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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

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

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-06-06T18:24:16Z No. of bitstreams: 1 ZILMAR FERNANDES NÓBREGA MELO - DISSERTAÇÃO PPGEA 2002..pdf: 14604622 bytes, checksum: 92a403c722a2a592fbb333f263c9b64f (MD5) / Made available in DSpace on 2018-06-06T18:24:16Z (GMT). No. of bitstreams: 1 ZILMAR FERNANDES NÓBREGA MELO - DISSERTAÇÃO PPGEA 2002..pdf: 14604622 bytes, checksum: 92a403c722a2a592fbb333f263c9b64f (MD5) Previous issue date: 2002-08 / Estudou-se o armazenamento de méis de abelhas africanizadas (Apis mellifera L.) coletados no brejo e cariri do estado da Paraíba. Utilizou-se, quanto à florada, dois tipos de mel de abelha (mel de florada silvestre e mel de florada de baraúna), armazenados sob 3 diferentes condições de embalagens (El - recipiente de polietileno opaco e exposto à luz e temperatura ambiente; E2 - recipiente de polietileno exposto à temperatura ambiente e ao abrigo da luz; E3 - recipiente em vidro translúcido exposto à temperatura e luz ambiente), ao longo de 180 dias, tendo sido estes submetidos a análises físico-químicas mensalmente, com a finalidade de observar as possíveis alterações nas características físico-químicas (°Brix, Umidade, Cinzas, Hidroximetilfurfural-HMF, Açúcares redutores, Sacarose aparente, Atividade diastásica e pH), objetivando verificar a eficiência destas embalagens no índice de qualidade dos méis. As análises físico-químicas dos méis foram feitas seguindo o recomendado pela Instrução Normativa do Ministério da Agricultura e do Abastecimento (Brasil, 2000). Os valores médios encontrados, ao longo do Armazenamento, para o °Brix do mel silvestre ficaram no intervalo de variação de 78,03 a 78,42 °Brix e o da florada de baraúna de 81,31 a 81,63 °Brix e não houve diferença significativa entre as embalagens ao longo do armazenamento. Os níveis de Umidade (%) para os méis silvestres e de baraúna foram 19,48 a 20,48 % e 16,25 a 16,70 %, respectivamente. Os valores Cinzas e de Sólidos Insolúveis não apresentara diferenças significativas no tempo e/ou nas embalagens. Os níveis no índice de HMF ficaram no intervalo de variação de 4,57 a 10,17 mg/kg de mel e 1,08 a 7,12 mg/kg, no mel Silvestre e no mel de baraúna, respectivamente. Pelas análises estatísticas, verificou-se, para os dois méis, diferença significativa a 1% de probabilidade e que as médias diferem entre si. Houve para o HMF, diferença significativa entre as embalagens e a E2 foi a mais eficiente no controle do aumento deste índice nos dois méis estudados. Os valores médios de Açúcares Redutores ficaram entre 74,09 e 76,41 % no mel Sivestre e entre 62,51 e 64,80 % para o mel de baraúna. Os valores de Sacarose Aparente ficaram entre 2,18 a 3,01 % para o mel silvestre e entre 2,40 e 2,89 % no mel de baraúna. Os méis sofreram pequena variação no tempo e as embalagens não influenciaram sobre a sacarose, nos dois méis. A Atividade Diastásica (DN) no mel Sivestre ficou no intervalo de variação de 13,37 a 18,71 DN e 9,14 a 13,25 DN no mel de baraúna. As análises estatísticas para os dois méis foram significativas ao nível de 1% de probabilidade e no teste de Tukey, as médias diferiram entre si, e a embalagem E2, nos dois méis analisados, mostrou-se mais eficiente na desaceleração do índice de Diástase em relação a El e E3. Quanto ao pH, o mel Sivestre foi mais ácido (3,42 a 3,55) do que o mel de baraúna (3,85 a 4,15). Todas as amostras encontram-se dentro dos padrões exigidos pelo Ministério da Agricultura e do Abastecimento. Do resultado final, concluiu-se que houve maior eficiência na embalagem E2, que contribuiu para a desaceleração do ritmo no índice de diástase. Observou-se que o índice de acidez livre aumentou ao longo do armazenamento. / The storage of african bees honeys (Aipis mellifera L.) which were collected from slough and cariri in Paraíba state was studied. Considering the bloomed, two kinds of bee honey (wild bloomed honey and baraúna bloomed honey) were used and storaged under three different conditions of packages ( Ei - opaque polyethylene container, that was under environment light and temperature; E2 - polyethylene container that was under environment temperature but it wasn't under light environment; E3 - translucent glass container which was exposed to environment temperature and light). These honeys stayed 180 days in packages and their physiochemical characteristics were analysed monthly to observe the possible alterations of them ( 0 Brix, Humidity, Ashes, Hydroximethylfurfurol - HMF, Reducers Sugar, Apparent Saccharose, Diastasic Activity and pH) to verify the efficiency of these packages in the honeys qualities index. The physiochemical analyses of the honeys were done according to the Normative Instruction of the Agriculture and Provisioning Ministry (Brazil, 2000). During the storage time, the médium found values to the 0 Brix of the wild bloomed honey were between 78.03 and 78.42 0 Brix and the ones of the baraúna bloomed honey were between 81.31 and 81.63 0 Brix and there wasn't significant difference among the packages during the storage period. The Humidity rates (%) were between 19.48 and 20.48% to the wild honey and between 16.25 and 16.70 to the baraúna honey. There were no significant differences of the Ashes and Insoluble Solids values on their time and packages. The rates in the HMF index varied from 4.57 to 10.17 mg/Kg of honey and from 1.08 to 7.12 mg/Kg, to the wild and baraúna honeys, respectively. According to the statistics analyses, a significant difference of 1% of probability for both honeys was verified, besides the averages are different among them. To the HMF, there was a significant difference among the packages and the E2 was the most efficient to the control of the increase of this index in both studied honeys. The médium values of the Reducers Sugar were between 74.09 and 76.41% to the wild honey and they were between 62.51 and 64.80% to the baraúna honey. The Apparent Saccharose values were between 2.18 and 3.01% to the wild honey and they were between 2.40 and 2.89 to de baraúna honey. The honeys had a little time variation and the packages had no influence on the saccharose, in both honeys. The Diastasic Activity (DN) on the Wild honey varied from 13.37 to 18.71 DN and the variation of the baraúna honey was from 9.14 to 13.25 DN. The statistics analyses were significative at 1% of probability and at the Tukey test for both honeys, the averages were diferent among them and the E2 package was more efficient than the Ei and E3 to the unacceleration of the diástase index for both honeys. Analysing the pH, the wild honey was more acid (3.42 to 3.55) than the baraúna honey (3.85 to 4.15). Ali the samples are according to the Agriculture and Provision Ministry patterns. According to the final result, it's conclued that there was a bigger efficiency of the E2 package and it contributed to the rhythm unaceleration in the distasic index. It was observed that the free acidity index increased during the storage period.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / A pesquisa objetivou investigar o processo de cointegraÃÃo espacial entre os principais mercados brasileiros exportadores de mel natural: Rio Grande do Sul, Santa Catarina, PiauÃ, Cearà e SÃo Paulo. Foram utilizados dados secundÃrios obtidos diretamente da base de dados do MinistÃrio de Desenvolvimento, IndÃstria e ComÃrcio Exterior, compreendendo a sÃrie de tempo entre janeiro de 2002 a julho de 2011. Para investigar as relaÃÃes entre os mercados supracitados, fez-se uso de metodologia baseada nas premissas da Lei do PreÃo Ãnico â LPU e modelagem que considera a presenÃa de custos de transaÃÃo. Os resultados indicam que, apesar da confirmaÃÃo da existÃncia de cointegraÃÃo entre os mercados brasileiros exportadores de mel natural, a LPU nÃo foi totalmente confirmada, uma vez que se rejeitou a hipÃtese nula de perfeita integraÃÃo espacial entre os mercados. O teste de exogeneidade fraca indica que o mercado de mel natural do Rio Grande do Sul atua como mercado central na formaÃÃo de preÃos. Hà a presenÃa de custos de transaÃÃo entre quase todos os mercados espacialmente separados, pois, segundo os resultados oriundos da modelagem threshold, rejeitou-se a hipÃtese nula de ajuste linear e simÃtrico entre os mercados investigados. / This study investigated the process of cointegration space between the main markets Brazilian exporters of natural honey Rio Grande do Sul, Santa Catarina, PiauÃ, Cearà and SÃo Paulo. Secondary data obtained directly from the database of the MinistÃrio de Desenvolvimento, IndÃstria e ComÃrcio Exterior, comprising the time series between January 2002 and July 2011. To investigate the relationship between the markets mentioned above, was made use of a methodology based on the premises of the Law of One Price- LPU and modeling that considers the presence of transaction costs. The results indicate that despite the confirmation of the existence of markets cointegration Brazilian exporting natural honey, the LPU has not been fully confirmed, since it is rejecting the null hypothesis of perfect integration space between the markets. The weak exogeneity test indicates that the market for natural honey of Rio Grande do Sul acts asthe central market pricing. There is the presence of transaction costs between almost allspatially separated markets, because, according to the results from the modelingthreshold, rejected the null hypothesis of linear and symmetric adjustment between markets investigated.

Lactobacillus kunkeei is one of the most abundant bacteria within the honey crop of the honey bee. Genome sequencing of L. kunkeei isolated from honey bees all over the world showed several genes unique for L. kunkeei. Among these orphan genes, an array of four to five highly conserved genes coding for giant extracellular proteins were found. Cryogenic electron microscopy imaging of a giant-protein preparation from L. kunkeei A00901 showed an overall structure similar to a long string with a knot at the end. Further analysis showed high similarity between the different giants at the N-terminus, and secondary structure predictions showed that the same region was rich in β-sheets.  These results, combined with the knowledge of other large extracellular proteins, led to the hypothesis that the “knot” domain is located at the N-terminus and that these proteins are used by the cell to latch on to the intestine lining or other cells in the honey crop. In this study, predictions were made to locate the N-terminal domains of two of these giant proteins. Four different constructs were made for each protein, where three constructs were designed for expression and purification of the N-terminal domain with different end-positions, and one construct was for a predicted β-solenoid domain located downstream from the N-terminal domain. The protein constructs were recombinantly produced in E. coli, and three of the N-terminal constructs from both proteins were purified. Thermal stability was tested using nano differential scanning fluorimetry (nanoDSF), Thermofluor, and circular dichroism (CD), which all showed characteristic melting curves at low melting temperatures, ranging from 33 °C to 44 °C, for all three constructs. During CD measurements, all three constructs showed refolding after thermal denaturation and a higher abundance of antiparallel β-sheets over α-helices. Looking at the protein structure, small angle X-ray scattering data indicated that all three proteins formed elongated structures. These results indicate that a folded domain has been found for both proteins. Although, further analysis will be required to determine the boundaries of the N-terminal domains, and to elucidate if these domains have anything to do with ligand binding and the L. kunkeei ability to latch onto the honey crop.

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