Orientador: Osmar Malaspina / Banca: Ademilson Espencer Egea Soares / Banca: Roberta Cornelio Ferreira Nocelli / Resumo: Muitos inseticidas atuam inibindo a ação de neurotransmissores específicos, causando a super atividade dos neurônios, como por exemplo, aqueles ligados ao aprendizado e a memória e, como conseqüência, podem alterar estruturas cerebrais. Atualmente, o inseticida fipronil é muito utilizado como defensivo agrícola, principalmente em culturas como cana-de-açúcar, soja e citrus, sendo que sua pulverização, que visa o controle de insetos-praga, vem causando sérios danos aos insetos não-alvo, como os polinizadores. Embora alguns estudos tenham sido realizados para os efeitos das doses subletais de inseticidas em abelhas A. mellifera, não existe nenhum trabalho documentado sobre esses efeitos para as abelhas africanizadas, híbrido predominante no país. Assim, esse projeto teve como objetivo estabelecer as doses subletais para o inseticida fipronil, administrado oralmente e por contato; os efeitos no comportamento de forrageamento das abelhas; e as possíveis alterações na morfologia do cérebro das abelhas (A. mellifera). Para isso, previamente foi calculado a DL50 oral e por contato, para que posteriormente estabelecesse os valores das doses subletais. Para as abelhas tratadas foram avaliados teste da atividade locomotora durante o forrageamento e de sensibilidade ao alimento, através da metodologia de Resposta e Extensão da Probóscide (REP), que é uma reprodução no que ocorre na interação abelha-planta. Por fim, foram realizados cortes histológicos no cérebro das abelhas tratadas a fim de detectar as possíveis alterações morfológicas destas estruturas e utilizar a técnica de imunohistoquímica para marcar e mapear as regiões cerebrais de atuação do inseticida. Os resultados mostraram-se promissores para as avaliações sobre o efeito de baixas doses do inseticida fipronil para as abelhas africanizadas, embora mais estudos deverão ser realizados e os testes melhores adaptados. / Abstract: Many pesticides act as an inhibitor of the action of specific neurotransmissors, causing a super activity of neurons, for example, those linked with learning and memory and, as consequence, they can alter cerebral structures. Nowadays, the pesticide fipronil is largely used as an agricultural defense, manly in sugar cane, soy and citrus cultures. Its use that targets the control of plague insects has been causing serious effects on other insects, such as pollinators. Although some studies have been done on the effects of sublethal doses of pesticides on bees Apis mellifera, there are no documented work on those effects on Africanized bees, a hybrid very abundant in this country. Therefore, this project has the objective of establishing the sublethal doses of the pesticide fipronil, administrated orally and by contact; the effects of the foraging behavior of work bees; possible alterations on the brain morphology of Africanized bees (A. mellifera). For that, the oral and by contact DL50 were calculated previously, so that the values of sublethal doses could be established later. For bees treated with sublethal doses of fipronil, tests of dislocation activity during foraging and sensitivity to food were evaluated through the PER methodology, which is a reproduction of what happens in the interaction bee/plant. At last, histological cuts were made in the brains of treated bees to detect possible morphological alterations on these structures and to use the imunehistochemistry technique to mark and map the cerebral regions affected by the pesticide. The results showed themselves very promising for the evaluations of small doses of fipronil pesticide on Africanized bees, although further studies should be made and tests more adapted. / Mestre

A diapausa é um fenômeno amplamente presente nos artrópodes e é considerada como primordial para o sucesso evolutivo da Classe Insecta, pois possibilita a sobrevivência em condições adversas, como estações frias e secas. Sabe-se que durante a diapausa ocorre o silenciamento de muitos genes e que outros são unicamente expressos nesta fase. Embora existam evidências de que o processo da diapausa tenha se mantido conservado durante a evolução das espécies, ainda há lacunas no conhecimento sobre o nível de conservação dos padrões metabólicos. Um bom modelo para se estudar a diapausa é Tetrapedia diversipes, uma espécie bivoltina de abelha solitária. Os indivíduos que nascem na primeira geração seguem o desenvolvimento desde ovo até adulto em tempo bem menor do que aqueles que nascem na segunda geração; estes retardam o desenvolvimento na fase larval. Além disso, essa espécie é de fácil obtenção no seu ambiente natural, pois apresenta alta taxa de nidificação em ninhos-armadilha. O objetivo deste trabalho foi comparar o perfil de expressão de genes entre as larvas da 1ª geração (que não entram em diapausa), larvas da 2ª geração (que entrariam em diapausa) e das larvas em diapausa. Foram identificados 196 genes diferencialmente expressos, destes 87 foram anotados. Muitos destes genes já foram descritos na literatura como relacionados à diapausa em outras espécies, no entanto, o padrão de expressão não é conservado. Os genes aqui identificados foram divididos em cinco grupos: relacionados à desintoxicação celular, cutícula e citoesqueleto, metabolismo de lipídeos e esteróis, ciclo celular e outros genes relacionados à diapausa / The diapause is broadly distributed among the arthropods and has had an important role for the evolutionary success of the Class Insecta, mainly because this process permits insects to explore adverse conditions, such as cold and dry seasons. It is known that there are many genes being silenced and others being uniquely expressed during diapause. And although there are evidences that the diapause process has remained conserved during the evolution of species, it is still not clear how conserved are the metabolic patterns involved in this behavior. Tetrapedia diversipes is a solitary bee and a good model to study diapause. Individuals from the first generation do not enter in diapause and develop faster than individuals from the second generation, which enter in diapause during the winter. Moreover, this species is easy to capture in natural conditions due to the high rate of nesting in trap nests. The aim of this work was to compare the gene expression profile among non-diapause larvae from first and second generation (about to enter diapause) and larvae already in diapause, trough transcriptome data. One hundred ninety-four genes were identified as differentially expressed and 87 of them were annotated. Many of these genes have already been described as related to diapause in others species, but the expression pattern was not conserved. These genes were divided in five groups: related to cellular detoxification, cuticle and cytoskeleton, lipids and steroids metabolism, cell cycle and other genes related to diapause

The European honey bee, Apis mellifera, is a primarily beneficial insect for mankind. It has been utilized by humans for thousands of years for the products and services it provides. Crop pollination and honey production are two of the most economically beneficial activities of the honey bee. Though they have been important for many centuries and immeasurable amounts of effort have been expended investigating the methods and means to harness their natural abilities, a far lesser amount of attention has been directed towards exploring their molecular makeup. These experiments involve identifying modification of gene transcription as a result of exposure to a pheromone or a parasite. This data will provide information on the general types of transcripts involved in the biochemical response of the honey bee to the two stimuli and will also provide specific candidates for further investigation of their potential role in downstream behavioral events.

The European honey bee, Apis mellifera, is a primarily beneficial insect for mankind. It has been utilized by humans for thousands of years for the products and services it provides. Crop pollination and honey production are two of the most economically beneficial activities of the honey bee. Though they have been important for many centuries and immeasurable amounts of effort have been expended investigating the methods and means to harness their natural abilities, a far lesser amount of attention has been directed towards exploring their molecular makeup. These experiments involve identifying modification of gene transcription as a result of exposure to a pheromone or a parasite. This data will provide information on the general types of transcripts involved in the biochemical response of the honey bee to the two stimuli and will also provide specific candidates for further investigation of their potential role in downstream behavioral events.

No description available.

The foraging behavior of native solitary and primitively social bees was analyzed by identifying scopal pollen loads. In all species individual bees specialized on one pollen type during single foraging bouts. Generalized foraging behavior at the species level may result from switching pollens on sequential foraging bouts in individuals or from the individuals of a colony simultaneously gathering different pollens. Foraging behavior at the species level had a bimodal distribution, indicating a functional division between specialists and generalists. Though approximately 40% of the generalist species switched pollen preferences between years, no specialist species switched preferences between years. Generalist species have longer seasonal activity periods than specialists. All specialists were found in families (Andrenidae, Colletidae, Megachilidae) or subfamilies (Dufoureinae) in which most species are known to be strictly solitary. Only generalists were found in the subfamily Halictinae which has both social and solitary species. Seasonal variability in flower abundance and phenology was related to foraging preferences of bees. Solitary and primitively social bees, that are univoltine and cannot easily track between-year variation in resources, preferred species with simple flowers and low variability in flower abundance. Bumblebees, with greater behavioral flexibility than solitary bees, used the more abundant and variable flowers when they are available. Foraging behaviors observed in solitary and primitively social bees may result from selection to minimize uncertainty where floral resources are variable and unpredictable between years. The persistence of different foraging behaviors and social behaviors in a bee community may be maintained by the complementary costs and benefits of each behavior. Generalists have greater flexibility in responding to temporal variation, but this flexibility is obtained at the expense of less efficient use of individual floral resources. Specialists do not switch resources and may therefore have greater foraging efficiency, but they will be at a disadvantage when there is high year-to-year variability. Social species can retain both flexibility and efficiency if individual colony members specialize on different resources. However, social bees require a longer period to produce reproductives than do solitary bees, and may have lowered fecundity if the blooming season is unusually short.

The honey bee is one of the most familiar insects in the world, and plays an important role in the global economy providing essential pollination services to crops, fruit trees and vegetables. However, honey bee health is severely threatened by the ectoparasitic mite Varroa destructor, which feeds on the hemolymph of pupal and adult bees, resulting in loss of nutrients and circulatory fluids, decreased overall body weight and eventually the death of the bees. To investigate the molecular defense mechanisms of the honey bee against varroa mite infestation, we employed DNA microarray analysis to compare gene expression of two contrasting honey bee colony phenotypes selected from the Saskatraz breeding program. One designated as G4 is susceptible to the varroa mite, while the other designated as S88 is highly tolerant to the varroa. Total RNAs were isolated from bees at two different stages, dark-eyed pupa and adult worker, infected or non-infected with varroa mites, and used for DNA microarray analysis. The results showed that distinct sets of genes were differentially regulated in the varroa-tolerant and varroa-susceptible honey bee phenotypes, with and without varroa infestation. In both phenotypes, there were more differentially-expressed genes identified at the pupal stage than at the adult stage, indicating that at the pupal stage honey bees are more responsive to the varroa infestation than adult bees. In the phenotype comparisons, substantially more differentially-expressed genes were found in the tolerant than susceptible line, indicating that the tolerant phenotype has an increased capacity to mobilize the expression of the genes in response to varroa mite infestation. Based on function, the differentially-expressed genes could be classified into groups that are involved in olfactory signal transduction, detoxification, metabolism and exoskeleton formation, implying several possible mechanisms for the host-parasite interaction and resistance. Quantitative RT-PCR was used to confirm the data obtained from the DNA microarray hybridization. Eleven out of twelve genes selected based on the microarray data showed consistent expression patterns measured by both methods. Overall, comprehensive evaluation of the gene expression of honey bees in response to the mite infestation by DNA microarray has revealed several possible molecular mechanisms for the host defense against the pest. Identification of highly differentially expressed genes between the two phenotypes provides potential biomarkers that can be used for breeding honey bees resistant to the varroa mite.

The goal of this study was to examine the impact of Africanization on the genetic structure of the Welder Wildlife Refuge feral honey bee population by scoring mtDNA and microsatellite polymorphisms. Adult honey bee workers, collected between 1991 and 2001, were screened for mtDNA using the cytochrome b/BglII, ls rRNA/EcoRI, and COI/HinfI PCR-based assays. The procedure allowed identification of four mitotypes: eastern European, western European, A. m. lamarckii, and A. m. scutellata. The relative frequencies of the four mitotypes changed radically during the 11-year period. Prior to immigration of Africanized honey bees, the resident population was essentially of eastern European maternal ancestry. The first colony of A. m. scutellata mitotype was detected in 1993. Between 1995 and 1996 there was a mitotype turnover in the population from predominantly eastern European to predominantly A. m. scutellata. From 1997 onward, most colonies (69 %) were of A. m. scutellata mitotype. The temporal change in mtDNA was paralleled by nuclear DNA. The 12 microsatellite loci analyzed indicated (1) the mechanism of Africanization of the Welder population involved both maternal and paternal bi-directional gene flow (hybridization) between European and Africanized honey bees; and (2) the resident panmitic European population was replaced by panmitic asymmetrical admixtures of A. m. scutellata and European genes. The steepest increase in the proportion of introgressed A. m. scutellata nuclear alleles occurred between 1994 and 1997. The post-Africanization gene pool was composed of a diverse array of recombinant classes with a substantial European genetic contribution (mean proportion of European-derived alleles was 37 % as given by mR estimator or 25 % as given by mY estimator, for 1998-2001). If European genes continue to be retained at moderate frequencies, then the Africanized population is best viewed as a "hybrid swarm" instead of "pure African". The most radical change in the genetic structure of the Welder Wildlife Refuge feral honey bee population (observed between 1995 and 1997) coincided with arrival of the parasitic Varroa mite. We suggest that Varroa likely hastened the demise of European honey bees and had a major role in restructuring the Welder Wildlife Refuge feral honey bee population.

Biological and cultural control tactics are available for many agricultural pests but insecticides still play an important role in the rapid reduction of pest incidence when damage reaches economic levels. Laboratory and field toxicities of the reduced-risk products spinetoram and flubendiamide to Itame argillacearia (blueberry spanworm) was compared to deltamethrin, a conventional synthetic pyrethroid insecticide. In laboratory experiments, I. argillacearia larvae were highly susceptible to spinetoram and flubendiamide, and efficacy in the field was comparable to that of deltamethrin. Lethal and sublethal effects of the biopesticide formulations of Beauveria bassiana and Bacillus subtilis, and spirotetramat, a new tetramic acid insecticide, to bumble bees, Bombus impatiens, were also assessed. When ingested, field rates of spirotetramat caused high mortality after a week, and B. subtilis significantly reduced drone production. Field rates of spirotetramat, when applied topically, reduced drone production, but drone production varied following topical treatments of either biopesticide.

Red sorrel is a perennial weed in wild blueberry fields that decreases yield. Multiple experiments were conducted to evaluate its impact on blueberry pollination, Botrytis blight incidence, and berry yield. Kerb applications did not significantly impact blueberry stem or floral bud formation. Removal of red sorrel with Kerb increased blueberry yield at both sites. However a double application had no difference than one application. Blueberry and red sorrel flowering overlapped and red sorrel pollen grains were found on blueberry flowers in both years at all sites. Red sorrel pollen grains increased the incidence of germinating spores in Petri dishes and this relationship was adequately modeled with a three parameter, exponential rise to a maximum. Red sorrel pollen significantly increased disease incidence on immature blueberry flowers. Honey bees foraged from blueberry and red sorrel flowers, but there was no evidence to suggest that they favored red sorrel flowers over blueberry flowers.