Correlation between mandibular gland secretion and cuticular hydrocarbons in the stingless bee Melipona quadrifasciata

We investigated whether Melipona quadrifasciata worker mandibular gland secretions contribute directly to their cuticular hydrocarbon profile. The mandibular gland secretion composition and cuticular surface compounds of newly emerged worker bees, nurse bees, and foragers were determined by gas chromatography and mass spectrometry and compared. Both the mandibular gland secretions and the cuticular surface compounds of all worker stages were found to be composed almost exclusively of hydrocarbons. Although the relative proportion of hydrocarbons from the cuticular surface and gland secretion was statistically different, there was a high similarity in the qualitative composition between these structures in all groups of bees.

Acceptance Threshold Hypothesis is Supported by Chemical Similarity of Cuticular Hydrocarbons in a Stingless Bee, Melipona asilvai

The ability to discriminate nestmates from non-nestmates in insect societies is essential to protect colonies from conspecific invaders. The acceptance threshold hypothesis predicts that organisms whose recognition systems classify recipients without errors should optimize the balance between acceptance and rejection. In this process, cuticular hydrocarbons play an important role as cues of recognition in social insects. The aims of this study were to determine whether guards exhibit a restrictive level of rejection towards chemically distinct individuals, becoming more permissive during the encounters with either nestmate or non-nestmate individuals bearing chemically similar profiles. The study demonstrates that Melipona asilvai (Hymenoptera: Apidae: Meliponini) guards exhibit a flexible system of nestmate recognition according to the degree of chemical similarity between the incoming forager and its own cuticular hydrocarbons profile. Guards became less restrictive in their acceptance rates when they encounter non-nestmates with highly similar chemical profiles, which they probably mistake for nestmates, hence broadening their acceptance level.

The role of wax and resin in the nestmate recognition system of a stingless bee, Tetragonisca angustula

Recent research has shown that entrance guards of the stingless bee Tetragonisca angustula make less errors in distinguishing nestmates from non-nestmates than all other bee species studied to date, but how they achieve this is unknown. We performed four experiments to investigate nestmate recognition by entrance guards in T. angustula. We first investigated the effect of colony odours on acceptance. Nestmates that acquired odour from non-nestmate workers were 63% more likely to be rejected while the acceptance rate of non-nestmates treated with nestmate odour increased by only 7%. We further hypothesised that guards standing on the wax entrance tube might use the tube as an odour referent. However, our findings showed that there was no difference in the acceptance of non-nestmates by guards standing on their own colony's entrance tube versus the non-nestmate's entrance tube. Moreover, treatment of bees with nestmate and non-nestmate resin or wax had a negative effect on acceptance rates of up to 65%, regardless of the origin of the wax or resin. The role of resin as a source of recognition cues was further investigated by unidirectionally transferring resin stores between colonies. Acceptance rates of nestmates declined by 37% for hives that donated resin, contrasting with resin donor hives where acceptance of non-nestmates increased by 21%. Overall, our results confirm the accuracy of nestmate recognition in T. angustula and reject the hypothesis that this high level of accuracy is due to the use of the wax entrance tubes as a referent for colony odour. Our findings also suggest that odours directly acquired from resin serve no primary function as nestmate recognition cues. The lack of consistency among colonies plus the complex results of the third and fourth experiments highlight the need for further research on the role of nest materials and cuticular profiles in understanding nestmate recognition in T. angustula.

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.

Enhanced production of parthenocarpic cucumbers pollinated with stingless bees and Africanized honey bees in greenhouses

Crops have different levels of dependence on pollinators; this holds true even for cultivars of the same species, as in the case of cucumber (Cucumis sativus). The aim of this research was to assess the attractiveness of flowers of three Japanese parthenocarpic cucumber cultivars and evaluate the importance of Africanized bees (Apis mellifera), and the Brazilian native stingless bees, Jata (Tetragonisca angustula) and Ira (Nannotrigona testaceicornis) on fruit production. Several parameters, including frequency of bee visits to flowers as well as duration of nectar collection and fruit set were examined; additionally, fruit weight, length and diameter were evaluated. Three greenhouses located in Ribeirao Preto, SP, were used for planting three cucumber cultivars (Hokushin, Yoshinari and Soudai). The female flowers were more attractive than male flowers; however, Jata bees were not observed visiting the flowers. The Africanized and the Ira bees collected only nectar, with a visitation peak between 10 and 12h. Visits to female flowers had a longer duration than visits to male flower visits in all three cultivars. Africanized bee colonies declined due to loss of bees while in the greenhouse; the native stingless bee colonies did not suffer these losses. When bees were excluded, fruit set was 78%; however, when bees had access to the flowers, fruit set was significantly (19.2%) higher. Fruit size and weight did not differ with and without bees. This demonstrates that even in parthenocarpic cucumber cultivars, which do not require pollination in order to from fruits, fruit production is significantly increased by bee pollination.

Behavioral suites mediate group-level foraging dynamics in communities of tropical stingless bees

Competition for floral resources is a key force shaping pollinator communities, particularly among social bees. The ability of social bees to recruit nestmates for group foraging is hypothesized to be a major factor in their ability to dominate rich resources such as mass-flowering trees. We tested the role of group foraging in attaining dominance by stingless bees, eusocial tropical pollinators that exhibit high diversity in foraging strategies. We provide the first experimental evidence that meliponine group foraging strategies, large colony sizes and aggressive behavior form a suite of traits that enable colonies to improve dominance of rich resources. Using a diverse assemblage of Brazilian stingless bee species and an array of artificial ""flowers"" that provided a sucrose reward, we compared species` dominance and visitation under unrestricted foraging conditions and with experimental removal of group-foraging species. Dominance does not vary with individual body size, but rather with foraging group size. Species that recruit larger numbers of nestmates (Scaptotrigona aff. depilis, Trigona hyalinata, Trigona spinipes) dominated both numerically (high local abundance) and behaviorally (controlling feeders). Removal of group-foraging species increased feeding opportunities for solitary foragers (Frieseomelitta varia, Melipona quadrifasciata and Nannotrigona testaceicornis). Trigona hyalinata always dominated under unrestricted conditions. When this species was removed, T. spinipes or S. aff. depilis controlled feeders and limited visitation by solitary-foraging species. Because bee foraging patterns determine plant pollination success, understanding the forces that shape these patterns is crucial to ensuring pollination of both crops and natural areas in the face of current pollinator declines.

A method for harvesting unfermented pollen from stingless bees (Hymenoptera, Apidae, Meliponini)

Pollen traps used for harvesting pollen from Apis mellifera do not work for stingless bees, as most species have small entrances and rapidly deposit large quantities of propolis at any barrier in front of the nest. Some stingless beekeepers harvest pollen by removing it directly from pollen pots, but this pollen is normally fermented and unpalatable. The aim of this study was to test a new method for harvesting pollen from stingless bee colonies before it begins to ferment. Colonies of Scaptotrigona depilis were removed and replaced by empty hives, which were occupied by the returning foragers and used for storing pollen and nectar. After one week, the pollen and honey were harvested directly from the storing pots and weighed. On average, the colonies produced 8.7 g of honey and 54.2 g of unfermented pollen (n = 10). This method is a viable option for harvesting unfermented pollen from stingless bees, especially with species that harvest large amounts of pollen. The unfermented pollen of S. depilis was well received in taste tests, receiving higher scores than fermented pollen, and similar scores to A. mellifera pollen, so could have great commercial possibilities. It is also a good method for studying the foraging of stingless bees because the amount of harvested food can be easily and precisely quantified.

Bees at war: Interspecific battles and nest usurpation in stingless bees

We provide the first evidence for interspecific warfare in bees, a spectacular natural phenomenon that involves a series of aerial battles and leads to thousands of fatalities from both attacking and defending colonies. Molecular analysis of fights at a hive of the Australian stingless bee Tetragonula carbonaria revealed that the attack was launched by a related species, Tetragonula hockingsi, which has only recently extended its habitat into southeastern Queensland. Following a succession of attacks by the same T. hockingsi colony over a 4-month period, the defending T. carbonaria colony was defeated and the hive usurped, with the invading colony installing a new queen. We complemented our direct observations with a 5-year study of more than 260 Tetragonula hives and found interspecific hive changes, which were likely to be usurpation events, occurring in 46 hives over this period. We discuss how fighting swarms and hive usurpation fit with theoretical predictions on the evolution of fatal fighting and highlight the many unexplained features of these battles that warrant further study.

Anti-staphylococcal activity of C-methyl flavanones from propolis of Australian stingless bees (Tetragonula carbonaria) and fruit resins of Corymbia torelliana (Myrtaceae)

Propolis of Australian stingless bees (Tetragonula carbonaria, Meliponini) originating from Corymbia torelliana (Myrtaceae) fruit resins was tested for its antimicrobial activities as well as its flavonoid contents. This study aimed at the isolation, structural elucidation and antibacterial testing of flavanones of C. torelliana fruit resins that are incorporated into stingless bee propolis. Flavanones of this study were elucidated by spectroscopic and spectrometric methods including UV, 1D and 2D NMR, EI-MS, ESI-MS and HR-MS. The results indicated known C-methylated flavanones namely, 1 (2S)-cryptostrobin, its regioisomer 2 (2S)- stroboponin, 3 (2S)- cryptostrobin 7-methyl ether, and 6 (2S)- desmethoxymatteucinol, and known flavanones 4 (2S)- pinostrobin and 5 (2S)- pinocembrin as markers for C. torelliana fruit resins and one propolis type. Ethanolic preparations of propolis were shown to be active against Staphylococcus aureus (ATCC 25923) and to a lesser extent against Pseudomonas aeruginosa (ATCC 27853). C. torelliana flavanones inhibited the growth of S. aureus therefore contributing to the antibacterial effects observed for Australian stingless bee propolis extracts.

Morphological and molecular characters reveal differentiation in a Neotropical social bee, Melipona beecheii (Apidae : Meliponini)

Morphometrics and DNA microsatellites were used to analyse the genetic structure of populations of the stingless bee M. beecheii from two extremes of its geographic range. The results showed that populations from Costa Rica and Yucatan exhibit substantial phenotypic and molecular differentiation. Bees from Yucatan were smaller and paler than those from Costa Rica. The value of multilocus F-ST = 0.280 (P <0.001) confirmed that there were significant molecular genetic differences between the two populations. Populations showed significant deviation from Hardy Weinberg equilibrium and the values of FIS (the inbreeding coefficient) were positive for Costa Rica = 0.416 and the Yucatan Peninsula = 0.193, indicating a lack of heterozygotes in both populations possibly due to inbreeding. The DNA sequence of 678 bp of the mitochondrial gene COI differed between populations by 1.2%. The results of this study should be considered in conservation programmes, particularly with regard to the movement of colonies between regions.

Selective preying of the sphecid wasp Trachypus boharti on the meliponine bee Scaptotrigona postica: potential involvement of caste-specific cuticular hydrocarbons

The specialist digger wasp Trachypus boharti Rubio-Espina preys exclusively on males of the stingless bee Scaptotrigona postica Latreille 1807, although the hunting attacks involve both male and worker bees of S. postica and members of its own species. To understand the mechanism of prey selection, the cuticular hydrocarbon patterns of workers and males of S. postica are analyzed in detail, and the mandibular secretion of males is examined. The cuticular profiles of males and workers are distinctively different. The major group of cuticular compounds, heptacosene isomers, is twice as abundant in workers as in males. There is no clear distinction between worker and male mandibular secretions. Such a distinct and straightforward caste-specific difference in cuticular hydrocarbons could function as a recognition cue by which T. boharti distinguishes between workers and males of S. postica.

Microdissection: a tool for bee chromosome studies

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

Electrophoretic analysis of the protein in the midgut of stingless bees (Apidae; Meliponinae) with a comparison of necrophagous and feeding pollen workers

SDS electrophoresis of midgut proteins from two Trigonini species with different feeding habits shows many similarities and the absence of unique protein in the necrophagous stingless bee Trigona hypogea, suggesting that biochemical adaptation to necrophagy, in this bee, occurs more in the qualitative than in quantitative level. Workers of different ages display some variations in the midgut protein pattern, suggesting that it is important to know the age in studies regarding to bee digestion.