Toxic effects of methanolic and dichloromethane extracts of flowers and peduncles of Stryphnodendron adstringens (Leguminosae : Mimosoideae) on Apis mellifera and Scaptotrigona postica workers

Laboratory bioassays were conducted to evaluate toxicity of methanolic and dichloromethane extracts obtained from Stryphnodendron adstringens to Apis mellifera and Scaptotrigona postica workers. The extracts were incorporated into the diet of the bees for evaluation of mortality rates. The ingestion bioassays were made with three concentrations (0.002mg/g, 0.005mg/g and 0.01 mg/g) for each bee species. The workers were kept in cages, with twenty workers per cage for each concentration tested. All bioassays had sixty workers in three cages that where maintained in a biological oxygen demand incubator with controlled temperature and humidity. The data obtained in the toxicity bioassays were analyzed statistically by Log Rank test and all methanolic and clichloromethane extracts showed significant (P < 0.0001) toxic effects in all tested concentrations.

Insect Venom Peptides

The insects of the order Hymenoptera ( bees, wasps, and ants) are classified in two groups, based on their life history: social and solitary. The venoms of the social Hymenoptera evolved to be used as defensive tools to protect the colonies of these insects from the attacks of predators. Generally they do not cause lethal effects but cause mainly inflammatory and/or immunological reactions in the victims of their stings. However, sometimes it is also possible to observe the occurrence of systemic effects like respiratory and/or kidney failure. Meanwhile, the venoms of solitary Hymenoptera evolved mainly to cause paralysis of the preys in order to permit egg laying on/within the prey's body; thus, some components of these venoms cause permanent/transient paralysis in the preys, while other components seem to act preventing infections of the food and future progenies. The peptide components of venoms from Hymenoptera are spread over the molar mass range of 1400 to 7000 da and together comprise up to 70% of the weight of freeze-dried venoms. Most of these toxins are linear polycationic amphipatic peptides with a high content of alpha-helices in their secondary structures. These peptides generally account for cell lysis, hemolysis, antibiosis, and sometimes promote the delivery of cellular activators/mediators through interaction with the G-protein receptor, and perhaps some of them are even immunogenic components. In addition to these peptides, the Hymenopteran venoms also may contain a few neurotoxins that target Na+ and/or Ca+2 channels or even the nicotinic ACh receptor. This review summarizes current knowledge of the biologically active Hymenoptera venoms.

Ultrastructure of the midgut epithelium of Meliponinae larvae with different developmental stages and diets

The comparative study of the ultrastructure of the midgut epithelium of stingless bee larvae that eat plant protein (pollen) and animal protein (carrion) throughout the larval phase, shows variations in the digestive cells that are only relative to larval aging and not to the type of larval diet. The cells of older larvae present a cytoplasm with empty spaces that result from emptying of lipid and glycogen stocks, and the presence of autophagic vacuoles. These results are discussed in relation to the hypothesis that variations in the digestive tract of insects may be associated with different diets or phylogeny. We conclude that different diets do not determine cell morphology adaptations in the studied species. As the variations in the ultrastructure of the midgut epithelium are the same in all studied species, including the necrophagous species Trigona hypogea, throughout the larval stage, this sequence of changes seems to be due to different physiological state during larval development.

Behavioral responses evoked in honey bee workers by Dufour gland extracts (Hymenoptera : Apidae)

A series of experiments was performed to assess possible roles of the honey bee Dufour gland secretion. Bioassays with extracts of queen and worker glands from two colonies were made under artificial conditions, in which nestmate and non-nestmate forager workers were tested. The results demonstrate that forager workers display behavioral responses when exposed to Dufour gland extracts of nestmates, but remain indifferent when exposed to non-nestmate extracts. Also, the results demonstrate that forager workers are attracted by virgin queen gland extracts, and repelled by forager worker extracts. The data demonstrate that the Dufour gland secretion is colony- and caste-specific. The attractant remains in the nest. The repellent effect of forager worker extracts is interpreted as an alarm-like pheromone. The attractant effect of virgin queen extracts could be useful in the swarming process to attract scout bees to the eventual founder virgin queen or to attract old foragers to the virgin queen that remains in the nest.

Morphological modifications induced by an artificial diet on the hypopharyngeal glands of Apis mellifera (Hymenoptera, apidae) during their degenerative process

The morphological aspects of the hypopharyngeal glands were analyzed in worker bees of Apis mellifera of 15 and 30 days of age. The individuals were kept in a room with controlled temperature at 32degreesC where they received water and either a protein or a high energy food. Nurse and foraging workers were used as a control for the experiment. The morphological results showed that the different diets modified the cell death characteristics and intensified its occurrence. Both diets caused precocious glandular degeneration. However, this anticipation of cell death was more pronounced in the glandular tissue of the workers who received the high energy diet when compared to the glands of the bees fed with the protein meal.The degenerative signs observed were an intense cytoplasmic vacuolization, with a loss of cytoplasm and of the cell boundaries, dilation or condensation of the cells and nuclei, and nuclear fragmentation. At the end of the degenerative process, we observed the extrusion of nuclei and, finally, the dissolution of the glands. The hypopharyngeal glands' remains were found in the haemolymph.

Toxicity of barbatimao to Apis mellifera and Scaptotrigona postica, under laboratory conditions

Two species of plants commonly known as barbatim (a) over tildeo occur in Brazil, Stryphnodendron adstringens (Fabaceae) (true barbatim (a) over tildeo) and Dimorphandra mollis (Caesalpiniaceae) (false barbatim (a) over tildeo). These two species have a similar flowering period and are considered by beekeepers to cause bee mortality during this period. Flowers were collected from both species, dehydrated, ground and incorporated into an experimental diet for bees of two different species, Apis mellifera and Scaptotrigona postica. Both plant species were toxic to A. mellifera, reducing their median survival. D. mollis was toxic to S. postica, and Stryphnodendron adstringens reduced median survival of this bee species even when used at a concentration of 2.5%. In a choice experiment carried out with A. mellifera and the two plant species, the honey bees could choose not to feed on the diets containing the flowers, and feed on sugar and honey instead, but they did not. This shows us that the flowers of S. adstringens were not repellent to the bees. The plants were more toxic to A. mellifera than to Scaptotrigona postica, a result that can be explained by the fact that A. mellifera was introduced into Brazil whereas S. postica is a native stingless bee.

Morphological features and occurrence of degenerative characteristics in the hypopharyngeal glands of the paper wasp Polistes versicolor (Olivier) (Hymenoptera : Vespidae)

Different histochemical techniques were applied to examine the morphological features of the secretory cells of hypopharyngeal glands in the wasp Polistes versicolor. The results showed that most analyzed individuals present active glands with secretion stored in the cytoplasm. In some glands, morphological analyses revealed the presence of degenerative characteristics. Analyses of cellular integrity, however, did not detect dead cells. The results showed that, in P. versicolor, the development and regression of the hypopharyngeal glands were not age related, unlike glands of social bees. (c) 2006 Elsevier Ltd. All rights reserved.

Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera L)

Different modes of cell death have been revealed in the regressing hypopharyngeal glands of worker honey bees. The hypopharyngeal gland, which is well developed in young nursing bees to produce protein for larval food, was seen to regress naturally in foraging adult worker bees. A range of techniques including histology, cytochemistry, in situ TUNEL, Annexin V and Comet assays indicated that cells within the gland demonstrate progressive symptoms of apoptosis, necrosis and a vacuolar form of programmed cell death. The latter mode of cell death did not display chromatin margination, but was accompanied by an enhanced level of autophagic and hydrolytic activity in which a cytosolic source of acid phosphatase became manifest in the extra-cisternal spaces. Normal and annexin-positive cells were found to occur in the younger nursing bees, whilst necrosis and an aberrant vacuolar type of apoptosis predominated in the older foraging bees. The relevance of these results to the classification of programmed cell death is discussed. (C) 2000 Academic Press.

Enzymatic activity of hypopharyngeal gland extractsfrom workers of Apis mellifera (Hymenoptera, Apidae, Apinae)

This research presents a comparative study of enzymatic activity of the hypopharyngeal gland extracts from workers of Apis mellifera in three physiologic stages: newly emerged, nurse and forager workers, with the objective of contributing to the comprehension of the gland function. In order to determinate the enzymes present in the extracts, the Api Zym kit (Bio Merieux) was used to test the activity of 19 different enzymes. The enzymes found in larger amounts only in the hypopharyngeal glands from certain individuals were the following: in newly emerged workers, the N-acetyl-double down arrow-glucosaminidase that may be digesting the chitin of some food ingested by the bee; in forager workers, the acid phosphatase that is likely acting in authophagic processes, the a-glucosidase, in the processing of nectar into honey, and the double down arrow-glucosidases, in the pollen digestion.

Jelleines: a family of antimicrobial peptides from the Royal Jelly of honeybees (Apis mellifera)

Four antimicrobial peptides were purified from Royal Jelly of honeybees, by using reverse phase-HPLC and sequenced by using Q-Tof-MS/MS: PFKLSLHL-NH2 (Jelleine-I), TPFKLSLHL-NH2 (Jelleine-II), EPFKLSLHL-NH2 (Jelleine-III), and TPFKLSLH-NH2 (Jelleine-IV). The peptides were synthesized on-solid phase, purified and submitted to different biological assays: antimicrobial activity, mast cell degranulating activity and hemolysis. The Jelleines-I-III presented exclusively antimicrobial activities against yeast, Gram+ and Gram- bacteria; meanwhile, Jelleine-IV was not active in none of the assays performed. These peptides do not present any similarity with the other antimicrobial peptides from the honeybees; they are produced constitutively by the workers and secreted into Royal Jelly. (C) 2004 Elsevier B.V. All rights reserved.

Ocorrência e morfologia de glândulas tegumentares no abdome de algumas abelhas (Hymenoptera: Apidae): um estudo comparado

No presente trabalho foi estudada a ocorrência, distribuição e morfologia de glândulas tegumentares do abdome em Exomalopsis auropilosa Spinola, Centris fuscata Lepeletier, Epicharis flava Friese e Xylocopa (Neoxylocopa) suspecta Moure & Camargo. Os resultados mostraram a ocorrência de glândulas epiteliais (classe I) e unicelulares (classe III) com distribuição dorsal (tergais) e ventral (esternais) sem que se pudesse caracterizar um padrão relacionável com a posição filogenética ou grau de sociabilidade. No entanto, verificou-se uma tendência para maior número de glândulas em espécies com algum grau de sociabilidade como E. auropilosa e X. suspecta.

Tegumentary epithelial glands in the abdomen of virgin and physogastric queens of the stingless bee Scaptotrigona postica Latreille (Meliponini: Trigonina)

Nas operárias das abelhas eussociais, regiões do epitélio tegumentar do abdome podem hipertrofiar-se e tornarem-se glandulares, sendo responsáveis pela produção de cera usada na contrução dos alvéolos de cria. Nos meliponíneos, epitélio com essas características também foi observado nas rainhas, porém sua função ainda continua desconhecida. Teria esse epitélio função homóloga nas operárias? As rainhas, além da função reprodutiva, desempenhariam funções na colônia até então consideradas exclusivas das operárias? Para tentar colaborar no esclarecimento dessas questões realizou-se um estudo histológico e ultra-estrutural das glândulas epiteliais tegumentares do terceiro tergito em rainhas virgens e fisogástricas de Scaptotrigona postica Latreille. Os resultados morfológicos mostraram que o epitélio glandular existe e é facilmente discernível no terceiro tergito das rainhas, sendo muito mais desenvolvido nas fisogástricas do que nas virgens. A ultra-estrutura mostrou que há diferenças no tipo de organização do retículo endoplasmático liso entre as rainhas, o que, juntamente com os resultados da histologia e morfometria, indicam poder haver diferenças funcionais desse epitélio entre elas. Como já observado na literatura, nas rainhas virgens tal epitélio pode estar envolvido na produção de cera e confecção de alvéolos de cria, porém sua função nas rainhas fisogástricas é totalmente desconhecida. A hipótese lançada no presente estudo é de que esse epitélio pode estar envolvido na produção de feromônios que irão atuar nas interações sociais da rainha poedeira.

Ocorrência, morfologia e ultra-estrutura da glândula de Dufour de Scaptotrigona postica Latreille (Hymenoptera: Apidae)

A glândula de Dufour é uma glândula acessória do aparelho reprodutivo feminino das abelhas. Nas abelhas neotropicais sem ferrão, tem sido pouca estudada sob todos os aspectos: morfológico, ontogenético e bioquímico. Na tentativa de colaborar com o conhecimento dessa glândula em abelhas sem ferrão, foi realizado um estudo da sua ocorrência, morfologia e desenvolvimento em Scaptotrigona postica Latreille. Os resultados mostraram que ela se encontra ausente nas operárias, como ocorre em muitas outras espécies desse grupo. Nas rainhas, as células glandulares parecem mais ativas nas virgens, possuindo uma desenvolvida rede de retículo endoplasmático liso tubular, grânulos de secreção e polirribossomos dispersos no citoplasma, além de apresentarem núcleos maiores do que os das células glandulares das fisogástricas. Nas rainhas fisogástricas há dois tipos de células glandulares, ambas aparentemente inativas sinteticamente. As glândulas das rainhas fisogástricas são claramente capazes de captar substâncias da hemolinfa, provavelmente lipídios, que não penetram nas células, mas passam pelos espaços intercelulares e, através da cutícula, chegam diretamente à luz da glândula. A bem desenvolvida dupla camada de lâmina basal ao redor da glândula pode atuar no processo de captação de substâncias da hemolinfa. A secreção, e conseqüentemente sua função, pode ser diferente nas duas classes de rainhas.

Dufour glands in the hymenopterans (Apidae, Formicidae, Vespidae): a review

Anexas ao aparelho do ferrão dos himenópteros aculeados encontram-se as glândulas de veneno e as de Dufour. A glândula de veneno é originada das glândulas associadas ao ovopositor dos himenópteros ancestrais não aculeados, já a glândula de Dufour é menos derivada, homóloga das glândulas colateriais dos outros insetos, sendo encontrada em todas as fêmeas dos himenópteros. Nestes insetos sua função é, em grande parte, desconhecida, mas, em formigas, parece estar envolvida com a comunicação e a defesa e, nas abelhas não sociais, com a construção e a proteção do ninho. Nas vespas pode estar relacionada ao reconhecimento parental. Foram observadas diferenças morfológicas e na composição química da secreção da glândula de Dufour entre as espécies, bem como na mesma espécie, entre as castas dos himenópteros sociais e entre indivíduos da mesma casta desempenhando diferentes funções ou pertencentes a ninhos diferentes. Portanto, nos himenópteros, sua função original de produzir substâncias para proteger os ovos ou favorecer a ovoposição parece ter sido substituída ou complementada com a função de produzir semioquímicos com função na comunicação.

Nuclear alterations associated to programmed cell death in larval salivary glands of Apis mellifera (Hymenoptera : Apidae)

The silk glands of bees are a good model for the study of cell death in insects. With the objective to detect the nuclear features during glandular regression stage, larvae at the last instar and pre-pupae were collected and their silk glands were dissected and processed for ultrastructural analysis and histologically for cytochemical and imunocytochemical analysis. The results showed that the cellular nuclei exhibited characteristics of death by atypical apoptosis as well as autophagic cell death. Among the apoptosis characteristic were: nuclear strangulation with bleb formation in some nuclei, DNA fragmentation in most of the nuclei and nucleolar fragmentation. Centripetal chromatin compaction was observed in many nuclei, forming a perichromatin halo differing from typical apoptotic nuclei. With regards to the characteristics of autophagic-programmed cell death, most relevant was the delay in the collapse of many nuclei. (C) 2006 Elsevier Ltd. All rights reserved.