European honey bee (Apis mellifera) is major pollinator for agriculture and vital for food production. Large number of viruses infecting A. mellifera have been discovered over the years, but it isn't yet known if they are pathogenic for their host. However, presence of non-viral pathogens like Varroa destructor can greatly increase their virulence and have fatal consequences for the colony. The aim of this study was to test and verify robustness of the method for virome detection on healthy honey bees from the Czech Republic. Last but not least we aimed to detect non-viral parasites and correlate their presence with detected viruses. We have successfully identified large number of viral sequences from different viral families. Viral composition was found to be influenced mainly by colony from where the honey bees were collected. That was mainly given by a large amount of bacteriophages in the samples. However, analysis of individual viruses, known to infect honey bee, indicated that viral prevalence and viral loads of specific viruses is quite different among individual honey bees from the same colony. Interestingly we were able to find highly diverse Lake Sinai viruses. We were able to observe correlations either between individual viruses or viral other non-viral pathogens. Further analysis is...

Apis mellifera Linnaeus (1758) è un insetto eusociale conosciuto in tutto il mondo sia per la produzione di miele sia per il suo ruolo di impollinatore, uno dei servizi ecosistemici fondamentali per la biodiversità del pianeta. Durante la sua attività di foraggiamento, l’ape è esposta agli inquinanti ambientali tra cui il particolato atmosferico aerodisperso (PM). Il particolato atmosferico può depositarsi sul corpo dell’insetto e infine contaminare anche i prodotti apistici come polline e miele. Il PM può avere diverse dimensioni (es. PM10, PM2.5, PM0.1), composizione chimica, morfologia e fonti di emissione (naturale o antropica). Nel presente elaborato di tesi, tecniche di microscopia elettronica a scansione (SEM-EDX) sono state utilizzate per caratterizzare la contaminazione da PM di origine antropica del corpo dell’ape e dei suoi prodotti (Capitolo 2 e Capitolo 3) e analisi molecolari per studiare gli eventuali effetti sub-letali sul microbiota intestinale di api esposte ai PM per via orale (Capitolo 4). / Apis mellifera Linnaeus (1758) order Hymenoptera family Apidae, is a eusocial insect widely known for its role in pollination, a fundamental ecosystem service for plant biodiversity and ultimately for the planet. During flight and foraging activity, the honey bee can collect airborne particulate matter (PM) on their own body, especially on the forewings, and can also contaminate bee products as pollen and honey. Particulate matter can originate from natural or anthropic sources, and is characterised by size (e.g., PM10, PM2.5, PM0.1), chemical composition, and morphology. In this thesis, honey bee, pollen and honey were used as bioindicator of PM – from coarse to ultrafine – in industrial areas of the Po Valley, Italy (Chapter 2 and Chapter 3). The (sub-lethal) effects of Titanium dioxide – a widespread airborne PM1 pollutant – on the honey bee through oral exposure was then investigated (Chapter 4). The technique used to analyse the PM contaminating bees and bee products is the scanning electron microscopy (SEM) coupled with X-ray spectrometer (EDX). EDX spectra allowed us to obtain chemical information from specimens, while backscattered-electron (BSE) imaging and elemental mapping provided both compositional and topographic information of PM.

Paenibacillus larvae is a gram-positive spore-forming bacterium that affects and kills the larvae of the honey bee (Apis mellifera) and causes the American foulbrood disease. Adults bees do not become infected, but they transmit tenacious spores within the hive and between hives and can infect larvae while caring for them. It is not allowed by law to treat bees in the Czech Republic, but the recommended preventive method for reducing the amount of spores in the hive is the shook swarm method (bees are moved to a new clean hive and the old hive is destroyed with all brood and supplies). The aim of this work was to detect and quantify P. larvae in bee workers using the quantitative real-time polymerase chain reaction (qPCR). In the first experiment, the two set of samples were taken - bees before and after the shook swarm method, but the expected decrease in spores in the samples taken after shook swarm was not confirmed, and conversely, non-specific products were amplified. In the second experiment, the presence of P. larvae spores in samples from heavily infected hives (with clinical symptoms of American foulbrood) and from hives with almost no findings of P. larvae spores, both originating from the same habitat, were compared. In this case, the differences were clearly visible. There were not...

Lactobacillus kunkeei is the most common and dominant bacterium in the honey stomach of honeybees. L. kunkeei has been isolated from honeybees all over the world. Genome sequencing has identified 5 genes for exceptionally large proteins in the genome of L. kunkeei. These proteins do not show any similarity to sequences of proteins with a known structure. These giant proteins all have a conserved region of 60 amino acids in their C-terminus. This conservation led to the hypothesis that the C-terminal domains of the giant proteins are important for their function with possibly a role in the attachment to the cell wall. In this study, a total of eight different constructs were made for two of these giant proteins. The boundaries for the constructs were determined based on bioinformatic predictions. The eight constructs all have different start positions and all end at the very C-terminal end of the protein. These constructs were cloned into an expression vector. One of the full-length giant protein was cloned into an expression vector as well.  The C-terminal constructs and the full-length proteins were recombinantly produced in Escherichia coli. Expression of six C-terminal constructs was observed and an attempt was made to purify two of the C-terminal constructs. Expression of the full-length giant protein was observed as well and purification was attempted. Neither the C-terminal constructs nor the full-length giant protein could be purified at full length. The results for the C-terminal constructs show that no folded C-terminal domain has been found for the giant proteins. A purified protein construct of the N-terminal of one of the giant proteins was available. This protein was analyzed using biophysical techniques. Circular dichroism was used to test the thermal stability. The construct did not refold after being thermally denatured. Circular dichroism measurements indicated that the N-terminal construct is composed of a mixture of α-helices and ß-sheets. Small-angle X-ray scattering data indicated that the N-terminal construct had an elongated shape with knot-like parts. Protein crystals have been obtained for the N-terminal construct and these will be analyzed using X-ray diffraction.

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Chez de nombreuses Angiospermes, la pollinisation croisée est nécessaire pour le succès de la reproduction. La plupart de ces plantes ont évolué afin de favoriser la pollinisation entomophile, principalement assurée par les abeilles. C’est le cas des Cucurbitacées, dont le melon (Cucumis melo L.), importante culture du sud de la France et le concombre d’âne (Ecballium elaterium (L.) A. Rich.) plante spontanée endémique du bassin méditerranéen, les deux modèles de cette étude. Afin de mieux comprendre les relations plante-pollinisateur,nous avons choisi de caractériser le couple pollen de Cucurbitacées/abeille domestique (Apis mellifera L.) à travers la production de pollen, sa présentation sur la fleur et son transport sur l’abeille. Nous avons montré que, chez ces plantes, l’ontogenèse du pollen s’accompagne d’erreurs développementales conduisant à la formation de près de 3% de grains de pollen vides. De plus, nous avons mis en évidence que chez E. elaterium, des erreurs méiotiques entraînaient la formation de grains de pollen génétiquement anormaux qui pourraient être source d’autopolyploïdie. Chez C. melo, nos résultats ont révélé que la mise en contact du pollen avec le milieu environnant entraîne une baisse de 30% de sa viabilité, mais que celle-ci reste stable jusqu’à la fin de l’anthèse. L’aptitude à germer, quant à elle, finit d’être acquise juste avant anthèse et décroît de 12% en fin de période d’anthèse. Nous avons montré que le pollen de C. melo transporté sur le corps de l’abeille pouvait soit avoir une viabilité et une aptitude à germer préservée, probablement par protection contre la déshydratation, soit perdre quasi-totalement sa viabilité et son aptitude à germer. Enfin, nous avons détecté des composés biochimiques spécifiques des abeilles protégeant ou diminuant la qualité reproductrice du pollen / For many flowering plant species cross pollination is necessary to ensure reproductive success. Most of these plants have evolved to encourage insect pollination which is mainly carried out by bees. Such is the case of plants in the Cucurbitaceae family, including the cantaloupe (Cucumis melo L.), an important crop of the South of France, and squirting cucumber (Ecballium elaterium (L.) A. Rich.), a wild plant restricted to the Mediterranean Basin, the two vegetal models used in this study. To better understand plant-pollinator relations, we proposed to characterise the interaction between Cucurbitaceae pollen and the honey bee (Apis mellifera L.) through pollen production, its presentation and its transport on the honey bee body. Our results showed that developmental anomalies took place during pollen ontogenesis in these species leading to 3% of the pollen grains produced to be empty. Moreover, we demonstrated that meiotic abnormalities in E. elaterium leading to the production of genetically abnormal pollen grains could be a source of autopolyploidy. At anthesis in C. melo, our results showed that pollen viability decreased to 30% when pollen was exposed to environmental conditions, then remained stable during the period of anthesis.The ability for pollen to germinate was completed just before anthesis then decreased to 12% at the end of anthesis. We determined that viability and germinability of cantaloupe pollen carried on the honey bee body can be enhanced or decreased according to which specific honey bee biochemical compounds were detected

La malnutrition est identifiée comme l’un des facteurs potentiellement responsables des mortalités élevées de colonies d’abeilles des dernières années au Québec. Pour contrer cela, les apiculteurs donnent des suppléments de pollen à leurs colonies, mais les impacts d’une telle pratique à diverses périodes sont méconnus. Les effets de la disponibilité du pollen sur le développement de colonies d’abeilles ont été mesurés pendant 3 différentes périodes : au printemps, durant la pollinisation de la canneberge et à la fin de l’été. À chacune des périodes correspondait une expérience distincte utilisant 40 colonies. Pour chaque expérience, des conditions d’abondance de supplément de pollen et de restriction de pollen naturel étaient créées chez les colonies pendant un mois selon un plan d’expérience factorielle 2x2. L’élevage du couvain et la récolte de miel ont été mesurés jusqu’à la fin de l’été (début de l’été suivant pour l’expérience de fin d’été). Au printemps, les colonies restreintes en pollen naturel ont élevé 18% moins de couvain (p<0.05) pendant la période de restriction et 11% de moins à la fin de l’été alors que l’utilisation du supplément n’a eu aucun effet (p>0.05). Les colonies supplémentées durant la pollinisation des canneberges ont élevé moins de couvain (p<0.05) à la fin de l’été. Pour l’expérience de fin d’été, les colonies supplémentées ont eut une meilleure reprise printanière (p<0.05) de l’élevage du couvain (60% de plus) alors qu’une restriction en pollen naturel avait un effet négatif (p>0.05). Les récoltes de miel ont été augmentées (p<0.05) de 1,3 kg pendant la pollinisation de la canneberge alors qu’elles ont été diminuées (p<0.05)par une restriction en pollen naturel de 4,2 kg à la fin de l’été et de 15 kg au printemps. / The use of pollen supplement is a countermeasure to honey bee malnutrition which is identified as one of the factors causing high colonies losses over the past few years in Quebec. There is little documentation on the results of using pollen supplement during different periods. The effects of pollen availability and supplementation on the development of honey bee colonies were examined during 3 different periods: in spring, during cranberry pollination and in late summer. Each period was a distinct study using 40 different colonies. In each study, pollen supplemented and pollen restricted conditions were created for one month in 10 colonies per treatment group in a 2x2 factorial design experiment. Brood rearing and honey yield were monitored until the end of summer for the spring and the cranberry pollination studies and until the end of the following spring for the late summer study. In the spring study, pollen restricted colonies reared 18% less brood (p<0.05) during the restriction period and 11% less brood (p<0.05) by the end of summer while pollen supplement had no effect (p>0.05). Colonies supplemented during cranberry pollination study reared less brood (p<0.05) by the end of summer. In the late summer study, supplemented colonies had a greater (p<0.05) spring build-up (60% more brood) and pollen restriction negatively influence (p<0.05) brood rearing. Honey yield was decreased (p<0.05) by 15 kg in colonies exposed to a pollen restriction in spring. It was increased by 1.3 kg (p<0.05) in pollen supplemented colonies during the cranberry pollination study and was reduced by 4.2 kg (p<0.05) in pollen restricted colonies in the late summer study. In conclusion, pollen supplement improved colonies population when fed in late summer and not during spring or in cranberry pollination and was without impact on honey yield.

Immunodeficient honey bee (Apis mellifera) colonies suffer from broad range of parasites including eukaryotic protozoa. Despite this fact, the eukaryotic parasites are still poorly documented in the Czech Republic. The presence of eukaryotic parasites (Nosema ceranae, Nosema apis, Crithidia mellificae and Apicystis bombi) was observed in different apiaries in the Czech Republic. The samples were taken in 9 apiaries in 53 beehives during the 2014/2015 season. From each beehive, 10 adult of honey bees were taken from the peripheral comb in triplicate. DNA was isolated from every sample of honey bees. The parasites were detected by polymerase chain reaction (PCR) with specific primers. The treatment fall of parasitic mite Varroa destructor was obtained from beekeepers for season of 2014. Crithidia mellificae was detected by 5 types of specific primers (SEF, SER; SSU, SSU rRNA, Cyt b, Tryp cyt b) and positive amplicons were cloned and sequenced. The obtained sequences were compared with GeneBank and showed similarity from 98-100% to sequences of Lotmaria passim (Trypanosomatid). Crithidia mellificae was not detected. L. passim had prevalence of 79,2% and is reported in the Czech Republic for the first time. Primer Tryp-cyt b is recommended for the routine detection of L. passim. Nosema ceranae was...

Nosema ceranae est un parasite émergeant d’Apis mellifera décrit dans certaines régions comme la cause majeure de la mortalité des abeilles. Dans d’autres cas, il est soupçonné d’affaiblir les colonies par l’interaction avec d’autres facteurs de pression de l’environnement. Dans le cadre du phénomène global de la mortalité des abeilles, nous avons orienté nos recherches vers l’étude des effets N. ceranae, en faisant l’hypothèse que ce parasite est capable d’induire des changements comportementaux chez A. mellifera dus à des altérations physiologiques, ce qui pourrait éventuellement perturber l’organisation sociale et aboutir à la mort de la colonie. Etant donné cette hypothèse, trois domaines d’étude ont été inclus dans notre recherche, (i) les effets de N. ceranae sur l’organisation sociale de la colonie, (ii) les mécanismes moléculaires à la base des effets chez les abeilles parasitées, et (iii) les différences en virulence d’isolats de N. ceranae ce qui pourrait expliquer la variation des effets du parasite chez l’abeille. Nous avons obtenu trois résultats majeurs. D’abord, nous avons constaté des modifications dans la structure sociale des abeilles après l’infection. Ces changements sembleraient contribuer à la survie de la colonie constituant probablement un mécanisme d’immunité sociale. Ce mécanisme géré par un signal phéromonal, permettrait de diminuer la transmission du parasite au sein de la colonie et prolonger la survie des abeilles saines. Ensuite, nous avons mis en évidence des effets sur la physiologie de l’intestin de l’abeille qui pourraient causer sa mort : l’induction du stress oxydatif et l’inhibition du renouvellement cellulaire de l’épithélium. Finalement, nos résultats suggèrent que certaines caractéristiques de l’hôte et conditions environnementales augmenteraient la probabilité de N. ceranae d’induire la mort. En conclusion, N. ceranae a le potentiel de causer la mort des abeilles, cependant, la colonie pourrait contrer l’infection, par exemple, par de mécanismes d’immunité sociale, or, la réponse générale à l’infection dépendrait des caractéristiques de l’hôte en combinaison avec les conditions de l’environnement. Le phénomène d’effondrement de colonies à l’échelle mondiale a mis en évidence la fragilité du système colonie d’abeilles – environnement. L’étude de chaque facteur participant au système, en autres, parasites, pesticides, changements dans l’environnement, pratiques apicoles, est essentielle pour une meilleure compréhension de toutes les interactions qui maintiennent l’équilibre écologique des colonies / Nosema ceranae is an emergent parasite of the honey bee Apis mellifera. In some regions it has been found to be the main reason for bee mortality, while in others it is suspected of weakening honey bee colonies by interacting with other environmental stressors. In the context of worldwide colony losses, we focus our research on the study of N. ceranae, with the hypothesis that this parasite is able to induce behavioral changes in bees through physiological modifications, which could alter social organization and cause colony death. Given this hypothesis, the program of study falls into three areas; (i) N. ceranae effects on colony social organization, (ii) molecular mechanisms of N. ceranae infection underlying observed effects, and (iii) differences in virulence of N. ceranae strains which could explain the diversity of parasite effects. We obtained three main results. First, we observed modifications in honey bee social structure after infection. This mechanism under pheromone control, would reduce parasite transmission within the colony and increase the lifespan of healthy bees. These changes may contribute to colony survival as part of a mechanism of social immunity. Second, we found two mechanisms whereby the pathogen affects the physiology of bee midgut epithelium that could lead to host mortality: oxidative stress and the inhibition of cellular renewal. Finally, our results suggest that certain host and environmental conditions increase the probability that N. ceranae will cause bee mortality. In conclusion, N. ceranae has the potential to cause bee death, however at colony level bees might counteract infection through, for example, social immunity mechanisms; although, overall honey bee response to infection would depend on characteristics of the host in combination with environmental conditions. Worldwide colony losses phenomenon have highlighted the fragility of the “honey bee colony – environment” system. The study of each factor involve in this system, including parasites, pesticides, environmental changes and beekeeping practices, is essential to better understand all of the interactions that maintain the ecological balance of honey bee colonies