A morphologically specialized soldier caste improves colony defense in a neotropical eusocial bee

Division of labor among workers is common in insect societies and is thought to be important in their ecological success. In most species, division of labor is based on age (temporal castes), but workers in some ants and termites show morphological specialization for particular tasks (physical castes). Large-headed soldier ants and termites are well-known examples of this specialization. However, until now there has been no equivalent example of physical worker subcastes in social bees or wasps. Here we provide evidence for a physical soldier subcaste in a bee. In the neotropical stingless bee Tetragonisca angustula, nest defense is performed by two groups of guards, one hovering near the nest entrance and the other standing on the wax entrance tube. We show that both types of guards are 30% heavier than foragers and of different shape; foragers have relatively larger heads, whereas guards have larger legs. Low variation within each subcaste results in negligible size overlap between guards and foragers, further indicating that they are distinct physical castes. In addition, workers that remove garbage from the nest are of intermediate size, suggesting that they might represent another unrecognized caste. Guards or soldiers are reared in low but sufficient numbers (1-2% of emerging workers), considering that <1% usually perform this task. When challenged by the obligate robber bee Lestrimelitta limao, an important natural enemy, larger workers were able to fight for longer before being defeated by the much larger robber. This discovery opens up opportunities for the comparative study of physical castes in social insects, including the question of why soldiers appear to be so much rarer in bees than in ants or termites.

Habitat Fragmentation Effects on the Orchid Bee Communities in Remnant Forests of Southeastern Brazil

The effect of habitat fragmentation on the structure of orchid bee communities was analyzed by the investigation of the existence of a spatial structure in the richness and abundance of Euglossini species and by determining the relationship between these data and environmental factors. The surveys were carried out in four different forest fragments and one university campus. Richness, abundance, and diversity of species were analyzed in relation to abiotic (size of the area, extent of the perimeter, perimeter/area ratio, and shape index) and biotic characteristics (vegetation index of the fragment and of the matrix of each of the locations studied). We observed a highly significant positive correlation between the diversity index and the vegetation index of the fragment, landscape and shape index. Our analysis demonstrated that the observed variation could be explained mainly by the vegetation index and the size of the fragment. Variations in relative abundance showed a tendency toward an aggregated spatial distribution between the fragments studied, as well as between the sampling stations within the same habitat, demonstrating the existence of a spatial structure on a small scale in the populations of Euglossini. This distribution will determine the composition of species that coexist in the area after fragmentation. These data help in understanding the differences and similarities in the structure of communities of Euglossini resulting from forest fragmentation.

Pollination services at risk: Bee habitats will decrease owing to climate change in Brazil

Native bees are important providers of pollination services, but there are cumulative evidences of their decline. Global changes such as habitat losses, invasions of exotic species and climate change have been suggested as the main causes of the decline of pollinators. In this study, the influence of climate change on the distribution of 10 species of Brazilian bees was estimated with species distribution modelling. We used Maxent algorithm (maximum entropy) and two different scenarios, an optimistic and a pessimistic, to the years 2050 and 2080. We also evaluated the percentage reduction of species habitat based on the future scenarios of climate change through Geographic Information System (GIS). Results showed that the total area of suitable habitats decreased for all species but one under the different future scenarios. The greatest reductions in habitat area were found for Melipona bicolor bicolor and Melipona scutellaris, which occur predominantly in areas related originally to Atlantic Moist Forest. The species analysed have been reported to be pollinators of some regional crops and the consequence of their decrease for these crops needs further clarification. (C) 2012 Elsevier B.V. All rights reserved.

PLGA microspheres containing bee venom proteins for preventive immunotherapy

Bee venom (BV) allergy is potentially dangerous for allergic individuals because a single bee sting may induce an anaphylactic reaction, eventually leading to death. Currently, venom immunotherapy (VIT) is the only treatment with long-lasting effect for this kind of allergy and its efficiency has been recognized worldwide. This therapy consists of subcutaneous injections of gradually increasing doses of the allergen. This causes patient lack of compliance due to a long time of treatment with a total of 30-80 injections administered over years. In this article we deal with the characterization of different MS-PLGA formulations containing BV proteins for VIT. The PLGA microspheres containing BV represent a strategy to replace the multiple injections, because they can control the solute release. Physical and biochemical methods were used to analyze and characterize their preparation. Microspheres with encapsulation efficiencies of 49-75% were obtained with a BV triphasic release profile. Among them, the MS-PLGA 34 kDa-COOH showed to be best for VIT because they presented a low initial burst (20%) and a slow BV release during lag phase. Furthermore, few conformational changes were observed in the released BV. Above all, the BV remained immunologically recognizable, which means that they could continuously stimulate the immune system. Those microspheres containing BV could replace sequential injections of traditional VIT with the remarkable advantage of reduced number of injections. (C) 2011 Elsevier B.V. All rights reserved.

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome

Abstract Background The ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers. Results Of the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes. Conclusions The versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.

Antimicrobial and antiproliferative activities of stingless bee Melipona scutellaris geopropolis

Background Geopropolis is a type of propolis containing resin, wax, and soil, collected by threatened stingless bee species native to tropical countries and used in folk medicine. However, studies concerning the biological activity and chemical composition of geopropolis are scarce. In this study, we evaluated the antimicrobial and antiproliferative activity of the ethanolic extract of geopropolis (EEGP) collected by Melipona scutellaris and its bioactive fraction against important clinical microorganisms as well as their in vitro cytotoxicity and chemical profile. Methods The antimicrobial activity of EEGP and fractions was examined by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against six bacteria strains as well as their ability to inhibit Streptococcus mutans biofilm adherence. Total growth inhibition (TGI) was chosen to assay the antiproliferative activity of EEGP and its bioactive fraction against normal and cancer cell lines. The chemical composition of M. scutellaris geopropolis was identified by reversed-phase high-performance liquid chromatography and gas chromatography–mass spectrometry. Results EEGP significantly inhibited the growth of Staphylococcus aureus strains and S. mutans at low concentrations, and its hexane fraction (HF) presented the highest antibacterial activity. Also, both EEGP and HF inhibited S. mutans biofilm adherence (p < 0.05) and showed selectivity against human cancer cell lines, although only HF demonstrated selectivity at low concentrations. The chemical analyses performed suggest the absence of flavonoids and the presence of benzophenones as geopropolis major compounds. Conclusions The empirical use of this unique type of geopropolis by folk medicine practitioners was confirmed in the present study, since it showed antimicrobial and antiproliferative potential against the cancer cell lines studied. It is possible that the major compounds found in this type of geopropolis are responsible for its properties.

Variation morphogeometrics of Africanized honey bees (Apis mellifera) in Brazil

The morphometrics of the honey bee Apis mellifera L., 1758 has been widely studied mainly because this species has great ecological importance, high adaptation capacity, wide distribution and capacity to effectively adapt to different regions. The current study aimed to investigate the morphometric variations of wings and pollen baskets of honey bees Apis mellifera scutellata Lepeletier, 1836 from the five regions in Brazil. We used geometric morphometrics to identify the existence of patterns of variations of shape and size in Africanized honey bees in Brazil 16 years after the classic study with this species, allowing a temporal and spatial comparative analysis using new technological resources to assess morphometrical data. Samples were collected in 14 locations in Brazil, covering the five geographical regions of the country. The shape analysis and multivariate analyses of the wing allowed to observe that there is a geographical pattern among the population of Apis mellifera in Brazil. The geographical variations may be attributed to the large territorial extension of the country in addition to the differences between the bioregions.

Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.

Ornithophily in the genus Salvia L. (Lamiaceae)

This dissertation presents for the first time a survey of bird pollinated (ornithophilous) Salvia species. Within the approximately 1000 species of the worldwide distributed genus roughly 20% (186 spp.) are bird pollinated. Excepting four species in the Old World (South Africa and Madagascar), ornithophilous species are restricted to the New World where they represent about one third of the species. They occur mainly in higher altitudes (1500-3000m) and usually grow as shrubs or perennial herbs (97%). The bilabiate to tubular flowers are often red (at least 49%), averaging 35mm (7-130mm) in length and produce a large to medium volume of nectar with rather low sugar concentration. Pollination by sunbirds and white-eyes is documented in a South African species, and that by hummingbirds in 16 species of the New World (USA, Mexico, Guatemala and Bolivia). Beside pollinator observations, the functionality of the staminal levers, the process of pollen transfer and the fitting between flowers and birds are tested by inserting museum skins and metal rods into fresh flowers. The most surprising result is the finding of two different main pollen transfer mechanisms. In at least 54% of the species an active staminal lever mechanism enables pollen deposition on the birds body. This is illustrated in detail in the South African S. lanceolata at which birds were observed to release the lever mechanism and became dusted with pollen. In contrast, the lever mechanism in about 35% of the New World species is reduced in different ways. Pollen transfer by inactive ‘levers’ is demonstrated in detail in S. haenkei in Bolivia, at which four pollinating hummingbird species could be observed. The tubular corolla forced the birds in a specific position, thereby causing pollen transfer from the exserted pollen-sacs to the birds body. With respect to the floral diversity and systematic affiliation of the species, parallel evolution of ornithophily and lever reduction is likely. Considering that bird pollinated species might have derived from bee pollinated species and that the staminal levers have become secondarily inactive, it is concluded that the shift in pollinators induced phenotypic changes even disabling such a sophisticated structure as the staminal lever mechanism.

Description of an ancient social bee trapped in amber using diagnostic radioentomology

The application of non-invasive imaging technologies using X-radiation (diagnostic radioentomology, ‘DR’) is demonstrated for the study of amber-entombed social bees. Here, we examine the external and internal morphology of an Early Miocene (Burdigalian) stingless bee (Apinae: Meliponini) from the Dominican Republic using non-destructive X-ray microtomography analysis. The study permits the accurate reconstruction of features otherwise obscured or impossible to visualize without destroying the sample and allows diagnosis of the specimen as a new species, Proplebeia adbita Greco and Engel.

Virus infections and winter losses of honey bee colonies (Apis mellifera)

Apiculturists have recently been confronted with drastic and inexplicable winter losses of colonies, and virus infections may be involved. Here, we surveyed 337 Swiss honey bee colonies in the winter of 2005 and 2006 and categorized their health status as: 1. dead (= no or few live bees left); 2. weak (= dwindling, high mortality of adult bees); or 3. healthy (= normal overwintering colony). From each colony, pooled adult workers were analyzed for deformed wing virus (DWV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and Kashmir bee virus (KBV). Neither KBV nor CBPV were found, but significantly higher ABPV and DWV infections were found in dead vs. weak vs. healthy colonies (except DWV in 2006 between weak and healthy). Moreover, ABPV and DWV loads were positively correlated with each other. This is the first report demonstrating statistically significant correlations between viruses associated with Varroa destructor and winter mortality.

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