteins (fed to developing larvae by nurse workers) and enzymatic changes in glucose oxidase (an enzyme involved in social immunity), which I hypothesise are linked with hypopharyngeal gland development. Despite these laboratory observations, no behavioural effects were observed in a field setting, monitored using Radio Frequency Identification transponders. Lastly, using RNA-Sequencing to investigate changes across the honey bee transcriptome, this thesis identified a suite of genes that were differentially expressed in adult workers in response to immune challenge and/or dietary neonicotinoid exposure. Wounding and bacterial-like infection led to upregulation of known immune genes, including a peptidoglycan recognition protein and antimicrobial effectors. Chronic exposure to thiamethoxam and imidacloprid led to downregulation of genes associated with several metabolic pathways, such as oxidative phosphorylation, pyruvate- and purine- metabolic pathways, as well as ribosomal activity. Some of these genes identified provide candidates for further study to elucidate functional effects mechanisms and better understand health outcomes, as well as potential new biomarkers for use in pesticide risk assessment. This thesis presents novel findings and offers opportunities for future research that will be of interest to a wide audience, including risk assessors and policy makers, as well as the broader biological community, including ecotoxicologists, insect physiologists and molecular biologists.

Pollination is an important ecosystem service necessary for 80% of global plant reproduction. This service can also improve fruit production and is essential for 84% of all crops grown in the EU. However, some pollinators have been declining for the last decades and the important honey bee is no exception. One of the reasons to this decline is believed to be agricultural use of chemicals (i.e. fungicides, herbicides and insecticides). One of the most commonly used herbicides is RoundUp, which include glyphosate as its active substance. In this study, I test if RoundUp affects honey bee behaviour by exposing them during a short-term period to one of three different concentrations (0 mg/l, 5 mg/l and 50 mg/l). The test was conducted in a wind tunnel and aimed to test the hypothesis that glyphosate affects searching abilities and flight time of honey bees. In contrast to my hypothesis, I was unable to detect effects on searching ability. However, method limitations might have been the reason only three bees, out of 147, found the food. My analysis showed neither any effects from RoundUp on fly time (P>0.05) or activity (P=0.066). However, in my data I could see a negative trend in activity and due to earlier data collected around glyphosate, I argue that exposure to glyphosate might have a negative long-term impact on honey bee survival.

Varroa destructor, in Czech language known as kleštík včelí, is a parasitic mite (of eastern bee and honeybee) which causes the disease called varroasis. Kleštík is like a black passenger on the body of bees and drones, which commonly penetrate foreign hives, and because of that they can be considered the main vector of parasite. In the Czech Republic we fight this parasite using methods every since its discovery in the early 80s of the 20th century. Some methods are defined by the Veterinary Institute in the Czech Republic and the others were disovered by the beekeepers themselves. The main purpose of this thesis was to determine what are current methods to suppress or to completely obliterate the infection caused by Varroa destructor. What methods have proved to be most effective, and what new methods were invented from the time of its discovery. I conducted a field experiment bee hives belonging to beekeeping organizations Trhové Sviny during the 2015 and the 2016. The practical part of my research ( i.e. occurrence and also the review of varroasis disease treatment) had little effect on normal activities of beekeepers. The process of Varroa destructor presence, its treatment and the methods of treatment researched. Based on the collected samples sent to Veterinary Administration for testing to find out whether are the Varroa counter measures effective or not. The conclusion of my reasearch ist he unificated treatment and detection of Varroa are of the main importance. That goes not only for the beekeeping associations but also by unregistered beekeepers. Because the amount of bee colonies here in the Czech Republic is huge, the use of alternative treatment and suppresing procedures as well as the intentional breeding for resistance against Varroa are rather unrealistic, the only viable option fort he moment , is to follow Veterinary Services guidelines.

Nosemosis is a serious disease of bees caused by microsporidia Nosema apis and Nosema ceranae. Both species are widely spread around the world and in the Czech Republic. The aim of this thesis was to evaluate the incidence and prevalence of Nosema spp., describe the species variability and assess the influence of the season. PCR method amplifying part of the gene encoding the small ribosomal subunit rRNA was used to identify the species of Nosema spp. A total 77 samples originated from 17 farmers were examined Out of them, 71% (55 samples) were positive for the presence of Nosema spp. Samples were collected in five seasons in 2011?2012. Both N. apis, and N. ceranae were detected in all breeds. In 2011, N. apis was detected as causative agent of nosemosis except one sample, where the mixed infection was detected. In 2012, N. ceranae was observed in mono- or mixed infections. Currently monoinfections of N. apis were not detected in 2012. Generally, the highest occurrence was detected in the autumn and winter months.

Diploma Thesis watch qualitative indicators of honey in dependence of different ways of nutrition of bee colony. Determined the influence on fragmentation of honey, content solids materials, acidity of honey, electrical conductivity of honey and using of refractometer to find out content of water.

Made available in DSpace on 2014-06-11T19:32:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-01-10Bitstream added on 2014-06-13T21:05:31Z : No. of bitstreams: 1 melo_crm_dr_botfm.pdf: 282857 bytes, checksum: b93322b1d4fd06e5dda5714db163ab21 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A restrição hospitalar de alimentação e fluido oral para parturientes é uma tradição obstétrica fortemente organizada, justificada pelo risco de regurgitação e aspiração do conteúdo gástrico durante a anestesia. Estudos demonstram que independente do tempo da última refeição, o estômago nunca está completamente vazio, pois o jejum não elimina o conteúdo estomacal; pelo contrário, aumenta a concentração de ácido clorídrico, podendo o jejum prolongado causar aumento do volume gástrico e da acidez. Embora a infusão intravenosa seja necessária, em muitas circunstâncias obstétricas, para administração de medicamentos ou anestesia, o emprego de fluidos intravenosos de rotina não pode ser considerada um substituto completamente seguro de alimento e líquidos no trabalho de parto. Estudos comparam o esforço do trabalho de parto com o desempenho atlético como correr uma maratona, porém há carência de informação das necessidades nutricionais da parturiente e seu feto A normatização das práticas durante a assistência ao parto normal reflete a promoção do parto e nascimento saudáveis, porém observam-se ainda atitudes desvinculadas dos últimos achados científicos. / Oral fluid and dietary restriction for parturients in hospital settings is a highly organized obstetric tradition aimed at preventing regurgitation and aspiration of gastric matter from taking place during anesthesia. Studies have shown that, independently from the time of the last meal, the stomach is never completely empty because fasting does not eliminate stomach contents. Quite the contrary, there is an increase in chloridric acid. Besides, prolonged fasting may raise the level of gastric volume and acidity. Intravenous infusion is necessary, but when it comes to medication or anesthesia management, routine intravenous fluids may not work as well as food and liquids during obstetric labor. Some studies show that giving birth takes as much effort as running a marathon. Nevertheless, information about the nutritional needs of parturient and fetus is scarce. Although less scientific forms of labor management can still be found, concrete measures towards promoting a healthier labor can be taken by the standardization of the assistance to the mother in normal labor.

Made available in DSpace on 2014-06-11T19:33:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-09-19Bitstream added on 2014-06-13T19:23:40Z : No. of bitstreams: 1 cardoso_kfg_dr_botfmvz.pdf: 274279 bytes, checksum: 4c63fe896bafffcb2b2627717e3c4439 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / O objetivo deste trabalho foi caracterizar a qualidade higiênico-sanitária do mel produzido na região do Pólo Cuesta do Estado de São Paulo, por meio da enumeração do Número Mais Provável de Coliformes Totais e Termotolerantes, contagem de bolores e leveduras e atividade de água. Foram coletadas 37 amostras de méis de diferentes floradas produzidas por abelhas Apis mellifera L. em apiários distribuídos na região. As amostras de mel foram coletadas no momento do beneficiamento, acondicionadas em embalagens plásticas do apiário e armazenadas à temperatura ambiente. Todas as análises foram realizadas segundo as recomendações do Ministério da Agricultura, Pecuária e Abastecimento no Laboratório de Microbiologia de Alimentos do Instituto de Biociências da UNESP de Botucatu. Todas as amostras revelaram-se negativas quanto à presença de coliformes totais e termotolerantes e 34 (91,9%) apresentaram contaminação por bolores e leveduras, variando entre 102 até 105 UFC/g. Quanto a atividade de água, os menores valores foram observados na florada de laranja com média de 0,575±0,01 Aa, diferindo significativamente das floradas de eucalipto (0,629±0,02 Aa) e silvestre (0,614±0,02 Aa). Conclui-se que a qualidade microbiológica do mel produzido na região do Pólo Cuesta do Estado de São Paulo apresenta-se satisfatória / The objective of this study was to characterize the hygienic quality of the honey produced in the Pólo Cuesta region of São Paulo state by enumerating the Most Probable Number of Total Coliforms and Thermotolerants, by counting yeasts and molds, and by water activity. Thirty-seven samples of honey of flowerings produced by Apis mellifera L. bees in apiaries spread throughout the region were collected. The 28 honey samples were collected at processing, kept in plastic packages of the apiary, and stored in room temperature. All the analyses were carried out according to the recommendations of the Ministry of Agriculture, Livestock and Supply in the Laboratory of Food Microbiology of the Bioscience Institute at UNESP - Botucatu. All the samples proved negative as to the presence of total coliforms and thermotolerants and 34 (91.9%) of them presented some contamination by yeasts and molds ranging from 102 to 105 UFC/g. As to water activity, the lowest values were observed in the orange flowering with an average of 0.575±0.01 Aa, which is significantly different from the eucalyptus flowering (0.629±0.02 Aa) and from the sylvestral one (0.614±0.02 Aa). It is concluded that the microbiological quality of the honey produced in the Pólo Cuesta region of São Paulo state proves satisfactory

abstract: Food is an essential driver of animal behavior. For social organisms, the acquisition of food guides interactions with the environment and with group-mates. Studies have focused on how social individuals find and choose food sources, and share both food and information with group-mates. However, it is often not clear how experiences throughout an individual's life influence such interactions. The core question of this thesis is how individuals’ experience contributes to within-caste behavioral variation in a social group. I investigate the effects of individual history, including physical injury and food-related experience, on individuals' social food sharing behavior, responses to food-related stimuli, and the associated neural biogenic amine signaling pathways. I use the eusocial honey bee (Apis mellifera) system, one in which individuals exhibit a high degree of plasticity in responses to environmental stimuli and there is a richness of communicatory pathways for food-related information. Foraging exposes honey bees to aversive experiences such as predation, con-specific competition, and environmental toxins. I show that foraging experience changes individuals' response thresholds to sucrose, a main component of adults’ diets, depending on whether foraging conditions are benign or aversive. Bodily injury is demonstrated to reduce individuals' appetitive responses to new, potentially food-predictive odors. Aversive conditions also impact an individual's social food sharing behavior; mouth-to-mouse trophallaxis with particular groupmates is modulated by aversive foraging conditions both for foragers who directly experienced these conditions and non-foragers who were influenced via social contact with foragers. Although the mechanisms underlying these behavioral changes have yet to be resolved, my results implicate biogenic amine signaling pathways as a potential component. Serotonin and octopamine concentrations are shown to undergo long-term change due to distinct foraging experiences. My work serves to highlight the malleability of a social individual's food-related behavior, suggesting that environmental conditions shape how individuals respond to food and share information with group-mates. This thesis contributes to a deeper understanding of inter-individual variation in animal behavior. / Dissertation/Thesis / Doctoral Dissertation Biology 2017

Nós avaliamos a atividade antimicrobiana do mel coletado de ninhos de 12 espécies de abelhas nativas sem ferrão comumente encontrados no Brasil e 25 amostras de mel de Apis mellifera não pasteurizados que foram identificadas como sendo unifloral e uma amostra de mel multifloral. A atividade antimicrobiana de cada amostra de mel foi testada contra cinco espécies de bactérias patogênicas, uma espécie de fungo patogênico e uma espécie de levedura patogênica, comparando esta atividade com o mel terapêutico de manuka produzido por abelhas Apis mellifera na Nova Zelândia a partir do néctar de Leptospermum scoparium (Myrtaceae). Cinco das treze amostras de mel das abelhas sem ferrão foram bactericidas e oito foram fungicidas contra o fungo patogênico Trichophyton rubrum. Somente a levedura Candida albicans foi resistente a todas as amostras de mel. As amostras de mel de Apis mellifera que apresentaram atividade bactericida foram caju, romã e cana, nenhuma das amostras de mel de A. mellifera afetou o fungo T. rubrum. Os meis de Nannotrigona testaceicornis, Plebeia remota, Tetragona clavipes e Scaptotrigona depilis todos com alto nível de atividade antimicrobiana, foram significativamente mais eficiente em termos de atividade antimicrobiana que o mel de manuka, especialmente contra Escherichia coli, Klebsiella pneumoniae e T. rubrum. O mel artificial feito com os principais açúcares encontrados no mel e com teor de água semelhante, não teve atividade antimicrobiana em nossas análises. Nós também fizemos análises de espectrometria de massas das amostras de mel e encontramos alguns \"fingerprint\" característicos que foram associadas com a atividade antimicrobiana. / We evaluated the antimicrobial activity of honey collected from nests of 12 species of native stingless bees commonly found in Brazil and 25 unpasteurized honey samples of Apis mellifera that were identified as being unifloral and one multifloral honey sample. The antimicrobial activity of each honey sample was tested against five species of pathogenic bacteria, a pathogenic fungal species and a pathogenic yeast species, comparing this activity with therapeutic manuka honey produced by Apis mellifera bees in New Zealand from the nectar of Leptospermum scoparium (Myrtaceae). Five of thirteen samples of honey from the stingless bees were bactericidal and eight were fungicidal against the pathogenic fungus Trichophyton rubrum. Only the yeast Candida albicans was resistant to all honey samples. The Apis mellifera honey samples that showed bactericidal activity were caju, romã e cana, none of the A. mellifera honey samples affected the fungus T. rubrum. The honeys from Nannotrigona testaceicornis, Plebeia remote, Tetragona clavipes and Scaptotrigona depilis all had a high degree of antimicrobial activity, they were significantly more efficient in terms of antimicrobial activity than manuka honey, especially against Escherichia coli, Klebsiella pneumoniae and T. rubrum. Artificial honey, made with the main sugars found in honey and with similar water content, had no antimicrobial activity in our analyses. We also made mass spectrometry analyses of the honey samples and found some fingerprint characteristics that were associated with antimicrobial activity.