L’acarien Varroa destructor est l’un des principaux responsables de l’effondrement des colonies d’abeilles domestique Apis mellifera. L’arsenal thérapeutique disponible pour lutter contre ce parasite ubiquiste apparait insuffisant à ce jour. Dans le cadre de cette thèse, la démarche de drug design, généralement utilisée en santé humaine, a été appliquée pour le développement de nouveaux médicaments vétérinaire à usage varroacide. Les travaux de cette thèse se sont focalisés sur deux cibles du système nerveux : l’acétylcholinestérase et les récepteurs à l’octopamine. Ces deux cibles ont déjà montré leur intérêt varroacide, notamment au travers des médicaments contenant du coumaphos ou de l’amitraze. Concernant l’acétylcholinestérase, un criblage fait avec le modèle 3D de l’enzyme, construit par homologie de séquences, a permis d’identifier deux composés de la chimiothèque du CERMN. Nous avons également exploré le potentiel varroacide d’acaricides de la famille des carbamates, démontrant l’intérêt du pirimicarbe comme varroacide. Concernant l’octopamine, l’étude de quatre dérivés de l’amitraze a montré l’intérêt de l’un d’entre eux. Un criblage par similarité de structure avec ce dérivé a mis en exergue une molécule issue de la chimiothèque du CERMN. Toutes les molécules pointées par ces travaux de thèse montrent de bons résultats lors de tests in vitro ou in vivo. Toutefois, afin de minimiser le risque pour l’abeille et maximiser l’efficacité anti-varroa, ces leads doivent être optimisés avant d’être ajouté à l’arsenal des médicaments varroacides. / The mite Varroa destructor is one of the main contributors to the collapse of honey bee colonies Apis mellifera. The therapeutic arsenal available against this ubiquitous parasite appears insufficient to date. In this thesis, the drug design approach, generally used in human health, was applied to the development of new varroacide veterinary drugs.The works of this thesis focused on two nervous system targets: acetylcholinesterase and octopamine receptors. These two targets have already shown their varroacide interest, especially through drugs containing coumaphos or amitraz. Regarding acetylcholinesterase, a screening made on the 3D model of the enzyme, built by sequence homology, allowed to identify two compounds from the CERMN compound library. We also explored the varroacide potential of carbamate acaricides, demonstrating the interest of pirimicarb as a varroacide. Regarding octopamine, the study of four derivatives of amitraz has shown the interest of one of them. Structural similarity screening with this derivative highlighted one hit from the CERMN compound library. All molecules pointed out by these thesis works show good results during in vitro or in vivo tests. However, to minimize the risk to honey bees and maximize their anti-varroa efficiency, these leads need to be optimized before being added to the arsenal of varroacide drugs.

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Host-associated microbial communities, also known as microbiomes, are essential to the health of their hosts, and disturbance of these communities can negatively impact host fitness. The honey bee gut microbiome is a relatively simple host-associated community that makes an excellent model system for studying microbiome stability. In addition, honey bees are essential agricultural pollinators, so factors that impact their health are important for food security. The presented research focused on the stability of the honey bee gut microbiome in response to disturbance from the antibiotic tetracycline. Tetracycline was chosen because it is the most commonly used antibiotic in beekeeping, and may have negative effects on bees through the disruption of their gut microbiomes. The first study presents a new fecal sampling method for studying the honey bee gut microbiome of individual bees over time. This method accurately represented bacterial community structure in the gut microbiome as determined with 16S rRNA gene amplicon sequencing, as fecal and whole gut samples did not differ significantly for individual bees. The fecal sampling technique was then used to examine changes to individual honey bee gut bacterial communities before and after tetracycline exposure. Minimal differences in gut community structure were detected prior to and five days after tetracycline treatment. However, there was variability in how individual gut microbiomes were affected by tetracycline treatment, highlighting the importance of intraspecific variation in response to disturbance. The second study investigated whether the timing of disturbance during a host's life impacts microbiome community stability. Newly emerged bees were treated with tetracycline, returned to their hive, and recollected 7 or 14 days later. The gut communities of the bees were then characterized using 16S rRNA gene amplicon sequencing. Gut microbiome structure of bees treated with tetracycline at emergence differed from controls both 7 and 14 days after emergence, with the antibiotic-treated bees having lower community richness overall. This study showed that early life disturbance of host-associated microbial communities can influence microbiome structure later in life. The final study describes the occurrence of antibiotic resistance genes (ARGs) in honey bee gut bacterial symbionts from hives across the US. Honey bee gut metagenomes were sampled from hives at 13 apiaries located in a transect from Virginia to Washington, and ARG presence was assessed across the sites. We also specifically quantified the abundances of two common tetracycline resistance genes (tet(B) and tet(M)) across apiaries. ARGs, both for antibiotics used in beekeeping and unrelated antibiotics, were detected in honey bee gut bacteria from all apiaries. Tetracycline resistance genes were the most common across all apiaries, and the abundance of two tetracycline resistance genes varied by apiary. Members of the honey bee gut microbiome contained different proportions of ARGs, but taxa within a single family contained similar proportions, possibly indicating phylogeny plays a role in ARG accumulation. In particular, Gilliamella and Frischella, both in the family Orbaceae, contained the highest percentages of ARGs. The results from this study suggest honey bee bacteria act as reservoirs of ARGs. Overall, the presented research contributes to the field of biology by highlighting the importance of intraspecific variation in host-associated microbial communities and presenting a new method for studying honey bee gut microbiome variation at the individual-level, showing that early life events in honey bees influence microbiome development, and suggesting that honey bee bacterial symbionts have adapted to deal with antibiotic disturbance through the accumulation of ARGs. / Doctor of Philosophy / Nearly all animals, including honey bees, have communities of bacteria that live on and in them. These communities, called microbiomes, are often essential to the health of their hosts. For instance, communities of gut bacteria can be important for breaking down food for digestion. Honey bees have approximately 10 bacterial species that consistently live in their guts and provide these types of services to their host. As with many bacterial communities, these beneficial bacteria can be impacted by exposure to antibiotics, even though antibiotics can also be important for treating or preventing dangerous bacterial infections. In honey bee hives, the antibiotic tetracycline is used to prevent bacterial disease. However, tetracycline may simultaneously be negatively impacting colony health through disruption of the honey bee gut microbiome. The goal of the presented work was to understand how tetracycline impacts the honey bee gut microbiome. In my first chapter, I demonstrate a new fecal sampling method that will allow us to understand how gut microbiomes from individual bees change over time. I first compared the bacteria found in fecal samples to those in the whole guts of bees and found that the bacterial communities of the fecal samples and guts were very similar, indicating that fecal sampling is a good method for studying the honey bee gut microbiome. I then used my fecal sampling method to determine how individual honey bee gut microbiomes respond to antibiotic disturbance over time. I collected fecal samples from adult bees prior to treatment, treated the bees with tetracycline, and after five days of being maintained in the lab, recollected fecal samples. My results showed few changes to the bacterial communities before and after treatment, suggesting some honey bee gut microbiomes may be resistant to tetracycline. In my second chapter, I addressed whether exposure to antibiotics early in life had long-term impacts on the gut microbiome. I treated bees at the start of adulthood with tetracycline, returned the bees to their hive for 7 or 14 days, and assessed their microbiome. Tetracycline treatment at the beginning of adulthood changed the gut microbiome later in life, as the microbiomes of tetracycline-treated bees and controls differed from one another both 7 and 14 days after exposure. This chapter shows that disturbances to microbiomes during early life can also affect microbiomes later. My third chapter addressed how honey bee bacteria have adapted to antibiotic use by identifying antibiotic resistance genes (ARGs) in honey bee gut bacteria from 13 hives located in a transect across the US from the state of Washington to Virginia. I found a variety of antibiotic resistance genes in honey bee gut bacteria, both associated with beekeeping and likely environmental contamination. The prevalence of antibiotic resistance genes in honey bee bacteria may help us track antibiotic resistance in the environment. Ultimately, my dissertation contributes to our understanding of how antibiotic use affects honey bees by changing their gut microbiome.

Honey bees, Apis mellifera Linn., provide vital economic and ecological services via pollination while concurrently facing multiple interconnected stressors impacting their health. Many crops like apples, peaches, and cherries that add diversity and nutrition to our diet are wholly or partially dependent upon the pollination services of insects. Orchard crops are self-incompatible and commonly regarded as crops reliant on the pollination services of insects, and while previous studies have focused on the impact of bees to orchard crops during bloom, fewer studies have examined the reciprocal relationship of the orchards on honey bees, particularly across the entire foraging season. Here we investigated the foraging dynamics of honey bees in an orchard crop environment in Northern Virginia, United States. We decoded, mapped, and analyzed 3,710 waggle dances, which communicate the location of a valuable resource in the environment, for two full foraging seasons (April-October, 2018-2019), and, concurrent to the dance filming, collected pollen from returning foragers. We found that bees forage locally the majority of the time (< 2 km) throughout the season, with some long-range distances occurring in May after bloom (both 2018 and 2019) and in fall (2019). The shortest communicated median distances (0.50 km and 0.53 km), indicating abundant food availability, occurred during September in both years, paralleling the bloom of an important late season resource, goldenrod (Solidago). We determined, through plotting and analyzing the communicated forage locations and from the collected pollen from returning foragers, that honey bees forage more within apple orchards after the bloom (29.4% and 28.5% foraging) compared to during bloom (18.6% and 21.4% foraging) on the understory of clover and plantain. This post bloom foraging also exposes honey bees to the highest concentration of pesticides across the entire foraging season (2322.89 ppb pesticides versus 181.8 during bloom, 569.84 in late summer, and 246.24 in fall). Therefore, post bloom apple orchards supply an abundance of forage, but also the highest risk of pesticide exposure, which may have important implications for management decisions of bees in orchards. / Master of Science in Life Sciences / Honey bee hives have been declining significantly in the United States, driven by a multitude of issues and stressors including pesticide exposure, disease, pests such as varroa mites, and poor nutrition caused by natural land being converted into development or agriculture. Agricultural landscapes, in particular, are often monocultures are saturated with pesticides creating a potentially hazardous environment, yet reliant on bees to provide pollination for crops. Because of this interconnected relationship between bees and flowers and the effects of stressors agricultural systems cause have with pollinators, it is necessary to understand how honey bees forage in these environments and what potential health risks they face. We investigated honey bees foraging dynamics in an apple orchard in Northern Virginia, United States by observing honey bee waggle dance behavior, where bees literally waggle back and forth for a certain time and at a certain angle telling their nestmates where a resource is, and collecting pollen from returning forager bees to better understand when, where, and upon what honey bees forage throughout the foraging season, which is when flowers are available and the weather warm enough (April – October). We found that bees mostly forage locally near the hive throughout the season, indicating that sufficient amount of food was available even after short bloom time of the apple (April to mid-May). We determined, through plotting and analyzing the waggle dance locations, that honey bees forage more within apple orchards after the bloom on mostly clover and plantain. This abundance of post bloom foraging also exposes honey bees to the highest amounts of pesticides across the entire foraging season. Post bloom apple orchards supply an abundance of forage, but also the highest risk of pesticide exposure to honey bees.

The honey bee is a widely managed crop pollinator that provides the agricultural industry with the sustainability and economic viability needed to satisfy the food and fiber needs of our society. Excessive use of agrochemicals such as the acaricides coumaphos and tau-fluvalinate, and the fungicide, chlorothalonil is implicated in the reduced number of managed bee colonies available for crop pollination services. Here, I report the effects of pesticide exposures on the nutritional and immune health of the honey bee. Total protein concentration was significantly reduced in the coumaphos- and chlorothalonil-treated individuals compared to the pesticide-untreated bees. Total carbohydrate concentration was significantly reduced in the tau-fluvalinate-, coumaphos-, and chlorothalonil-treated individuals compared to the pesticide-untreated bees. Total lipid concentration was significantly decreased in the chlorothalonil-treated individuals compared to the pesticide-untreated bees. Body weight was significantly reduced for the tau-fluvalinate-, coumaphos-, and chlorothalonil-treated individuals, respectively, compared to the pesticide-untreated bees. Head width was significantly reduced for the chlorothalonil-treated individuals whereas the wing length was significantly reduced for the coumaphos and chlorothalonil-treated individuals, respectively, compared to the pesticide-untreated bees. Phenoloxidase activity was significantly increased in the coumaphos-treated individuals compared to the pesticide-untreated bees. Glucose oxidase activity was significantly increased in the chlorothalonil-treated individuals compared to the pesticide-untreated bees. While more research is needed to verify the observed effects of the pesticides on the nutritional and immunity health of the honey bee, it is important for beekeepers to consider alternative methods for control of varroa mites and the use of fungicides near their colonies. / Master of Science in Life Sciences

The effects of miticides on the reproductive physiology of queens and drones were examined. The first study examined the effects of Apistan (fluvalinate), Check Mite+ (coumaphos), and Apilife VAR (74% thymol) on sperm production and viability in drones. Drones from colonies treated with each miticide were collected at sexual maturity. Sperm production was determined by counting the number of sperm in the seminal vesicles. Sperm for viability assays was analyzed by dual fluorescent staining. Apilife VAR and coumaphos significantly lowered (P<0.0001) sperm production and coumaphos treatments caused a significant decrease (P<0.0001) in the sperm viability. The effects of miticides on queens was examined by treating queen-rearing colonies and examining the number and viability of sperm in the spermathecae of newly mated queens. Queens from each treatment group were collected after mating and the spermathecae were removed and analyzed. Colonies treated with coumaphos failed to provide viable queens and were excluded. Apilife VAR was found to significantly decrease (P<0.0016) sperm viability. No significant differences in sperm numbers were found between treatments. The effect of miticides on sperm viability over time was also examined. Drones were reared as described, but the spermatozoa were collected as pooled samples from groups of drones. The pooled samples from each treatment were subdivided and analyzed periods of up to 6 weeks. Random samples were taken from each treatment (n = 6 pools) over a period of 6 weeks. The exposure of drones to coumaphos during development significantly reduced sperm viability for all 6 weeks, and caused a large decline in week 6. The potential impacts of these results on queen performance and failure are discussed. / Master of Science in Life Sciences

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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...