Relatively little is known about the temporal control of behavior of honey bee queens under natural conditions. To determine if mated honey bee queens possess diel rhythmicity in behavior, we observed them in glass-sided observation hives, employing two focal studies involving continuous observations of individual queens as well as a scan-sampling study of multiple queens. In all cases, all behaviors were observed at all times of the day and night. In four of the five queens examined in focal studies, there were no consistent occurrences of diel periodicity for any of the individual behaviors. A more encompassing measure for periodicity, in which the behaviors were characterized as active (walking, inspecting, egg-laying, begging for food, feeding, and grooming self) or inactive (standing), also failed to reveal consistent diel rhythmicity. Furthermore, there were no consistent diel differences in the number of workers in the queen's retinue. Behavioral arrhythmicity persisted across seasons and despite daily changes in both light and temperature levels. Both day and night levels of behavioral activity were correlated with daytime, but not with nighttime, ambient temperatures. The behavior of the one exceptional queen was not consistent: diurnal activity patterns were present during two 24-h observation sessions but arrhythmicity during another. Based on the behavior observed by all but one of the queens examined in this work, the arrhythmic behavior by the mated honey bee queen inside the colony appears to be similar to that exhibited by worker bees before they approach the age of onset of foraging behavior.

Entre todas las especies de brasicáceas, la de mayor importancia es Brassica napus L. (colza), que se destaca por su elevada participación en la producción mundial de aceites comestibles. Muchas especies de brasicáceas, cultivadas y silvestres, no sólo tienen importancia en la alimentación humana, sino que han sido mencionadas como fuentes de polen y néctar. Es decir, que el solo hecho de formar parte de la comunidad vegetal aumenta las posibilidades de alimento para un gran número de polinizadores y visitantes florales. La presencia de visitantes florales de brasicáceas en el centro-sur de la Provincia de Buenos Aires (República Argentina) fue evaluada en parcelas de colza y en parches de brasicáceas espontáneas ubicados en la localidad de Tres Arroyos, en la Chacra Experimental Integrada Barrow (-38.319305, - 60.239380). Para realizar los muestreos se utilizaron redes entomológicas, trampas de agua de colores y el método de observación directa. Además, con el objetivo de determinar el efecto de los polinizadores sobre el rendimiento y sus componentes, en las parcelas de colza se colocaron jaulas excluidoras de polinizadores. Otra parte del trabajo se realizó en dependencias del Departamento de Agronomía de la Universidad Nacional del Sur con el fin de evaluar la germinabilidad in vitro del polen expuesto a condiciones ambientales de la colmena. Para esto, se seleccionaron colmenas de Apis mellifera L. y se colocaron en su interior muestras de polen de colza que se obtuvieron de plantas cultivadas en macetas de 10 litros. En la zona de estudio se comprobó que numerosas especies de insectos, de diversas familias y órdenes, visitaron el cultivo de colza al igual que los parches de especies espontáneas. Todas las especies capturadas se encontraron tanto en el cultivo como en las malezas aledañas. Esto indica que los polinizadores utilizan ambos recursos. Las zonas con vegetación espontánea aledañas a las parcelas de colza presentaron una floración más extensa, brindando recursos a los visitantes florales antes, durante y después de la floración del cultivo. Los resultados de este trabajo muestran que la visita de insectos polinizadores en el cultivo de colza mejora la producción de semilla y esto se traduce en un mayor rendimiento, que es, en definitiva, el objetivo de todo productor. También se demostró que el aumento de rendimiento de semilla tuvo implicancia directa sobre la producción de aceite por hectárea, dado que la visita de los polinizadores mejoró la cantidad de semilla y no generó variaciones significativas en el tenor de materia grasa. Por otro lado, se demostró que las condiciones ambientales dentro de la colmena de Apis mellifera son adversas para la germinabilidad de los granos de polen de colza, independientemente de las condiciones ambientales externas. Las zonas de cría son las de mayor temperatura y en 24 h no se registraron granos de polen con capacidad de germinar. A partir de las 48 h la capacidad de germinabilidad del polen disminuyó significativamente y el bajo porcentaje que germinó presentó tubos completamente atrofiados. Aunque el polen que se ubicó alejado de las zonas de cría mantuvo más tiempo su capacidad de germinar, sólo lo hizo por menos de 72 h. Los resultados de este trabajo respaldan una reducción sustancial del tiempo de carencia que exigen las empresas semilleras y abren la posibilidad de que se utilice una misma colmena en dos servicios de polinización consecutivos sin riesgo de contaminación por polen no deseado. / Among all the species of brassicas, the most important one is Brassica napus L. (rapeseed), which stands out for its significant contribution to the global production of edible oils. Many species of brassicas, both cultivated and wild, are not only important for human consumption but have also been mentioned as sources of pollen and nectar. In other words, simply being part of the plant community increases the food possibilities for a large number of pollinators and floral visitors. The presence of brassica floral visitors in the central-southern region of Buenos Aires Province (Argentina) was evaluated in rapeseed plots and patches of spontaneous brassica plants located in Tres Arroyos, at the Chacra Experimental Integrada Barrow (- 38.319305, -60.239380). Entomological nets, colored water traps, and direct observation methods were used for sampling. Additionally, exclusion cages were placed in the rapeseed plots to determine the effect of pollinators on yield and its components. Another part of the study was conducted at the Department of Agronomy, Universidad Nacional del Sur, to assess the in vitro germinability of pollen exposed to hive environmental conditions. Honey bee (Apis mellifera L.) colonies were selected, and samples of rapeseed pollen obtained from plants cultivated in 10-liter pots were placed inside the hives. The study in the area confirmed that numerous insect species from different families and orders visited both the rapeseed crop and the patches of spontaneous species. All captured species were found in both the crop and the surrounding weeds, indicating that pollinators utilize both resources. The areas with spontaneous vegetation adjacent to the rapeseed plots exhibited more extensive flowering, providing resources for floral visitors before, during, and after the crop's flowering period. The results of this study demonstrate that the visitation of pollinating insects in the rapeseed crop improves seed production, ultimately resulting in higher yields, which is the goal of every farmer. It was also shown that the increased seed yield directly impacted oil production per hectare, as pollinator visitation improved seed quantity without generating significant variations in fat content. On the other hand, it was demonstrated that the environmental conditions within the Apis mellifera hive are unfavorable for the germinability of rapeseed pollen grains, regardless of external environmental conditions. The brood areas have the highest temperatures, and within 24 hours, no pollen grains capable of germinating were recorded. From 48 hours onward, the germination capacity of the pollen significantly decreased, and the low percentage that did germinate exhibited completely atrophied tubes. Although pollen placed away from the brood areas maintained its germination capacity for a longer period, it did so for less than 72 hours. The results of this study support a substantial reduction in the required waiting time demanded by seed companies and open up the possibility of using the same hive for two consecutive pollination services without the risk of contamination by undesired pollen.

The western honey bee, Apis mellifera, provide invaluable economic and ecological services while simultaneously facing stressors that may compromise their health. For example, agricultural landscapes, such as a row crop system, are necessary for our food production, but they may cause poor nutrition in bees from a lack of available nectar and pollen. Row crops are largely wind or self-pollinated, and while previous studies have focused on the impact of bees to row crops, fewer studies have examined the reciprocal relationship of the row crops on honey bees. Here we investigated the foraging dynamics of honey bees in a row crop environment. We decoded, mapped, and analyzed 3460 waggle dances, which communicate the location of where bees collected food, for two full foraging seasons (April – October, 2018-2019), and concurrently collected pollen from returning foragers. We found that bees foraged mostly locally (< 2 km) throughout the season. The shortest communicated median distances (0.48 and 0.32 km), indicating abundant food availability, occurred in July in both years, which was when our row crops were in full bloom. We determined, by plotting and analyzing the communicated locations, that most mid-summer foraging was in row crops, with at least 40% of honey bee recruitment dances indicating either cotton or soybean fields. Bees also largely foraged for nectar when visiting row crop fields, only returning to the hive with Glycine spp. pollen, and foraging on nearby trees and weeds for pollen. Foragers were exposed to thirty-five different pesticides throughout the foraging season, based on pesticide residues in collected pollen. Overall, row crop fields are contributing a surprising majority of mid-summer forage to honey bee hives and suggests that similar agricultural landscapes may also provide abundant, mid-summer forage opportunities for honey bees, however, at the risk of pesticide exposure. / Master of Science in Life Sciences / Declines in the number of honey bee hives have been observed in the United States and western Europe throughout the last century, driven by environmental stressors such as poor nutrition caused by anthropogenic landscape change and pesticide exposure. Agricultural landscapes, for example, contain monocultures and often necessitate pesticide use, which may be detrimental to bee health. Because of these effects, it is necessary to understand how honey bees forage in these systems and what potential health risks they face. We investigated honey bees foraging dynamics in a row crop environment, observing honey bee waggle dance recruitment behavior and gathering forager-collected pollen to better understand when, where, and what honey bees forage on throughout the season (April – October). We found that bees largely foraged near the hive throughout the season, indicating that sufficient resources were available, particularly in July when crops were in full bloom. During full bloom bees considerably foraged in cotton and soybean fields. We found that bees collected minimal row crop pollen, apart from soybean pollen, largely foraging on trees and flowering weeds for pollen. Through pollen foraging bees were exposed to thirty-five pesticides, ranging in toxicity and mode of action. Overall, honey bees foraging in a row crop system foraged substantially in row crop fields during the mid-summer. Row crops systems may be able to provide abundant forage during the mid-summer, but could come at the risk of exposure to pesticides.

Respiration rates and changes in the amount of nutrients in queen-laid and worker-laid eggs of the honey bee, <I>Apis mellifera</I> L., were determined for the 3 days of embryonic development. Respiration was quantified by measuring the amount of CO₂ produced during 13 hr of artificial incubation at four temperature treatments: 28°C, 31°C, 34°C, 36°C (± 0.5°C). The amounts of lipids and carbohydrates were also quantified in the eggs of queens and laying workers on day 1, 2 and 3 using high performance thin layer chromatography. The mean respiration rate for fertilized and unfertilized eggs from queens was 0.1 ± 0.0 µL CO₂/hr/egg, the same as the mean respiration rate obtained for unfertilized eggs from laying workers. The results of carbohydrate analysis showed a total of 2.4 ± 0.6 µg/egg total sugars in the fertilized eggs of queens, an equivalent of 8.3% on a dry weight basis, while unfertilized eggs contained a total of 1.4 ± 0.4 µg/egg total sugars, equivalent of 6.3% on a dry weight basis. Total lipids, excluding fatty acids, were 10.7 ± 6.1 µg/egg (37.4%) for fertilized eggs and 8.4 ± 1.3 µg/egg (40.8%) for unfertilized eggs. The respiration rate at 34°C was 0.17 ± 0.01 µL CO₂/hr/egg on day 1, the same as day 1 at 36°C. Day 2 respiration rates were 0.13 ± 0.04 µL CO₂/hr/egg and 0.15 ± 0.01 µL CO₂/hr/egg, respectively. On day three, 0.22 ± 0.01 µL CO₂/hr/egg was recorded at 34°C and 0.24 0.01 µL CO₂/hr/egg at 36°C. At low temperatures of 28°C and 31°C, a respiration rate of 0.12 ± 0.01 µL CO₂/hr/egg was recorded on day 1, for eggs held at both temperatures. Day 2 results were 0.07 ± 0.01 µL CO₂/hr/egg at 28°C and 0.11 ± 0.01 µL CO₂/hr/egg at 31°C, while 0.07 ± 0.01 and 0.12 ± 0.01 µL CO₂/hr/egg, respectively, were measured on day 3. Mortality results, as indicated by pre-emergence embryos, showed that 75% developed at 34°C compared to 37.5% at 36°C. Low temperatures of 28°C had 12.5% developing to pre-emergence stage while 50% developed at 31°C. Respiratory results showed significant differences (p=0.05) between the different days of incubation and temperature treatments, respectively. No significant difference was observed between the fertilized eggs and unfertilized eggs from queens at the same temperature treatment. The comparison of unfertilized eggs from queens and those from laying workers also showed no significant difference. The regression (R²=0.65) was significant (P=0.05) when CO₂ output on all the days and temperature treatments were compared. The amount of nutrients in the eggs of queens and those of laying workers, were significantly different (P=0.05) depending on egg type and age. No significant difference was observed between the colonies headed by queens or those of laying workers. Although the queen-laid eggs showed a relatively higher mean value for carbohydrates than worker-laid eggs, the reverse was the case for lipids. On comparing the amount of nutrients per unit weight for queen-laid and worker-laid eggs, no significant differences were observed. From the results obtained, inferences were made about the natural differences between the eggs from queens, and those produced by laying workers. / Master of Science

Throughout the last five decades, European Honey bee (Apis mellifera) colonies have been heavily damaged by invading Varroa mites (Varroa destructor), and, more recently, small hive beetles (Aethina tumida). These pests infest A. mellifera colonies throughout Virginia, with V. destructor feeding upon the lipids of their hosts and spreading viruses, and A. tumida feeding extensively on hive products and brood. Because V. destructor has historically demonstrated acaricide resistance, this study examined V. destructor resistance to three common acaricides (amitraz, coumaphos, and tau-fluvalinate) throughout the three geographic regions of Virginia using glass vial contact bioassays; the results showed no resistance in the sites tested. To gain better insights into A. tumida pharmacology, several known acetylcholinesterase (AChE) inhibitors and three novel insecticides (previously shown to have low mammalian toxicity) were tested against an A. tumida laboratory colony through in vivo and in vitro bioassays. The results of these bioassays indicated that coumaphos was most selective and topically effective against A. tumida, while only one experimental compound was selective against A. tumida, with 29-fold less potency than coumaphos. These results can help apiculturists in making informed pest management choices and can lead to future studies further examining V. destructor resistance and optimizing A. tumida insecticide treatments. / Master of Science in Life Sciences / Beekeepers throughout the world have experienced great economic loss and observed a troubling decline in European Honey bee colonies over the past fifty years due to Varroa mite infestations. Varroa mites feed on the fat body of bees, depriving them of nutrients and infecting them with various diseases. Attempts made to control Varroa mites with synthetic chemicals throughout the years have led to acaricide resistance. To look at resistance in Virginia’s mite populations, resistance testing was performed on Varroa mite populations throughout the three geographic regions of Virginia, and these studies showed that there was not resistance in these populations. Another significant hive pest that is increasingly prevalent in the United States is the small hive beetle (SHB), which feeds on bee brood and hive products. SHB management tactics are still being explored, and this project tested various known insecticide treatments against small hive beetles and bees, as well as three insecticide treatments that were designed for mosquito control and have low toxicity to mammals. The results of this study showed that, of the insecticides tested, coumaphos was the most selective against SHB. This information can help beekeepers in Virginia make informed choices when deciding how to treat Varroa mite infestations, and can add to the knowledge base of those fighting small hive beetle infestations.

Mecanismos que regem o desenvolvimento do olho composto e lóbulo óptico tem sido amplamente estudados em Drosophila melanogaster onde a retina é formada a partir de um disco imaginal anexado com o cérebro e os lóbulos opticos a partir do primórdio óptico externo. Através de histologia comparativa e análise de expressão gênica no desenvolvimento do sistema visual em Apis mellifera nós procuramos elucidar questões sobre plasticidade do desenvolvimento subjacente a fortes diferenças sexo- e casta-específico no olho assim como contribuir com aspectos evo-devo. O desenvolvimento dos lóbulos ópticos ocorre por dobramento neuroepitelial a partir de um centro de diferenciação no cérebro larval. Deste centro, a medula, lamina e lóbula surgem ao mesmo tempo em operárias e zangões. Dois passos marcam a diferenciação da lâmina (i) sua origem a partir da diferenciação de neuroblastos da camada mais externa da medula, isso coincidindo com o primeiro pico de expressão de roughest, e (ii) 24 horas mais tarde o aparecimento dos omatideos hexagonais coincidindo com o segundo pico de expressão de roughest. Com a inclusão de genes candidatos relacionados com o desenvolvimento do olho e lóbulos ópticos em insetos [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) e epidermal growth factor receptor (egfr)] nós encontramos distintos picos de expressão para sol, eya, mnb e so em níveis de transcritos e tempo de aparição do pico diferindo entre operárias e zangões. Enquanto estes quatro genes mostraram relativa sincronia durante o desenvolvimento em zangões, o mesmo não ocorreu em operárias. Além disso, em operárias sol é muito mais expresso na pré-pupa do que em zangões. Ambos os sexo mostraram padrões muito similares de expressão de elav, exceto por um atraso em zangões. Em contraste, a expressão de egfr ocorre antes em zangões. Durante a phase chave no desenvolvimento do sistema visual, uma análise global do transcriptoma, por meio de micro-arranjos mostrou vários genes relacionados com ciclo celular entre os diferencialmente expressos. Em conclusão, a relação entre tempo e eventos morfológicos com os padrões de expressão gênica revelou diferenças possivelmente relacionadas com mecanismos subjacentes ao desenvolvimento do sistema visual altamente dimorfico de Apis mellifera. / Developmental mechanisms governing compound eye development in insects have been broadly studied in Drosophila melanogaster, where the retina is formed from an imaginal disc attached to the larval brain. However little is known about eye development in other insects, most of which do not have such imaginal eye discs. Through a comparative histological and gene expression analysis of eye development in the honey bee, Apis mellifera, we intended to elucidate questions about developmental plasticity underlying the marked sex and castespecific differences in eye size, as well as to contribute to evo-devo aspects. Optic lobe development occurs by neuroepithelial folding initiating from a differentiation center in the larval brain. From this center, the medula, lamina and lobula arise at the same time in drones and workers. Two steps mark the differentiation of the lamina (i) its origin from neuroblasts differentiating in the outer layer of the medula, this coinciding with the first peak of roughest expression during the feeding stage of the fifth larval instar, and (ii) 24 hours later, the appearance of hexagonal ommatidia, coinciding with a second peak in roughest expression. Upon including further candidate genes related to insect eye development [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) and epidermal growth factor receptor (egfr)] we found distinct expression peaks for sol, eya, mnb and so, with timing and relative transcript levels differing between drones and workers. Whereas these four genes showed a relatively synchronous pattern of expression in drones in the fifth larval instar, this was not so in workers. Furthermore, in prepupae sol was higher expressed in workers than the other three genes, and also in comparison to drones. Both sexes showed a strikingly similar expression pattern for elav, except for some delay in drones. In contrast, egfr expression was found to occur earlier in drones. Through a global transcriptom analysis, done at a key step of larval development, several genes were reveled as diffetentially expressed, many of these regulating cell cycle steps. In conclusion, the relationship in the timing of morphological events with gene expression patterns revealed differences possibly related to mechanisms underlying development of the highly dimorphic compound eye in the honey bee.

O comportamento higiênico (CH) é uma característica muito utilizada para seleção em programas de melhoramento genético de abelhas Apis mellifera , em especial para o controle de doenças sem a necessidade de tratamentos químicos. Entretanto, o controle de qualquer comportamento é extremamente difícil sem que se conheçam os mecanismos que os determinam e quais os fatores ambientais que os influenciam. Os objetivos deste trabalho se constituíram em verificar se as abelhas forrageiras podem realizar o comportamento higiênico durante a noite, período no qual existe pouca ou nenhuma coleta de recursos e verificar o efeito das variáveis climáticas: temperatura, umidade relativa e em especial das precipitações pluviométricas no comportamento higiênico das abelhas africanizadas. Os experimentos foram realizados no Apiário Experimental do Departamento de Genética da Faculdade de Medicina de Ribeirão PretoUSP. Foram utilizadas seis colônias de abelhas africanizadas escolhidas aleatoriamente, colméias de observação e o sistema de monitoramento de uma Câmara Climática dotada de sensores de temperatura, umidade e registradores automáticos de atividades de vôo dotados de foto-células (Apidômetros) instalados no alvado das colônias. Próximo ao laboratório foi montada uma Estação Climatológica Modelo Vantage Pro-2 acoplada ao computador (com recepção wireless) para registro dos dados climáticos. Para o processamento estatístico dos dados dos experimentos utilizamos os testes One Way Repeated Measures (RM) ANOVA, RM ANOVA on Ranks, Paired t-test e o teste de Correlação de Spearman, levando-se em consideração a normalidade das amostras. Para avaliarmos a possível influência das abelhas forrageiras no CH realizamos três experimentos. No primeiro verificamos que as forrageiras realizam o CH na ausência de abelhas mais jovens. O segundo experimento foi realizado com quadros-testes de CH introduzidos nas colméias em horários distintos, sendo três repetições realizadas das 12h às 22h (6 horas durante o dia e 4 horas durante a noite) e das 24h às 10h (6 horas durante a noite e 4 horas durante o dia). As médias de células vazias foram respectivamente de 10,82% e 14,17%. Estes dados apresentaram diferença estatisticamente significante, sendo que o CH foi mais eficiente quando o quadro-teste permaneceu a maior parte do tempo (6 horas) na colméia durante a noite. As mesmas colônias foram utilizadas em mais três repetições realizadas das 18h às 4h (10 horas durante a noite) e das 6h às 16h (10 horas durante o dia). As médias de células vazias foram de 28,56% durante a noite e 23,90% durante o dia. Neste caso, embora não haja diferença estatística significante foi possível observar uma tendência do CH ser mais eficiente no período noturno. Contudo, como neste experimento não foi possível observar nenhuma abelha forrageira realizando o CH, um novo experimento foi realizado com uma colméia de observação para filmagens de abelhas de idade controlada e marcadas com etiquetas coloridas e numeradas. No entanto, nenhuma abelha observada forrageando anteriormente foi vista realizando o CH durante a noite. Constatamos que colônias constituídas por abelhas jovens apresentam melhor desempenho no CH do que colônias constituídas por abelhas de todas as idades. Quanto a influência das condições climáticas, realizamos testes de CH dois dias antes da chuva, durante a chuva e dois dias depois da chuva. Os testes de CH foram estatisticamente mais eficientes em dias chuvosos do que antes e depois da chuva quando realizados na primavera e no verão. Porém, durante o outono e o inverno os testes de CH não apresentaram nenhuma diferença estatísticamente significante. Mesmo não tendo sido observadas abelhas forrageiras realizando o CH não podemos descartar a possibilidade destas abelhas auxiliarem no CH em dias chuvosos e durante a noite quando a maior parte das campeiras estão no interior da colméia. Também podemos atribuir os resultados obtidos ao possível desvio de função de outras abelhas responsáveis pela recepção, evaporação e armazenamento de néctar e empacotamento de pólen, já que durante a noite e a chuva a coleta de recursos é extremamente reduzida ou não existe. A variável climática umidade relativa do ambiente comportou-se como um fator inversamente proporcional em relação ao CH, enquanto que a temperatura não apresentou nenhuma diferença estatísticamente significante em nenhum dos tratamentos. No entanto, como não foi possível obter dados de temperaturas mais extremas durante o período dos experimentos esta variável deve ser melhor pesquisada para se verificar o efeito dela no CH das abelhas africanizadas. / Hygienic behavior (HB) of honey bees (Apis mellifera ) is a useful and selectable characteristic for resistance to diseases. However, in order to efficiently evaluate and select for this behavior we need to understand the mechanisms involved and how environmental factors influence HB. We examined how time of the day, bee age and behavioral ontogeny, and climatic variables, including temperature, relative humidity and rainfall affect the HB of Africanized bees. We used six colonies of Africanized bees, observation hives and a hive temperature control chamber (colonies had free access to the outside), with temperature and relative humidity sensors and automatic flight activity recorders at the hive entrances. A climatic station placed near the hives was used to record the weather data. The data was analyzed with one way repeated measures ANOVA, ANOVA on ranks, paired t-tests and Spearman\'s correlation tests. We found that foraging bees can perform HB when the younger bees are removed from the colonies. When the HB tests were run from 12h to 22h (six hours during the day and four hours during the night), 10.8% of the brood was removed; when it was run from 24h to 10h (six hours during the night and four hours during the day, 14.2% of the brood was removed. These percentages were significantly different (three repetitions). The same tests were run from 18h to 4h (10 hours during the night; 28.6% removal) and 6h to 16h (10 hours during the day; 23.9% removal). In this case, there was no significant difference, though there appeared to be a tendency towards greater efficiency at night, similar to what was seen in the experiments with six versus four hours of night-time activity. We hypothesized that unoccupied forager bees may contribute at night; however, when we filmed the behavior of marked bees, those that were seen to make foraging trips did not perform HB at night. We also found that colonies formed only by young bees had more efficient HB than colonies formed by bees of all ages. To determine the influence of climatic conditions, we tested HB two days before rainy days, during rainy days and two days after rainy days; HB was significantly more efficient on rainy days than before and after during spring and summer (when most rain falls). However, during autumn and winter (normally dry seasons) there were no significant differences between days with and without rainfall. The variable relative humidity was inversely correlated with HB, while temperature was not significantly correlated with HB, though we did not test extreme temperatures.

Introdução: Hexamerinas são proteínas de estocagem sintetizadas pelo corpo gorduroso de larvas de insetos e secretadas na hemolinfa, onde se acumulam. A função canônica das hexamerinas consiste em servir de reserva de aminoácidos e energia para a reconstrução de tecidos e órgãos durante a metamorfose. Este trabalho teve como objetivo a busca por evidências de funções alternativas das hexamerinas durante o ciclo de vida de abelhas A. mellifera. Resultados: Os perfis temporais de expressão das quatro hexamerinas (HEX 70a, HEX 70b, HEX 70c e HEX 110), verificados por meio de SDS-PAGE e western blot, corroboram sua função canônica na metamorfose. Consistente com esta função, as quatro hexamerinas foram localizadas no citoplasma das células do corpo gorduroso utilizando-se anticorpos específicos e microscopia confocal. No entanto, funções adicionais puderam ser inferidas com base nos seguintes resultados: (1) Foci das quatro hexamerinas foram localizados nos núcleos de algumas células do corpo gorduroso em metamorfose, levando à hipótese de que têm função anti-apoptótica durante este período crítico do desenvolvimento; (2) Além disso, HEX 70a e HEX 110 foram localizadas no citoplasma e núcleo de células ovarianas e testiculares, indicando função no desenvolvimento e maturação das gônadas; (3) A co-localização de um análogo de timidina (EdU) e HEX 70a nos núcleos das células dos ovaríolos, sugeriu fortemente uma função na proliferação celular. O knockdown de HEX 70a in vivo por meio de injeção de anticorpo específico prejudicou o crescimento dos ovaríolos de rainhas, reforçando a hipótese de função na proliferação celular, (4) interferiu na esclerotização da cutícula de operárias, indicando função na formação do exoesqueleto e (5) provocou a antecipação da ecdise adulta, provavelmente em resposta à ausência (ou diminuição) dos aminoácidos derivados das hexamerinas. Foram investigados também aspectos da regulação dos genes de hexamerinas. A manipulação experimental da dieta alimentar e dos títulos do hormônio juvenil (HJ) interferiram claramente na expressão dos genes de hexamerinas. A potencial ação reguladora do HJ foi reforçada pelos resultados de análises por bioinformática da região 5 UTR de cada gene de hexamerina (Martins et al., 2010) que revelaram potencial motivo de ligação à proteína Ultraspiracle (Usp), um membro do complexo receptor do HJ no DNA. Procedimentos para expressar as hexamerinas in vitro em sistema de bactérias e purificá-las estão em progresso visando a caracterização da estrutura e de interações entre as subunidades. Conclusão: Estes resultados ressaltam que as hexamerinas têm outras funções no ciclo de vida de A. mellifera, além da função já bem estabelecida de reserva de aminoácidos para a metamorfose. / Background: Insect hexamerins are storage proteins synthesized by the larval fat body and secreted into the hemolymph, where they accumulate. The canonical function of hexamerins is to provide amino acids and energy for the reconstruction of tissues and organs during pupal-to-adult development. The aim of the current study was to search for evidence of alternative roles for the hexamerins in the life cycle of the honey bee, A. mellifera. Results: The canonical role of insect hexamerins received support from our data on the temporal expression profiles of the four honey bee hexamerin subunits (HEX 70a, HEX 70b, HEX 70c and HEX 110), as verified by SDS-PAGE and western blot using hemolymph and fat body samples. Consistent with the canonical function, the four hexamerins were localized in the cytoplasm of fat body cells, during metamorphosis, by using specific antibodies and confocal laser-scanning microscopy. However, additional functions could be inferred by the following findings: (1) The four hexamerins were also localized in the nuclei of some fat body cells, thus tentatively suggesting an anti-apoptotic role during metamorphosis; (2) Furthermore, HEX 70a and HEX 110 were localized in the cytoplasm and nucleus of ovarian and testicular cells, pointing to a role in gonad development and maturation. Co-labeling of the thymidine analog EdU and HEX 70a in the ovariole cell nuclei, strongly suggested a role in cell proliferation; HEX 70a depletion via injection of the specific antibody in queen pupae impaired ovariole growth, thus strengthening our hypothesis on a role in cell proliferation, (3) HEX 70a depletion also impaired cuticle sclerotization, indicating a function in exoskeleton formation, and (4) led to a precocious adult ecdysis, perhaps in response to the lack (or decrease) in hexamerin-derived amino acids. We also investigated aspects of the regulation of hexamerin genes. The experimental manipulation of diet consumption and juvenile hormone (JH) titer clearly interfered in the expression of hexamerin genes. Regulation by JH was also supported by a previous bioinformatics analysis of the 5 UTR region of each hexamerin gene (Martins et al., 2010), which revealed a potential binding site for Ultraspiracle (Usp), a member of the JH receptor complex in the DNA. Experiments are in progress for in vitro expression and purification of the four hexamerins aiming to further characterize their structures and interactions. Conclusion: Taken together, these results imply in novel roles for hexamerins in the life cycle of A. mellifera in addition to their well-established role as amino acids sources for metamorphosis.

O ciclo circadiano é um sistema adaptativo e vantajoso que permite a antecipação dos organismos e sincronização de suas atividades fisiológicas frente às variações ambientais que ocorrem ao longo do dia. Seu funcionamento molecular acontece pela geração dos ritmos circadianos, os quais surgem a partir da expressão cíclica dos genes do relógio em um feedback autoregulatório. Em insetos, os ritmos circadianos apresentam função importante em coordenar o timing do desenvolvimento e o comportamento. Nos últimos anos, estudos desvendaram que o relógio molecular de insetos sociais apresenta um funcionamento mais similar ao relógio de mamíferos do que com outros insetos como Drosophila melanogaster. Em especial, abelhas sociais têm sido ótimos modelos para investigar como os ritmos circadianos são modulados de acordo com as interações sociais entre os indivíduos, a plasticidade comportamental e a divisão social do trabalho entre as operárias. Operárias jovens (nutrizes) cuidam da cria no interior da colônia e geralmente apresentam uma atividade arrítimica ao longo de 24 horas, enquanto as abelhas mais velhas (forrageiras) são rítmicas e desenvolvem atividades complexas no ambiente externo. Nesse trabalho, nós caracterizamos os perfis de expressão dos genes do relógio period (per), cryptochrome mammalian-like (cry-m), clock (clk), cycle (cyc), timeout 2 (tim2), par domain protein 1 (pdp1), vrille (vri) e clockwork orange (cwo) durante todo o desenvolvimento de abelhas operárias de Apis mellifera. Verificamos que os genes do relógio são expressos antes mesmo da formação do sistema nervoso central no embrião e que seus transcritos podem ser herdados maternalmente. No desenvolvimento de larvas e pupas, revelamos que estes genes são diferencialmente expressos entre as fases investigadas e, com exceção dos genes cwo e tim2, todos respondem ao tratamento com o Hormônio Juvenil (HJ) na fase de pupa de olho branco. A resposta positiva dos genes clk, cyc e pdp1 frente ao tratamento hormonal pode estar relacionada com o envolvimento destes nas vias que respondem à sinalização do HJ, interagindo com os genes Kruppel (Kr-h1) e Methoprene-tolerant (MET). No desenvolvimento adulto, vimos que os genes per e cry-m são potenciais marcadores da plasticidade comportamental e divisão social do trabalho. Em um experimento usando single-cohort colony, estes genes apresentam níveis transcricionais que não oscilam em cabeças de operárias jovens (3 e 7 dias) ao longo de 24 horas, comparado aos níveis de expressão que oscilam de forma robusta em abelhas mais velhas (15 e 25 dias). Ainda, reconstruímos redes de interação proteína-proteína e miRNA-mRNA onde foram identificadas potenciais moléculas que atuam modulando os genes do relógio em nível póstranscricional e traducional. Dentre elas, validamos as interações entre os miRNA-34 e seus sítios de ligação que estão presentes nas 3`UTRS dos genes cyc e cwo, através do ensaio por luciferase, revelando que este miRNA é um regulador negativo da expressão desses genes. Pela primeira vez, realizamos uma análise ampla dos genes do relógio em um inseto social, além de identificar novas moléculas que podem atuar modulando os ritmos circadianos. Nosso trabalho demonstra a importâcia das abelhas sociais como modelos ideais para desvendar os mecanismos moleculares que regem os ritmos circadianos não só em abelhas, como também em outros organismos, inclusive mamíferos. / The circadian clock is an advantageous adaptive system that enables organisms to anticipate and syncronize their biological activities during the daily environmental changes. The circadian clock acts through the ontogeny of circadian rhythms, which are generated by the cyclic expression of the clock genes in an autregulatory feedback loop. In insects, the circadian rhythms have important roles in the coordination of the developmental timing and behavior, interacting with the endocrine system. In the last years, researchers revealed that the molecular clock of social insects is more similar to mammals than to insects. In particular, the social honeybee is an excellent model to investigate how the circadian rhythms are modulated accordingly to the social context, behavioral plasticity, and taskrelated activities. While young bees (nurses) work arrhythmically around the clock inside the colony in brood-care activities, old bees (foragers) need to be strongly rhythmic to develop complex tasks. In this work, we characterized the expression patterns of the clock genes period (per), cryptochrome mammalian-like (cry-m), clock (clk), cycle (cyc), timeout 2 (tim2), par domain protein 1 (pdp1), vrille (vri) e clockwork orange (cwo) in the entire development of Apis mellifera. Our results revealed that the clock genes are expressed before the formation of the central nervous system in embryos and that their transcripts might be inherited maternally. The clock genes are diferentially modulated during the larval and pupal development and, except for tim2 and cwo, all of them respond to the treatment with Juvenile Hormone (JH) in white-eyed pupae. The positive response to JH by clk, cyc and pdp1 might be related to the involvement of these genes on the pathways of the JH signaling, interacting with Kruppel (Kr-h1) and Methoprene-tolerant (MET) genes. In the adult development, the clock genes per and cry-m are potential molecular markers of the behavioral plasticity and division of labor in a single-cohort colony, once they did not exhibit transcriptional oscillations in heads of young bees (3 and 7 days-old) during 24h, compared to the robust transcriptional oscillation in old bees (15 and 25 days-old). Additionally, we reconstructed protein-protein and miRNA-mRNA interaction networks and identified putative molecules involved in the post-transcriptional and translational regulation of the clock genes. Among those molecules, we validated interactions between the miR-34 and its binding sites in the 3`UTR of cyc and cwo by luciferase assay, showing that this miRNA is a negative regulator of both clock genes. We showed for the first time a broad analysis of the circadian clock elements in a social insect, and also identified news molecules with potential to act as modulators of the circadian rhythms. This work expands the knowledge about the biological roles of the circadian clock in honeybees. Our work also contributes to highlight the importance of honeybees as an ideal model to uncover the molecular mechanisms that govern the circadian rhythms, not only in bees, but in other organisms, including mammals.

A relação entre nutrição e fenótipo é uma questão especialmente desafiadora em casos de polifenismo facultativo, como no caso das castas de insetos sociais, por exemplo, a abelha melífera Apis mellifera. Após estudos de vias de sensoriamento de nutrientes, modificações inesperadas nestas vias de sinalização revelaram a resposta à hipóxia como um possível mecanismo subjacente à regulação do tamanho corporal e crescimento de órgãos. Uma vez que essa resposta está intimamente ligada a condições metabólicas das células, o presente estudo foi concebido para investigar possíveis alterações no metabolismo mitocondrial e oxidativo no processo de diferenciação de castas em A. melífera na fase larval, com foco no corpo gorduroso, que é o centro metabólico dos insetos. Partindo da hipótese de que rainhas e operárias são criadas em células de cria abertas sob condições iguais de disponibilidade de oxigênio, nós investigamos o número e a distribuição mitocondrial, bem como as taxas de consumo de oxigênio em mitocondrias de células de corpo gorduroso durante estágios larvais críticos. Por meio de análises de imunofluorescência e microscopia eletrônica encontramos maior densidade de mitocôndrias no corpo gorduroso larval da rainha, dado corroborado pela quantificação de unidades funcionais mitocondriais por ensaio de citrato sintase. Medições de consumo de oxigênio por respirometria de alta resolução revelaram que as larvas de rainha têm capacidades máximas mais altas de produção de ATP, com menor demanda fisiológica, mas com mesma eficiência mitocondrial que operárias. A análise da expressão de fatores relacionados à mitogênese mostrou que os homólogos dos genes codificadores dos fatores de transcrição TFB1 e TFB2 e de um regulador nutricional, ERR, estão mais expressos em larvas de rainha. Apesar das diferenças encontradas na respiração mitocondrial entre as duas castas, as mesmas apresentaram níveis similares de produção de lactato e peróxido de hidrogênio, sem grandes alterações referentes à condições de estresse oxidativo e ao estado redox das célula. Encontramos altos níveis de expressão de genes codificadores das enzimas do sistema antioxidante MnSOD e catalase em células do corpo gorduroso larval de rainha, que garantem a redução de eventuais danos oxidativos. Estes resultados são fortes evidências de que a nutrição diferencial das larvas pelas operárias adultas, como estímulo externo da indução do desenvolvimento das castas, diferencialmente afeta a dinâmica e funcionalidade mitocondrial como elemento intrínseca da plasticidade fenotípica neste inseto social. / The connection between nutrition and phenotype is a particularly challenging issue in cases of facultative polyphenism, as for instance in the honeybee Apis mellifera. After studies on nutrient sensing pathways found unexpected modifications in these signaling pathways, a response to hypoxia was revealed as a possible mechanism underlying regulation of body size and organ growth. Since this response is closely linked to the metabolic conditions of the cells, the present study was designed to investigate the role of the mitochondrial and oxidative metabolism in the fat body of honeybee larvae, which is the metabolic center in insects, in the context of the caste differentiation process in A. mellifera. Based on the fact that honey bee larvae are reared in open brood cells, the queen and worker larvae should be exposed to equal oxygen diffusion conditions, and hence we investigated mitochondrial number and intracellular distribution, as well as rates of mitochondrial oxygen consumption in fat body cells during the larval stages critical for caste differentiation. By means of immunofluorescence and electron microscopy we found a higher density of mitochondria in the fat body of queen larvae. This result was corroborated by the quantification of mitochondrial functional units using a citrate synthase assay. Measurements of oxygen consumption obtained by high resolution respirometry revealed that queen larvae have higher maximum capacities of ATP production, with less physiological demand and higher mitochondrial efficiency than workers. Analysis of the expression of genes related to mitogenesis showed that Apis homologs of the transcription factors TFB1 and TFB2 and of the nutritional regulator, ERR are higher expressed in queen larvae. Despite differences in mitochondrial respiration, the two castes presented similar levels of lactate and hydrogen peroxide production, and without major chang in oxidative stress and cellular redox status. The high transcriptional levels of genes encoding enzymes of the antioxidant system, MnSOD and catalase observed in fat body cells of queen larvae guarantee that oxidative damage is reduced during larval development. These results are strong evidence that the differential nutrition of honey bee larvae by the adult worker, as the external stimulus for caste induction, differentially affects mitochondrial dynamics and functionality as an intrinsic element of phenotypic plasticity in this social insect.