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.

Isolation and characterization of microsatellite markers from the stingless bee Nannotrigona testaceicornis

Conservation of natural populations and handling of breeding programs would benefit from the availability of molecular markers. Stingless bees are one of the most important pollinators in several ecosystems. Thus, seventeen microsatellite markers were developed from an enriched genomic library of Nannotrigona testaceicornis. They were characterized using 50 samples. The expected and observed heterozygosities ranged from 0.59 to 0.89 and from 0.39 to 0.79, respectively. These markers will contribute to advance researches on the genetic conservation, characterization and preservation of the Brazilian native bees.

Farnesoic acid O-methyl transferase (FAMeT) isoforms: Conserved traits and gene expression patterns related to caste differentiation in the stingless bee, Melipona scutellaris

Farnesoic acid O-methyl transferase (FAMeT) is the enzyme that catalyzes the formation of methyl farnesoate (MF) from farnesoic acid (FA) in the biosynthetic pathway of juvenile hormone (JH). This work reports the cloning, sequencing, and expression of FAMeT gene from the stingless bee Melipona scutellaris (MsFAMeT). The MsFAMeT in silica analysis showed that greatest sequence similarity is found in Apis mellifera and other insects, while relatively less similarity is shown in crustaceans. Evidence of alternative splicing of a 27 nucleotide (nt) microexon explains the presence of the detected isoforms, 1 and 2. The expression analysis of the two isoforms showed a marked difference when castes were compared, suggesting that they could be involved differently in the JH metabolism in M. scutellaris, providing new insights for the comprehension of female plasticity.

Of mites and bees: A review of mite-bee associations in Australia and a revision of Raymentia Womersley (Acari : Mesostigmata : Laelapidae), with the description of two new species of mites from Lasioglossum (Parasphecodes) spp. (Hymenoptera : Halictidae)

Social bees have a diverse fauna of symbiotic mesostigmatic mites, including highly pathogenic parasites of the honeybee, but there are few reports of Mesostigmata phoretic on or inhabiting the nests of solitary or communal, ground-nesting bees. In south-eastern Australia, however, native bees in the family Halictidae carry what appears to be a substantial radiation of host-specific mesostigmatans in the family Laelapidae. Herein, we redescribe the obscure genus Raymentia , associated with Lasioglossum (Parasphecodes ) spp. bees (Halictidae) and describe two new species, R. eickwortiana from L. lacthium (Smith) and R. walkeriana from L. atronitens (Cockerell). The type species, R. anomala Womersley, is associated with L. altichum (Smith). In addition, we review the mites known to be associated with Australian bees, provide a key to differentiate them, and describe and illustrate acarinaria of the Halictinae. We also report on the first occurrences in Australia of the genera Trochometridium Cross (Heterostigmata: Trochometridiidae), from L. eremaean Walker (Halictidae), and Cheletophyes Oudemans (Prostigmata: Cheyletidae) from Xylocopa Latreille (Xylocopinae), and on the previously unknown association between a Neocypholaelaps Vitzthum (Mesostigmata: Ameroseiidae) and Lipotriches tomentifera (Friese) (Halictidae).

Airborne pollen of Brisbane, Australia: a five-year record, 1994-1999

The seasonal incidence of pollen in the atmosphere of Brisbane has been established from a near continuous. volumetric trapping program over the five-year period, July 1994-June 1999. Grass pollen accounts for 71.6% of the average annual pollen load with highest densities (up to 150 grains/m(3)) recorded in summer and autumn. Significant contributions were also made by taxa of the Cupressaceae (8.7%) and Urticaceae (1.8%) during spring and of the Pinaceae (4.5%) during winter. Pollen seasons of the Casuarinaceae (6.5%) and Myrtaceae (3.2%) are more extended, the former peaking in late winter and the latter in late spring. The onset and duration of the Poaceae and Urticaceae seasons varied from year to year, being later when precipitation levels were low in the late spring-early summer months. Total pollen numbers and grass pollen densities are substantially less than those recorded from southern Australia. Nevertheless, respiratory disease in Brisbane affects up to 10% of the population, and airborne pollen of Poaceae, Urticaceae, Cupressaceae, Pinaceae, and Myrtaceae have been implicated in the release of allergens.

Airborne Pinus pollen in the atmosphere of Brisbane, Australia and relationships with meteorological parameters

Relationships between weather parameters andairborne pollen loads of Pinus inBrisbane, Australia have been investigated overthe five-year period, June 1994–May 1999.Pinus pollen accounts for 4.5% of the annualairborne pollen load in Brisbane where thePinus season is confined to the winter months,July–early September. During the samplingperiod loads of 11–>100 grains m3 wererecorded on 24 days and 1–10 grains m3 on204 days. The onset and peak dates wereconsistent across each season, whereas the enddates varied. The onset of the Pinuspollen season coincided with the coolestaverage monthly temperatures (< 22°C),lowest rainfall (< 7mm), and four weeks afterdaily minimum temperatures fell to 5–9°Cin late autumn. Correlations obtained betweendaily airborne Pinus pollen counts andtemperature/rainfall parameters show thatdensities of airborne Pinus pollen arenegatively correlated with maximum temperature(p < 0.0001), minimum temperature (p < 0.0001)and rainfall (p < 0.05) during the mainpollination period. The mean duration of eachpollen season was 52 days; longer seasons wereshown to be directly related to lower averageseasonal maximum temperatures (r2 = 0.85,p = 0.025). These results signify that maximumand minimum temperatures are the majorparameters that influence the onset andduration of the Pinus pollen season inthe environs of Brisbane. Respiratory allergyis an important health issue in Brisbane,Australia, but it remains unknown whether ornot airborne Pinus pollen is acontributing factor.

The coincidence of thrips and dispersed pollen in PNRSV-infected stonefruit orchards - a precondition for thrips-mediated transmission via infected pollen

Recent laboratory studies have demonstrated that Prunus necrotic ringspot virus (PNRSV) (family Bromoviridae) can be readily transmitted when thrips and virus-bearing pollen are placed together on to test plants. For this transmission mechanism to result in stonefruit tree infection in the field, PNRSV-bearing pollen must be deposited onto surfaces of stonefruit trees on which thrips also occur. In a previous paper, we demonstrated that almost all pollen in a PNRSV-infected Japanese plum orchard in southeastern Queensland was deposited onto flowers, whereas few grains occurred on leaves and none on stems. Here, we present results of our investigation of thrips species composition, distribution and abundance on stonefruit trees in the same study area as our previous pollen deposition study. We collected a total of 2010 adult thrips from 13 orchards during the 1989, 1991 and 1992 flowering seasons of which all but 14 were in the suborder Terebrantia. Most (97.4%) terebrantian thrips were of three species, Thrips imaginis, Thrips australis and Thrips tabaci. Thrips tabaci as well as species mixtures that included T imaginis, T australis and T tabaci have been shown to transmit PNRSV via infected pollen in laboratory tests. Adult thrips were frequently collected from flowers but rarely from leaves and never from stems. Large and significant differences in numbers of T imaginis, T australis and T tabaci adults in flowers occurred among orchards and between seasons. No factor was conclusively related to thrips numbers but flowers of late-flowering stonefruit varieties tended to hold more thrips than those of early-flowering varieties. Our results indicate that the common thrips species present on stonefruit trees in the Granite Belt are also ones previously shown to transmit PNRSV via infected pollen in the laboratory and that these thrips are concentrated in stonefruit flowers where most stonefruit pollen is deposited. These results contribute to mounting circumstantial evidence that stonefruit flowers may be inoculated with PNRSV via an interaction of thrips with virus-bearing pollen and that this transmission mechanism may be an important cause of new tree infections in the field.

In vitro exposure of acer negundo pollen to atmospheric levels of SO2 and NO2: effects on allergenicity and germination

In the last years, a rising trend of pollen allergies in urban areas has been attributed to atmospheric pollution. In this work, we investigated the effects of SO2 and NO2 on the protein content, allergenicity, and germination rate of Acer negundo pollen. A novel environmental chamber was assembled to exposure pollen samples with SO2 or NO2 at two different levels: just below and two times the atmospheric hour-limit value acceptable for human health protection in Europe. Results showed that protein content was lower in SO2- exposed pollen samples and slightly higher in NO2-exposed pollen compared to the control sample. No different polypeptide profiles were revealed by SDSPAGE between exposed and nonexposed pollen, but the immunodetection assays indicated higher IgE recognition by all sera of sensitized patients to Acer negundo pollen extracts in all exposed samples in comparison to the nonexposed samples. A decrease in the germination rate of exposed in contrast to nonexposed pollen was verified, which was more pronounced for NO2-exposed samples. Our results indicated that in urban areas, concentrations of SO2 and NO2 below the limits established for human protection can indirectly aggravate pollen allergy on predisposed individuals and affect plant reproduction.

Cardiovascular profile after intravenous injection of Africanized bee venom in awake rats

The manifestations caused by Africanized bee stings depend on the sensitivity of the victim and the toxicity of the venom. Previous studies in our laboratory have demonstrated cardiac changes and acute tubular necrosis (ATN) in the kidney of rats inoculated with Africanized bee venom (ABV). The aim of the present study was to evaluate the changes in mean arterial pressure (MAP) and heart rate (HR) over a period of 24 h after intravenous injection of ABV in awake rats. A significant reduction in basal HR as well as in basal MAP occurred immediately after ABV injection in the experimental animals. HR was back to basal level 2 min after ABV injection and remained normal during the time course of the experiment, while MAP returned to basal level 10 min later and remained at this level for the next 5 h. However, MAP presented again a significant reduction by the 7th and 8th h and returned to the basal level by the 24th h. The fall in MAP may contribute to the pathogenesis of ATN observed. The fall in MAP probably is due to several factors, in addition to the cardiac changes already demonstrated, it is possible that the components of the venom themselves or even substances released in the organism play some role in vascular beds.

Observations on flower visitors to Bertholletia excelsa H.B.K. and Couratari tenuicarpa A.C. Sm. (Lecythidaceae)

Bees visiting flowers of Bertholletia excelsa. (Brazil Nut tree) and Couratari tenuicarpa were collected, their behavior described and the pollen found clinging to their dorsal thorax and stored on their legs was identified. Female bees of Xylocopa frontalis(Olivier) and males of Eulaema mocsaryi (Friese) are apparently effective pollinators of Couratari in igapó near Manaus. Female bees of Euplusia seabrai Moure in litt., Epicharis umbraculata (Fabricius), Epicharis rustica (Olivier) and Eulaema nigrita(Lepeletier), as well as male bees of Eulaema cingulata(Fabricius) and Eulaema nigrita are apparently effective pollinators of adult Brazil Nut trees in the Aleixo plantation near Manaus. Only large bees capable of uncurling the floral androecium can effectively pollinate Couratari or Bertholletia.Pollen analysis indicated that all bees captured carried pollen of the host tree in question and had been foraging on flowers of plant species common in secondary growth. Secondary growth near the Aleixo plantation supports a bee guild which appeared to effectively pollinate almost every flower on the Brazil Nut tree studied. Proximity to primary forest (and to those Euglossine bee species which occur only in primary forest) therefore does not appear to be necessary for pollination of Brazil Nut trees.In the Aleixo plantation chronic low fruit set is probably due to some factor other than pollination. Since natural occurrences of Brazil Nut trees to the north and south of Manaus are associated with a higher soil fertility, low production at the Aleixo plantation may be due to deficiencies of the soil.

A novel approach for honey pollen profile assessment using an electronic tongue and chemometric tools

Nowadays the main honey producing countries require accurate labeling of honey before commercialization, including floral classification. Traditionally, this classification is made by melissopalynology analysis, an accurate but time-consuming task requiring laborious sample pre-treatment and high-skilled technicians. In this work the potential use of a potentiometric electronic tongue for pollinic assessment is evaluated, using monofloral and polyfloral honeys. The results showed that after splitting honeys according to color (white, amber and dark), the novel methodology enabled quantifying the relative percentage of the main pollens (Castanea sp., Echium sp., Erica sp., Eucaliptus sp., Lavandula sp., Prunus sp., Rubus sp. and Trifolium sp.). Multiple linear regression models were established for each type of pollen, based on the best sensors sub-sets selected using the simulated annealing algorithm. To minimize the overfitting risk, a repeated K-fold cross-validation procedure was implemented, ensuring that at least 10-20% of the honeys were used for internal validation. With this approach, a minimum average determination coefficient of 0.91 ± 0.15 was obtained. Also, the proposed technique enabled the correct classification of 92% and 100% of monofloral and polyfloral honeys, respectively. The quite satisfactory performance of the novel procedure for quantifying the relative pollen frequency may envisage its applicability for honey labeling and geographical origin identification. Nevertheless, this approach is not a full alternative to the traditional melissopalynologic analysis; it may be seen as a practical complementary tool for preliminary honey floral classification, leaving only problematic cases for pollinic evaluation.

Evaluation of bioactivities of a propolis sample (Gerês) of Portuguese origin

Dissertação de mestrado em Genética Molecular