Nosema ceranae, a microsporidian, has been given much attention in recent years as it has been linked with Colony Collapse Disorder (CCD), which leads to the sudden deaths of honey bee colonies. It has been described that many pathogenic organisms secrete virulence factors in order to hijack its host`s cellular functions, but in most cases the underlying mechanisms of this process still remains to be deciphered. Cornman et al. (2009) have identified in N. ceranae a list of putative effector proteins (called secretome) destined to be secreted into the host, and I have taken this list for further investigation using a bioinformatical and experimental approaches. The principal aim of this project was to generate a N. ceranae ORFeome for genes predicted to be secreted, elucidate the function of effector candidates important for N. ceranae biology and/or pathogenicity, as well as to investigate any interactions between N. ceranae proteins and its host utilising two eukaryotic model organisms, budding yeast, S. cerevisiae, and fruit fly, D. melanogaster. A library of S. cerevisiae strains expressing N. ceranae proteins was generated utilising the Gateway® technology, and phenotypic and localisation screens were undertaken to investigate the N. ceranae secretome. Two N. ceranae ORFs, NcORF-15 (NcORF-02039) and NcORF-16 (NcORF-01159) encoding a putative thioredoxin and a hexokinase, respectively, were subjected to yeast complementation assays in order to assess their catalytic activity. NcORF-15, the putative thioredoxin, was able to rescue the sensitive phenotype of S. cerevisiae Δtrx2 under oxidative stress, whereas NcORF-16, the putative hexokinase, did not complement YSH7.4-3C, a triple knockout lacking hexokinase activity. A third N. ceranae effector candidate NcORF-4 (NcORF-00654), a putative proteasome subunit, was investigated for its nuclear localisation and protein interactions in both S. cerevisiae and D. melanogaster.

In this dissertation, we explore the properties of correlation structure for spatio-temporal point processes and a quantitative spatial process. Spatio-temporal point processes are often assumed to be separable; we propose a formal approach for testing whether a particular data set is indeed separable. Because of the resampling methodology, the approach requires minimal conditions on the underlying spatio-temporal process to perform the hypothesis test, and thus is appropriate for a wide class of models. Africanized Honey Bees (AHBs, Apis mellifera scutellata) abscond more frequently and defend more quickly than colonies of European origin. That they also utilize smaller cavities for building colonies expands their range of suitable hive locations to common objects in urban environments. The aim of the AHB study is to create a model of this quantitative spatial process to predict where AHBs were more likely to build a colony, and to explore what variables might be related to the occurrences of colonies. We constructed two generalized linear models to predict the habitation of water meter boxes, based on surrounding landscape classifications, whether there were colonies in surrounding areas, and other variables. The presence of colonies in the area was a strong predictor of whether AHBs occupied a water meter box, suggesting that AHBs tend to form aggregations, and that the removal of a colony from a water meter box may make other nearby boxes less attractive to the bees.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2009. / Härtill 4 uppsatser.

Orientador: Percília Cardoso Giaquinto / Resumo: As abelhas têm grande importância na polinização; no entanto, o desaparecimento e morte de colônias de Apis mellifera Linnaeus, 1758 (Hymenoptera: Apidae) têm aumentado em várias regiões do mundo, prejudicando a sobrevivência da espécie e a estrutura dos ecossistemas. O uso de agrotóxicos em cultivos agrícolas vem sendo apontado como um dos fatores responsáveis pela intensificação do desaparecimento das abelhas. No presente estudo, avaliou-se a toxicidade, as alterações comportamentais e motoras de abelhas campeiras de A. mellifera expostas aos herbicidas 2,4-D e glifosato, isolados ou associados, por testes de ingestão e contato. Para determinação da dose letal (DL50) foi analisada a mortalidade de abelhas expostas a diferentes doses dos herbicidas por contato e ingestão por 24 horas. A atividade motora das abelhas expostas aos herbicidas glifosato, 2,4-D e associação de ambos foi avaliada 4 e 24 horas após sua exposição utilizando caixa de observação comportamental. As DL50 encontradas dos herbicidas glifosato e 2,4-D foram respectivamente: 273,93 e 127,70 µg/abelha para ingestão e 255,73 e 97,09 µg/abelha para contato. As doses subletais dos herbicidas glifosato e 2,4-D foram, respectivamente: 5,47 e 2,55 µg/abelha para ingestão e 5,11 e 1,94 µg/abelha para contato. A dose letal de 4 horas e subletal de 24 horas foram altamente tóxicas para as abelhas causando alterações locomotoras, as quais em campo podem influenciar o forrageamento das campeiras. Dos testes de doses sub... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bees are very important for pollination, however, disappearance and death of Apis mellifera hives in many regions of the world has increased, damaging the species survival and ecosystems maintenance. The use of herbicides in agricultural crops has been accused as one of the factors responsible for this disappearance. This study aimed to evaluate the toxicity, behavioral and motor abnormalities on foraging honey bees Apis mellifera exposed to 2,4-D and glyphosate alone and combined. To determine the lethal dose (LD50), bees were exposed to different herbicides doses by contact or ingestion and mortality was analyzed. After 24 hours, the mortality results were analyzed. The glyphosate and 2,4-D LD50 found were, respectively: 273,93 and 127,70 μg/bee for ingestion and 255,73 and 97,09 μg/bee for contact. The sublethal doses for glyphosate and 2,4-D were, respectively: 5,47 and 2,55 μg/bee for ingestion and 5,11 and 1,94 μg/bee for contact. The bee’s motor activity was assessed 4 and 24 hours after herbicides and combination exposure, using a behavioral observation box. The 24 hours lethal and sublethal doses were highly toxic to bees causing locomotors changes, which in the field can influence the bees foraging activity. Only the 4h sublethal herbicide association test showed additive effect, however this fact deserves attention because when carried to the colony, the herbicides can influence other bees behavior and cause weakening or even death of the hive. / Mestre

abstract: Neurotoxicology has historically focused on substances that directly damage nervous tissue. Behavioral assays that test sensory, cognitive, or motor function are used to identify neurotoxins. But, the outcomes of behavioral assays may also be influenced by the physiological status of non-neural organs. Therefore, toxin induced damage to non- neural organs may contribute to behavioral modifications. Heavy metals and metalloids are persistent environmental pollutants and induce neurological deficits in multiple organisms. However, in the honey bee, an important insect pollinator, little is known about the sublethal effects of heavy metal and metalloid toxicity though they are exposed to these toxins chronically in some environments. In this thesis I investigate the sublethal effects of copper, cadmium, lead, and selenium on honey bee behavior and identify potential mechanisms mediating the behavioral modifications. I explore the honey bees’ ability to detect these toxins, their sensory perception of sucrose following toxin exposure, and the effects of toxin ingestion on performance during learning and memory tasks. The effects depend on the specific metal. Honey bees detect and reject copper containing solutions, but readily consume those contaminated with cadmium and lead. And, exposure to lead may alter the sensory perception of sucrose. I also demonstrate that acute selenium exposure impairs learning and long-term memory formation or recall. Localizing selenium accumulation following chronic exposure reveals that damage to non-neural organs and peripheral sensory structures is more likely than direct neurotoxicity. Probable mechanisms include gut microbiome alterations, gut lining damage, immune system activation, impaired protein function, or aberrant DNA methylation. In the case of DNA methylation, I demonstrate that inhibiting DNA methylation dynamics can impair long-term memory formation, while the nurse-to- forager transition is not altered. These experiments could serve as the bases for and reference groups of studies testing the effects of metal or metalloid toxicity on DNA methylation. Each potential mechanism provides an avenue for investigating how neural function is influenced by the physiological status of non-neural organs. And from an ecological perspective, my results highlight the need for environmental policy to consider sublethal effects in determining safe environmental toxin loads for honey bees and other insect pollinators. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2016

Submitted by Juliana Sartori Lunardi (jusartorilunardi@gmail.com) on 2018-04-26T20:50:59Z No. of bitstreams: 1 Juliana Sartori Lunardi -Dissertação mestrado REPOSITÓRIO.pdf: 1404393 bytes, checksum: 14768aa62b64729cfed9d15a41a9a557 (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-04-27T13:47:53Z (GMT) No. of bitstreams: 1 lunardi_js__me_bot_int.pdf: 1404393 bytes, checksum: 14768aa62b64729cfed9d15a41a9a557 (MD5) / Made available in DSpace on 2018-04-27T13:47:53Z (GMT). No. of bitstreams: 1 lunardi_js__me_bot_int.pdf: 1404393 bytes, checksum: 14768aa62b64729cfed9d15a41a9a557 (MD5) Previous issue date: 2018-02-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / As abelhas têm grande importância na polinização; no entanto, o desaparecimento e morte de colônias de Apis mellifera Linnaeus, 1758 (Hymenoptera: Apidae) têm aumentado em várias regiões do mundo, prejudicando a sobrevivência da espécie e a estrutura dos ecossistemas. O uso de agrotóxicos em cultivos agrícolas vem sendo apontado como um dos fatores responsáveis pela intensificação do desaparecimento das abelhas. No presente estudo, avaliou-se a toxicidade, as alterações comportamentais e motoras de abelhas campeiras de A. mellifera expostas aos herbicidas 2,4-D e glifosato, isolados ou associados, por testes de ingestão e contato. Para determinação da dose letal (DL50) foi analisada a mortalidade de abelhas expostas a diferentes doses dos herbicidas por contato e ingestão por 24 horas. A atividade motora das abelhas expostas aos herbicidas glifosato, 2,4-D e associação de ambos foi avaliada 4 e 24 horas após sua exposição utilizando caixa de observação comportamental. As DL50 encontradas dos herbicidas glifosato e 2,4-D foram respectivamente: 273,93 e 127,70 µg/abelha para ingestão e 255,73 e 97,09 µg/abelha para contato. As doses subletais dos herbicidas glifosato e 2,4-D foram, respectivamente: 5,47 e 2,55 µg/abelha para ingestão e 5,11 e 1,94 µg/abelha para contato. A dose letal de 4 horas e subletal de 24 horas foram altamente tóxicas para as abelhas causando alterações locomotoras, as quais em campo podem influenciar o forrageamento das campeiras. Dos testes de doses subletais em 4h, foram significativos os de ingestão do 2,4-D e a associação dos herbicidas a qual demonstrou efeito aditivo. A associação dos herbicidas merece atenção, pois em campo essa combinação será tóxica as abelhas. Com este estudo, espera-se contribuir para a compreensão do efeito de herbicidas, assim como auxiliar no estabelecimento de medidas que reduzam a exposição de polinizadores a agrotóxicos prejudiciais. / Bees are very important for pollination, however, disappearance and death of Apis mellifera hives in many regions of the world has increased, damaging the species survival and ecosystems maintenance. The use of herbicides in agricultural crops has been accused as one of the factors responsible for this disappearance. This study aimed to evaluate the toxicity, behavioral and motor abnormalities on foraging honey bees Apis mellifera exposed to 2,4-D and glyphosate alone and combined. To determine the lethal dose (LD50), bees were exposed to different herbicides doses by contact or ingestion and mortality was analyzed. After 24 hours, the mortality results were analyzed. The glyphosate and 2,4-D LD50 found were, respectively: 273,93 and 127,70 μg/bee for ingestion and 255,73 and 97,09 μg/bee for contact. The sublethal doses for glyphosate and 2,4-D were, respectively: 5,47 and 2,55 μg/bee for ingestion and 5,11 and 1,94 μg/bee for contact. The bee’s motor activity was assessed 4 and 24 hours after herbicides and combination exposure, using a behavioral observation box. The 24 hours lethal and sublethal doses were highly toxic to bees causing locomotors changes, which in the field can influence the bees foraging activity. Only the 4h sublethal herbicide association test showed additive effect, however this fact deserves attention because when carried to the colony, the herbicides can influence other bees behavior and cause weakening or even death of the hive. / CNPq: 132451/2016-6. 7

Made available in DSpace on 2017-07-10T17:48:11Z (GMT). No. of bitstreams: 1 Fernando_da_Cunha.pdf: 1663334 bytes, checksum: 747cba01a82289ae88e1bcc8c569db5f (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The direct and indirect effects of agricultural pesticides has been the focus of increasing numbers of studies by different entities and in different locations. The Apis mellifera bee is an insect that are part of the natural environment, producing honey from its interaction with vegetation. Brazilian legislation seldom refers to the concentration of pesticides in food, and honey, a product produced by bees, has had few cases of contamination by pesticides used for the control of pests in agriculture. The maximum pesticide residue limit (MRLs) established in legislation principally relate to those products used in large-scale crop production. Organophosphate and organochloride standards were used on samples and validated. In this way the identification of pesticide residues in honey generated by bees acquired in native trees and planted cultures in a 1,500m radius of the hives, by way of chromatography (CGMS), was estimated over two seasons in fifteen apiaries in the city of Santa Helena-PR. On average, the areas utilised for the study corresponded to 58.92% under agricultural production, 22.08% in native forest, 9.52% in water, and 6.04% in buildings and dwellings. There was however no significant effect in the study area on the percentage of contamination in the samples. In september to november of 2013, 26.66% of the samples were contaminated. In december to january of 2014 the figure was 8.88%. There was significant effect in the period (p<0.05) on the Fenclorfós, Chloropyrifos, Mitotane and Bicylo, with a greater occurrence and concentration in september to november of 2013. Those pesticides with residue limits outside of the legislation, did not have significant effect in the period (p>0.05) on the occurrence of contaminated samples with Parathion Methyl, Chloropyrifos, and DDE. Parathion Methyl and Chloropyrifos were found at levels above those specified in the legislation in both periods of the study. This would suggest the overuse of these pesticides at above recommended levels by agricultural producers to control pests. In conclusion, the Apis mellifera bees were efficient in the detection of pesticide residues in honey, that were used in agricultural production near the apiaries between September 2013 and December 2014. This reinforces the potential of these insects as bioindicators of environmental quality / O efeito direto e indireto dos agrotóxicos utilizados na agricultura tem sido, cada vez mais, alvo de estudo nos diferentes ambientes e organismos vivos com os quais têm contato. As abelhas Apis mellifera são insetos que estão diretamente relacionadas aos ambientes naturais, produzindo o mel com características específicas dessa relação com os vegetais, nos quais buscam alimento. A legislação brasileira pouco se refere à concentração de pesticidas nos alimentos, e o mel apresenta especificações de limites máximos de resíduos de poucos princípios ativos utilizados na agricultura. Assim, buscou-se identificar os pesticidas presentes no mel de Apis mellifera e relacionar os agrotóxicos encontrados às possíveis fontes de contaminação, utilizando o mel como bioindicador ambiental. Foram utilizados padrões de pesticidas organoclorados e organofosforados, recuperados nas amostras e validados, a identificação dos pesticidas no mel foi realizada por meio das análises cromatográficas, em 86 amostras coletadas de 15 apiários e em duas épocas diferentes. Esses apiários foram georreferenciados e a partir das imagens foi estimado o uso e ocupação do solo, onde em média, o uso do solo por área agrícola correspondente a 58,92%, área de mata (22,08%), área de água (9,52%) e área com edificações (6,04%). No entanto, não houve efeito significativo de área sobre a porcentagem de contaminação das amostras. No período de setembro a novembro de 2013, 26,66% das amostras foram contaminadas, e no período de dezembro a janeiro de 2014, 8,88%. Houve efeito significativo de época (p<0,05) sobre o Fenclorfós, Chlorpirifós, Mitotane e Bicyclo, tendo maior ocorrência e concentração de setembro a novembro de 2013. Com relação aos pesticidas com limites de resíduos fora da legislação, não houve efeito significativo de época (p>0,05) sobre a ocorrência de amostras contaminadas com Paration Metílico, Chlorpirifós e DDE. Paration Metílico e Clorpirifós foram encontrados acima do preconizado pela legislação em ambas as épocas de estudo, revelando o uso indevido destes compostos para as culturas e possivelmente o número excessivo de aplicações para o controle de pragas agrícolas. Contudo, o mel de Apis mellifera se mostrou um bioindicador eficiente de resíduos empregados nas culturas agronômicas de entorno dos apiários da pesquisa entre setembro de 2013 e janeiro de 2014, reforçando a importância destes insetos polinizadores para a avaliação da qualidade ambiental

The daily patterns of task performance in honey bee colonies during behavioral development were studied to determine the role of circadian rhythmicity in age-related division of labor. Although it is well known that foragers exhibit robust circadian patterns of activity in both field and laboratory settings, we report that many in-hive tasks are not allocated according to a daily rhythm but rather are performed 24 h per day. Around-the-clock activity at the colony level is accomplished through the performance of some tasks by individual workers randomly with respect to time of day. Bees are initially arrhythmic with respect to task performance but develop diel rhythmicity, by increasing the occurrence of inactivity at night, prior to becoming foragers. There are genotypic differences for age at onset of rhythmicity and our results suggest that these differences are correlated with genotypic variation in rate of behavioral development: genotypes of bees that progressed through the age polyethism schedule faster also acquired behavioral rhythmicity at an earlier age. The ontogeny of circadian rhythmicity in honey bee workers ensures that essential in-hive behaviors are performed around the clock but also allows the circadian clock to be engaged before the onset of foraging.

Forager honey bees exhibit a robust time memory, based on an endogenous circadian clock, enabling them to schedule their flights to coincide with the nectar presentation of known food sources. They retain this time memory for several consecutive days even in the absence of nectar rewards. Recent work has identified 2 classes of forager: "persistent" foragers that reconnoiter a known food source to ascertain its status and "reticent" foragers that apparently wait in the hive for a waggle dance confirming source availability. Surprisingly, a foraging group contains 40-90% persistent foragers, depending on experience at the source. What is the benefit in sending so many foragers to investigate a source when only a few foragers are required to reactivate the entire group? We used an agent-based software model to test the energetics underlying several different ratios of persistent and reticent individuals in the foraging group while varying 6 ecological factors: forager group size, source distance, source sucrose concentration, source availability in hours, number of days the source is known to the colony, and the rate at which new unemployed foragers appear on the dance floor. Our model demonstrates 2 primary explanations. First, a large number of persistent foragers are needed to ensure that at least some foragers will reconnoiter their source early in its availability, thus enabling the group to effectively exploit the source. Second, the cost of a reconnaissance flight is negligible compared with even a single successful foraging trip.