SammanfattningBinäringen i Sverige kan tyckas vara ett smalt område, men trots detta så är kommunikationenmellan de olika aktörerna inom området bristfällig. Detta orsakar i sig att det är svårt att bildasig en uppfattning om Apis Mellifera skall betraktas som skyddsvärd. Syftet med denna studieär främst att undersöka om A. Mellifera drabbas av massdöd och konsekvenser av detta.Studien undersöker även de hotbilder som kan leda till eventuell massdöd och hur dessa kanbekämpas både preventivt och progressivt.De metoder som används i studien är sekundäranalys och semistrukturerade kvalitativaintervjuer. Metoderna valdes ut för att tydligt sammanställa och redovisa vad tidigareforskning genererat för resultat och sedan jämföra resultaten med intervjusvaren från experterinom den svenska binäringen och forskningssamhället för att utfinna vilka eventuella likheteroch skiljaktigheter. Materialet i metoddelen har främst analyserats med hjälp av AlanBrymans (2011) bok om kvalitativa och kvantitativa metoder.Resultaten visar att det finns en viss samstämmighet i somliga statiska variabler som monetäraförluster och vinster av A. Melliferas och andra pollinatörers ekosystemtjänster och att derasvikt för biologisk mångfald samt att det finns ett antal hot och försvårande omständigheter närdet gäller bevarandet av honungsbin. Resultaten visar också en stor skepticism från bådeforskare och andra experter, både inom litteraturen och aktuell forskning. För att uppnå enkonsensus för hållbar utveckling inom detta område som gynnar miljön, A. Mellifera och sistmen inte minst jordbrukarna rent ekonomisk behövs ett tydligare ramverk och lagar samtincitament för att kompensera jordbrukare som övergår till ekologisk odling.Nyckelord: Apis Mellifera, bin, massdöd, jordbruk, monokulturer, varroa destructor,pesticider, insekticider, Imidakloprid, neonikotinoider / AbstractThe Swedish bee-industry may seem like a small field, but despite of that the communicationbetween the different actors in the industry is inadequate. This makes it difficult to form anopinion wheter A. Mellifera should be considered to be in need of protection or not. Thepurpose of this study has primarily been to examine if A. Mellifera is suffering frommassdeath and eventual consequenses of this. This study also examines the threats that couldcause massdeath and how they aret o be managed in a preventive and progressive way.The study was conducted as a mix of a secondary analysis and semistructuredqualitative interviews, this to compile and present what results have come out of recentresearch and then compare that reserach with the answers from a mix of experts in theswedish bee-industry and the scientific society to try to find the out what eventual similaritiesand differences there are. The material in the methodpart has mainly been analyzed with thehelp of Alan Brymans (2011) book on qualitative and quantitative methods.The results show that there is some consistency in some static variables that monetarylosses and gains by A. Melliferas and other pollinators ecosystem services and theirimportance for biological diversity and that there are a number of threats and aggravatingcircumstances in the conservation of honeybees. At the same time, the results show a greatskepticism from scientists and other experts, both in literature and current research in relationto each other. In order to achieve a consensus for sustainable development in this area thatbenefit the environment, A. mellifera and last both not least the farmers in an economic waythere is a need for a clearer framework and laws and incentives to compensate farmers makingan ecological transition.Keywords: Apis Mellifera, bees, massdeath, agriculture, monocultures, varroa destructor,pesticides, insecticides, neonicotinoids

The loss of managed honey bee colonies, resulting from their unintentional exposure to pesticides, is a topic of concern for the agricultural and apicultural industry. Current methods for reducing pesticide exposure to bees involve the application of pesticides before crop bloom or in the evening when foraging bees are less likely to be exposed to these applications. There is an urgent need for additional protection procedures to reduce the annual losses of managed bee colonies. Another method for protecting these pollinators is the use of chemical deterrents to reduce the interaction times of foraging bees with pesticide-treated crops. Historically, insect repellents (IRs) have been used to prevent the spread of deadly human diseases by arthropod vectors. However, it has been shown that bees can be repelled from pesticide-treated crops using DEET and bee pheromonal compounds. Here, I report the toxicological and deterrent effects of bee pheromonal compounds, as well as the deterrent effects of heterocyclic amines (HCAs) on bees. The results of this study indicate that the bee pheromonal compounds, at 8, 20, 60 and 100% concentrations, are toxic to bees and inhibit the feeding of bees within a confined space. Additionally, the pheromonal compounds and the HCAs are as efficacious as DEET in deterring bees from treated food sources. The HCA piperidine was observed to effectively deter bee foragers from a sugar feeder in a high-tunnel experiment as well as from melon flowers and knapweed in field experiments. Electroantennogram recordings were conducted to verify an olfactory response of the bees to the tested compounds. Pheromonal compounds were readily detected by bee antennae; whereas, the HCAs did not elicit significant responses in the bee antennae. These data suggest that bee pheromonal compounds, as well as HCAs, may serve as candidates for the further investigation as repellents to protect bees from unintentional pesticide exposures. / Ph. D.

The European honey bee, Apis mellifera, is an important pollinator of horticultural and agricultural field crops, providing ≈ 90% of all commercial pollination services (Genersch et al. 2010). The recent rise in colony loss due to Colony Collapse Disorder (CCD) has been a source of concern for both beekeepers and the apiculture industry. One of the factors implicated in CCD is infestation by the ectoparasitic mite, Varroa destructor. Initial efforts to control the mite relied heavily on regular application of miticides without regard to actual mite infestation levels. This approach has led to problems of resistance in the mite and contamination of the hive and hive-products. Because it is unlikely that miticides will be removed as an option for mite management, a precision mite management (PMM) approach using information on the spatiotemporal distribution of the mite to improve sampling and treatments is seen as a viable option, particularly with respect to treatment costs and impacts on the environment. The primary objective of this study was to develop an understanding of the spatiotemporal distribution of the Varroa mite and bee brood within hives for the purpose of developing a PMM approach for the mite. Varroa mite populations were sampled from May to June, 2012 and February to October, 2013. Sampling was conducted with three commonly used sampling methods: soapy water roll (SWR), brood uncapping, and a modified sticky board; brood uncapping, however, was discontinued during the study due to hive the labor cost and harmful effects of this method to the hives. Similar trends in mite population levels were observed using the soapy water roll and sticky board sampling methods. Spearman's nonparametric analysis showed that there was a significant correlation (ρ = 0.47, P<0.001) in mite population levels for the soapy water roll and sticky board methods for sampling conducted from February to September, 2013 (the SWR method was not used in October). This was despite the fact that there was no significant correlation (ρ= -0.03, P = 0.8548) between the two sampling methods during the spring sampling period from February to April, 2013. The observed lack of correlation between the two sampling methods in early spring was likely due to the low population of brood in the hive, which caused the majority of the mites to remain on adult bees. Mites per 100 adult bees, therefore, appear to reflect mite population levels within the hive more closely than mite fall on sticky borad during the February to April sampling period. This suggests that the soapy water roll method is a better method for estimating mite population levels within the hive in the early spring compared with the sticky board method. Geospatial analyses of the distributions of mite fall on the sticky boards were conducted using geostatistics and Spatial Analysis by Distance IndicEs (SADIE). Both analyses showed that mite fall on the sticky board was generally aggregated and the aggregation increased with mite population levels. The average range of the variogram from geostatistical analysis was estimated at 4 sticky board cells; this range value was increased to 5 cells and was used to develop a systematic outside-range sampling protocol for mites on a sticky board. The results showed that the accuracy of the systematic outside-range sampling compared well with that of the traditional sticky board counting method in estimating total mite fall, but required only 60% of the effort (i.e., counting 63 instead of 105 cells). SADIE analysis showed that there is an overall association between the distribution of mite fall on a sticky board and the distribution of brood within a hive. A greater degree of correspondence was also observed in the association of drone and mite distributions during May to June; greater correspondence in worker brood and mite associations was observed in August and September. These differences may be due to relative amounts of the two types of brood present within the hive. A test of the efficacy of precision application of Varroa mite treatment based on the association between drone brood and mite fall resulted in a significantly greater reduction in mite levels on the sticky board using a traditional miticide treatment method compared with the control and precision treatments (𝜒2 =362.571; df = 2; P <0.0001); mite population levels with the precision method, however, were significantly reduced compared with the control. / Master of Science in Life Sciences

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.

In the eusocial insect honeybee (Apis mellifera), many sterile worker bees live together with a reproductive queen in a colony. All tasks of the colony are performed by the workers, undergoing age-dependent division of labor. Beginning as hive bees, they take on tasks inside the hive such as cleaning or the producing of larval food, later developing into foragers. With that, the perception of sweetness plays a crucial role for all honeybees whether they are sitting on the honey stores in the hive or foraging for food. Their ability to sense sweetness is undoubtedly necessary to develop and evaluate food sources. Many of the behavioral decisions in honeybees are based on sugar perception, either on an individual level for ingestion, or for social behavior such as the impulse to collect or process nectar. In this context, honeybees show a complex spectrum of abilities to perceive sweetness on many levels. They are able to perceive at least seven types of sugars and decide to collect them for the colony. Further, they seem to distinguish between these sugars or at least show clear preferences when collecting them. Additionally, the perception of sugar is not rigid in honeybees. For instance, their responsiveness towards sugar changes during the transition from in-hive bees (e.g. nurses) to foraging and is linked to the division of labor. Other direct or immediate factors changing responsiveness to sugars are stress, starvation or underlying factors, such as genotype. Interestingly, the complexity in their sugar perception is in stark contrast to the fact that honeybees seem to have only three predicted sugar receptors. In this work, we were able to characterize the three known sugar receptors (AmGr1, AmGr2 and AmGr3) of the honeybee fully and comprehensively in oocytes (Manuscript II, Chapter 3 and Manuscript III, Chapter 4). We could show that AmGr1 is a broad sugar receptor reacting to sucrose, glucose, maltose, melezitose and trehalose (which is the honeybees’ main blood sugar), but not fructose. AmGr2 acts as its co-receptor altering AmGr1’s specificity, AmGr3 is a specific fructose receptor and we proved the heterodimerization of all receptors. With my studies, I was able to reproduce and compare the ligand specificity of the sugar receptors in vivo by generating receptor mutants with CRISPR/Cas9. With this thesis, I was able to define AmGr1 and AmGr3 as the honeybees’ basis receptors already capable to detect all sugars of its known taste spectrum. In the expression analysis of my doctoral thesis (Manuscript I, Chapter 2) I demonstrated that both basis receptors are expressed in the antennae and the brain of nurse bees and foragers. This thesis assumes that AmGr3 (like the Drosophila homologue) functions as a sensor for fructose, which might be the satiety signal, while AmGr1 can sense trehalose as the main blood sugar in the brain. Both receptors show a reduced expression in the brain of foragers when compared with nurse bees. These results may reflect the higher concentrated diet of nurse bees in the hive. The higher number of receptors in the brain may allow nurse bees to perceive hunger earlier and to consume the food their sitting on. Forager bees have to be more persistent to hunger, when they are foraging, and food is not so accessible. The findings of reduced expression of the fructose receptor AmGr3 in the antennae of nurse bees are congruent with my other result that nurse bees are also less responsive to fructose at the antennae when compared to foragers (Manuscript I, Chapter 2). This is possible, since nurse bees sit more likely on ripe honey which contains not only higher levels of sugars but also monosaccharides (such as fructose), while foragers have to evaluate less-concentrated nectar. My investigations of the expression of AmGr1 in the antennae of honeybees found no differences between nurse bees and foragers, although foragers are more responsive to the respective sugar sucrose (Manuscript I, Chapter 2). Considering my finding that AmGr2 is the co-receptor of AmGr1, it can be assumed that AmGr1 and the mediated sucrose taste might not be directly controlled by its expression, but indirectly by its co-receptor. My thesis therefore clearly shows that sugar perception is associated with division of labor in honeybees and appears to be directly or indirectly regulated via expression. The comparison with a characterization study using other bee breeds and thus an alternative protein sequence of AmGr1 shows that co-expression of different AmGr1 versions with AmGr2 alters the sugar response differently. Therefore, this thesis provides first important indications that alternative splicing could also represent an important regulatory mechanism for sugar perception in honeybees. Further, I found out that the bitter compound quinine lowers the reward quality in learning experiments for honeybees (Manuscript IV, Chapter 5). So far, no bitter receptor has been found in the genome of honeybees and this thesis strongly assumes that bitter substances such as quinine inhibit sugar receptors in honeybees. With this finding, my work includes other molecules as possible regulatory mechanism in the honeybee sugar perception as well. We showed that the inhibitory effect is lower for fructose compared to sucrose. Considering that sugar signals might be processed as differently attractive in honeybees, this thesis concludes that the sugar receptor inhibition via quinine in honeybees might depend on the receptor (or its co-receptor), is concentration-dependent and based on the salience or attractiveness and concentration of the sugar present. With my thesis, I was able to expand the knowledge on honeybee’s sugar perception and formulate a complex, comprehensive overview. Thereby, I demonstrated the multidimensional mechanism that regulates the sugar receptors and thus the sugar perception of honeybees. With this work, I defined AmGr1 and AmGr3 as the basis of sugar perception and enlarged these components to the co-receptor AmGr2 and the possible splice variants of AmGr1. I further demonstrated how those sugar receptor components function, interact and that they are clearly involved in the division of labor in honeybees. In summary, my thesis describes the mechanisms that enable honeybees to perceive sugar in a complex way, even though they inhere a limited number of sugar receptors. My data strongly suggest that honeybees overall might not only differentiate sugars and their diet by their general sweetness (as expected with only one main sugar receptor). The found sugar receptor mechanisms and their interplay further suggest that honeybees might be able to discriminate directly between monosaccharides and disaccharides or sugar molecules and with that their diet (honey and nectar). / Beim dem eusozialen Insekt Honigbiene (Apis mellifera) leben tausende sterile Arbeitsbienen zusammen mit einer fortpflanzungsfähigen Königin in einem Volk. Alle Aufgaben in der Kolonie werden von diesen Arbeiterinnen erledigt, während sie eine altersabhängige Arbeitsteilung durchlaufen. Als Stockbienen beginnend übernehmen sie Aufgaben im Stock wie die Reinigung oder die Produktion von Larvenfutter und entwickeln sich später zu Sammlerinnen. Das Wahrnehmung von Süße spielt für alle Honigbienen eine entscheidende Rolle, egal ob sie auf den Honigvorräten im Stock sitzen oder nach Nahrung suchen. Ihre Fähigkeit Süße zu wahrzunehmen ist zweifellos notwendig, um Nahrungsquellen zu identifizieren und zu bewerten. Viele der Verhaltensentscheidungen bei Honigbienen basieren auf ihrer Zuckerwahrnehmung, entweder auf individueller Ebene für die Nahrungsaufnahme oder für soziales Verhalten wie beispielsweise das Sammeln oder Verarbeiten von Nektar. Honigbienen zeigen auf vielen Ebenen ein komplexes Spektrum bei der Wahrnehmung von Süße. Sie können mindestens sieben Zuckerarten wahrnehmen und sammeln diese für ihren Stock. Darüber hinaus scheinen sie zwischen diesen Zuckern unterscheiden zu können oder zeigen zumindest klare Präferenzen beim Sammeln. Außerdem ist die Zuckerwahrnehmung bei Honigbienen nicht starr. Ihre Zuckerwahrnehmung ändert sich, wenn sie von einer Stockbiene (z. B. Ammen) zum Nahrungssammeln außerhalb des Stockes übergehen, und ist somit mit ihrer Arbeitsteilung verbunden. Andere direkte oder unmittelbare Faktoren, die die Reaktion auf Zucker verändern, sind Stress, Hunger oder zugrunde liegende Faktoren wie der Genotyp. Interessanterweise steht die Komplexität der Zuckerwahrnehmung in starkem Kontrast zu der Tatsache, dass Honigbienen bisher anscheinend nur drei mögliche Zuckerrezeptoren haben. In dieser Arbeit konnten wir die drei bekannten Honigbienenzuckerrezeptoren (AmGr1, AmGr2 und AmGr3) in Xenopus-Oozyten vollständig und umfassend charakterisieren (Manuscript II, Chapter 3 und Manuscript III, Chapter 4). Wir konnten zeigen, dass AmGr1 ein breitdetektierender Zuckerrezeptor ist, der auf Saccharose, Glukose, Maltose, Melezitose und Trehalose (der Hauptblutzucker bei Honigbienen), aber nicht auf Fruktose reagiert. AmGr2 fungiert als ein Co-Rezeptor, der die Spezifität von AmGr1 verändert. AmGr3 ist ein spezifischer Fruktoserezeptor und wir haben die Heterodimerisierung der Rezeptoren überprüft. Mit meinen Studien konnte ich die gefundene Ligandenspezifität der Zuckerrezeptoren in vivo reproduzieren und vergleichen, indem ich Rezeptormutanten mit CRISPR/Cas9 generierte. Dabei konnte ich AmGr1 und AmGr3 als die Basisrezeptoren von Honigbienen definieren, die bereits alle Zucker ihres bekannten Geschmacksspektrums detektieren können. In der Expressionsanalyse meiner Doktorarbeit (Manuscript I, Chapter 2) konnte ich zeigen, dass beide Basisrezeptoren in den Antennen und im Gehirn von Ammenbienen und Sammlerinnen exprimiert werden. Diese Arbeit geht davon aus, dass AmGr3 (wie das Homologe in Drosophila) als Sensor für Fruktose fungiert, die das Sättigungssignal sein könnte, während AmGr1 Trehalose als Hauptblutzucker im Gehirn wahrnehmen kann. Beide Rezeptoren zeigen eine reduzierte Expression im Gehirn von Sammlerinnen im Vergleich zu Ammenbienen. Diese Ergebnisse könnten die höher konzentrierte Ernährung der Ammenbienen im Stock widerspiegeln. Die höhere Anzahl an Rezeptoren im Gehirn könnte es den Ammenbienen ermöglichen frühzeitiger Hunger wahrzunehmen und die Nahrung, auf der sie sitzen aufzunehmen. Sammelbienen dagegen müssen beim Sammeln und dem reduzierten Nahrungsangebot ausdauernder sein. Die gemessene reduzierte Expression des Fruktoserezeptors AmGr3 in den Antennen von Ammenbienen entsprechen meinen anderen Ergebnissen, wonach Ammenbienen im Vergleich zu Sammelbienen an den Antennen auch weniger empfindlich auf Fruktose reagieren (Manuscript I, Chapter 2). Dies ist möglich, da Ammenbienen eher auf reifem Honig sitzen, der nicht nur einen höheren Zuckergehalt, sondern auch vermehrt Monosaccharide (wie Fructose) enthält, während Sammelbienen weniger konzentrierten Nektar bewerten müssen. Meine Untersuchungen zur Expression von AmGr1 in den Antennen von Honigbienen ergaben keine Unterschiede zwischen Ammenbienen und Sammlerinnen, obwohl Sammlerinnen empfindlicher auf den entsprechenden Zucker Saccharose reagieren. Angesichts unserer Ergebnisse, dass AmGr2 der Co-Rezeptor von AmGr1 ist, kann die Hypothese aufgestellt werden, dass AmGr1 und der vermittelte Saccharose-Geschmack möglicherweise nicht direkt durch seine Expression, sondern indirekt durch seinen Co-Rezeptor reguliert werden. Meine Dissertation zeigt somit deutlich, dass die Zuckerwahrnehmung bei Honigbienen mit Arbeitsteilung verbunden ist und direkt oder indirekt über die Expression geregelt zu werden scheint. Der Vergleich mit einer anderen Charakterisierungsstudie, durchgeführt an anderen Bienenrassen und damit einer alternativen Proteinsequenz von AmGr1, zeigt, dass die Co-Expression verschiedener AmGr1-Varianten mit AmGr2 die Zuckerantwort unterschiedlich verändert. Daher liefert diese Arbeit erste wichtige Hinweise darauf, dass alternatives Spleißen auch bei Honigbienen einen wichtigen Regulationsmechanismus für die Zuckerwahrnehmung darstellen könnte. Des Weiteren habe ich herausgefunden, dass der Bitterstoff Chinin die Qualität der Belohnung in Lernexperimenten für Honigbienen senkt (Manuscript IV, Chapter 5). Bisher wurde kein Bitterrezeptor im Genom von Honigbienen gefunden und diese Arbeit deutet darauf hin, dass Bitterstoffe wie Chinin Zuckerrezeptoren in Honigbienen hemmen. Mit dieser Erkenntnis schließt meine Dissertation auch andere Moleküle als mögliche Regulationsmechanismen in die Zuckerwahrnehmung der Honigbiene ein. Wir haben gezeigt, dass die hemmende Wirkung bei Fruktose im Vergleich zu Saccharose geringer ist. Unter der Berücksichtigung, dass Zuckersignale bei Honigbienen möglicherweise unterschiedlich attraktiv verarbeitet werden, kommt meine Arbeit zu dem Schluss, dass die Hemmung der Zuckerrezeptoren durch Chinin bei Honigbienen abhängig ist von der verwendeten Konzentration, der Bedeutung bzw. Attraktivität des Zuckers und seiner Konzentration. Mit meiner Doktorarbeit konnte ich das Wissen über die Zuckerwahrnehmung der Honigbiene insgesamt erweitern und einen komplexen, umfassenden Überblick formulieren. Ich konnte den mehrdimensionalen Mechanismus aufzeigen, der die Zuckerrezeptoren und damit die Zuckerwahrnehmung von Honigbienen reguliert. Ich konnte AmGr1 und AmGr3 als Basis der Zuckerwahrnehmung definieren und diese Komponenten auf den Co-Rezeptor AmGr2 und die möglichen Spleißvarianten von AmGr1 erweitern. Ich habe außerdem gezeigt, wie diese Zuckerrezeptorkomponenten funktionieren, interagieren, und dass sie eindeutig an der Arbeitsteilung bei Honigbienen beteiligt sind. Zusammenfassend beschreibt meine Dissertation die Mechanismen, die es Honigbienen ermöglichen, Zucker auf komplexe Weise wahrzunehmen, selbst wenn sie eine begrenzte Anzahl von Zuckerrezeptoren besitzen. Meine Daten deuten stark darauf hin, dass Honigbienen Zucker und ihre Nahrung nicht nur aufgrund ihrer generellen Süße unterscheiden können (wie dies mit nur einem Hauptzuckerrezeptor zu erwarten wäre). Die gefundenen Zuckerrezeptormechanismen und deren Zusammenspiel legen nahe, dass Honigbienen möglicherweise direkt zwischen Monosacchariden und Disacchariden bzw. Zuckermolekülen und damit zwischen ihrer Nahrung (Honig und Nektar) unterscheiden können.

Comme de nombreuses espèces naturelles, l’abeille européenne (Apis mellifera) est confrontée à une pression croissante de facteurs biotiques et abiotiques : environnement, diversité génétique, parasitisme, etc. Chacun de ces facteurs peut potentiellement influencer les communautés de micro-organismes qui constituent le microbiote de l’abeille et évoluent avec elle. L’objectif de cette thèse était de comprendre la dynamique et la structuration du microbiote intestinal de la lignée évolutive M de l’abeille européenne en fonction de facteurs biogéographiques, génétiques et temporels des colonies d’abeilles. Cette analyse a été réalisée dans sept conservatoires de cette lignée évolutive, répartis au Portugal (A. m. iberiensis), en Espagne et en France (A. m. mellifera), selon un gradient Nord-Sud et Est-Ouest. Dans un premier temps, mon travail a permis de décrire un facteur jusqu’ici mal connu : l’humidité dans les ruches. Celle-ci s’est révélée stable, entre 50 et 60 % d’humidité relative toute l’année, suggérant une hygrorégulation par les colonies d’abeilles, quelle que soit la période de l’année et la dynamique populationnelle au sein des nids (ici, la ruche). Par la suite, nous avons développé un protocole permettant le suivi spatio-temporel de la charge virale des abeilles par cytométrie en flux. Son application sur nos abeilles a montré que le temps influence moins la charge virale que le lieu géographique. Enfin, une analyse métagénomique sur un gène ciblé (ARNr 16s) a confirmé l’effet de la localisation géographique des ruches, cette fois sur la structuration des communautés bactériennes peuplant les intestins des abeilles qui appartiennent à la lignée évolutive M. Par ailleurs, cette dernière analyse a montré l’importance de prendre en compte l’interaction entre les facteurs, qui peuvent avoir plus d’impact pris ensemble que séparément. Enfin, des perspectives sont envisagées, telles que la réalisation d’un réseau d’inter-actants permettant de comprendre la part de chaque facteur sur les communautés bactériennes mais également les pathogènes. / Like many natural species, the European bee (Apis mellifera) faces a threat of biotic and abiotic factors: environment, genetic diversity, parasitism, etc. Each of these factors can potentially influence the communities of microorganisms that constitute the bee's microbiota and evolve with it. The aim of this work was to understand the dynamics and structure of the gut microbiota of the M evolutionary line of A. mellifera according to biogeographic, genetic and temporal factors of bee colonies. This analysis was carried out in the conservatories of this evolutionary line, located in Portugal (A. m. iberiensis), Spain and France (A. m. mellifera) along a North-South and East-West gradient. First, my work has described a hitherto barely understood factor: humidity in hives. It appeared to be stable in our beehives, between 50 and 60% relative humidity all year long, which suggests a hygroregulation by honeybee colonies whatever the periode of year and the populational dynamics inside the nest (i.e. the beehive). We developed a protocol allowing spatio-temporal monitoring of the viral load of bees by flow cytometry. After using it on our colonies, we showed that time influences less viral load than geographical location. Finally, a metagenomic analysis on a target gene (16s rRNA) confirmed the importance of the geographical location of beehives, this time on the structuring of bacterial communities living in guts of honeybees belonging to the M evolutive lineage. In addition, this last analysis has shown the importance to take into account the interaction between factors, which can have a bigger effect when taken together. Finally, perspectives are envisaged, such as the realization of a network of interactants allowing to understand the part of each factor on the bacterial but also pathogens’ communities.

Inúmeros aspectos da reprodução em Apis mellifera já foram extensamente divulgados, no entanto, os mecanismos reguladores da manutenção do estado estéril das operárias, bem como aqueles que permitem a ativação de seus ovários, ainda estão para serem descobertos. Por exemplo, a organização dos folículos ovarianos em crescimento e a arquitetura e papel das células foliculares neste processo. Além disso, para compreender o processo de ativação dos ovários em um contexto mais amplo, também é necessária uma investigação da síntese e maturação de diferentes classes de RNAs as quais modelam redes de interações gênicas extremamente complexas. Portanto, neste doutorado, tivemos como objetivo realizar 1- uma análise morfológica dos ovários ativos de operárias de A. mellifera obtidos em condições orfandade, com ênfase nas células foliculares e 2- um estudo aprofundado da regulação da expressão gênica (genes estruturais e reguladores) que é de fundamental importância para ligar os genótipos aos fenótipos. A análise morfológica dos ovários de operárias de A. mellifera foi realizada em microscópio de fluorescência ou confocal (priorizou a contagem das células foliculares) e microscópio eletrônico de transmissão, que permitiu a descrição e caracterização, pela primeira vez, da patência em ovários de operárias A. mellifera. Paralelamente, por meio da técnica de RNAseq, foi possível analisar o transcriptoma (miRNAs e mRNAs) de amostras específicas de ovários, em diferentes estados fisiológicos, em rainhas e operárias. Os mRNAs e miRNAs que se destacaram em nossas análises in silico foram validados experimentalmente por RT-PCR com alto grau de reprodutibilidade e em harmonia com o estado fisiológico dos ovários. Os transcritos altamente expressos nos ovários ativados foram: fpps5, cad, obp7, yellow-g e aqueles representados pelo GB42182 e GB44975. Acreditamos estes genes possam fazer parte da rede que regula o processo de ativação dos ovários em A. mellifera. Os miRNAs que se destacaram em nossas análises foram: A) miR-306 e miR-317 - altamente expressos nas amostras de ovários funcionais e B) miR-71 pelo fato de, nas análises in silico, ser o mais forte candidato a alvejar a vitelogenina, e na análise experimental, apresentarem, microRNAs e mRNAs, perfis de expressão antagônicos. A construção de bibliotecas de microRNAs e mRNAs a partir de ovários funcionais e não funcionais de abelhas operárias e rainhas, a análise de expressão, bem como a predição de uma rede de integração nos deu um retrato do sensível equilíbrio reprodutivo que mantém ambas as castas em aparente harmonia dentro da colônia aonde elas assumem, no momento certo, seus papéis nesta sofisticada sociedade empreendendo ou não a reprodução. / Countless aspects of reproduction in Apis mellifera have been widely published, however, the regulatory mechanisms for the maintenance of the sterile state of workers as well as those that allow the activation of their ovaries are still to be discovered, as much as the organization of growing ovarian follicles, the architecture and the role of follicular cells during this process. Furthermore, to understand the activation process of the ovaries in a broader context, it is also necessary to investigate the synthesis and maturation of different classes of RNAs which exemplify networks of gene interactions, extremely complex. Therefore, PhD project, we aimed to approach: 1 - A morphological analysis of active ovaries of A. mellifera workers obtained in queenless conditions, with emphasis on the follicular cells and 2 - A detailed study of the regulation of gene expression (structural and regulatory genes) that is crucial for linking genotypes to phenotypes. Morphologic analysis of workers ovaries of A. mellifera was performed under a fluorescence microscope or confocal (prioritized follicular cell count) and transmission electron microscope, which allowed, for the first time, a description and characterization of the patency of worker ovaries in A. mellifera. Similarly, by RNAseq technique, it was possible to analyze the transcriptome (miRNAs and mRNAs) of specific samples of ovaries at different physiological states, in queens and workers. mRNAs and miRNAs that stood out in our in silico analysis were experimentally validated by RT-PCR with high reproducibility and in harmony with ovaries physiologic state. Transcripts highly expressed in activated ovaries were fpps5, cad, obp7, yellow-g and those represented by GB42182 and GB44975. We believe these genes may be part of the network that regulates ovaries activation process in A. mellifera. miRNAs that stood out in our analysis were: - a) - miR-306 and miR-317 - highly expressed in samples of active ovaries and b) -miR-71 by the fact that the in silico analysis, was the strongest candidate to target vitellogenin, and in experimental analysis, presented antagonistic profile of expression when microRNAs and mRNAs were contrasted. The construction of microRNAs and mRNAs libraries from active and inactive ovaries of worker bees and queens, the analysis expression, as well as the prediction of a integrative network has given us a portrait of the sensitive reproductive balance that keeps both castes of bees in apparent harmony within the colony, where they take each one, at the right time, their roles in this sophisticated society, undertaking or not the reproduction.

O desenvolvimento em insetos é coordenado majoritariamente pela ação de dois grandes hormônios: HJ e 20E. Esses hormônios desencadeiam cascatas gênicas resultando em mudanças fenotípicas, fisiológicas e comportamentais. Além dos hormônios, os miRNAs também atuam sobre a expressão desses genes componentes das cascatas de resposta aos hormônios durante o desenvolvimento nos insetos. Neste estudo objetivamos analisar a relação entre hormônios e genes, hormônios e miRNAs e genes e miRNAs, em estágios finais do desenvolvimento de Apis mellifera. O perfil de expressão dos fatores de transcrição Usp, ftz-f1, EcR, chd64, inr2, Kr-h1, gce e os regulares putativos miR-34, miR-281 e miR-252b foram avaliados a partir do 5 estagio larval até adultos recém emergidos, por meio de RT-qPCR. Também avaliamos o efeito de doses exógenas de HJ III e 20E sobre o desenvolvimento pupal (Pw e Pb). A maioria dos genes testados mostraram responder as variações hormonais nos estágios pupais tal qual respondem em estágios larvais. Contudo, gce e chd64 mostraram responder diferente as variações hormonais em estágios pupais, sugerindo assim, que estes podem desempenhar papel diferente nos estágios finais do desenvolvimento. O gce que é um receptor nuclear de HJ em insetos, mostrou rápida resposta ao tratamento com 20E e não respondeu ao tratamento com HJ, semelhantemente o chd64 também não respondeu aos tratamentos com HJ. Nossos dados ainda apontam Usp como um gene de resposta imediata (early gene). O miR-34 e o miR-281 são fortes candidatos a reguladores chave na metamorfose, uma vez que estes apresentam interações (in silico) com a maioria dos genes aqui estudados, além de responderem ao tratamento hormonal para ambos os hormônios. Este estudo caracteriza componentes da rede regulatória do desenvolvimento pupal em abelhas melíferas. / The development in honeybees is mainly controlled by the action of two major hormones, juvenile hormone (JH) and 20-hidroxyecdysone (20E). These hormones trigger gene cascades, which results in phenotypic, physiological and behavioral changes. Besides hormones, a class of non-coding RNAs, the microRNAs, regulates gene expression at a post-transcriptional level during insect development. In this study we aimed to analyze the relationship between developmental genes and morphogenetic hormones, in final stages of the development of Apis mellifera. The expression profile of the orphan nuclear receptor Usp, ftz-f1, EcR, chd64, inr2, Kr-h1), gce, early-trypsin, and their putative regulators miRNA-34, miRNA-281, miRNA-252a and miRNA-252b were assessed from 5? instar larvae to newly emerged adults by qPCR. The effect of exogenous doses of both hormones applied on white eyed pupae (Pw) and brown eyed pupae (Pb) was also tested. Most of the genes seem to respond to hormonal variation in pupal stages as they do in larval stages. However, gce and chd64 showed a different response to hormonal treatment in pupal states, thus suggesting they play different roles in final stages of development. Unexpectedly gce, which is a nuclear receptor of JH in insects, showed a quick response to 20E treatment and no response to JH in pupal stages of honeybees, as well as chd64 which also responded only to the 20E treatment. In addition, we recognized Usp as an Immediate Early Gene, for it responded rapidly to hormonal treatments and quickly restored its level. In addition, we find the miR-34 and miR-281 as strong candidates of regulators since they presented many putative interactions in the 3\'UTR of the candidate genes and showed to be affected by the hormonal treatment. This study describes new components to the regulatory network that regulates bee development.

SILVA, I. C. Efeito tóxico de Stryphnodendron polyphyllum (Fabaceae) para as abelhas Apis mellifera, Tetragonisca angustula, Scaptotrigona aff. depilis e Nannotrigona testaceicornis (Apidae). 2012. 101 f. Tese de Doutorado - Faculdade de Filosofia Ciências e Letras, Universidade de São Paulo, Ribeirão Preto, 2012. No Brasil, estudos realizados sobre o pólen de Stryphnodendron polyphyllum (barbatimão) mostraram um efeito tóxico quando incorporado em dietas oferecidas para larvas de abelhas africanizadas. Esse pólen é o agente causador da Cria Ensacada Brasileira (CEB), caracterizada por mortalidade de pré-pupas. Doenças em Apis mellifera tem sido amplamente estudadas. Entretanto, há poucos relatos sobre a sua ocorrência em abelhas sem ferrão. Portanto, este trabalho teve por objetivos gerais: avaliar se abelhas africanizadas apresentam diferenças na sobrevivência, na quantidade de pólen de barbatimão coletado e se existem diferenças na coleta desse pólen entre as colônias de apiários diferentes; verificar se as abelhas Scaptotrigona aff. depilis, Tetragonisca angustula e Nannotrigona testaceicornis coletam esse pólen e se ele causa mortalidade de crias; subsequentemente realizar a criação in vitro de larvas de A. mellifera, S. aff. depilis, T. angustula e N. testaceicornis para avaliar a toxicidade desse pólen para as crias. Para isso, colônias de A. mellifera, S. aff. depilis, T. angustula e N. testaceicornis foram transportadas para apiários localizados em Altinópolis/SP, durante o período de florescimento do barbatimão (outubro a dezembro). Nas colônias de abelhas africanizadas foi coletado o pólen do favo e avaliado a sobrevivência das crias com idade controlada. Nas abelhas sem ferrão foi coletado o pólen da corbícula e do interior das colméias. As amostras de pólen foram preparadas pelo método padrão européu, sendo contados 300 grãos de pólen em três lâminas diferentes para verificar a porcentagem de grãos de pólen de barbatimão. As abelhas africanizadas apresentam diferenças na coleta de pólen dentro e entre apiários; apenas três colônias não coletaram e uma coletou pouco pólen de barbatimão, entre 92 colônias analisadas. As crias dessas colônias não apresentaram sintomas da CEB. As rainhas filhas dessas colônias foram inseminadas instrumentalmente e fecundadas naturalmente. As colônias contendo essas rainhas foram analisadas em 2011 juntamente com as matrizes; houve diferenças nas sobrevivências, dependendo da localização do apiário. Foram encontradas diferenças na preferência de coleta do pólen de barbatimão. Os resultados obtidos poderão contribuir para que novas pesquisas sejam realizadas com o objetivo de compreender o mecanismo de baixa preferência para a coleta do pólen e para que programas de seleção sejam realizados visando reduzir os problemas causados por este pólen tóxico. Nas abelhas sem ferrão encontramos o pólen de barbatimão nos potes de alimento e nas corbículas. Algumas crias mortas foram encontradas nas células dos favos durante o mesmo período em que foi observado a mortalidade em abelhas africanizadas com sintomas da CEB. Os resultados sugerem que a mortalidade observada pode ser devida ao consumo desse pólen. As larvas das abelhas sem ferrão foram criadas in vitro e alimentadas com o alimento larval contendo diferentes quantidades de pólen de barbatimão. A sobrevivência de S. aff. depilis e N. testaceicornis não foi afetada quando ingeriram o alimento contendo o pólen de barbatimão, porém para T. angustula a sobrevivência foi significativamente maior quando as larvas receberam apenas o alimento larval (controle) em comparação com aquelas alimentadas com o alimento contendo 1% do pólen de barbatimão. As larvas de A. mellifera também foram criadas in vitro e alimentadas com dietas contendo 1, 2 e 3% do pólen de barbatimão. Todas as larvas alimentadas com o polen de barbatimão morreram com os sintomas semelhantes aos observados em condições naturais. De acordo com os resultados obtidos concluímos que essas abelhas sem ferrão são fisiologicamente mais resistentes a este pólen tóxico. / In Brazil, studies conducted on the Stryphnodendron polyphyllum (barbatimão) pollen have shown its toxic effect when it is incorporated into diets offered to Africanized bees larvae. This pollen is the causative agent of Brazilian Sac Brood (BSB), characterized by pre-pupae mortality. Diseases in Apis mellifera haves been extensively studied. However, there are a few reports on their occurrence in stingless bees. Thus, the main objective of this work was to evaluate whether Africanized bees present differences in survival, amount of barbatimão pollen collected and whether there are differences in the collections of this pollen between colonies of different apiaries; to verify whether Scaptotrigona aff. depilis, Tetragonisca angustula and Nannotrigona testaceicornis bees collect this pollen and whether it leads to brood mortality; and subsequently to evaluate the toxicity of this pollen to bees brood by rearing A. mellifera, S. aff. depilis, T. angustula and N. testaceicornis larvae in vitro. For this A. mellifera, S.aff. depilis, T. angustula and N. testaceicornis colonies were taken to apiaries located in Altinópolis/SP, during babartimão flowering period (October to December). In the Africanized bee colonies pollen was collected from the comb and the survival of the brood with controlled age was evaluated. In the stingless bee colonies pollen was collected from the corbicula of the bees and also from the interior of the hive. The pollen samples were prepared by the standard European method, counting 300 pollen grains in three different slides to check the percentage of barbatimão pollen grains. Africanized bees presented differences in pollen collections within and among apiaries; only three colonies did not collect and one collected little barbatimão pollen, among 92 colonies analyzed. The brood of these colonies did not present symptoms of BSB. The daughter queens of these colonies were instrumentally inseminated or naturally fecundated. These colonies were analyzed in 2011, together with their parental colonies; there were differences in survival depending on the apiary location. We found differences in the collection preference of barbatimão pollen. These results will contribute to the planning of new studies with the aim of understanding the low preference mechanism for pollen collection and selection programs aimed at reducing problems with this toxic pollen. In the stingless bees, we found barbatimão pollen in the food pots and in the corbiculae. Some dead brood was found in the brood cells, during the same period in which mortality was observed in Africanized bees with BSB symptoms. The results suggest that the mortality observed may be due to the consumption of this pollen. Larvae of stingless bees were reared in vitro and fed larval food containing different amounts of barbatimão pollen. The survival of S. aff. depilis and N. testaceicornis was not affected when ingesting food containing barbatimão pollen, but survival of T. angustula was significantly higher when the larvae received only larval food (control) compared to those fed larval food containing 1% barbatimão pollen. The larvae of A. mellifera were also reared in vitro and fed diets containing 1, 2 and 3% barbatimão pollen. All larvae fed barbatimão pollen died with symptoms similar to the ones observed under natural conditions. We conclude that these stingless bees are physiologically more resistant to this toxic pollen.

A produção de pólen apícola vem experimentando uma rápida e empolgante expansão desde o final da década de 1990. Tal crescimento pode ser explicado pelo aumento do interesse popular em alimentos de alto valor nutricional, bem como outras inúmeras aplicações desse produto na produção animal. Atualmente em torno de 1.500 toneladas de pólen são produzidas anualmente em todo o mundo. Apesar da importância econômica, social e ecológica assumida por esse setor da cadeia produtiva apícola, poucos estudos vêm abordando o tema, desde o fim da década de 1960. Tal desinteresse científico fez com que diversos aspectos relacionados às técnicas de manejo mais adequadas permanecessem pouco entendidos ou até mesmo nunca validados. Como agravante, um acentuado declínio populacional vem sendo percebido nas espécies de polinizadores, em especial A. mellifera. Apesar da gravidade e recorrência, tal fenômeno ainda é pouco entendido. Inúmeros agentes parecem exercer algum tipo de influencia negativa na biologia e fisiologia das abelhas melíferas (pesticidas, patógenos, ectoparasitas e perda de habitat), porém nenhum desses fatores foi comprovadamente identificado como desencadeador. Hoje a hipótese emergente, mais aceita entre os pesquisadores da área, trata do possível efeito sinergístico desses fatores como possível desencadeador dessa síndrome. É nesse contexto que estudos relativos à sanidade dessas abelhas se tornaram essenciais para a conservação da espécie e de seus serviços ecológicos. O pólen como imunoefetor e mitigador do estresse oxidativo se tornou, portanto, uma das mais proeminentes alternativas para proteção das abelhas A. mellifera. A pesquisa teve como principal objetivo a obtenção de informações que propiciassem o melhoramento das práticas vigentes de produção de pólen apícola, além do melhor entendimento do efeito do pólen no sistema imunológico de abelhas melíferas (analisado através do estudo da prevalência de patógenos) e no sistema de defesa antioxidante desses insetos. Este estudo traz informações importantes sobre o desenvolvimento de um sistema produtivo de pólen apícola mais eficaz. A utilização de populações coloniais de tamanhos médios (entre 19.000 e 30.000 indivíduos), combinada com suplementação de carboidrato, foi capaz de produzir resultados produtivos vantajosos. Também concluímos que apiários de produção devem ser prioritariamente instalados em regiões de temperaturas tropicais (com menores amplitudes térmicas), cujo ponto de orvalho também é elevado (alta umidade média) e com períodos moderados de precipitação (porém com frequência regular). Também concluimos que o uso de rainhas inseminadas deve ser considerado pelos apicultores com o objetivo de melhorar a suas produções, uma vez que tal técnica é capaz de produzir resultados mais rápidos e potencialmente mais duradouros do que as técnicas de seleção unilaterais mais comumente usadas nos sistemas produtivos de pólen no Brasil. Os dados obtidos nessa pesquisa também nos mostram indícios de que a expressão relativa dos genes PRX5, SOD2 e GSTS4 respondem significativamente à exposição ao herbicida Paraquat. Por este motivo, novos estudos são necessários para elucidar a real possibilidade dos mesmos serem utilizados como marcadores biológicos para a intoxicação causada por esse herbicida. Modificações significativas também foram observadas na prevalência de patógenos como Nosema, DWV e SBV (quando abelhas foram expostas ao Paraquat). Essa pesquisa destaca os efeitos mitigativos do pólen na redução do estresse oxidativo causado pela exposição ao Paraquat. As taxas de sobrevivência, promovidas por dietas ricas em pólen, parecem também estar ligadas à regulação positiva de genes antioxidantes como os CYPs, SODs, VG e GSTs assim como a menores cargas de IAPV, Nosema sp, e espécies tripanosomatídeas. Este é um indicativo da eficácia do pólen como antioxidante e imunoefetor, assim como uma possível alternativa para a manutenção da saúde das abelhas no mundo todo. / The demand for bee pollen has significantly increased in the last two decades. This growing consuption can be explained by the increasing incorporation of this product into human diets, as well as the efficiency of this product in improving livestock production. Currently around 1,500 tonnes of pollen are produced annually worldwide. Despite the economic, social and ecological importance assumed by this sector of the beekeeping industry chain, few studies have approached the subject since 1960. As a consequence of such scientific negligency, several aspects related to productive management remain poorly understood or even never validated. To make matters worse, a sharp population decline has been noticed in pollinator species, especially A. mellifera, since 2006. No single cause has been identified for these dramatic losses, but rather multiple interacting factors are likely responsible (such as pesticides, malnutrition, habitat loss, and pathogens). In this context, studies addressing the health of honey bees have become essential for the conservation of this pollinator as well as its ecological services. Thus, Pollen as imunoefetor and potencial source of antioxidants has became one of the most prominent alternatives for honey bees protection. This research aimed to obtain information able to foster the improvement of pollen production. We also tested the effects of a widely used herbicide on the physiology of honey bees, specifically focusing the effects of Paraquat on the expression of antioxidant-related genes. In doing so, we simultaneously identify the dynamics of pathogen prevalence when bees are exposed to different concentrations of Paraquat. Finally, we investigate the effect of pollen (added to the diet of tested bees) on both oxidative stress and pathogen loads. This study provides important information for the development of a more eficiente pollen productive system. The use of mid-sized swarms (e.g. 19,000 to 30,000 individuals) combined with carbohydrate supplementation is able to provide advantageous productive results. We also concluded that apiaries should prioritarily be settled in regions which present tropical weather (narrow temperature range), the humidity is high and the precipitation is frequent. We were able to conclude that the use of inseminated queens must be considered by beekeepers in order to improve their production, once this technique is able to produce result improvement faster than other unilateral breeding techniques. The data obtained in this study also showed us evidences that the relative expression of genes such PRX5, SOD2 and GSTS4 respond to exposure to the herbicide Paraquat. Therefore, further studies are required to better elucidate the real possibility of those antioxidante related genes to be applied as biological markers for intoxication produced by this herbicide. Significant changes were also observed in the found levels of pathogens such as Nosema, DWV and SBV (when bees were exposed to Paraquat). This research highlights the mitigating effects of pollen on reducing the oxidative stress caused by Paraquat exposure. Survival rates promoted by pollen-rich diets also seems to be linked to upregulation of genes such as CYPs, SODs, VG and GSTs, as well as reduced prevalence of IAPV, Nosema sp and tripanosomatyd species. This research sheds light into the antioxidant and imunoefetor effects of pollen as well as the promising use of those pollerich diets as alternative for improving honey bee\'s health.