Honey bees play an important role in the ecological environment. Regrettably, a decline in honey bee colonies caused by chemical insecticides has occurred throughout the world. Potential stereoselective toxicity of chiral insecticides may be a hidden source of danger to bee colonies. In this study, the stereoselective exposure risk and mechanism of malathion and its chiral metabolite malaoxon were investigated. The absolute configurations were identified using an electron circular dichroism (ECD) model. Ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for chiral separation. In pollen, the initial residues of malathion and malaoxon enantiomers were 3571-3619 and 397-402 mu g/kg, respectively, and R-malathion degraded relatively slowly. The oral LD50 values of R-malathion and S-malathion were 0.187 and 0.912 mu g/bee with 5 times difference, respectively, and the malaoxon values were 0.633 and 0.766 mu g/bee. The Pollen Hazard Quotient (PHQ) was used to evaluate exposure risk. R-malathion showed a higher risk. An analysis of the proteome, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and subcellular localization, indicated that energy metabolism and neurotransmitter transport were the main affected pathways. Our results provide a new scheme for the evaluation of the stereoselective exposure risk of chiral pesticides to honey bees.

Eragrostis japonica (Thunb.) Trin, a kind of grass weeds in paddy rice fields, has recently been developing rapidly as the most harmful weed for rice production in addition to Echinochloa crusgalli and Leptochloa chinensis in partial areas of Anhui, Jiangsu, and Zhejiang provinces (Zhou et al.). In September 2019, inflated sori in the ovaries of E. japonica were identified in fields in Chuzhou County, Anhui Province. The disease incidence was approximately 50% at the survey site. Smut sori were in some spikelets of infected inflorescence destroying the inner most floral organs. Sori were ovoid and initially covered with a thin peridium (Figure 1). The sori busted after maturation, and the black powdery spores spread to the plant and soil. The sori were crushed using the sterilized tweezers. The black powdery spores were suspended with sterilized water and spread onto potato dextrose agar (PDA) medium. Spores were covered with spines, and the shape of spores varied from globose to ovoid. Scanning electron microscope was used to observe the morphology of spores. The spores were 7.5-12.3 μm × 5.8-11.8 μm (n=50). The surface of spores was covered with spines, and dense verruca were identified between the spines (Figure 2). For molecular identification, the primers ITS1/ITS4 and GAPDH-F/R were employed to amplify the rDNA region. The resulting sequences from the studied material were submitted to Genbank (MW819938 and MZ508441). BLASTn analysis revealed that ITS sequence shared 99% similarity with EF040584 (719/728) the ITS sequence from type specimen of Ustilago planetella. Based on the above results, the pathogen was identified as Ustilago planetella (Vánky, 2007). To verify the Koch's postulates, a pathogenicity test was performed by infiltrating the inflorescence with microspore suspensions of the strain SMZF-2 (1×106 spores/mL) when the E. japonica flowers for 30 minutes. The plants were inoculated in a moist chamber (with a relative humidity >80%) with a 12-h light cycle at 28℃. Inflated sori were observed after inoculated for 2 weeks, and U. planetella was isolated and identified with the methods described above. No sori were observed in the water-inoculated control plants. U. planetella had been reported to infect Eragrostis japonica in Thailand (Vánky, 2007). To our knowledge, this is the first report of U. planetella causing Smut on E. japonica (Thunb.) Trin in central region of China. The current study may help solve the negative effect of E. japonica in paddy rice production via using U. planetella in the future.