O desempenho das atividades na colônia de abelhas Apis mellifera está relacionado com o funcionamento do sistema de glândulas que as abelhas possuem. No entanto, a presença de patógenos na população pode causar alterações comportamentais que prejudicam o bom desenvolvimento da colônia. O trabalho objetivou verificar se há relação entre a secreção proteica glandular e cerebral nas diferentes atividades desempenhadas por operárias adultas, em A. m. scutellata (Africanizada) e A. m. ligustica (Europeia), e como o vírus deformador de asas (DWV) poderia interferir no desempenho dessas atividades. Foram realizados: testes de proteínas pelo método Bradford no cérebro e nas glândulas salivar torácica, salivar cefálica, mandibular e hipofaríngea de abelhas Africanizadas e Europeias com idades de 0 (recém-emergidas), 5, 10, 15, 20, 25 e 30 dias; medição da área dos acinos das glândulas salivar cefálica e hipofaríngea dessas abelhas; avaliação do desenvolvimento de atividade realizada pelas abelhas e reversão do processo de oviposição por operárias em abelhas Africanizadas; teste para a presença do DWV por PCR em tempo real, em abelhas Africanizadas e em cérebro e glândulas salivar torácica, salivar cefálica, mandibular e hipofaríngea em abelhas Europeias com 5 e 30 dias de idade; e avaliação morfológica dos danos causados pela infecção aguda do DWV no cérebro e glândulas estudadas. As abelhas Africanizadas e Europeias apresentaram teores estatisticamente significativos de proteínas (p>0,05) para todas as estruturas e idades estudadas. As áreas dos acinos das glândulas salivar cefálica e hipofaríngea apresentaram alterações de acordo com o desenvolvimento dessas glândulas nas diferentes idades estudadas. Foi observado que enxames de abelhas de mesma idade são capazes de executar diferentes atividades necessárias para a sobrevivência de sua colônia, sendo que a principal atividade, desempenhada nas diferentes idades estudadas, era indicada pelos teores significativos de proteínas encontrados (p>0,05). As abelhas Europeias de mesma idade apresentaram desenvolvimento dos acinos da glândula salivar cefálica superior ao dos acinos da hipofaríngea nas idades iniciais avaliadas, diferindo da área dos acinos das mesmas glândulas quando comparadas com abelhas que tiveram o seu desenvolvimento em colônia normal. As operárias podem controlar o estado zanganeiro da colônia. As abelhas Africanizadas e Europeias apresentaram resultados positivos para o vírus DWV, resultado também observado nas glândulas salivar torácica e mandibular das abelhas com cinco dias de idade. Foi observado que a infecção aguda do DWV causa alterações morfológicas nas glândulas das abelhas,as quais apresentam plasticidade no desenvolvimento das atividades, não sendo estas restritas a uma determinada idade. Além disso, a infecção pelo vírus DWV não é limitante em abelhas Africanizadas, mas é em Europeias. / The performance of tasks in the Apis mellifera colony is related to the functioning of their gland system. However, the presence of pathogens in the population may lead to behavior modifications that can harm the proper development of the colony. This work aimed to verify whether there is relation between gland and brain protein secretion for the different tasks performed by the adult worker honeybees, in A. m. scutellata (Africanized) and A. m. ligustica (European), and how the deformed wing virus (DWV) could interfere with the performance of these tasks. The following analysis were carried out: protein tests (Bradford method) in the brain and in mandibular, hypopharyngeal, and thoracic and cephalic salivary glands, in Africanized and European honeybees at the ages of 0 (newborn), 5, 10, 15, 20, 25 and 30 days; measurements of the acini area of cephalic salivary and hypopharyngeal glands in those honeybees; evaluation of the development of tasks performed by honeybees and reversion of the laying workers of Africanized honeybees; test to detect DWV using real-time PCR in Africanized honeybees and in the brain and in mandibular, hypopharyngeal, and thoracic and cephalic salivary glands of European honeybees at the ages of 5 and 30 days; and morphological evaluation of the damages caused by the DWV acute infection in the brain and in the studied glands. Africanized and European honeybees showed statistically significant protein contents (p>0.05) for all the structures and ages studied. The acini areas of cephalic salivary and hypopharyngeal glands showed modifications according to the development of those glands at the different studied ages. It was observed that the swarms of bees with the same age are capable to perform different tasks needed to the survival of their colony, being the principal task, performed at the different studied ages, indicated by the significant protein contents found (p>0.05). The European honeybees at the same age showed higher acini development in the cephalic salivary gland than in the hypopharyngeal one at the initial studied ages, differing from the acini area in the same glands of honeybees grown in a regular colony. The worker honeybees can control the laying workers of the colony. Africanized and European honeybees showed positive results for DWV, which was also observed in thoracic salivary and mandibular glands in five-day-old honeybees. It was observed that the acute DWV infection causes morphological modifications in the honeybees glands, which show plasticity in the performance of tasks, not being them restricted to a particular age. Moreover, the DWV infection is not limiting for Africanized honeybees, but it is for the European ones.

O desempenho das atividades na colônia de abelhas Apis mellifera está relacionado com o funcionamento do sistema de glândulas que as abelhas possuem. No entanto, a presença de patógenos na população pode causar alterações comportamentais que prejudicam o bom desenvolvimento da colônia. O trabalho objetivou verificar se há relação entre a secreção proteica glandular e cerebral nas diferentes atividades desempenhadas por operárias adultas, em A. m. scutellata (Africanizada) e A. m. ligustica (Europeia), e como o vírus deformador de asas (DWV) poderia interferir no desempenho dessas atividades. Foram realizados: testes de proteínas pelo método Bradford no cérebro e nas glândulas salivar torácica, salivar cefálica, mandibular e hipofaríngea de abelhas Africanizadas e Europeias com idades de 0 (recém-emergidas), 5, 10, 15, 20, 25 e 30 dias; medição da área dos acinos das glândulas salivar cefálica e hipofaríngea dessas abelhas; avaliação do desenvolvimento de atividade realizada pelas abelhas e reversão do processo de oviposição por operárias em abelhas Africanizadas; teste para a presença do DWV por PCR em tempo real, em abelhas Africanizadas e em cérebro e glândulas salivar torácica, salivar cefálica, mandibular e hipofaríngea em abelhas Europeias com 5 e 30 dias de idade; e avaliação morfológica dos danos causados pela infecção aguda do DWV no cérebro e glândulas estudadas. As abelhas Africanizadas e Europeias apresentaram teores estatisticamente significativos de proteínas (p>0,05) para todas as estruturas e idades estudadas. As áreas dos acinos das glândulas salivar cefálica e hipofaríngea apresentaram alterações de acordo com o desenvolvimento dessas glândulas nas diferentes idades estudadas. Foi observado que enxames de abelhas de mesma idade são capazes de executar diferentes atividades necessárias para a sobrevivência de sua colônia, sendo que a principal atividade, desempenhada nas diferentes idades estudadas, era indicada pelos teores significativos de proteínas encontrados (p>0,05). As abelhas Europeias de mesma idade apresentaram desenvolvimento dos acinos da glândula salivar cefálica superior ao dos acinos da hipofaríngea nas idades iniciais avaliadas, diferindo da área dos acinos das mesmas glândulas quando comparadas com abelhas que tiveram o seu desenvolvimento em colônia normal. As operárias podem controlar o estado zanganeiro da colônia. As abelhas Africanizadas e Europeias apresentaram resultados positivos para o vírus DWV, resultado também observado nas glândulas salivar torácica e mandibular das abelhas com cinco dias de idade. Foi observado que a infecção aguda do DWV causa alterações morfológicas nas glândulas das abelhas,as quais apresentam plasticidade no desenvolvimento das atividades, não sendo estas restritas a uma determinada idade. Além disso, a infecção pelo vírus DWV não é limitante em abelhas Africanizadas, mas é em Europeias. / The performance of tasks in the Apis mellifera colony is related to the functioning of their gland system. However, the presence of pathogens in the population may lead to behavior modifications that can harm the proper development of the colony. This work aimed to verify whether there is relation between gland and brain protein secretion for the different tasks performed by the adult worker honeybees, in A. m. scutellata (Africanized) and A. m. ligustica (European), and how the deformed wing virus (DWV) could interfere with the performance of these tasks. The following analysis were carried out: protein tests (Bradford method) in the brain and in mandibular, hypopharyngeal, and thoracic and cephalic salivary glands, in Africanized and European honeybees at the ages of 0 (newborn), 5, 10, 15, 20, 25 and 30 days; measurements of the acini area of cephalic salivary and hypopharyngeal glands in those honeybees; evaluation of the development of tasks performed by honeybees and reversion of the laying workers of Africanized honeybees; test to detect DWV using real-time PCR in Africanized honeybees and in the brain and in mandibular, hypopharyngeal, and thoracic and cephalic salivary glands of European honeybees at the ages of 5 and 30 days; and morphological evaluation of the damages caused by the DWV acute infection in the brain and in the studied glands. Africanized and European honeybees showed statistically significant protein contents (p>0.05) for all the structures and ages studied. The acini areas of cephalic salivary and hypopharyngeal glands showed modifications according to the development of those glands at the different studied ages. It was observed that the swarms of bees with the same age are capable to perform different tasks needed to the survival of their colony, being the principal task, performed at the different studied ages, indicated by the significant protein contents found (p>0.05). The European honeybees at the same age showed higher acini development in the cephalic salivary gland than in the hypopharyngeal one at the initial studied ages, differing from the acini area in the same glands of honeybees grown in a regular colony. The worker honeybees can control the laying workers of the colony. Africanized and European honeybees showed positive results for DWV, which was also observed in thoracic salivary and mandibular glands in five-day-old honeybees. It was observed that the acute DWV infection causes morphological modifications in the honeybees glands, which show plasticity in the performance of tasks, not being them restricted to a particular age. Moreover, the DWV infection is not limiting for Africanized honeybees, but it is for the European ones.

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.

4 Summary Altogether, the six most common bee viruses which infect the honey bee (Apis mellifera) were monitored in the territory of the Czech Republic between 2006 and 2009. Parallel infections of viruses (DWV, ABPV and BQCV) in bee adults and parallel co- infection of viruses with fungal diseases caused by Nosema apis and Nosema ceranae were confirmed by PCR tests. A new sensitive method of detection of the originator of the American foulbrood (Paenibacillus larvae) from bee debris was developed for the practical use of detection of AFB disease in bee populations. Various approaches for the extraction of spores from bee debris and lyses of spores were compared. The sensitivity of PCR tests for the presence of Paenibacillus larvae in debris was compared with the classic cultivation method. The PCR method for the detection American foulbrood was further studied and developed to be more efficient. A new method, based on a matrix-like sample re-arrangement and a use of pooled samples, has been developed for testing 1000 samples in 35 PCR reactions. Another goal was to develop a robust and fast screening method for American foulbrood based on the cultivation test using paper sheets RIDA®COUNT with a specific cultivation medium, specific selection conditions for Paenibacillus larvae and chromogen visualization...

L’abeille mellifère (Apis mellifera) est une espèce clé dans son aire d’origine tant pour les services écosystémiques rendus que pour les productions apicoles. Toutefois, ses populations sont soumises à différents facteurs de stress biotiques responsables de mortalités importantes. Dans les îles du Sud- Ouest de l’Océan Indien (SOOI) la sous-espèce d’abeille endémique est A. m. unicolor. Toutefois, aucune étude n’avait été réalisée sur sa pathosphère et ce n’est que depuis 2010 où l’ectoparasite Varroa destructor a envahi certaines de ces îles, menaçant cette sous-espèce, que de premières études ont été conduites. Dans ce travail de thèse, nous avons réalisé un état des lieux général de la santé des cheptels d’abeilles grâce à des enquêtes épidémiologiques descriptives dans la majorité des îles : La Réunion, Maurice, Rodrigues, Madagascar et les Seychelles. Nous avons pu montrer des faciès épidémiologiques assez similaires entre les îles, marqués par une dominance de Nosema ceranae particulièrement dans les petites îles (80-100%) et la présence de 3 virus : BQCV (4-89%), CBPV (2-51% excepté à Rodrigues) et DWV (4-40% excepté à La Réunion). D’autres agents pathogènes n’ont été retrouvés que sur certains territoires comme Aethina tumida, Braula pretoriensis, Acarapis sp. ou Melissococcus plutonius. L’analyse de la diversité génétique réalisée sur les 3 virus a montré une proximité des souches virales au sein du SOOI. Les enquêtes réalisées dans un contexte sans varroa ont montré une bonne santé des colonies avec une absence de signes cliniques. Les mortalités constatées n’ont concernées que les territoires envahis par varroa : Madagascar, Maurice, La Réunion. Varroa semble donc être le principal responsable des mortalités aiguës de colonies dans la zone bien avant les autres agents pathogènes ou les causes environnementales. Son impact sur les populations d’abeilles et in fine sur les écosystèmes indigènes sera à évaluer dans les années futures. / The honeybee (Apis mellifera) is a key species in its native range for both ecosystem services offerred and for bee products. However, its populations are subject to various biotic stressors responsible for significant mortalities. In the South-West Indian Ocean region the endemic bee subspecies is A. m.unicolor. However, no studies had been carried out on its pathosphere and it is only since 2010 where the ectoparasite Varroa destructor invaded some of these islands, threatening this subspecies, that first studies were conducted. In this thesis work, we carried out a general inventory of the health of honeybee herds through descriptive epidemiological surveys in the majority of islands: Réunion, Mauritius, Rodrigues, Madagascar and Seychelles. We were able to show quite similar epidemiological facies between islands, characterized by a dominance of Nosema ceranae especially in small islands (80-100%) and the presence of 3 viruses: BQCV (4-89%), CBPV (2- 51% except in Rodrigues) and DWV (4-40% except in Reunion). Other pathogens have only been found in certain territories such as Aethinatumida, Braula pretoriensis, Acarapis sp. or Melissococcus plutonius. The analysis of the genetic diversity carried out on the 3 viruses showed a proximity of viral strains within the SOOI. Surveys conducted in a context without varroa showed good colony health with no clinical signs. The observed mortalities concerned only the territories invaded by varroa: Madagascar, Mauritius, Reunion. Varroa therefore appears to be the main cause of acute colony mortality in the area long before other pathogens or environmental causes. Its impact on bee populations and ultimately on native ecosystems will be evaluated in future years.

L’abeille domestique (Apis mellifera) est un insecte qui contribue à l’agriculture par sa pollinisation. Le taux élevé des mortalités hivernales des colonies est préoccupant depuis des décennies au Canada. Plusieurs facteurs sont impliqués, particulièrement Varroa destructor; un parasite qui se nourrit du corps gras de l’abeille. Le développement d’outils adaptés permettrait un meilleur suivi des colonies. Le projet consiste à corréler l’infestation de varroa avec les concentrations en sucres sériques et les co-infections (virales et bactériennes). Cette étude compare dans le temps six ruches fortement infestées et six ruches traitées (témoins). Un prélèvement d’hémolymphe a été effectué pour mesurer les concentrations en sucres en utilisant un glucomètre humain préalablement validé. Les concentrations en sucres (glucose et tréhalose) dans l’hémolymphe étaient significativement plus faibles (p<0.001) dans les ruches fortement infestées que les témoins en septembre. L’analyse RT-PCR multiplexe de six virus (DWV A/B, BCQV, KBV, IAPV et ABPV) a démontré que les ruches fortement infestées présentent une infection simultanée virale avec des charges plus élevées que chez les ruches témoins (p<0.05) pour la majorité des virus, sauf pour ABPV. Chez les ruches fortement parasitées, les charges virales pour DWVA et BQCV sont plus élevées en septembre qu’en juillet (p≤0.0001). Serratia marcescens a été seulement détectée dans une ruche infestée et une ruche témoin. Une exposition continue et élevée à varroa occasionne, en automne, une augmentation des charges virales et une diminution des sucres, suggérant une altération de l’immunité, du métabolisme et des réserves. Ces paramètres provoquent une faiblesse et une mortalité des colonies. / The European honeybee (Apis mellifera) contributes to the agriculture by its pollination; however, the mean overwintering loss rate of colonies over the last decades in Canada is worrisome. Varroa destructor, which feeds on the fat bodies of honeybees, is considered one of the most important causes of bee colony declines. The development of adapted diagnostic tools would improve the monitoring of honeybee health. This project aims to correlate the infestation by varroa to the hemolymph sugar concentrations (trehalose and glucose) and bacterial and viral coinfections. Six highly infested and six treated hives were compared over time. Pooled hemolymph of honeybees was collected for sugar concentration measurements using a previously validated portable glucometer. The hemolymph samples were also submitted for bacteriology. Multiplex RT-PCR analyses were performed on pooled honeybees for six viruses: Deformed wing virus A and B (DWV-A/B), Bee Queen Cell Virus (BQCV), Acute Bee Paralysis Virus (ABPV), Kashmere Bee Virus (KBV), Israeli Acute Paralysis Virus (IAPV). The results show that, in September, sugar concentrations in hemolymph were significantly lower in highly infested hives (p<0.001). Infested hives showed markedly higher viral loads (p<0.05), except for ABPV. Viral loads were significantly higher (p≤0.0001) in September than in July for DWV-A and BQCV. Serratia marcescens was only detected in one infested hive and one control. Overall, a continued and severe exposure to varroa leads to increased viral charges and decreased sugar concentrations, suggesting alterations in immunity, metabolism and reserve mobilization. All these parameters contribute to the weakening and mortality of the colonies.