Excessive honey bee colony losses all over the world are believed to be caused by multiple stressors. In this thesis, I characterized and quantified pathogen levels in honey bee colonies, studied their interactions with each other and with their associated parasite vectors, examined factors that influence their combined impacts on honey bees and developed methods to manage honey bee viruses so that colony losses can be minimized. My baseline study of virus prevalence and concentration in healthy and unhealthy (showing visible signs of disease) colonies in Canada showed that seven economically important viruses (DWV, BQCV, IAPV, KBV, SBV, ABPV, and CBPV) were all widely distributed in Canada. Differences in concentration and prevalence of some viruses were found between unhealthy and healthy colonies but these differences may have been due in part to seasonal or regional effects. Studies of the impact of viruses on worker bee populations over winter showed different factors were correlated with bee loss in different environments. Spring concentrations of DWV and mean abundance of Varroa (Varroa destructor) were positively correlated with bee loss and negatively correlated with spring population size in outdoor-wintered colonies. Fall concentration of IAPV was negatively correlated with spring population size of colonies in indoor-wintering environments but not in outdoor-environments. My study showed that it is important to consider location of sampling when associating pathogen loads with bee loss with Nosema and BQCV. Seasonal patterns of parasites and pathogens were characterized for each wintering methods (indoor and outdoor). My results revealed lower ABPV and Nosema ceranae prevalence and lower DWV concentration in genetically diverse than genetically similar colonies. I showed that within colony genetic diversity may be an important evolutionary adaptation to allow honey bees to defend against a wide range of diseases. In laboratory studies, I showed that feeding DWV to larvae in the absence of Varroa causes wing deformity and decreased survival rates of adult bees relative to bees not fed DWV. Finally, I showed that RNA silencing can be used to reduce DWV concentrations in immature and adult bees, reduce wing deformity in emerging adults, and increase their longevity relative to controls.

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.

Nosematosis belongs to the intestinal disease of bees, which is widely spread both in the world and in the Czech Republic. It is caused by Nosema apis and Nosema ceranae. The aim of this diploma thesis was to evaluate the occurrence of the Nosema dispute in the territory of ČSV Bystřice, to describe species variability and to assess the effect of the season on the occurrence of Nosema apis and Nosema ceranae. To detect infection of colonies, 5 bees from selected hives were examined under the microscope. In positive specimens on nosematosis, the species of Nosema spore was molecularly examined. In total, 93 samples from 15 breeders were examined. Samples were taken in the spring and summer of 2017. The sampling took place both for malachists and for farmers with more than 150 bee colonies. During the summer sampling more positive bees were found than in spring sampling. Beekeepers were further assessed by the number of hives. Following the analysis of the samples obtained using the PCR method, Nosema ceranae disputes were present in the vast majority of colonies.

Le parasite microsporidien Nosema ceranae est un pathogène émergent de l’abeille européenne (Apis mellifera). Il provoque une maladie appelée nosémose qui peut induire de fortes mortalités dans les colonies. La présence de N. ceranae dans les ruches n’est pas toutefois pas systématiquement associée à des symptômes ou à une dépopulation, ce qui suggère une variabilité de sa virulence. Une hypothèse proposée pour expliquer cette variation repose sur l'existence potentielle de variants parasitaires de niveaux de virulence différents. Ce travail a eu pour objectif d’évaluer le polymorphisme de N. ceranae par une approche multilocus, dans le but de savoir s’il est possible de différencier des isolats parasitaires. La diversité nucléotidique de dix marqueurs génétiques a été évaluée dans des abeilles géographiquement éloignées. L’analyse du polymorphisme de ces gènes a révélé un fort contenu allélique au sein même d'un individu hôte mais une absence de divergence entre les populations parasitaires issues d'hôtes distincts. Ces données montrent que cette approche multilocus ne permet de pas de différencier des isolats de N. ceranae, mais que des populations parasitaires similaires infectent des abeilles géographiquement distantes. Ces données sont en accord avec l'hypothèse d'une colonisation récente d'A. mellifera par N. ceranae mais posent de nombreuses questions quand à l'origine de la diversité parasitaire au sein d'un seul individu. Le second volet de cette thèse a eu pour objectif de rechercher dans le génome de N. ceranae des gènes codant de potentiels facteurs de virulence puis de produire des protéines recombinantes et des anticorps dirigés contre ces facteurs. Ces anticorps devaient permettre de localiser ces protéines d'intérêt au niveau subcellulaire dans des tissus infectés. / The microsporidian parasite Nosema ceranae is an emergent pathogen of the Western honeybee (Apis mellifera). It is associated to a disease called nosemosis that can lead to high mortality of honybees in colonies. Its presence in hives has not been systematically linked to symptoms or depopulation, suggesting a variation in its virulence. Thus, the existence of several N. ceranae variants with different virulence levels has been proposed. In this work aimed to assess N. ceranae polymorphism through a multilocus approach to test whether is it possible to discriminate between parasite taxa. Thus the nucleotide diversity of ten marker genes has been measured in parasite populations isolated from single A. mellifera individuals in distant locations. While high nucleotide diversity and allele content have been observed for all genes in single individuals, the absence of isolate differentiation precluded any taxa discrimination. These data support the hypothesis of a recent host-jump to A. mellifera and suggest that similar populations of parasites infect honeybees in distant locations. However they question the origin of such polymorphism within one host. In the second part of this work genes encoding putative virulence factors have been searched within N. ceranae genome, in order to produce recombinant proteins and then specific antibodies. Such antibodies would allow the subcellular localization of those proteins in infected tissues.

Western honey bees (Apis mellifera; hereafter honey bees) provide vital pollination services to global agriculture and biodiversity. However in recent years they have experienced severe population declines in many regions of the northern hemisphere. Although causes of these honey bee declines are not well understood, multiple pressures such as changes in land-use and climate, management issues, and introduced parasites are believed to be responsible. First described in honey bees in 2006 during a period of high colony mortalities, the microsporidian gut parasite Nosema ceranae became of great concern. In this dissertation I investigated the distribution, management, virulence, and inter-specific interactions of this introduced species. First, I described and clarified the multiple pressures believed to influence honey bee health, including N. ceranae, especially in relation to the mysterious phenomenon Colony Collapse Disorder. I then surveyed colonies in Maritime Canada for N. ceranae and the historic honey bee microsporidian Nosema apis. Although both species were present at a regional scale, intensive sampling in Nova Scotia revealed that N. ceranae was highly prevalent compared to the historic congener. Next, I investigated two potential management options for the parasite. Chemotherapy using the fungicide fumagillin reduced N. ceranae spore intensity but had no effect on colony survival, and indoor over-wintering did not reduce spore intensity but was associated with increased colony survivorship in spring. Using a comparative approach, I observed that N. ceranae infection significantly reduced honey bee longevity in the laboratory but did not influence overall colony health or strength in the field. Last, a laboratory study demonstrated reduced spore production during N. ceranae and N. apis co-infection, possibly due to inter-specific competition that has resulted in the displacement of the historic Nosema species by N. ceranae in many global regions. This dissertation provides crucial information on biology and management of N. ceranae that can be used towards the development of an integrated pest management strategy, and for future studies investigating factors that may influence the parasite’s distribution, virulence, and inter-specific interactions.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-03-13T13:17:05Z No. of bitstreams: 1 FranklinAS_DISSERT.pdf: 1406892 bytes, checksum: 31ccee360ab84a9af3e75803363344c4 (MD5) / Made available in DSpace on 2017-03-13T13:17:05Z (GMT). No. of bitstreams: 1 FranklinAS_DISSERT.pdf: 1406892 bytes, checksum: 31ccee360ab84a9af3e75803363344c4 (MD5) Previous issue date: 2016-02-27 / Africanized honey bees (Apis mellifera) from the region of the Semiarid Potiguar has two main diseases: varroatose, whose agent is the mite Varroa destructor and, the Nosemosis disease, recently introduced in the state, whose agent was previously identified in the area of our experimental apiaries as being the microsporide Nosema ceranae. The objectives of this study were to evaluate the effects of direct insolation and shading on the hives, in relation to the mite infestation rate in the adult bees and in the worker brood; on hygienic behavior and on the Nosemosis disease. The experiment was conducted in CETAPIS / UFERSA in the Mossoró-RN city. It were used 10 beehives installed under a metallic structure covered by dry leaves of coconut, it was built on the east-west direction, and with a capacity to absorbed about 90% of the direct insolation. Ten other beehives were installed under the direct effect of insolation, about 15 meters in relation to the former group. The results showed that during the dry season period the hives installed in the shade had an average infestation rate (%) in adult bees significantly lower (P <0.01) than in hives installed in the sun. Although the results show a smaller infestation trend on the bee brood of hives installed in the shade, no significant differences were observed (P = 0.253). Already hygienic behavior was very low, considering that the bees were Africanized bees and it were captured in the region during the swarming season. However, there was a significant negative correlation between the hygienic behavior and the mite infestation rate in adult bees. The nosemosis disease showed one prevalence of 100% in the experimental apiary, however, the average number of spores per bee can be considered very low, being significantly higher during the harvest period than in the off season period's, however, this results did not show significant differences between the hives receiving direct insolation and that ones under shade / As abelhas africanizadas (Apis mellifera) da região do Semiárido Potiguar apresentam duas doenças principais: A varroatose cujo agente é o ácaro Varroa destructor, e a nosemose, recentemente introduzida no estado, cujo agente foi identificado anteriormente na região dos apiários experimentais como sendo o microsporídio Nosema ceranae. Os objetivos desta pesquisa foram avaliar os efeitos diretos da insolação e do sombreamento sobre colmeias em relação à taxa de infestação do ácaro em abelhas adultas e nas crias; sobre o comportamento higiênico e sobre a doença nosemose. O experimento foi realizado no CETAPIS/UFERSA em Mossoró, RN, onde, utilizou-se 10 colmeias instaladas sob uma latada construída no sentido leste-oeste, coberta com folhas de coqueiro que absorvia cerca de 90% da insolação e, outras 10 sob efeito direto da insolação. As coletas foram feitas no período da safra (chuvoso), nos dias 30/01, 20/03, 09/4 e 10/05, e no período da entressafra (seca), nos dias 18/08, 23/09, 07/10 e 30/11. Os resultados mostraram que durante o período de entressafra as colmeias instaladas na sombra apresentaram uma taxa de infestação média (%) (6,54 ± 0,59) em abelhas adultas menor (P<0.01) do que nas colmeias instaladas no sol (9,71 ± 1,02). Embora os resultados tenham mostrado uma tendência de infestação menor nas crias em colmeias instaladas na sombra, em relação as instaladas no sol, não foram observadas diferenças significativas (P=0.253). Já o comportamento higiênico (%) foi muito baixo para as colmeias na sombra e no sol (57, 61 ± 6,44 e 57,33 ± 7,96) respectivamente. No entanto, observou-se uma correlação negativa significativa entre este comportamento e a taxa de infestação do ácaro em abelhas adultas. Quanto à nosemose, foram encontrados esporos em todas as colmeias tanto no sol quando na sombra, mas no entanto, o número médio de esporos por abelha pode ser considerado muito baixo, sendo significativamente maior no período de safra (200.000 ± 40. 869) do que na entressafra (31.250 ± 3.900), no entanto, não se obteve diferenças significativas entre as colmeias na sombra e no sol / 2017-03-13

De nombreuses études suggèrent que le déclin des colonies d’abeilles domestiques (Apis mellifera) serait dû à l’action combinée de plusieurs facteurs de stress, et notamment des agents pathogènes et des pesticides. Nous avons précédemment démontré qu’une co-exposition des abeilles au parasite intestinal Nosema ceranae et à l'insecticide fipronil, administré chroniquement en doses sublétales, entraînait une forte augmentation de la mortalité des abeilles. De plus, des études suggèrent que l'infection par N. ceranae pourrait augmenter la capacité antioxydante des cellules intestinales de l'abeille. Nous nous sommes demandé si l'élévation du taux de mortalité dans un contexte d'infection, combiné à une intoxication au fipronil, pourrait être le résultat d'une production d'espèces réactives de l'oxygène (ERO). Nos résultats indiquent une diminution de la quantité des ERO, mais aussi de la quantité de protéines oxydées en présence de N. ceranae. Ceci pourrait être la résultante d’une augmentation des activités enzymatiques antioxydantes. Lorsque les abeilles ont été traitées avec les deux facteurs de stress (N. ceranae et fipronil), nous n’avons cependant pas mesuré d’augmentation des ERO, tandis que l’oxydation des protéines était significativement augmentée. Ainsi, la présence du parasite semble perturber la balance oxydative des cellules intestinales et pourrait augmenter la toxicité du fipronil. Des études complémentaires ont également été menées in vitro sur des cellules humaines HFF, infectées avec une autre espèce microsporidienne, Encephalitozoon cuniculi, et/ou exposées au fipronil. Les résultats ont montré que la présence du parasite limitait l’augmentation des ERO induite par le fipronil. De plus, des résultats préliminaires tendent à montrer une augmentation de l’activité métabolique des mitochondries dans les cellules infectées par le parasite. Enfin, dans le but de mieux comprendre le dialogue N. ceranae/abeille/microbiote intestinal, nous avons analysé par une approche de séquençage d’amplicons d’ADNr et d’ARNr 16S la composition et l’abondance des communautés microbiennes de l’intestin après infection et/ou intoxication chronique avec différents pesticides. N. ceranae semble perturber l’activité de plusieurs groupes bactériens, et la présence de pesticides accroît fortement ces perturbations. Ainsi, l’impact d’une co-exposition N. ceranae/pesticides sur le microbiote intestinal pourrait être l’un des éléments clés du déclin des colonies. / Many studies suggest that the observed decline of Apis mellifera honeybee colonies would be due to the combined action of multiple stressors, including both pathogens and pesticides. We previously demonstrated that the honeybee co-exposure to the gut parasite Nosema ceranae and the fipronil insecticide, administered chronically in sublethal doses, highly increased the bee mortality. Moreover, studies suggest that the infection by N. ceranae may increase the antioxidant capacity of the bee intestinal cells. We wondered whether the increase in mortality rate when infection is combined with fipronil intoxication could be the result of reactive oxygen species (ROS) production. Our results indicate that both the ROS amount and the concentration of oxidized proteins decreased upon infection. This could be the result of an increased antioxidant enzymatic activities. When bees were co-exposed to both stressors (N. ceranae and fipronil), we did not measured any increase in ROS level, but the amount of oxidized proteins was significantly increased. Thus, the presence of the parasite seems to disrupt the oxidative balance of the intestinal cells and could increase the toxicity of fipronil. Complementary studies were also conducted in vitro with human cells (HFF), infected with a different microsporidian species, Encephalitozoon cuniculi, and/or treated with fipronil. The results showed that the presence of the parasite reduced the increase in ROS induced by fipronil. In addition, preliminary results showed an increase in mitochondrial metabolic activity in cells infected with the parasite. Finally, in order to better understand the N. ceranae/honeybee/intestinal microbiota dialogue, we analysed the composition and the abundance of microbial communities in the gut after infection and/or intoxication with different pesticides using a next generation sequencing of both rDNA and rRNA 16S amplicons. N. ceranae seems to upset the activity of different groups of bacteria, and the presence of pesticides greatly increased these disturbances. Thus, the impact of N. ceranae/pesticide co-exposure on the intestinal microbiota may be one of the key elements in the decline of honey bee colonies.

Nosema ceranae, a microsporidian, has been given much attention in recent years as it has been linked with Colony Collapse Disorder (CCD), which leads to the sudden deaths of honey bee colonies. It has been described that many pathogenic organisms secrete virulence factors in order to hijack its host`s cellular functions, but in most cases the underlying mechanisms of this process still remains to be deciphered. Cornman et al. (2009) have identified in N. ceranae a list of putative effector proteins (called secretome) destined to be secreted into the host, and I have taken this list for further investigation using a bioinformatical and experimental approaches. The principal aim of this project was to generate a N. ceranae ORFeome for genes predicted to be secreted, elucidate the function of effector candidates important for N. ceranae biology and/or pathogenicity, as well as to investigate any interactions between N. ceranae proteins and its host utilising two eukaryotic model organisms, budding yeast, S. cerevisiae, and fruit fly, D. melanogaster. A library of S. cerevisiae strains expressing N. ceranae proteins was generated utilising the Gateway® technology, and phenotypic and localisation screens were undertaken to investigate the N. ceranae secretome. Two N. ceranae ORFs, NcORF-15 (NcORF-02039) and NcORF-16 (NcORF-01159) encoding a putative thioredoxin and a hexokinase, respectively, were subjected to yeast complementation assays in order to assess their catalytic activity. NcORF-15, the putative thioredoxin, was able to rescue the sensitive phenotype of S. cerevisiae Δtrx2 under oxidative stress, whereas NcORF-16, the putative hexokinase, did not complement YSH7.4-3C, a triple knockout lacking hexokinase activity. A third N. ceranae effector candidate NcORF-4 (NcORF-00654), a putative proteasome subunit, was investigated for its nuclear localisation and protein interactions in both S. cerevisiae and D. melanogaster.

This report investigates the presence of Nosema in Sweden. Nosema is an intracellular parasite within the order Microsporidia and is considered a global threat. The most recent academic study on the presence of Nosema in Sweden was published in 2013, however in that study no presence of Nosema in the north of Sweden was reported. The aim of this study was to explore the presence of Nosema in Sweden, particularly in the north of Sweden. Samples were gathered from different locations in Sweden, and thereafter analysed (n=74), 54 samples were analysed under microscope. A geographical map was created to establish the range of Nosema on a global perspective. PCR primers and FISH gene probes for molecular identification were evaluated and tested both in SILVA and in an own database created in the bioinformatics software package ‘ARB’. Main findings of the study were that Nosema were detected in two samples in Sweden and that further studies with more sophisticated methods and better sequencing needs to be developed in order to fully investigate the presence of Nosema in the north of Sweden.

Managed honey bee colonies face severe winter losses in northern climates. In my studies, interactions between genotypes of bees (genetically selected stock and unselected stock) with different levels of resistance and tolerance to varroa mites were assessed under a variety of treatment combinations to quantify effects of queen pheromone, acaricide treatment, wintering method, ventilation condition and pathogen infection on the costs and benefits associated with mite removal and mite-tolerance behaviors. In most of the experiments, mite-resistance caused greater varroa mite mortality within selected stock relative to unselected stock. Artificial and natural sources of queen pheromone caused greater varroa mite mortality within honey bee colonies relative to queenless colonies. While mite resistance had significant benefits, I showed that when producers selected colonies containing some mite resistance traits, it was traits associated with mite-tolerance and not mite-resistance were maintained and contributed to wintering success. Tolerance was effective at two levels of mites as obtained by late autumn treatment of colonies with oxalic but treatment did not improve wintering performance of either stock. Selected stock showed greater colony size, survival and resulted in more viable colonies in spring in comparison to unselected stock with similar initial mite levels (0.16 mites per bee). Selected stock showed greater relative wintering success than unselected stock when wintered indoors than when wintered outdoors but indoor wintering improved colony survival in both stocks relative to outdoor wintering. Carbon dioxide level increased within the winter bee cluster when colonies were maintained under restricted-ventilation (mean 3.82±0.031%, range 0.43-8.44%) and restricted ventilation increased mite mortality by 138% relative to standard-ventilation (mean 1.29±0.031%, range 0.09-5.26%), but restricted-ventilation did not affect bee mortality in comparison to standard-ventilation. In a laboratory study, I showed that Nosema inoculation (with co-infections of N. ceranae and N. apis) suppressed the effectiveness of mite removal behavior within selected bees relative to unselected bees. N. ceranae was more abundant than N. apis. Bees with greater mite removal capacities had higher costs associated with varroa-resistance as indicated by greater bee mortality rates when inoculated with varroa but bee mortality was not affected in Nosema inoculated bees.