This thesis is focused on the research of the varroatosis disease. In the theoretical part of the thesis I describe the parasitic mite Varroa destructor and its way of infestation. The goal was to document any knowledge gathered on the subject so far and the possibilities of bee vaccination. The practical part of this work included Varroa destructor occurrence monitoring within single hives. The monitoring was carried out in three localities. Localities was called Nedrahovice, Rybník and Kvašťov. The goal was to identify singular hive tolerance to the mite in the various localities based on the strength of the parasitic mite attack. While the hives of the Rybník locality were found to be the most tolerant, the hives of the Nedrahovice locality were found to be the least tolerant of all.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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Varroa destructor, principal ectoparasite de l'abeille, est l'un des responsables majeurs des phénomènes de perte de colonies observés partout dans le monde. Depuis son changement d'hôte et l'infestation de colonies d'Apis mellifera dans les années 1970, des traitements essentiellement chimiques ont été proposés pour limiter l'infestation. Pourtant ces dernières années, ces traitements ont fait l'objet d'études critiques à la fois à cause de la résistance émergente du parasite aux différents acaricides et du fait de la sensibilité des abeilles à l'accumulation de produits chimiques dans les différentes matrices de la ruche. De nouveaux traitements alternatifs contre la varroose sont donc nécessaires et très attendus afin d'enrayer rapidement les effets délétères du parasitisme acarien. Cependant, la recherche de ces nouvelles cibles thérapeutiques est limitée par l'absence d'un système d'élevage synchronisé de l'hôte et de son parasite en laboratoire. L'objectif technique de ce travail de thèse était donc de pallier à ce manque en développant une méthode pour coupler l'élevage de larves d'abeilles in vitro avec le transfert à la fois de la phase phorétique et de la phase de reproduction du parasite (respectivement sur les adultes et sur les pupes) dans des conditions de laboratoire. Le système mis au point lors de ces travaux de thèse a permis une étude plus approfondie de l'impact de facteurs variables chez l'hôte sur plusieurs points clés du cycle parasitaire. Les effets de la nutrition larvaire chez l'abeille, la longueur de la phase phorétique, le type d'abeilles adultes sur lequel Varroa reste au cours de cette phase ou encore l'âge des stades larvaires ont ainsi été étudiés. L'analyse des impacts potentiels de ces facteurs a porté principalement sur certains paramètres de la reproduction, du comportement et de la virulence de V. destructor. Les résultats obtenus ont montré que la nutrition larvaire, ou plus précisément la supplémentation de la nourriture larvaire avec des pollens monofloraux et polyfloraux, a un faible impact sur la reproduction du parasite, mais présente une tendance à influencer le poids de l'abeille ainsi qu'à potentiellement augmenter la tolérance de l'abeille à certains des symptômes de la varroose, tels que la perte de poids. La reproduction du parasite n'a pas non plus été affectée par les variations induites dans la phase phorétique. Ni la fertilité du parasite, ni l'expression des deux gènes de la vitellogénine n'ont fluctué lorsque la durée ou l'âge des adultes parasités pendant la phase phorétique variaient. En revanche, la durée du séjour sur les abeilles adultes avant la reproduction est corrélée positivement à la charge virale chez le parasite, ce qui explique pourquoi les varroas ayant vécu une longue phase phorétique induisent plus fréquemment des malformations. Enfin, l'âge des stades juvéniles des abeilles sont tous attrayants pour Varroa destructor, bien que certaines préférences aient été mises en évidence dans notre étude. Si l'attraction pour le 5ème stade larvaire et les abeilles nouvellement écloses est déjà connu, l'attractivité des nymphes aux yeux blancs est un phénomène nouveau qui pourrait être d'importance majeure pour la compréhension du cycle parasitaire. Des analyses approfondies pour isoler les signaux responsables de cette attraction restent nécessaires. En conclusion, les travaux de cette thèse offrent une méthode d'élevage innovante et ouvrent ainsi, pour l'ensemble de la communauté apicole, de nouvelles avenues de recherche passionnantes afin de développer nos connaissances à la fois sur l'abeille, sur le parasite et sur les virus qu'il transporte. Ce travail pourrait permettre d'identifier de nouvelles cibles thérapeutiques pour la conception de traitements alternatifs ciblant spécifiquement le parasite sans nuire à l'abeille. / Varroa destructor is the principal acarian parasite of the honey bee, involved in the phenomenon of colony loss which has been reported worldwide. Since its host shift from Apis cerana to Apis mellifera in the 1970's, chemical treatments have been set up to limit infestation. For many years however, these treatments have been the subject of critical studies both because of the resistance of the parasite to acaricides and of the sensitivity of bees to chemical accumulation in hive matrices. New alternative ways of varroatosis treatment are therefore urgently needed to limit the deleterious effects of the acarian parasitism. However, the search for these alternative therapeutic targets is limited by the lack of a laboratory system to transfer and synchronize the cycles of V. destructor and the honey bee under controlled conditions. The methodological objective of this Phd work was to overcome this issue by developing a method to couple the rearing of honey bee larvae in vitro to the transfer of both the parasite phoretic and reproductive phases (respectively on adults and on pupae) in laboratory conditions. Our system allowed further investigation regarding the impact of variable parameters in the host on several key points of the parasite cycle. The effects of several factors -such as the bee larval nutrition, the length of the phoretic phase, the type of adult bees on which Varroa stays during this phase or the age of pupal development- were studied. The potential impact in the parasite cycle focused mainly on reproductive, behavioural and virulence related parameters. The results obtained from our experiments showed that larval nutrition, tested via the supplementation of the larval food with pollen, had no impact on the reproduction of the parasite but a tendency to influence the weight of the bee, potentially increasing the bee tolerance to some of the symptoms of varroatosis, such as the weight loss. The reproduction of Varroa was not impacted by variations induced in the phoretic phase either. Neither the fertility of the parasite nor the expression of its two vitellogenin genes were impacted by the duration or the age of adults parasitized during the phoretic phase. On the other hand, the length of the stay on adult bees before reproduction positively correlates with the viral loads in the parasite, which explains why varroas experiencing a long phoretic phase induce malformation more frequently in parasitized pupae. Finally, the age of juvenile stages of the honey bees are all attractive to Varroa destructor, although some preferences have been highlighted in our study. If the attraction for 5th instar larvae and newly emerged bees have already been described, the attractions towards the white eyed pupae is a new phenomenon that could be of major importance in the understanding of the parasite cycle. Further analysis to isolate and shed light on the attraction signals involved in the process is needed. Altogether, this thesis has supplied, for the whole scientific beekeeping community, new exciting research avenues and methodologies to develop our knowledge about the bee, the parasite and the viruses it carries. It might allow us, in the future, to design alternative treatment specifically targeting the parasite without injuring the honey bee.

The aim of the thesis was to evaluate the influence of mite Varroa destructor on the health of honeybees and the occurrence of viral diseases of bees. The presence of Nosema was also explored. The occurrence of deformed wing virus and acute bee paralysis virus was detected by RT-PCR method. Coprological examination proved the incidence of spore Nosema spp.

Submitted by Élida Maeli Fernandes Quirino (maely_sax@hotmail.com) on 2018-05-15T13:05:55Z No. of bitstreams: 1 FRNACISCO ARICLENES OLINTO - DISSERTAÇÃO PPGSA PROFISSIONAL 2014..pdf: 1640106 bytes, checksum: b19b02a5c2c2ea22def9e997924518db (MD5) / Made available in DSpace on 2018-05-15T13:05:55Z (GMT). No. of bitstreams: 1 FRNACISCO ARICLENES OLINTO - DISSERTAÇÃO PPGSA PROFISSIONAL 2014..pdf: 1640106 bytes, checksum: b19b02a5c2c2ea22def9e997924518db (MD5) Previous issue date: 2014-12-16 / A apicultura é uma das poucas atividades agropecuárias que atende aos três requisitos da sustentabilidade: o econômico, o social e o ecológico. Sendo assim, fornece renda para o apicultor, ocupa mão de obra familiar ou contratada e contribui para a preservação da flora nativa. A abelha é importante para a economia mundial como polinizadora, aumentando a produção de frutos e sementes, e como fornecedora de mel, cera, geleia real, própolis, pólen e veneno (apitoxina). Esses produtos são muito procurados para a matéria-prima das indústrias farmacêutica, alimentícia e cosmética e para consumo em forma natural. A sanidade pode afetar o desenvolvimento da apicultura, pois a Apis mellifera como qualquer outro organismo vivo, é susceptível a doenças causadas por bactérias, vírus, fungos e outros parasitas e as desordens metabólicas, nutricionais e hormonais, além de intoxicações diversas. Assim, em virtude da necessidade de estudos a respeito da sanidade apícola, objetiva-se estudar o comportamento higiênico e a identificação da varroatose e nosemose em colônias de abelhas Apis mellifera em apiários localizados no Sertão do Estado da Paraíba. A pesquisa foi realizada no período de março, abril e maio de 2014, em cinco apiários localizados nos municípios de Condado, Pombal, Jericó, São Bentinho e São Domingos, ambos situados na Mesorregião do Sertão do Estado da Paraíba, com um total de 25 colmeias avaliadas. O teste de comportamento higiênico foi realizado com base no método de perfuração das células de crias. Para análise da infestação por ácaro Varroa destructor, as abelhas adultas foram coletadas e acondicionadas em frascos de vidro contendo álcool a 70%, após 24 horas foi realizada a contagem dos ácaros. Para a identificação de Nosema spp. foi utilizado o método de nível de infecção da colônia, sendo selecionadas dez abelhas adultas para retirada do aparelho intestinal e posterior leitura em microscópio óptico. O percentual de comportamento higiênico foi semelhante em ambos os apiários, principalmente em Condado (93,96%), Pombal (94,30%), Jericó (87,63%) e São Domingos (95,20%), ocorrendo apenas uma ligeira diferença no apiário de São Bentinho com uma média de 76,31%. No apiário de Condado foram encontrados 59 ácaros Varroa destructor, nas colmeias do apiário de Pombal obteve-se apenas seis ácaros, em Jericó foram encontrados 19 ácaros, no apiário de São Bentinho, foi observado o maior número de ácaros entre os apiários, 62 varroas no total e no apiário de São Domingos foram observados 48 ácaros. A frequência de Nosema spp. em colmeias manejadas na região do estudo foi de 36% de um total de 25 enxames avaliados. O apiário localizado no município de Pombal obteve os melhores resultados, apresentando índice elevado de comportamento higiênico e valores baixos para varroatose e nosemose. A africanização das abelhas e as altas temperaturas do Sertão Paraibano tendem a possibilitar melhor resistência das colmeias a enfermidades. / Beekeeping is one of the few agricultural activities that meets the three requirements of sustainability: economic, social and ecological. Therefore, provides income for the beekeeper, occupies family labor or hired and contributes to the preservation of native flora. The bee is important for the world economy as a pollinator, increasing the production of fruits and seeds, and as a supplier of honey, beeswax, royal jelly, propolis, pollen and venom (bee venom). These products are in high demand for raw materials in the pharmaceutical, food and cosmetics industries and for consumption in natural form industries. Sanity may affect the development of beekeeping because Apis mellifera like any other living organism is susceptible to diseases caused by bacteria, viruses, fungi and other parasites and metabolic, nutritional and hormonal disorders, and several poisoning. Thus, because of the need for studies concerning the apiculture health, this study focuses on hygienic behavior and the identification of varroatose and nosemosis in Apis mellifera honeybee colonies in apiaries located in the backlands of the state of Paraíba. The survey was conducted from March, April and May 2014, in five apiaries located in the cities of Condado, Pombal, Jericó, São Bentinho and São Domingos, both located in the Greater Region of the Backlands of the State of Paraíba, with a total of 25 evaluated hives. The hygienic behavior test was conducted based on the method of drilling the brood. For analysis of the mite Varroa destructor infestation, the adult bees were collected and placed in glass vials containing 70% alcohol, after 24 hours was performed to count the mites. For the identification of Nosema spp. we used the colony of infection level method, selecting ten adult bees to remove the intestinal and later reading device in an optical microscope. The hygienic behavior percentage was similar in both apiaries, especially in Condado (93.96%), Pombal (94.30%), Jericó (87.63%) and São Domingos (95.20%); there was one slight difference in the apiary of São Bentinho with an average of 76.31%. In Condado apiary found 59 mites Varroa destructor in the apiary hives of Pombal was obtained only six mites Jericó found 19 mites in the apiary of São Bentinho, it was observed the largest number of mites between apiaries, 62 varroas in total and in the apiary of São Domingos were observed 48 mites. The frequency of Nosema spp. In the hives managed in the region of the study was 36% of a total of 25 clusters evaluated. Apiary located in the city of Pombal obtained the best results, with high level of hygienic behavior and low values for varroatose and nosemosis. The Africanization of bees and the high temperatures of the Paraiba’s hinterlands tend to provide improved resistance to diseases of the hives.

Arsenophonus is vertically and horizontally transmitted parasitic bacteria and strengthens its transfer through phenomenon called son killing. Arsenophonus has been detected in the arthropod hosts, insects, ticks and the garden spider Araneus diadematus. The aim of this study is detection parasitic bacteria Arsenophonus in honeybees and its parasite Varroa destructor. We find out that bacteria Arsenophonus is present in both hosts and it is identified as Arsenophonus nasoniae. Detection of bacteria Arsenophonus is discovered for the first time in Varroa destructor mites. We proved that the frequency of bacteria Arsenophonus in mites Varroa destructor is significantly higher than in honeybees. This study shows that the mite Varroa destructor could act as a vector for transmission parasitic bacteria Arsenophonus among honeybees. Results of this study could lead to the future application of Arsenophonus as a biological control for the mite Varroa destructor.

The mite ectoparasite Varroa destructor, poses a serious threat for the survival of the Apis mellifera colonies. The intensive use of acaricidal products is one of the most common methods for defending bees from Varroa that can cause the contamination of the wax foundation used in beekeeping. The natural honeycomb management could provide a solution for this problem, because it involves the use of frames without wax foundation which allows the bees to build a complete comb ex novo. On the other hand, colonies which are free to build cells of their choice, usually build a number of drone cells higher than colonies managed with the wax foundation. This could potentially lead to several negative consequences because the V. destructor reproductive success is greater on drone broods than on worker broods. The aim of the present study was to examine the colony development, to evaluate the honey production and to monitor the growth of V. destructor infestations and associated virus infections in Apis mellifera colonies managed by using natural honeycombs compared with the conventional management. Several colony parameters were measured in spring and summer. The strength of the colony was used to estimate the worker and drone populations. In order to measure the V. destructor infestations were used several methods, such as the natural mite fall, the powdered sugar roll, the soapy water and the brood cell uncapping. Molecular analysis was performed in order to measure the viral load of five Apis mellifera viruses. The honey produced was measured by collecting the honey stored in the supers, which are boxes placed on a beehive for bees to store. The results showed that the higher presence of drone brood in the colonies managed using the natural honeycomb did not negatively affect the colony development nor the mite V. destructor population compared to control colonies. The molecular analysis showed that the DWV was the most common virus found in bee samples, and its viral load was more influenced from the mite infestation rate than from the treatment. The analysis carried out in this study showed that the natural honeycomb management can represent a valid alternative to the wax foundation. This kind of colony management thus appears to contradict our primary hypothesis which was that letting the bees build their own honeycomb would have led to a significant increase in the V. destructor infestation. Productivity data did not provide reliable results about the difference between the natural honeycomb and the conventional colony management due to climatic adversities. Further studies will be performed to better investigate this aspect. Data about the natural mite fall and the estimation of the mite population in the phoretic/reproductive phases provided a useful starting point for further studies on the correct timing to carry out acaricide treatments both in conventional and natural honeycomb managed colonies.

Laboratory bioassay was used to determine the pathogenicity of nine isolates of fungi of the genera Metarhizium, Beauveria and Clonostachys to the parasitic mite, Varroa destructor. All nine isolates were pathogenic to V. destructor with Metarhizuim anisopliae UAMH 9198, Clonostachys rosea UAMH 9161 and Beauveria bassiana GHA being the most pathogenic within their respective species. Metarhizium anisopliae UAMH 9198 was more lethal to V. destructor than B. bassiana GHA and C. rosea UAMH 9161 with LC50 values of 1.6 x 10(5), 9.6 x 10(6) and 5.4 x 10(6) conidia/mL, respectively. Metarhizium anisopliae and B. bassiana significantly affected brood and adult honey bee survivorship and their immune responses. They were lethal to the bees with LC50s of 3.70 x 10(6) and 2.62 x 10()5 conidia/mL, respectively. The effect of temperature and thymol on conidia germination, production and colony growth of the fungal isolates was determined. Temperature significantly affected conidia germination, production and colony growth (P < 0.05) but thymol did not. Efficacy of the two most promising isolates (M. anisopliae UAMH 9198 and B. bassiana GHA) as potential bio-control agents against V. destructor in hives was evaluated. Fungal inocula were applied as dry formulation, with corn flour as carrier, using dispenser tray and dusting applications. Treatments were either applied alone or in combination with thymol to determine any synergistic effects. All treatments significantly increased mite mortality (P < 0.05), however, the mite control efficacy varied between fungal treatments and application methods. Combined treatments of fungi and thymol caused significantly higher mite mortality than single fungal treatments, which showed control levels of ≤61%. Significant differences in mite mortality were found between the two delivery methods with the differences depending on the fungal isolate. The results suggest that M. anisopliae UAMH 9198 would be a more effective bio-control agent for the management of V. destructor in honey bee colonies than the other isolates tested when dispensed continuously in hives using delivery methods such as a dispenser tray. However, future research is needed to improve delivery methods and investigate the effect of carriers used in the formulation on the efficacy of such entomopathogenic fungi. / Ontario Ministry of Agriculture and Food (OMAF), University of Guelph

O objetivo desse estudo foi estimar parâmetros genéticos para características produtivas e comportamentais em uma população de abelhas Apis mellifera africanizadas por meio de inferência Bayesiana. Os dados foram submetidos a análises uni e bicaracterística utilizando o programa MTGSAM. Os modelos consideraram os efeitos (fixos) de local do apiário, mês-ano ou estação-ano e o número de caixilhos com abelhas aderentes como covariável linear. As estimativas de herdabilidade apresentaram magnitudes de moderada a alta para comportamento higiênico (0,81 ± 0,17), produção de própolis (0,83 ± 0,16), produção de mel (0,37 ± 0,22) e taxa de coleta de xarope (0,39 ± 0,22) e magnitude baixa para a percentagem de ácaros em abelhas adultas (0,12 ± 0,13). A rapidez de coleta de xarope apresentou correlação genética de 0,21 ± 0,51 com produção de mel, de 0,45 ± 0,33 com produção de própolis, e de 0,05 ± 0,43 com comportamento higiênico. As correlações genéticas entre produção de mel, produção de própolis e comportamento higiênico foram de 0,20 ± 0,43, de -0,11 ± 0,41 e de 0,23 ± 0,31, respectivamente. As correlações genéticas foram negativas entre percentagem de ácaros em abelhas adultas e as características produção de mel (-0,63 ± 0,39), produção de própolis (-0,07 ± 0,50), comportamento higiênico (-0,19 ± 0,51) e rapidez de coleta de xarope (- 0,41 ± 0,51). As características produção de mel, produção de própolis e comportamento higiênico apresentam potencial para seleção genética. A menor percentagem de ácaros em abelhas adultas está relacionado a maior produção de mel e maior comportamento higiênico, mas não deve ser usado como único critério de seleção devido a baixa herdabilidade. A seleção de abelhas que coletam xarope mais rapidamente, prevendo maior produção de mel, promoverá pequeno ganho genético. Ao selecionar abelhas que produzem mais própolis haverá pequenos ganhos genéticos para comportamento higiênico ou maior produção de mel. / This study was carried out to estimate genetic parameters for productive and behavioural traits in Africanized honey bees Apis mellifera. The data were submitted uni and bicharacter analysis using the software MTGSAM. The fixed effects considered in the models were localization of the hive, month-year or season-year and number of frames covered with bees as covariate. The heritability estimates were moderate to high for hygienic behaviour (0,81 ± 0,17), propolis production (0,83 ± 0,16), honey production (0,37 ± 0,22) and syrup-collection rate (0,39 ± 0,22) and lower for percentage of mites on adult bees (0,12 ± 0,13). Syrup-collection rate showed genetic correlation values of 0,21 ± 0,51 with honey production, 0,45 ± 0,33 with propolis production and 0,05 ± 0,43 with hygienic behaviour. Genetic correlation between honey and propolis was 0,20 ± 0,43, between honey production and hygienic behaviour was -0,11 ± 0,41 and between propolis production and hygienic behaviour was 0,23 ± 0,31. Genetic correlations were negative between percentage of mites on adult bees and other traits honey production (-0,63 ± 0,39), propolis production (-0,07 ± 0,50), hygienic behaviour (-0,19 ± 0,51) and syrup-collection rate. Honey production, propolis production and hygienic behavior traits have potential for genetic selection. The lower percentage of mites on adult bees increase honey production or hygienic behaviour, but it is not recommended as the only criterion for selection, due to its low heritability. Selection for syrupcollection rate will promote small genetic gain for honey production. Propolis production is positively correlated to hygienic behaviour or honey production.