Originally described from the Asian honey bee, Apis cerana, the microsporidian Nosema ceranae is an obligate, intracellular parasite that has recently been discovered infecting the western honey bee, Apis mellifera. More research on the biology of N. ceranae as well as on the impact it may have on A. mellifera is greatly needed. However, conducting studies on N. ceranae is not only dependent on seasonal availability of Nosema spores, but also on reliable methods for determining spore viability. This study presents the results of using cryogenics to provide long term storage of viable N. ceranae spores and a differential staining procedure that details how to use bright field microscopy with the fluorescent viability dye, propidium iodide (PI), and the fluorescent stain, 4', 6-diamidino-2-phenylindole (DAPI) to differentiate viable and nonviable spores. Using these methods, it was found that freezing N. ceranae at -70 °C in 10% glycerol yielded the lowest mean rate of spore mortality after thawing (24.2% ± 2.2).

Nosema ceranae is a recently described, widespread microsporidian parasite of Apis mellifera that has raised concerns as to whether it is contributing to increased colony losses. To better understand this parasite, investigations were made into the seasonality of infections, alternative transmission mechanisms, and potential control approaches. All studies used real-time PCR with specific primers and probes for N. ceranae, as well as traditional spore analysis. Monthly colony monitoring in Virginia showed that N. ceranae was present yearlong with the highest levels observed in April-June and lower levels through the fall and winter. There was no difference in infection levels among bees sampled from different areas of the hive regardless of the time of year. Additionally, N. ceranae infects all castes of the colony. Drones of different ages, including pupae, in-hive, and flying drones, were found to be infected at low levels with infections most prevalent during peak annual levels in April-June. Approximately 5% of flying drones had moderate to high levels of infection indicating that flying drones, which would be the most likely age group to drift, could assist in the horizontal transmission of N. ceranae both within and between apiaries. Immature and mated queens were also found to be infected at low levels. Infection in the ovaries and spermathecae suggests the possibility for vertical transmission. Finally, control of N. ceranae is thought to improve the health of bees and to reduce colony losses. Fall fumagillin treatments and winter stimulative pollen feeding were compared. Neither treatment significantly lowered N. ceranae levels in colonies sampled 3-6 months later, nor did they significantly improve colony survival. Due to the high cost of treatment and the time required, we do not recommend either treatment for N. ceranae infections during the fall. Colony winter losses due solely to N. ceranae seem unlikely because levels of N. ceranae were low. Impacts from N. ceranae infections were also minimal during the summer as productive colonies had some of the highest levels of infection. Although N. ceranae is prevalent throughout hives, it does not seem to be a major cause of colony losses. / Ph. D.

Apis mellifera est un insecte pollinisateur jouant un rôle économique et écologique majeur. Depuis plus d’une vingtaine d’années, d’importantes pertes de colonies d’abeilles ont été recensées à l’échelle mondiale. L’origine de ce phénomène impliquerait de nombreux facteurs de stress qui pourraient en outre interagir entre eux. Ce travail de thèse a eu pour but d’évaluer l’impact sur la santé de l’abeille de l’association entre un facteur biotique, le parasite microsporidien Nosema ceranae, et un facteur abiotique, des insecticides neurotoxiques à faibles doses. Des études en laboratoire ont montré que l’association N. ceranae-insecticide entraine une surmortalité significative, et plus précisément un effet synergique sur la mortalité des abeilles. Cet effet synergique semble indépendant de l’ordre d’exposition des abeilles aux deux facteurs de stress. De plus, lorsqu’ils ont été appliqués dès l’émergence des abeilles, ces facteurs ont eu un impact plus fort sur la mortalité. La réponse de l’abeille à N. ceranae et aux insecticides a ensuite été analysée à l’échelle transcriptomique. L’analyse du transcriptome de l’intestin a été réalisée en combinant une approche globale de séquençage à haut débit (RNA-Seq) et un suivi de l’expression d’une sélection de gènes par qRT-PCR. L’exposition à N. ceranae et aux insecticides a entraîné des modifications de l’expression de gènes impliqués dans les défenses de l’abeille (immunité, détoxication) et dans les métabolismes du tréhalose et de la chitine. De nombreuses perspectives à ce travail sont envisageables dans le but de mieux appréhender la réponse de l’abeille à différents facteurs de stress, notamment en combinant des expérimentations en laboratoire avec des études de terrain. / Apis mellifera is a pollinator insect playing of major economical and ecological importance. For more than two decades, severe honeybee colony losses have been reported worldwide. The origin of this phenomenon is thought to involve numerous stressors that could interact with each other. The objective of this work was to evaluate the impact on honeybee health of the association between a biotic stressor, namely the microsporidian parasite Nosema ceranae, and an abiotic stressor, here low doses of neurotoxic insecticides. Laboratory studies showed that the N. ceranae-insecticide association leads to a higher mortality in honeybees, and more precisely to a synergistic effect. This effect seemed to be independent of the exposure sequence to stressors. Moreover, when applied at the emergence of honeybees, these stressors had a higher impact on individuals’ mortality. The honeybee response to N. ceranae and to insecticides has also been analysed at a transcriptional level. A midgut transcriptome analysis has been performed combining a global approach, using high-throughput sequencing (RNA-Seq), and a quantitative RT-PCR monitoring of the expression of selected genes. The exposure to N. ceranae and to insecticides led to modifications in the expression of genes involved in honeybee defenses (immunity, detoxification) and in trehalose and chitin metabolisms. Following this work, several prospects can be initiated in order to improve our understanding regarding the honeybee response to various stressors, combining laboratory experiments with field studies.

Lors de la mise en place d’une interaction hôte-parasite, les principales barrières à franchir sont les mêmes quel que soit l’hôte considéré. Il faut que le parasite rencontre l’hôte puis qu’il soit capable d’échapper à ses systèmes de défenses. Pour cela, au-cours de la coévolution, les parasites ont ainsi développé des stratégies moléculaires leur permettant de pirater les réseaux de l’hôte, menant à l’établissement d’un dialogue moléculaire. Les microsporidies, qui sont des parasites intracellulaires obligatoires, ont, du fait de leur forte dépendance vis-à-vis de leur hôte, probablement développé des stratégies très poussées de piratage. L’objectif de cette thèse a été d’initier le décryptage du dialogue moléculaire qui s’établit entre une microsporidie et son hôte à deux niveaux d’intégration. Au niveau cellulaire, l’étude de la réponse protéique de cellules fibroblastiques humaines à l’infection par Anncaliia algerae a permis de suggérer l’existence d’une stratégie originale de leurre de l’hôte grâce à l’expression d’un élément transposable. Au niveau tissulaire, l’étude de la réponse protéique d’intestin d’abeilles infectées par Nosema ceranae a révélé une perturbation de l’homéostasie du tissu intestinal pouvant être à l’origine d’un impact négatif de l’infection sur le taux de renouvellement de l’épithélium. Un suivi du taux de multiplication des cellules souches intestinales lors d’une cinétique d’infection nous a permis de conforter cette hypothèse. Le suivi de l’expression de gènes impliqués dans les voies de signalisation contrôlant ce taux de renouvellement a confirmé une perturbation de l’homéostasie intestinale de l’abeille. Cependant, les mécanismes par lesquels les microsporidies arrivent à se développer chez leurs hôtes ne sont pas connus et méritent d’être explorés. / Within the host-parasite interaction, the parasite need to cross the same barriers whatever the host considered. First, the parasite has to meet its host and to escape its defense systems. For this purpose, the parasites have developed, during coevolution, molecular strategies allowing them to hijack the host networks, leading to the set-up of a real molecular crosstalk. Microsporidia, which are obligate intracellular parasites, have probably developed very sophisticated strategies to hijack their host cell functions as they are strongly dependent to their hosts. The objective of this thesis was to pave the way to the deciphering of the molecular dialogue that takes place during the interaction between a microsporidia and its host, at two different integration levels. At the cellular level, the study of the proteome response of human fibroblast cells to the infection by Anncaliia algerae allowed us to suggest the existence of a lure strategy used by A. algerae to bypass the host response. At the tissue level, the study of the midgut proteome response of honeybees infected by Nosema ceranae revealed a disturbance of the intestinal homeostasis. These results lead us to the hypothesis of a negative impact of the infection on the midgut epithelium renewal rates. This assumption was confirmed by a monitoring of the multiplication rate of intestinal stem cells during a kinetics of infection and of the expression of genes implicated in the signaling pathways controlling this renewal. However, the underlying mechanisms allowing microsporidia to develop in hosts are not known and deserve to be explored.

The nosem infection is a worldwide spread bee illness caused by two microsporidia (Nosema apis and Nosema ceranae). The aim of this thesis is to follow the occurrence of Nosema spp. and density of infection in the selected bee colonies considering the relation between N. apis, N. ceranae, climatic conditions and the condition of each bee colony concerned. In my thesis, I have used two ways of originator identification. The first was microscopy, using which we have discovered significant changes in the number of spores in one bee colony within one year. After the experiment had finished, we evaluated the number of measured spores in connection with the outside temperature, humidity, number of colonized frames, extenders, gentleness of bees and their sitting on the honey combs. The second way was using the PCR method. Through this method we have identified the percentage of positive bee colonies on the selected locations. Furthermore, we have confirmed the occurrence of each of the originators and evaluated if there is a mutual influence of Nosema spp. between bee 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.

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.

Microsporidia are a unique phylum of ubiquitous fungal pathogens that are able to infect a wide variety of hosts, including economically and ecologically important organisms. Recently, global declines of the Western honeybee (Apis mellifera) have been associated with infections of the microsporidian pathogen Nosema ceranae. This species was originally described in the Asiatic honeybee (A. cerana), and its identification in global A. mellifera hives could result from a recent host transfer. Recent genome studies have found that global populations of this parasite from A. mellifera hives are polyploid and that humans may have fueled their global expansion. In this thesis, I investigate the genetic diversity of N. ceranae populations from within their native range (Thailand) and among different hosts (A. mellifera, A. cerana), putting them in context with other previously sequenced global populations. Using both PCR and genome-based methods, my findings reveal that Thai populations of N. ceranae exhibit interesting genetic differences from other global pathogen populations but also have some similarities. Thai N. ceranae populations share many single nucleotide polymorphisms (SNPs) with other global populations and appear to be clonal. However, in stark contrast with previous studies, these populations carry many SNPs not found in other global populations of this parasite, indicating that these populations have evolved in their current geographic location for some time. This genome analysis also indicates the potential presence of diploidy within Thai populations of N. ceranae and possible host-specific loss of heterozygosity. Overall, my findings begin to reveal interesting patterns of genetic diversity in N. ceranae populations that bring us one step closer to understanding the biology and genetics of this important honeybee pathogen.