Desenvolvimento de um plataforma analítica para análise qualitativa e quantitativa de peptídeos de cérebros de abelhas Apis mellifera

As abelhas da espécie Apis mellifera são conhecidas por sua organização social, comportamento devotado e aparente complexidade comportamental, sendo também consideradas excelentes sistemas-modelo para estudos de comunicação, aprendizado e formação de memória. Uma vez que o comportamento social dos insetos é resultante de interações complexas em diferentes níveis de organização biológica, torna-se importante estudar os peptídeos envolvidos nesse processo, já que estes são indispensáveis em muitos processos fisiológicos, funcionando como neurotransmissores, hormônios, toxinas, antibióticos, defensinas, neuromoduladores e neurohormônios. Desta forma, o objetivo do presente trabalho foi padronizar uma metodologia analítica visando à determinação do perfil peptídico do cérebro de abelhas da espécie Apis mellifera utilizando como ferramentas a espectrometria de massas e a cromatografia líquida. Foram coletadas operárias de Apis mellifera com 20 dias de idade, cujos cérebros foram dissecados e devidamente processados visando a posterior separação e identificação dos peptídeos. Foram testadas duas estratégias distintas para a separação e identificação dos peptídeos, denominadas on-line e off-line. Ambas baseavam-se na utilização de um sistema de cromatografia líquida sob fase reversa e um espectrômetro de massas do tipo electrospray/Ion Trap-Time of Flight, mas na primeira essas ferramentas estavam acopladas, enquanto na segunda os dois processos foram realizados separadamente. Tais procedimentos permitiram identificar 19 peptídeos pertencentes às principais famílias de neuropeptídeos de cérebros de abelhas já descritos na literatura, entre elas as Alatostatina, Apidaecina, Corazonina e Taquicininas. Os resultados obtidos indicam que a estratégia off-line foi melhor em comparação à estratégia on-line, devido à possibilidade de se trabalhar com cada fração obtida separadamente para as...

Avaliação dos efeitos do Imidaclopride, sobre o sistema nervoso de Apis mellifera africanizada, através da expressão da proteína FOS

As abelhas Apis mellifera africanizadas são consideradas importantes agentes polinizadores que estão freqüentemente expostos à ação tóxica de inseticidas aplicados em cultivos. O imidaclopride é um inseticida sistêmico do grupo dos neonicotinóides e atua como agonista da acetilcolina nas sinapses do sistema nervoso central. Em abelhas foi verificado, através do método de resposta de extensão da probóscide (REP), que doses subletais de imidaclopride provocam deficiência no aprendizado olfatório e prejudicam a memória. A proteína Fos, expressa em neurônios, tem sua transcrição alterada por diversos estímulos como estresse, lesões, exposição a toxinas ou a predadores. Dessa forma, este trabalho teve por objetivo avaliar os efeitos neurotóxicos do inseticida imidaclopride, em operárias de Apis mellifera africanizadas, através da análise da expressão de Fos. Abelhas recém-emergidas e campeiras foram tratadas com 10, 20, 40 e 80 ng/abelha de imidaclopride e seus cérebros dissecados 15 minutos, 30 minutos, 1 hora e 4 horas após a ingestão do composto. Houve uma marcação positiva para Fos nos ocelos e na região dos olhos, principalmente na lâmina e na retina. Tal resultado era esperado, uma vez que o as abelhas foram expostas a luminosidade, durante a realização dos ensaios. Os lobos antenais são constituídos por prolongamentos de células sensoriais das antenas e pelos neurônios motores e os corpos pedunculados são tidos como os centros de processamento dos estímulos sensoriais recebidos pelos olhos e pelas antenas, assim, esperava-se que houvesse marcação positiva nestas regiões. Porém isto não foi observado no presente trabalho. Analisando-se os valores quantitativos da expressão da proteína Fos percebeu-se que não houve um padrão de ...(Resumo completo, clicar acesso eletrônico abaixo)

Ovary development in honeybee (Apis Mellifera L.) workers under co2 narcosis, caged outside of the colony

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of the collection season on production, size, and chemical composition of bee pollen produced by apis mellifera l.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Energetic feedings influence beeswax production by Apis mellifera L. honeybees

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Utilização dos parâmetros físico-químicos e redes neurais artificiais na identificação dos méis de abelhas (Apis mellifera L.) produzidos no verão e inverno na microrregião de Campos de Jordão, São Paulo

The objective of this work was to typify, through physicochemical parameters, honey from Campos do Jordão’s microrregion, and verify how samples are grouped in accordance with the climatic production seasonality (summer and winter). It were assessed 30 samples of honey from beekeepers located in the cities of Monteiro Lobato, Campos do Jordão, Santo Antonio do Pinhal e São Bento do Sapucaí-SP, regarding both periods of honey production (November to February; July to September, during 2007 and 2008; n = 30). Samples were submitted to physicochemical analysis of total acidity, pH, humidity, water activity, density, aminoacids, ashes, color and electrical conductivity, identifying physicochemical standards of honey samples from both periods of production. Next, we carried out a cluster analysis of data using k-means algorithm, which grouped the samples into two classes (summer and winter). Thus, there was a supervised training of an Artificial Neural Network (ANN) using backpropagation algorithm. According to the analysis, the knowledge gained through the ANN classified the samples with 80% accuracy. It was observed that the ANNs have proved an effective tool to group samples of honey of the region of Campos do Jordao according to their physicochemical characteristics, depending on the different production periods.

Study of visceral antinociceptive potential of bee Apis mellifera venom

Pain is one of the most common reasons for patients to seek medical care. Bee Apis mellifera venom (AMV) has traditionally been used to treat inflammatory diseases and the alleviation of pain. Herein, we aimed to investigate the visceral antinociceptive potential of A. mellifera bee venom and its possible mechanism of action. Acetic acid-induced writhing assay was used in mice to determine the degree of visceral antinociception. Visceral antinociceptive activity was expressed as the reduction in the number of abdominal constrictions. Mice received an intraperitoneal injection of acetic acid after administration of AMV (0.08 or 0.8 mg/kg; intraperitoneally (i.p.)). In mechanistic studies, separate experiments were realized to examine the role of α2-receptors, nitric oxide, calcium channels, K+ATP channel activation, TRPV1 and opioid receptors on the visceral antinociceptive effect of AMV (0.8 mg/kg), using appropriate antagonists, yohimbine (2 mg/kg), L-NG-Nitroarginine methyl ester (L-NAME, 10 mg/kg), verapamil (5 mg/kg), glibenclamide (5 mg/kg), ruthenium red (3 mg/kg) or naloxone (2 mg/kg). AMV presented visceral antinociceptive activity in both doses tested (0.08 and 0.8 mg/Kg). Visceral antinociceptive effect of AMV was resistant to all the antagonists used. Mice showed no significant alterations in locomotion frequency, indicating that the observed antinociception is not a consequence of motor abnormality. Although AMV efficient diminished the acetic acid-evoked pain-related behavior, its mechanism is unclear from this study and future studies are needed to verify how the venom exerts its antinociceptive action.

Identificação de regiões polimórficas para alta produtividade de mel e sequênciamento genômico em abelhas Apis mellifera africanizadas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera

Abstract Background In honeybees, differential feeding of female larvae promotes the occurrence of two different phenotypes, a queen and a worker, from identical genotypes, through incremental alterations, which affect general growth, and character state alterations that result in the presence or absence of specific structures. Although previous studies revealed a link between incremental alterations and differential expression of physiometabolic genes, the molecular changes accompanying character state alterations remain unknown. Results By using cDNA microarray analyses of >6,000 Apis mellifera ESTs, we found 240 differentially expressed genes (DEGs) between developing queens and workers. Many genes recorded as up-regulated in prospective workers appear to be unique to A. mellifera, suggesting that the workers' developmental pathway involves the participation of novel genes. Workers up-regulate more developmental genes than queens, whereas queens up-regulate a greater proportion of physiometabolic genes, including genes coding for metabolic enzymes and genes whose products are known to regulate the rate of mass-transforming processes and the general growth of the organism (e.g., tor). Many DEGs are likely to be involved in processes favoring the development of caste-biased structures, like brain, legs and ovaries, as well as genes that code for cytoskeleton constituents. Treatment of developing worker larvae with juvenile hormone (JH) revealed 52 JH responsive genes, specifically during the critical period of caste development. Using Gibbs sampling and Expectation Maximization algorithms, we discovered eight overrepresented cis-elements from four gene groups. Graph theory and complex networks concepts were adopted to attain powerful graphical representations of the interrelation between cis-elements and genes and objectively quantify the degree of relationship between these entities. Conclusion We suggest that clusters of functionally related DEGs are co-regulated during caste development in honeybees. This network of interactions is activated by nutrition-driven stimuli in early larval stages. Our data are consistent with the hypothesis that JH is a key component of the developmental determination of queen-like characters. Finally, we propose a conceptual model of caste differentiation in A. mellifera based on gene-regulatory networks.

Genes involved in thoracic exoskeleton formation during the pupal-to-adult molt in a social insect model, Apis mellifera

Background: The insect exoskeleton provides shape, waterproofing, and locomotion via attached somatic muscles. The exoskeleton is renewed during molting, a process regulated by ecdysteroid hormones. The holometabolous pupa transforms into an adult during the imaginal molt, when the epidermis synthe3sizes the definitive exoskeleton that then differentiates progressively. An important issue in insect development concerns how the exoskeletal regions are constructed to provide their morphological, physiological and mechanical functions. We used whole-genome oligonucleotide microarrays to screen for genes involved in exoskeletal formation in the honeybee thoracic dorsum. Our analysis included three sampling times during the pupal-to-adult molt, i.e., before, during and after the ecdysteroid-induced apolysis that triggers synthesis of the adult exoskeleton. Results: Gene ontology annotation based on orthologous relationships with Drosophila melanogaster genes placed the honeybee differentially expressed genes (DEGs) into distinct categories of Biological Process and Molecular Function, depending on developmental time, revealing the functional elements required for adult exoskeleton formation. Of the 1,253 unique DEGs, 547 were upregulated in the thoracic dorsum after apolysis, suggesting induction by the ecdysteroid pulse. The upregulated gene set included 20 of the 47 cuticular protein (CP) genes that were previously identified in the honeybee genome, and three novel putative CP genes that do not belong to a known CP family. In situ hybridization showed that two of the novel genes were abundantly expressed in the epidermis during adult exoskeleton formation, strongly implicating them as genuine CP genes. Conserved sequence motifs identified the CP genes as members of the CPR, Tweedle, Apidermin, CPF, CPLCP1 and Analogous-to-Peritrophins families. Furthermore, 28 of the 36 muscle-related DEGs were upregulated during the de novo formation of striated fibers attached to the exoskeleton. A search for cis-regulatory motifs in the 5′-untranslated region of the DEGs revealed potential binding sites for known transcription factors. Construction of a regulatory network showed that various upregulated CP- and muscle-related genes (15 and 21 genes, respectively) share common elements, suggesting co-regulation during thoracic exoskeleton formation. Conclusions: These findings help reveal molecular aspects of rigid thoracic exoskeleton formation during the ecdysteroid-coordinated pupal-to-adult molt in the honeybee.

Genetic characterization of slow bee paralysis virus of the honeybee (Apis mellifera L.)

Complete genome sequences were determined for two distinct strains of slow bee paralysis virus (SBPV) of honeybees (Apis mellifera). The SBPV genome is approximately 9 5 kb long and contains a single ORF flanked by 5'- and 3'-UTRs and a naturally polyadenylated 3' tail, with a genome organization typical of members of the family Iflaviridae The two strains, labelled `Rothamsted' and 'Harpenden', are 83% identical at the nucleotide level (94% identical at the amino acid level), although this variation is distributed unevenly over the genome. The two strains were found to co-exist at different proportions in two independently propagated SBPV preparations The natural prevalence of SBPV for 847 colonies in 162 apiaries across five European countries was <2%, with positive samples found only in England and Switzerland, in colonies with variable degrees of Varroa infestation