Field populations of native Indian honey bees from pesticide intensive agricultural landscape show signs of impaired olfaction.

Little information is available regarding the adverse effects of pesticides on natural honey bee populations. This study highlights the detrimental effects of pesticides on honey bee olfaction through behavioural studies, scanning electron microscopic imaging of antennal sensillae and confocal microscopic studies of honey bee brains for calcium ions on Apis cerana, a native Indian honey bee species. There was a significant decrease in proboscis extension response and biologically active free calcium ions and adverse changes in antennal sensillae in pesticide exposed field honey bee populations compared to morphometrically similar honey bees sampled from low/no pesticide sites. Controlled laboratory experiments corroborated these findings. This study reports for the first time the changes in antennal sensillae, expression of Calpain 1(an important calcium binding protein) and resting state free calcium in brains of honey bees exposed to pesticide stress.

Biodegradation of profenofos by Bacillus subtilis isolated from grapevines (Vitis vinifera).

The biodegradation of profenofos, an organophosphorus insecticide, by four Bacillus subtilis strains, namely, DR-39, CS-126, TL-171, and TS-204, isolated from grapevines or grape rhizosphere was studied in liquid culture, on grape berries, and in vineyard soil. Each of the four B. subtilis strains enhanced the degradation of profenofos in all three matrices. Degradation rate constants were best obtained by first + first-order kinetics module. In nutrient broth spiked with 5 µg/mL profenofos, inoculation with B. subtilis strains DR-39, CS-126, TL-171, and TS-204 reduced the half-life (DT50) of profenofos to 4.03, 3.57, 2.87, and 2.53 days, respectively, from the DT50 = 12.90 days observed in the uninoculated control. In Thompson Seedless grapes sprayed with profenofos at a field dose of 1250 mL ai/ha, the DT50 values were 1.07, 1.00, 2.13, and 2.20 days in grapes inoculated with B. subtilis strains DR-39, CS-126, TL-171, and TS-204, respectively, as compared to 2.20 days in uninoculated grapes. These four B. subtilis strains also enhanced the degradation of profenofos in autoclaved soil (DT50 = 5.93, 7.47, 6.00, and 4.37 days) and in nonautoclaved soil (DT50 = 0.87, 2.00, 2.07, and 2.43 days) amended with 5 µg/g profenofos from the half-lives of 17.37 and 14.37 days in respective uninoculated soils. Growth dynamic studies indicated that all four B. subtilis strains were able to establish and proliferate on berries and soil equally well in the presence or absence of profenofos. Degradation product 4-bromo-2-chlorophenol was identified by GC-MS. Strain DR-39 was most effective in the natural environments of grape and soil.

Essential oil of Indian propolis: chemical composition and repellency against the honeybee Apis florea.

Hitherto unknown biological properties and the chemical composition of the essential oil isolated from propolis of Indian origin were established. GC/MS Analysis of the essential oil revealed the presence of 32 constituents, of which ten were major compounds, nine had intermediate contents, and 13 were minor compounds. With the exception of six minor constituents, that could not be identified, their identification was based on the comparison of their mass spectra and Kovats retention indices with those listed in the NIST and Wiley mass spectral libraries. Their structural assignment was confirmed by GC/MS co-injection of the essential oil with authentic compounds. Quantification of the components was done by GC-FID analyses. Moreover, the essential oil was shown to possess repellent activity against the honeybee Apis florea. The activity was found to be dose dependent. The average repellency (ΔR) increased with increasing essential-oil concentration up to 24 µg/ml and remained constant for the formulation with the higher concentration. These findings established the chemical constitution of the essential oil and might be useful to beekeepers for the improvement of the bee management.

Multiresidue analysis of multiclass plant growth regulators in grapes by liquid chromatography/tandem mass spectrometry.

A selective and rapid multiresidue analysis method is presented for simultaneous estimation of 12 plant growth regulators (PGRs), namely, auxins (indol-3-acetic acid, indol-3-butyric acid, and naphthyl acetic acid), cytokinins (kinetin, zeatin, and 6-benzyladenine), gibberellic acid (GA3), abscisic acid, and synthetic compounds, namely, forchlorfenuron, paclobutrazole, isoprothiolane, and 2,4-dichlorophenoxy acetic acid (2,4-D) in bud sprouts and grape berries at the development stages of 2-3 and 6-8 mm diameters, which are the critical phases when exogenous application of PGRs may be necessary to achieve desired grape quality and yield. The sample preparation method involved extraction of plant material with acidified methanol (50%) by homogenization for 2 min at 15000 rpm. The pH of the extract was enhanced up to 6 by adding ammonium acetate, followed by homogenization and centrifugation. The supernatant extract was cleaned by SPE on an Oasis HLB cartridge (200 mg, 6 cc). The final extract was measured directly by LC/MS/MS with electrospray ionization in positive mode, except for 2,4-D, GA3, and abscisic acid extracts, which required analysis in negative mode. Quantification by multiple reaction monitoring (MRM) was supported with full-scan mass spectrometric confirmation using "information-dependent acquisition" triggered with MRM to "enhanced product ionization" mode of the hybrid quadrupole-ion trap mass analyzer. The LOQ of the test analytes varied between 1 and 10 ng/g with associated recoveries of 80-120% and precision RSD <25% (n = 8). Significant matrix-induced signal suppression was recorded when the responses for pre- and postextraction spikes of analytes were compared; this could be resolved by using matrix-matched calibration standards. The method could successfully be applied in analyzing incurred residue samples and would, therefore, be useful in precisely deciding the necessity and dose of exogenous applications of PGRs on the basis of measured endogenous levels.

Essential oil of Terminalia chebula fruits as a repellent for the indian honeybee Apis florea.

Plant-based repellent formulations for honeybees play an important role in the bee management. For this purpose, the essential oil of an Indian medicinal plant, Terminalia chebula, commonly known as Myrobalan, was isolated for the first time. Hitherto unknown chemical constituents of the essential oil were determined by GC/MS. The repellent activity of formulations of the essential oil, tested towards the Indian honeybee Apis florea, was found to be dose dependent. The repellency (DeltaR) increased with the concentration of essential oil in the formulations, reaching a maximum for the formulation containing 50 mg/ml of oil. A further increase in the oil concentration was found to reduce the DeltaR. The screening of formulations of the major essential-oil components identified indicated that formulations of furfural, 5-methylfurfural, tetradecanoic acid, palmitic acid, and oleic acid elicited no response in honeybees. In contrast, the formulations of phenylacetaldehyde were repellent, while those of ethyl cinnamate were attractant. These findings might be an asset for beekeepers to improve the bee management. Attractant formulations are effective to attract bees to the desired areas, thus improving the efficiency of pollination. Repellent formulations are used to repel honeybees, especially when toxic insecticides are sprayed on the fields.

Regulation of reproduction in the primitively eusocial wasp Ropalidia marginata: on the trail of the queen pheromone.

Queens and workers are not morphologically differentiated in the primitively eusocial wasp, Ropalidia marginata. Upon removal of the queen, one of the workers becomes extremely aggressive, but immediately drops her aggression if the queen is returned. If the queen is not returned, this hyper-aggressive individual, the potential queen (PQ), will develop her ovaries, lose her hyper-aggression, and become the next colony queen. Because of the non-aggressive nature of the queen, and because the PQ loses her aggression by the time she starts laying eggs, we hypothesized that regulation of worker reproduction in R. marginata is mediated by pheromones rather than by physical aggression. Based on the immediate loss of aggression by the PQ upon return of the queen, we developed a bioassay to test whether the queen's Dufour's gland is, at least, one of the sources of the queen pheromone. Macerates of the queen's Dufour's gland, but not that of the worker's Dufour's gland, mimic the queen in making the PQ decrease her aggression. We also correctly distinguished queens and workers of R. marginata nests by a discriminant function analysis based on the chemical composition of their respective Dufour's glands.

A fast, inexpensive, and safe method for residue analysis of meptyldinocap in different fruits by liquid chromatography/tandem mass spectrometry.

An analytical method is reported for residue analysis of the fungicide meptyldinocap in different fruit matrixes that involves extraction with ethyl acetate, hydrolysis of the residues with ethanolamine, and determination by LC/MS/MS. The method involves extraction of 10 g sample with 10 mL ethyl acetate; evaporation of the ethyl acetate phase to dryness, and subsequent hydrolysis of the residues to 4,6-dinitro-2-(1-methylheptyl) phenol on reaction with 1% ethanolamine. The pH of this hydrolyzed product was neutralized with formic acid and analyzed by LC/MS/MS. The hydrolysis reaction followed pseudo-first-order kinetics, and the reaction product was spectroscopically confirmed as 2-(1-methylheptyl)-4,6-dinitrophenol. The method offered > 80% recoveries at an LOQ of 10 ng/g for grape and mango, 25 ng/g for pomegranate with intralaboratory Horwitz ratio < 0.5, and measurement uncertainties < 10% at LOQ levels. Considering first-order rate kinetics, activation energy, enthalpy of activation, and entropy of activation varied as solvent > mango > grape > pomegranate. Free energy of activation at 298 K was higher than at 280 K and was similar for solvent and three matrixes at both temperatures.