Techniques for the In Vitro Production of Queens in Stingless Bees (Apidae, Meliponini)

Considering the ecological importance of stingless bees as caretakers and pollinators of a variety of native plants makes it necessary to improve techniques which increase of colonies' number in order to preserve these species and the biodiversity associated with them. Thus, our aim was to develop a methodology of in vitro production of stingless bee queens by offering a large quantity of food to the larvae. Our methodology consisted of determining the amount of larval food needed for the development of the queens, collecting and storing the larval food, and feeding the food to the larvae in acrylic plates. We found that the total average amount of larval food in a worker bee cell of E varia is approximately 26.70 +/- 3.55 mu L. We observed that after the consumption of extra amounts of food (25, 30, 35 and 40 mu L) the larvae differentiate into queens (n = 98). Therefore, the average total volume of food needed for the differentiation of a young larva of F. varia queen is approximately 61.70 +/- 5.00 mu L. In other words; the larvae destined to become queens eat 2.31 times more food than the ones destined to become workers. We used the species Frieseomelitta varia as a model, however the methodology can be reproduced for all species of stingless bees whose mechanism of caste differentiation depends on the amount of food ingested by the larvae. Our results demonstrate the effectiveness of the in vitro technique developed herein, pointing to the possibility of its use as a tool to assist the production of queens on a large scale. This would allow for the artificial splitting of colonies and contribute to conservation efforts in native bees.

Mixed colonies of two species of congeneric stingless bees (Hymenoptera: Apinae, Meliponini) display environmentally-acquired and endogenously-produced recognition signals

Nestmate recognition is fundamental for the maintenance of social organization in insect nests. It is becoming well recognized that cuticle hydrocarbons mediate the recognition process, although the origin of recognition cues in stingless bees remains poorly explored. The present study investigates the effects of endogenously-produced and environmentally-acquired components in cuticular hydrocarbons in stingless bees. The tests are conducted using colonies of Plebeia droryana Friese and Plebeia remota Holmberg. Recognition tests are performed with four different groups: conspecific nestmates, conspecific non-nestmates, heterospecifics and conspecific, genetically-related individuals that emerge in a heterospecific nest. This last group is produced by introducing brood cells of P. droryana into a P. remota colony, and the resulting adult bees are tested for acceptance 10 days after emergence. For all groups, 15 individuals are sampled for chemical analysis. The results show the acceptance of all conspecific nestmates, and the rejection of almost every conspecific non-nestmate and every heterospecific bee. Genetically-related individuals emerging from heterospecific nests present intermediate rejection (66.7% rejection). Chemical analysis shows that P. droryana individuals emerging in a P. remota nest have small amounts of alkene and diene isomers found in P. remota cuticle that are not found in workers from the natal nest. The data clearly show that the majority of the compounds present in P. droryana cuticle are endogenously produced, although a few unsaturated compounds are acquired from the environment, increasing the chemical differences and, consequently, the rejection percentages.

First record of chemical signals from the queen during the oviposition process in stingless bees

In stingless bees, the cell provisioning and oviposition process consists of several integrated behavioral sequences and several stereotyped queen-worker interactions. This study aims to demonstrate that chemical signals originating from the queen may contribute as cues for the sequence of the oviposition process in Melipona marginata. For this, we analyzed the cell before and after queen laying, and compared them with the cuticular hydrocarbons of the queen's abdomen, using a gas-chromatography and mass spectrometry system.

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.

Composition and antioxidant activity of honey from Africanized and stingless bees in Alagoas (Brazil): a multivariate analysis

The purpose of this study was to evaluate the antioxidant activity of honey from different entomological sources which were harvested in the dry season of 2008-2009 from distinct mesoregions of the State of Alagoas in the North East of Brazil. Honey produced by five different species of bees, even from the same region and season, showed a statistically significant difference (p <0.05) in the content of phenols, flavonoids and antioxidants, with higher levels of these compounds found in honey produced by Plebeia spp. and A. mellifera. Honey from stingless bees was quite different from that of A. mellifera, especially from the Plebeia spp. A dendrogram of the five species of bees showed the formation of 3 groups, one being formed by Apis mellifera, one by the genus Melipona (M. subnitida, M. quadrifasciata and M. scutellaris) and another formed by Plebeia spp.

Respiratory rhythms in stingless bee workers: circadian and ultradian components throughout adult development

The workers of the stingless bee, Melipona quadrifasciata, assume different tasks during their adult life. Newly emerged individuals remain inside the nest, without contact with the external environment. Maturing workers go to more peripheral regions and only the oldest, the foragers, leave the nest. As this diversity of activities implies different metabolic patterns, oxygen consumption has been measured in workers of three different ages: 24-48 h (nurses), 10-15 days (builders), and older than 25 days (foragers). Oxygen consumption of individually isolated workers was determined by intermittent respirometry, under constant darkness and temperature of 25 +/- 1 degrees C. Sets of 24-h measurements were obtained from individuals belonging to each of the three worker groups. Rhythmicity has been assessed in the daily (24 h) and ultradian (5-14 h) domains. This experimental design allowed detection of endogenous rhythms without the influence of the social group and without inflicting stress on the individuals, as would be caused by their longer isolation from the colony. Significant 24-h rhythms in oxygen consumption were present in nurses, builders and foragers; therefore, workers are rhythmic from the age of 24-48 h. However, the amplitude of the circadian rhythm changed according to age: nurses showed the lowest values, while foragers consistently presented the largest ones, about ten times larger than the amplitude of nurses` respiratory rhythm. Ultradian frequencies were detected for all worker groups, the power and frequencies of which varied little with age. This means that the ultradian strength was relatively larger in nurses and apparently maintains some relationship with the queen`s oviposition episodes.

Botanical Preferences of Africanized Bees (Apis mellifera) on the Coast and in the Atlantic Forest of Sergipe, Brazil

Pollen analysis in honey can be used as an alternative method to research into flowers visited by bees in an area. This study aimed to indentify the main floral families in honey from apiaries in the Atlantic Forest and Sergipe state coast. Honey samples from these apiaries were studied, as well as plants that grow around them, which can be used as a source of foraging for bees. The palynological technique was used to compare the pollen content of honey samples with the pollen grains from leaves of plants found in the vicinity of the apiaries to assess whether they had been visited by bees. The results of studies in both sites were similar in terms of incompatibility of families found in the apiary vicinity and honey. Thus, it was possible to observe that in honey samples from the coast and in the remaining Atlantic forest, the number of families was greater than the number of families found in the apiary vicinity, which highlights the diversity of plants visited by bees and a possible expansion of the visited area for food search. This diversity suggests an adaptive foraging behavior to plant resources available in the environment, which may facilitate the pollination of these botanical families and consequently improve their genetic quality.

Characterization of cuticular hydrocarbons of diploid and haploid males, workers and queens of the stingless bee Melipona quadrifasciata

Males, queens and workers of stingless bees show differences in external morphology, behaviour and roles within a colony. In addition, each individual has a cuticular chemical signature responsible for mutual communication that is essential for maintaining the integrity of the colony. In this paper we characterize the cuticular hydrocarbon composition of newly emerged diploid and haploid males, workers and virgin queens of Melipona quadrifasciata by gas chromatography-mass spectrometry (GC/MS) analysis. This is the first time that the cuticular profile of diploid males in a species of stingless bee has been characterized. We found differences in the cuticular hydrocarbon composition among males, workers and virgin queens, recording both qualitative and quantitative differences among individuals of different phenotypes. However, no compound was found exclusively in diploid males. The cuticular chemical profiles of haploid and diploid males were very similar to those of workers. Moreover, the cuticular lipids of males and workers were significantly different from those of queens. Tricosane, pentacosene-2 and 7-methyl-heptacosane were the compounds responsible for this significant separation. This result correlates with the behavioural and morphological differences among these phenotypes.

Gene copy number and differential gene expression in haploid and diploid males of the stingless bee, Melipona quadrifasciata

Complementary sex determination in Hymenoptera implies that heterozygosity at the sex locus leads to the development of diploid females, whereas hemizygosity results in haploid males. Diploid males can arise through inbreeding. In social species, these pose a double burden on colony fitness, from significant reduction in its worker force and through being less viable and fertile than haploid males. Apart from being "misfits", diploid males are of interest to assess molecular correlates for possibly ploidy-related bionomic differences. Herein, we generated suppression subtractive cDNA libraries from newly emerged haploid and diploid males of the stingless bee Melipona quadrifasciata to enrich for differentially expressed genes. Gene Ontology classification revealed that in haploid males more DEGs were related to stress responsiveness, biosynthetic processes, reproductive processes and spermatogenesis, whereas in diploid ones differentially expressed genes were associated with cellular organization, nervous system development and amino acid transport were prevalent. Furthermore, both libraries contained over 40 % ESTs representing possibly novel transcripts. Quantitative RT-PCR analyses confirmed the differential expression of a representative DEG set in newly emerged males. Several muscle formation and energy metabolism-related genes were under-expressed in diploid males. On including 5-day-old males in the analysis, changes in transcript abundance during sexual maturation were revealed.

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.

Desempenho de cultivares de morango submetidas a diferentes tipos de polinização em cultivo protegido

Resumo – O objetivo deste trabalho foi avaliar o efeito de diferentes tipos de polinização sobre a qualidade de frutos de cultivares de morangueiro e sua contribuição isolada para a massa dos frutos, bem como determinar o potencial de Plebeia nigriceps (Hymenoptera: Apidae, Meliponini) como agente polinizador em ambiente protegido. As cultivares Aromas, Diamante e Cegnidarem foram submetidas a tratamentos com autopolinização, polinização por P. nigriceps e polinização livre. Os experimentos foram conduzidos em estufa tipo pampeana, coberta com polietileno transparente e desprovida de telas anti-insetos nas laterais, com 1.344 plantas. Para as avaliações, foram marcadas 56 flores primárias em botão, de cada cultivar, e considerou-se cada planta uma repetição. Avaliaram-se massa de matéria fresca, peso, diâmetro, comprimento e presença de deformação nos frutos. A polinização entomófila tem contribuição variada à massa dos frutos, de acordo com a cultivar. As cultivares apresentam sensibilidade variada à autopolinização, no que se refere à incidência de frutos deformados. Ainterferência da polinização entomófila na produtividade do morangueiro está mais relacionada à redução do percentual de frutos deformados do que ao aumento da massa dos frutos em si. O comportamento de P. nigriceps indica que a espécie apresenta potencial para polinização da cultura do morangueiro em ambiente protegido.

Impact of Aqueous Plant Extracts on Trigona spinipes (Hymenoptera: Apidae)

The stingless bees are an important component of the insect biomass in many tropical areas, due to their collection of nectar and pollen. Trigona spinipes is a widely distributed species in South America, and described as a pollinator of many crops that can be used in a commercial pollinating system. The effects of plant extracts on insects are studied because of the demand for organic food and their selectivity to natural enemies. Plant insecticides are reported as a potential agent for the control of insect pests, however little is known about their impact on beneficial insects. This study investigated the survival of Trigona spinipes (Hymenoptera: Apidae, Meliponini) Fabricius, after exposure to the leaf extracts of Azadiracha indica (Meliaceae), Lippia sidoides (Verbenaceae), Sapindus saponaria (Sapindaceae), Anonna squamosa (Anonnaceae) Cymbopogon winterianum (Poaceae), Corimbia citriodora (Myrtaceae), Jatropha curcas (Euphorbiaceae) and Ricinus communis (Euphorbiaceae) and of seeds of Azadiracha indica, Ricinus communis Nordestina and AL Guarany varieties and Jatropha curcas. The extracts that had the greatest influence on the survival of the bees were A. indica at 3% and 7% of concentration, A. squamosa at a concentration of 10% with 68.89% survival and green leaf of R. communis at a concentration of 7%. The results show that although the extracts were effective in controlling pests, they may also affect the pollinator Trigona spinipes.

Cell-sealing efficiency and reproductive workers in the species Melipona bicolor (Hymenoptera, Meliponini): double standard and possible rogue conduct

In many hymenopteran insect societies, selfish workers are policed, as selfishness can negatively affect the average inclusive fitness of one or both castes by reducing either the degree of average relatedness to the colony's male offspring or colony efficiency. In stingless bees, the rapid capping of brood cells could aid in controlling selfishness; to this end, we studied cell-sealing efficacy in Melipona bicolor. Execution of cell sealing was found to be both rapid and almost continuous. Comparing the performance of reproductive and non-reproductive workers, the former sealed the cells more efficiently when they contained their own eggs, but less so when the queens' eggs were involved. We argue that the occurrence of disruptions in cell sealing through self-serving reproductive workers is capable of undermining sealing efficacy as a policing instrument, thus making reproductive workers potential rogue individuals.

Can climate shape flight activity patterns of Plebeia remota (Hymenoptera, Apidae)?

Flight activity of foragers of four colonies of Plebeia remota (Holmberg, 1903) was registered from December 1998 to December 1999, using an automated system (photocells and PLC system). The colonies originated from two different regions: Cunha, state of Sao Paulo, and Prudentopolis, state of Parana, Brazil. Flight activity was influenced by different climatic factors in each season. In the summer, the intensity of the correlations between flight activity and climatic factors was smaller than in the other seasons. During the autumn and winter, solar radiation was the factor that most influenced flight activity, while in the spring, this activity was influenced mainly by temperature. Except in the summer, the various climatic factors similarly influenced flight activity of all of the colonies. Flight activity was not affected by geographic origin of the colonies. Information concerning seasonal differences in flight activity of P. remota will be useful for prediction of geographic distribution scenarios under climatic changes.

Geopropolis from Melipona scutellaris decreases the mechanical inflammatory hypernociception by inhibiting the production of IL-1 beta and TNF-alpha

Ethnopharmacological relevance: The pharmacological activity of geopropolis collected by stingless bees (important and threatened pollinators), a product widely used in folk medicine by several communities in Brazil, especially in the Northeast Region, needs to be studied. Objective: The aim of this study was to evaluate the antinociceptive activity of Melipona scutellaris geopropolis (stingless bee) using different models of nociception. Material and methods: The antinociceptive activity of the ethanolic extract of geopropolis (EEGP) and fractions was evaluated using writhing induced by acetic acid, formalin test, carrageenan-induced hypernociception, and quantification of IL-1 beta and TNF-alpha. The chemical composition was assessed by quantification of total flavonoids and phenolic compounds. Results: EEGP and its hexane and aqueous fractions showed antinociceptive activity. Both EEGP and its aqueous fraction presented activity in the mechanical inflammatory hypernociception induced by the carrageenan model, an effect mediated by the inhibition of IL-1 beta and TNF-alpha. The chemical composition of EEGP and its hexane and aqueous fractions showed a significant presence of phenolic compounds and absence of flavonoids. Conclusion: Our data indicate that geopropolis is a natural source of bioactive substances with promising antinociceptive activity. (C) 2012 Elsevier Ireland Ltd. All rights reserved.