Rearing Africanized honey bee (Apis mellifera L.) brood under laboratory conditions

We developed a method for rearing larvae of Africanized bees under laboratory conditions to determine the amount of diet needed during larval development to obtain a worker bee. We started with larvae 18-24 h old, which were transferred to polyethylene cell cups and fed for five days. We found that the amount of diet needed for successful larval development was: 4, 15, 25, 50, and 70 mu L during the first to fifth days, respectively. The survival rate to the adult stage was 88.6% when the larvae received the daily amount of diet divided into two feedings, and 80% when they received only one feeding per day. The adult weight obtained in the laboratory, when the larvae received the daily amount of diet in a single dose, did not differ from those that were developed under field conditions (our control). All adults that we obtained in laboratory appeared to be normal. This technique has the potential to facilitate studies on brood pathogens, resistance mechanisms to diseases and also might be useful to test the impacts of transgenic products on honey bee brood.

Palynological and physicochemical characterization of Apis mellifera L. bee pollen in the Southern region of Brazil

Bee pollen has been used for many years in both traditional medicine and supplementary nutrition, as well as in alternative diets, mainly due to its nutritional properties and health benefits. Bee pollen production is a recent activity in Brazil, having begun in the late 1980s. However, the country has the potential of being a large world producer of high quality pollen, particularly because of the great diversity of tropical flora and the resistance of the Brazilian Apis mellifera bee races. Thirty-six samples of bee pollen from the Southern region of Brazil were analyzed regarding pollen types and physicochemical and nutritional composition. Only one sample was considered monofloral, which was exclusively composed by pollen from the Asteraceae family). The State of Parana showed a greater variety of pollen types, 18 in total, representing 82% of the total number identified in this study. The bee pollen in the States of Rio Grande do Sul and Parana showed a higher number of samples with humidity content above the standard permitted by the Brazilian legislation, i.e. over 4%. The bee pollen was characterized by its high protein content with average values of 20.47%. The analysis regarding humidity, lipids and sugar showed no statistical differences among the samples (p<0.05). The pollen samples had a high concentration of reducible sugars (48%). The predominant minerals in the samples PR, SC and RS were phosphorus (7102.29, 6873.40, 6661.73 mg/kg of pollen), followed by potassium (5383.73, 4997.77, 4773.26 mg/kg of pollen), calcium (1179.05, 961.93, 848.36 mg/kg of pollen) and magnesium (818.02, 679.01, 725.89 mg/kg of pollen). Statistical analysis (Tukey test) demonstrated no significant difference between the contents of calcium, copper, iron, phosphorus and sodium in the pollen samples of the South of Brazil. However, the samples from the State of Parana contained the highest contents of potassium and differed statistically from the samples of the State of Rio Grande do Sul.

Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays

Tight control over circulating juvenile hormone (JH) levels is of prime importance in an insect`s life cycle. Consequently, enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs), play major roles during metamorphosis and reproduction. In the highly eusocial Hymenoptera, JH has been co-opted into additional functions, primarily in the development of the queen and worker castes and in age-related behavioral development of workers. Within a set of 21 carboxylesterases predicted in the honey bee genome we identified one gene (Amjhe-like) that contained the main functional motifs of insect JHEs. Its transcript levels during larval development showed a maximum at the switch from feeding to spinning behavior, coinciding with a JH titer minimum. In adult workers, the highest levels were observed in nurse bees, where a low JH titer is required to prevent the switch to foraging. Functional assays showed that Amjhe-like expression is induced by JH-III and suppressed by 20-hydroxyecdysone. RNAi-mediated silencing of Amjhe-like gene function resulted in a six-fold increase in the JH titer in adult worker bees. The temporal profile of Amjhe-like expression in larval and adult workers, the pattern of hormonal regulation and the knockdown phenotype are consistent with the function of this gene as an authentic JHE. (C) 2008 Elsevier Inc. All rights reserved.

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies. (C) 2011 Elsevier Ltd. All rights reserved.

Cytoplasmic and nuclear localization of cadherin in honey bee (Apis mellifera L.) gonads

Cadherins are crucial molecules mediating cell-cell interactions between somatic and germline cells in insect and mammalian male and female gonads. We analysed the presence and localization of cadherins in ovaries of honeybee queens and in testes of drones. Transcripts representing two classical cadherins, E-cadherin (shotgun) and N-cadherin, as well as three protocadherins (Starry night, Fat and Fat-like) were detected in gonads of both sexes. Pan-cadherin antibodies, which most probably detect a honeybee N-cadherin, were used in immunolocalization analyses. In the germarium of ovarioles, cadherin-IR (cadherin immunoreactivity) was evidenced as homogeneously distributed in the cytoplasm and as nuclear foci, in both germline and somatic cells. It was also detected in polyfusomes and ring canals. In testiolar tubules, cadherin-IR showed a cytoplasmic and nuclear distributon alike in ovaries. The unexpected nuclear localization and cytoplasmic distribution in ovaries and testes were corroborated by immunogold electron microscopy, which revealed cadherin aggregates associated with electron-dense nuclear structures. With respect to cadherin localization, the honeybee differs from Drosophila, the model for gametogenesis in insects, raising the question as to how differences among solitary and social species may be built into and generated from the general architecture of polytrophic meroistic ovaries. It also indicates the possibility of divergent roles for cadherin in the functional architecture of insect gonads, in general, especially in taxa with high reproductive output.

Differential expression of hypoxia pathway genes in honey bee (Apis mellifera L.) caste development

Diphenism in social bees is essentially contingent on nutrient-induced cellular and systemic physiological responses resulting in divergent gene expression patterns. Analyses of juvenile hormone (JH) titers and functional genomics assays of the insulin-insulin-like signaling (IIS) pathway and its associated branch, target-of-rapamycin (TOR), revealed systemic responses underlying honey bee (Apis mellifera) caste development. Nevertheless, little attention has been paid to cellular metabolic responses. Following up earlier investigations showing major caste differences in oxidative metabolism and mitochondrial physiology, we herein identified honey bee homologs of hypoxia signaling factors, HIF alpha/Sima, HIF beta/Tango and PHD/Fatiga and we investigated their transcript levels throughout critical stages of larval development. Amsima, Amtango and Amfatiga showed correlated transcriptional activity, with two peaks of occurring in both queens and workers, the first one shortly after the last larval molt and the second during the cocoon-spinning phase. Transcript levels for the three genes were consistently higher in workers. As there is no evidence for major microenvironmental differences in oxygen levels within the brood nest area, this appears to be an inherent caste character. Quantitative PCR analyses on worker brain, ovary, and leg imaginal discs showed that these tissues differ in transcript levels. Being a highly conserved pathway and linked to IIS/TOR, the hypoxia gene expression pattern seen in honey bee larvae denotes that the hypoxia pathway has undergone a transformation, at least during larval development, from a response to environmental oxygen concentrations to an endogenous regulatory factor in the diphenic development of honey bee larvae. (C) 2010 Elsevier Ltd. All rights reserved.

Mars is close to venus - Female reproductive proteins are expressed in the fat body and reproductive tract of honey bee (Apis mellifera L.) drones

Vitellogenin (Vg) and lipophorin (Lp) are lipoproteins which play important roles in female reproductive physiology of insects. Both are actively taken up by growing oocytes and especially Vg and its receptor are considered as female-specifically expressed. The finding that the fat body of in honey bee (Apis mellifera) drones synthesizes Vg and is present in hemolymph has long been viewed as a curiosity. The recent paradigm change concerning the role played by Vg in honey bee life history, especially social division of labor, has now led us to investigate whether a physiological constellation similar to that seen in female reproduction may also be represented in the male sex. By means of Western blot analysis we could show that both Vg and Lp are present in the reproductive tract of adult drones, including the accessory (mucus) glands, but apparently are not secreted. Furthermore, we analyzed the transcript levels of the genes encoding these proteins (vg and lp), as well as their putative receptors (Amvgr and Amlpr) in fat body and accessory glands. Whereas lp, vg and Amlpr transcript levels decreased with age in both tissues. Amvgr mRNA levels increased with age in fat body. To our knowledge this is the first report that vitellogenin and its receptor are co-expressed in the reproductive system of a male insect. We interpret these findings as a cross-sexual transfer of a social physiological trait, associated with the rewiring of the juvenile hormone/vitellogenin circuitry that occurred in the female sex of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.

Characterization of a chemsensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

Análise do pólen encontrado em amostras de mel de Apis mellifera L. (Hymenoptera, Apidae) em uma área de savana de Roraima, Brasil

Foram analisadas amostras de mel de um apiário localizado na Aldeia do Contão, Roraima, Brasil. As amostras foram obtidas das colheitas nos meses de outubro e dezembro de 1996 e janeiro, fevereiro e março de 1997. Foram identificados um total de 20 tipos polínicos distribuídos em 18 gêneros e 13 famílias. As famílias: Mimosaceae (4 espécies), Anacardiaceae (3 espécies), Sterculiaceae (2 espécies), Caesalpiniaceae (2 espécies) e Amaranthaceae (2 espécies) foram as mais representadas, as demais por uma única espécie. Os tipos polínicos mais frequentes foram: Mimosa polydactyla H.B.K (outubro e dezembro de 1996), Curatella americana L. (janeiro, fevereiro e março de 1997). Encontrou-se três correlações significativas entre as frequências dos tipos polínicos de: Curatella americana L. X Mimosa polydactyla H.B.K (r = -0,99), Curatella americana L. X Astronium sp (r = 0,95) e Mimosa polydactyla H.B.K e Astronium sp (r = -0,91)

Avaliação do período de florescimento das plantas apícolas no ano de 1960, através do polem contido nos méis e dos coletados pelas abelhas (Apis Mellifera L.)

Neste trabalho apresentamos os resultados da análise polínica de 11 amostras de mel e de polem coletados pelas abelhas Apis mellifera L., durante o período de março de 1960, no Apiário da Escola Superior de Agricultura "Luiz de Queiroz". Através dessa análise, pudemos comprovar e identificar como plantas nectaríferas e poliníferas as seguintes espécies: Eucalyptus sp., Dombeya sp., Agave sisalana, Vernonia sp., Montanoa bipinnatificada, Persea americana, Baccharis sp.. Citrus sp., e como plantas poliníferas as que seguem: Bidens piíosus, Cosmus sulphureus, Pirostegia venusta, Mel-linis minutiflora. Pudemos, assim, avaliar de modo indireto o período de florescimento das plantas apícolas nos arredores do apiário da Escola e comparar essa avaliação com as observações diretas de KERR & AMARAL (196°) sobre o período de florescimentos das plantas apícolas no planalto Paulista. Daí podermos afirmar ser a análise polínica dos méis e a análise dos polens coletados pelas abelhas um método prático de valor para estudos de fenologia das plantas apícolas. Poderemos assim aplicar a análise polínica em nossos futuros estudos sobre a fenologia das plantas apícolas em diferentes regiões do Estado de São Paulo.

Dois ácaros associados à abelha (Apis mellifera L.) no Perú

Larvas de Leptus sp. (Acari, Prostigmata, Erythraeidae) foram coletadas das membranas intersegmentares dos tergitos e esternitos abdominais de abelhas (Ápis mellifera L.) em Cerro de Pasco, Peru. Não foram relatados danos por este ectoparasito. Fêmeas de Blattisoeius dentriticus (Berlese, 1918) (Acari, Mesostigmata, Ascidae) foram coletadas de colméias de abelhas, junto com Tyrophagus putrescentiae (Schrank, 1781) (Acari, Astigmata, Acaridae), em Lima, Peru.

Taudinaiheuttajien, lääkkeiden ja vierasaineiden vaikutuksista mehiläisen (Apis mellifera L.) tartunnanvastaiseen puolustukseen

Summary: Immunotoxic and immunomodulatory aspects of pathogens, drugs and xenobiotics in protection of the honey bee (Apis mellifera L.) to infections

Flora de importância polinífera para Apis mellifera (L.) na região de Viçosa, MG

Procurou-se conhecer a flora de importância polinífera para Apis mellifera (L.) na região de Viçosa, MG, em período de entressafra de mel, entre agosto e dezembro de 2005. O experimento foi realizado em dois apiários distintos, cada um com cinco colmeias. As cargas retidas nos coletores de pólen instalados nas colmeias foram analisadas quanto à origem botânica. As plantas em floração no entorno dos apiários foram coletadas e identificadas. A maioria das plantas de importância polinífera para abelhas na região de Viçosa era nativa, localizada em jardins e com hábito arbóreo. Pela análise palinológica, verificou-se que espécies como Anadenanthera colubrina, Arecaceae sp., Baccharis dracunculifolia, B. melastomaefolia, Coffea spp., Emilia sagittata, Eugenia uniflora, Mikania cordifolia, M. hirsutissima, Myrcia fallax, Psidium guajava, Vernonia condensata, V. diffusa, V. lanuginosa e V. mariana são potenciais recursos poliníferos a serem utilizados no período de entressafra do mel. Os resultados indicaram a importância de plantas localizadas em áreas abertas para o forrageamento de pólen por A. mellifera e confirmaram o potencial polinífero da região estudada, durante o período de entressafra do mel.

Plantas visitadas por Apis mellifera L. no vale do rio Paraguaçu, Município de Castro Alves, Bahia

As espécies vegetais visitadas por Apis mellifera no município de Castro Alves-BA (12°45'S e 39°26'W), região do vale do rio Paraguaçu, foram identificadas entre janeiro de 1994 a fevereiro de 1995. A comunidade de plantas apícolas foi caracterizada mediante o emprego dos índices de freqüência, constância, abundância, diversidade, uniformidade e dominância, baseando-se no número de abelhas coletadas em cada espécie vegetal. Um total de 48 espécies e 28 famílias de plantas foram visitadas por A. mellifera durante o período estudado e as principais espécies da flora apícola na área estudada foram Cissus simsiana Roem. & Schult. (Vitaceae), Melochia tomentosa L. (Sterculiaceae) e Portulaca elatior Mart. (Portulacaceae).