Supercritical fluid extraction for pesticide multiresidue analysis in honey: determination by gas chromatography with electron-capture and mass spectrometry detection

An analytical procedure using supercritical fluid extraction (SFE) and capillary gas chromatography with electron-capture detection was developed to determine simultaneously residues of different pesticides (organochlorine, organophosphorus, organonitrogen and pyrethroid) in honey samples. Fortification experiments were conducted to test conventional extraction (liquid-liquid) and optimize the extraction procedure in SFE by varying the CO2-modifier, temperature, extraction time and pressure. Best efficiency was achieved at 400 bar using acetonitrile as modifier at 90 degreesC. For the clean-up step, Florisil cartridges were used for both methods LLE and SFE. Recoveries for majority of pesticides from fortified samples of honey at fortification level of 0.01-0.10 mg/kg ranged 75-94% from both methods. Limits of detection found were less than 0.01 mg/kg for ECD and confirmation of pesticide identity was performed by gas chromatography-mass spectrometry in selected-ion monitoring mode. The multiresidue methods in real honey samples were applied and the results of developed methods were compared. (C) 2004 Elsevier B.V. All rights reserved.

Orchid bee baits attracting bees of the genus Megalopta (Hymenoptera, Halictidae) in Bauru region, São Paulo, Brazil: abundance, seasonality, and the importance of odors for dim-light bees

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Multiresidue determination of pesticides in honey samples by gas chromatography-mass spectrometry and application in environmental contamination

A simple and fast multiresidue method has been developed to determine 48 pesticides within the major groups of pesticides (organohalogen, organophosphorous, pyrethroids and organonitrogen) in representative samples of locally produced honey, in Bauru (State of São Paulo, Brazil) during 2003-2004. The recovery results found ranged from 76% to 95% and the limits of detection were lower than 0.01 mg/kg for gas chromatography with electron impact mass spectrometric detection in the selected ion monitoring mode (GC-MS-SIM). The results indicated that most pesticides found in the samples belonged to the organohalogen and organophosphorous groups and lower levels of residues of some organonitrogen and pyretroids were also detected. Malathion residues were detected in all the samples, in a high concentration, owing to its applications to control dengue mosquitoes in the area studied. (c) 2005 Elsevier Ltd. All rights reserved.

Honey consumption in the state of São Paulo: a risk to human health?

Infantile botulism was recognized in 1976 as a paralyzing disease caused by the ingestion of viable spores that would germinate and colonize the intestinal tract of infants, with local production and absorption of Clostridium botulinum toxin. The possible origins of botulinic spores are dust and honey, which has been identified as a dietary risk factor for infantile botulism. The objectives of the present study were to investigate 100 honey samples obtained in the state of São Paulo (Brazil) in terms of incidence of botulinic spores and of microbiologic quality, in agreement with Decree 367/9. All 100 samples analysed were negative for the presence of Salmonella, Shigella, total coliforms. C botulinum spores were present in 3 samples (3%) and molds and yeasts, in 64 samples (64%), but only 25 (25%) exceeded established criteria, with counts ranging from zero to 1.5 x 10(5) CFU/g. The presence of small sporogenic Gram-positive rods was observed in 42 (42%) of the 100 samples tested but these bacteria were not identified. (C) 2003 Elsevier Ltd. All rights reserved.

Changes in the morphology and ultrastructure of the Dufour's gland during the life cycle of the bumble bee queen, Bombus terrestris L-(Hymenoptera : Bombini)

The Dufour's gland is found closely associated with the sting apparatus of all female hymenopterans, playing multiple roles among bees. In some species of Bombus the gland may be involved in production of nestmate recognition pheromones, but in B. terrestris its function is not certain yet. The morphology of the :Dufour's gland of B. terrestris queens and the ultrastructural features of its cells were studied in different ages and behavioural stages using routine transmission electron microscopy. Measurements of the length and the diameter of the gland in the same conditions were also made. The Dufour's gland of the queen increases significantly in size (both in length and in diameter) with age and reproductive activity the ultrastructural features of the gland show electrondense material that comes from the haemolymph. This material is also present in the intercellular spaces, and is conducted to the subcuticular space, to be released directly into the glandular lumen. Hence at least part of the secretion is probably taken up directly from the haemolymph. The ultrastructural features indicate a more active phase of the gland corresponding to the period of egg-laying of the queen, and a decrease in activity when the queen is in hibernation as well as after the competition point. In conclusion, the gland is probably involved in reproduction, more specifically, in the marking off eggs.

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.

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.

Secretory cycle of the Dufour's gland in workers of the bumble bee Bombus terrestris L-(Hymenoptera : Apidae, Bombini)

The Dufour's gland of Bombus terrestris workers, of different ages and with varying degrees of ovary development, was studied with the aim to verify its involvement in reproduction. Measurements of the diameter and the length of the gland were made using an ocular micrometer adapted to a microscope. Transmission electron microscopy was used to study the morphology of the secretory cells and to analyze the secretory cycle. The glandular cells were considered to be near type II cells of NOIROT and QUENNEDEY (1974), a type that has not been described before in Hymenopterans. The results show that there is a correlation between the degree of ovary development and Dufour's gland activity of workers. The diameter of the gland and the secretory cell activity increased with increasing oocyte size in the ovary. Regressive conditions of the gland were observed, which are probably related to increasing worker age. To elucidate the production and releasing process of the secretion and to establish its precise function, a comparative analysis of the secretion process of the Dufour's gland of queens and workers is needed.

Ovarian ultrastructure in virgin queens of Apis mellifera L. narcotized by CO2

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

Bee Visitors of Quaresmeira Flowers (Tibouchina granulosa Cogn.) in the Region of Dourados (MS-Brasil)

This research was developed in the center-urban area at the city of Dourados (MS), in Tibouchina granulosa trees, a plant popularly known as quaresmeira in Brazil. The floral visitors of this species were recorded in three daily periods: 7 - 8 a.m., 1 - 2 p.m. and 4 - 5 p. m. Bee visitors of those plants were collected with an entomological net when they landed on the flowers. The bees were anesthetized in a closed camera containing etila acetate, conserved in Dietrich ' s fixative and, soon afterwards, transferred to 70% ethyl alcohol for subsequent identification. Almost 300 specimens of Africanized Apis mellifera, Trigona spinipes and Tetragonisca angustula were collected. The presence of these three species of bees in the flowers of T. granulosa suggests that they can be considered the main floral visitors of that vegetal species. T. angustula workers did not visit the flowers of T. granulosa between 7 and 8 a.m., but they were the main floral visitors from 4 to 5 p.m. The workers of T. spinipes presented a very aggressive behavior against the Africanized A. mellifera workers on the quaresmeira flowers, defending the food sources with strength and efficiency and provoking a drastic reduction in the number of honeybee visits between 1 and 2 p.m. Nevertheless, they did not interfere in the opportunistic activity of foraging by T. angustula workers, mainly between 4 and 5 p.m. The most important result of this research was the detection of the Competitive Exclusion Principle between A. mellifera and T. spinipes species in relation to the foraging behavior, when they exploit the floral resources of T. granulosa.

A comparative cytochemical study of the dufour gland in the eusocial bee Apis mellifera Linne, 1758 and Melipona bicolor Lepeletier, 1836

Dufour glands of Apis mellifera and Melipona bicolor were studied under light and transmission electron microscopy, using the cytochemical techniques of mercury bromophenol blue for protein detection, imidazole-buffered osmium tetroxide selective staining of unsaturated lipids, lanthanum nitrate for intercellular junction identification and zinc-iodide-osmium tetroxide for cytoplasmic endomembrane visualization. The results in both species corroborated the lipid nature of the gland secretion and showed in A. mellifera the poverty of the synthetic machinery in the worker gland cells in comparison with the queen, as expected by previous biochemical analyses. The pathway of the exogenous compounds of the secretion is intracellular, since substances can penetrate the cell folds and intercellular junctions, but their access to the, gland lumen is barred by the apical intercellular junctions.

Influence of the insecticide pyriproxyfen on the flight muscle differentiation of Apis mellifera (Hymenoptera, Apidae)

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

Ultrastructural aspects of the intercellular bridges between female bee germ cells

No ovário das abelhas as células germinativas e as células foliculares são interconectadas por pontes intercelulares mantidas abertas por reforços do citoesqueleto na membrana plasmática. As pontes entre as células germinativas têm comportamento dinâmico e provavelmente atuam na determinação do ovócito entre as células do clone formado pelas mitoses pré meióticas formando posteriormente uma via de transporte para que os produtos sintetizados pelas células nutridoras atinjam o ovócito durante sua maturação. Os elementos do citoesqueleto presentes nas pontes intercelulares das gônadas das abelhas são basicamente microfilamentos e microtúbulos, mas nas pontes entre os cistócitos pré-meióticos outro tipo de filamento (espesso de natureza não definida, associado a elementos do retículo endoplasmático) está presente, atravessando a ponte e prendendo-se através dos microfilamentos à membrana plasmática. Estes filamentos aparentemente controlam o vão da ponte. Terminada a fase de proliferação os cistócitos tomam a forma de uma roseta e um fusoma, formado pela convergência das pontes, aparece no centro desta. Nesta conformação os filamentos grossos não estão presentes. Nova mudança ocorre com a diferenciação do ovócito e das células nutridoras, com a reorientação de todas as pontes de maneira a canalizar o conteúdo das futuras células nutridoras para o ovócito.

Determinação das castas em Scaptotrigona postica (Latreille) (Hymenoptera, Apidae, Meliponini): diferenciação do ovário

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Metabolic, hygric and ventilatory physiology of a hypermetabolic marsupial, the honey possum (Tarsipes rostratus)

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