The Orchid Bee Fauna (Hymenoptera: Apidae: Euglossini) of a Neotropical Savanna: an Efficient Protocol to Assess Bee Community and Diversity Along Elevational and Habitat Complexity Gradients.

Males of euglossine bees, also known as orchid bees, are attracted to aromatic compounds and this feature has been exploited to collect them. Here we sampled orchid bee males using a rapid passive method in a Neotropical savanna ecosystem in Rio Preto State Park (PERP), southeastern Brazil. The sampling protocol consisted of 5-day surveys using scent traps at the beginning and end of the rainy seasons. PERP is inserted in a Brazilian savanna characterized by a mosaic of vegetation types, and elevational gradients. We also analyzed whether the habitat complexity and elevational gradients influence the richness and abundance of these bees. We collected a total of 996 individuals belonging to 14 species and four genera (Eufriesea Cockerell 1908, Euglossa Latreille 1802, Eulaema Lepeletier 1841, and Exaerete Hoffmannsegg 1817). Comparing our results with previous sampling protocols at PERP, higher abundance and richness were observed using traps than insect nets to survey these bees. In addition, the orchid bee fauna in PERP was successfully assessed by our rapid passive protocol, showing that this protocol seems to be useful for collecting orchid bees in open vegetation environments. We observed that the abundance of bees was lower at higher elevations, and both abundance and richness increased in more complex habitat. This may be due to the characteristics of Brazilian savannas where the lower elevations have higher habitat complexity, with more places for nest construction and resources for feeding and nesting.

European Foulbrood in stingless bees (Apidae: Meliponini) in Brazil: Old disease, renewed threat.

Stingless bees (Apidae: Meliponini) are a group of bees with vestigial stings showing a high level of social organization. They are important pollinators in tropical and subtropical regions, and, in the last decades, stingless beekeeping has increased rapidly in Brazil. Bee-collected pollen and honey of Apis mellifera can be an important source of disease when used as supplements to feed stingless bee colonies, a common and increasing practice adopted by stingless beekeepers. Here, we aimed to investigate the presence of pathogens commonly found in honey bees in diseased colonies of Melipona species in Espírito Santo and São Paulo States, Southeast Brazil. We detected, for the first time, the bacterium Melissococcus plutonius and symptoms of European foulbrood in Melipona spp., associated with brood death and colony losses in some cases. In addition, we tested for the presence of the bacterium Paenibacillus larvae and the fungus Aschosphaera apis, as well as the six more common honey bee viruses in Brazil (BQCV, ABPV, DWV, KBV, IAPV, CBPV) and the microsporidia Nosema apis and Nosema ceranae. However, only one sample of brood was infected with N. ceranae and all other pathogens, with the exception of Melissococcus plutonius, were absent in the analyzed brood. Lastly, we looked for toxic pollen in all food fed to diseased colonies, but none was present.

Immunosenescence in honey bees (Apis mellifera L.) is caused by intrinsic senescence and behavioral physiology.

Young honey bee workers (0 to 2-3 weeks old) perform tasks inside the colony, including brood care (nursing), whereas older workers undergo foraging tasks during the next 3-4 weeks, when an intrinsic senescence program culminates in worker death. We hypothesized that foragers are less able to react to immune system stimulation than nurse bees and that this difference is due to an inefficient immune response in foragers. To test this hypothesis, we used an experimental design that allowed us to uncouple chronological age and behavior status (nursing/foraging). Worker bees from a normal age demography colony (where workers naturally transit from nursing to foraging tasks as they age) and of a single-cohort colony setup (composed of same-aged workers performing nursing or foraging tasks) were tested for survival and capability of activation of the immune system after bacterial injection. Expression of an antimicrobial peptide gene, defensin-1 (def-1), was used to assess immune system activation. We then checked whether the immune response includes changes in the expression of aging- and behavior-related genes, specifically vitellogenin (vg), juvenile hormone esterase (jhe), and insulin-like peptide-1 (ilp-1). We found a significant difference in survival rate between bees of different ages but carrying out the same tasks. Our results thus indicate that the bees' immune response is negatively affected by intrinsic senescence. Additionally, independent of age, foragers had a shorter lifespan than nurses after bacterial infection, although both were able to induce def-1 transcription. In the normal age demography colony, the immune system activation resulted in a reduction in the expression of vg, jhe and ilp-1 genes in foragers, but not in the nurse bees, demonstrating that age and behavior are both important influences on the bees' immune response. By disentangling the effects of age and behavior in the single-cohort colony, we found that vg, jhe and ilp-1 response to immune system stimulation was independent of behavior. Younger bees were able to mount a stronger immune response than older bees, thus highlighting age as an important factor for immunity. Taken together, our results provide new insights into how age and behavior affect the honey bee's immune response.

Pollen diet for in vitro rearing of africanized honey bee larvae, Apis mellifera (Hymenoptera: Apidae) / Dieta polínica para criação in vitro de larvas de abelhas melíferas africanizadas, Apis mellifera (Hymenoptera: Apidae)

Pollen is the major protein source for honey bees, Apis mellifera. It is essential for the adults to produce royal jelly to feed the larvae. Young larvae receive the brood food, whereas the older (over 3 days old) larvae receive pollen in addition to brood food. The nutritional value of pollen has been investigated only in adults or at the colony level. Protocols for rearing Africanized honey bee larvae in vitro using diets with mixtures of pollen had not been established. We examined different concentrations (2.5, 5 and 10%) of two mixtures of pollen in the larval diet. The effects of pollen diets on larval development were assessed. The survival and development of larvae fed with 10% pollen was impaired; this concentration should be avoided. Concentrations of 2.5 and 5% pollen did not show significant changes in survival, weight, development or the hemolymph protein profile when compared to the controls (without pollen). However, differences in larval survival were observed between the two pollen mixtures (pollen blends 1 and 2), suggesting that a diet with a superior digestibility and greater familial diversity of pollen (blend 2) is more nutritionally adequate.

O pólen é a principal fonte de proteína para as abelhas melíferas, Apis mellifera. Ele é essencial para que os adultos produzam geleia real para nutrir as larvas. As larvas jovens recebem geleia real, enquanto que as larvas mais velhas (mais que 3 dias de idade) recebem pólen juntamente com a geleia real. O valor nutricional do pólen tem sido investigado apenas em adultos ou ao nível de colônia. Protocolos de criação de larvas de abelhas africanizadas in vitro utilizando dietas com misturas de pólen não foram estabelecidas. Nós examinamos diferentes concentrações (2,5, 5 e 10%) de duas misturas de pólen na dieta de larvas. O efeito das dietas de pólen no desenvolvimento larval foi avaliado. A sobrevivência e o desenvolvimento das larvas alimentadas com 10% de pólen foram prejudicados; esta concentração deve ser evitada. Concentrações de 2,5 e 5% de pólen não mostraram uma mudança significativa na sobrevivência, no peso, no desenvolvimento ou no perfil proteico da hemolinfa, quando comparado com os controles (sem pólen). No entanto, diferenças na sobrevivência das larvas foram observadas entre duas misturas de pólen (mistura 1 e 2), sugerindo que uma dieta com uma digestibilidade superior e maior diversidade de famílias de pólens (mistura 2) é nutricionalmente mais adequada.

Potential costs of bacterial infection on storage protein gene expression and reproduction in queenless Apis mellifera worker bees on distinct dietary regimes.

Insects are able to combat infection by initiating an efficient immune response that involves synthesizing antimicrobial peptides and a range of other defense molecules. These responses may be costly to the organism, resulting in it exploiting endogenous resources to maintain homeostasis or support defense to the detriment of other physiological needs. We used queenless worker bees on distinct dietary regimes that may alter hemolymph protein storage and ovary activation to investigate the physiological costs of infection with Serratia marcescens. The expression of the genes encoding the storage proteins vitellogenin and hexamerin 70a, the vitellogenin receptor, and vasa (which has a putative role in reproduction), was impaired in the infected bees. This impairment was mainly evident in the bees fed beebread, which caused significantly higher expression of these genes than did royal jelly or syrup, and this was confirmed at the vitellogenin and hexamerin 70a protein levels. Beebread was also the only diet that promoted ovary activation in the queenless bees, but this activation was significantly impaired by the infection. The expression of the genes encoding the storage proteins apolipophorins-I and -III and the lipophorin receptor was not altered by infection regardless the diet provided to the bees. Similarly, the storage of apolipophorin-I in the hemolymph was only slightly impaired by the infection, independently of the supplied diet. Taken together these results indicate that, infection demands a physiological cost from the transcription of specific protein storage-related genes and from the reproductive capacity.

Molecular characterization of a cDNA encoding prophenoloxidase and its expression in Apis mellifera.

Phenoloxidase (PO), a melanin-synthesizing enzyme known to play an important role in insect defense, is found as a zymogen (ProPO) in hemolymph and cuticle, where it is activated by proteolysis. We characterized the first proPO cDNA in an eusocial insect, the Apis mellifera honey bee. The AmproPO cDNA contains an ORF of 2079 bp encoding 693 amino acids, and is composed of 9 exons and 8 introns. Southern blot of digested genomic DNA suggested that only one copy of the proPO gene is present in A. mellifera. The molecular mass of the deduced ProPO and the active enzyme was predicted to be 80.1 and 74.4 kDa, respectively. The calculated pI was 6.28. BLASTp search of the deduced amino acid sequence, and neighbor-joining analysis, showed similarity with ProPOs from other insects, ranging from 47% to 63%. Protein signature analyses revealed four conserved regions, including the two copper binding sites characteristic of arthropod ProPOs. RT-PCR and Southern blot showed the highest amount of AmproPO transcripts in workers whole body, followed by queens and drones. Expression was also detected in hemocytes and integument. Real time RT-PCR showed higher amounts of AmproPO transcripts in adults and older pupae than in younger pupae and larvae, suggesting a function of AmproPO in adult exoskeleton melanization and differentiation.