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Hu Y.-Y.,Chinese Academy of Agricultural Sciences | Xu S.-F.,Chinese Academy of Agricultural Sciences | Wubie A.J.,Chinese Academy of Agricultural Sciences | Wubie A.J.,Institute of Amhara Agricultural Research | And 4 more authors.
Applied Entomology and Zoology | Year: 2013

Sensory neuron membrane protein (SNMP) is an olfactory receptor with photoaffinity analogs, capable of binding the pheromone membrane protein receptor deduced from receptor membrane protein with the pheromone-pheromone binding protein complex. However, this hypothesis has not yet been experimentally verified. In this experiment, the cDNA sequence encoding an open reading frame (ORF) of the SNMP gene AccSNMP1 (GenBank, KC012595) was cloned from Chinese honey bee, Apis cerana cerana Fabricius. Results from sequence analysis showed that this gene is 1,563 bp long, and that the ORF encodes 520 amino acids with a predicted molecular weight of 58.02 kDa, and has a theoretical isoelectric point of 5.83. Furthermore, there are two putative transmembrane domains. Multiple sequence alignment indicated that the AccSNMP1 gene from A. cerana cerana had different degrees of identity with the corresponding genes in nineteen other insects at the amino acid level. Phylogenetic analysis of the aligned sequences showed that A. cerana cerana is closely related to Apis mellifera Linnaeus and Bombus impatiens Cresson. Its distribution in tissues, as quantified using real-time RT-PCR, indicated that AccSNMP1 is highly expressed in the antennae and legs of A. cerana cerana, and there was a significant difference (p < 0.05) in gene expression between those tissues and tissues in the thorax, abdomen, snout, and head (not including antennae). Western blotting also confirmed the existence in the antennae of AccSNMP1 with an M W of 58.0 kDa, which is the same as the expected value of 58.02 kDa. An immunohistochemistry study showed that AccSNMP1 is expressed in the trichoid sensilla of A. cerana cerana antenna. Therefore, the results of this study provide the basis for further studies of the function of SNMP from A. cerana cerana. © 2013 The Japanese Society of Applied Entomology and Zoology.

He X.,Chinese Academy of Agricultural Sciences | Wubie A.J.,Chinese Academy of Agricultural Sciences | Wubie A.J.,Institute of Amhara Agricultural Research | Diao Q.,Chinese Academy of Agricultural Sciences | And 5 more authors.
Chemosphere | Year: 2014

REMI (restriction enzyme-mediated integration) technique was employed to construct Trichoderma atroviride strain T23 mutants with degrading capability of neonicotinoid insecticide, imidacloprid. The plasmid pBluescript II KS- hph used for integration in REMI mutants was confirmed by PCR and Southern hybridization. Among 153 transformants, 57% of them have showed higher neonicotinoid insecticide, imidacloprid, degradation ability than the wild strain T23 (p<. 0.01). More specifically, seven single-copied T. atroviride T23 transformants have confirmed a 30% higher degradation rate than their parent isolate. Among all transformed mutants, a 95% imidacloprid degradation rate was identified as the highest. This study, thus, provided an effective approach for improving neonicotinoid insecticide-degrading capability using REMI transformed T. atroviride mutants. © 2014 Elsevier Ltd.

Wubie A.J.,Chinese Academy of Agricultural Sciences | Wubie A.J.,Institute of Amhara Agricultural Research | Xu S.,Chinese Academy of Agricultural Sciences | Hu Y.,Chinese Academy of Agricultural Sciences | And 4 more authors.
Journal of Food, Agriculture and Environment | Year: 2013

Chalkbrood is an invasive mycosis in honeybees caused by Ascosphaera apis (Maasen ex Claussen) L. S. Olive & Spiltoir, that exclusively affects honeybee brood. The disease causes about 5 - 37% reduction in honey production in the world and 80% brood death. The severity of the disease, these days, has increased pesticide use and frequent long-distance transportation of colonies which may have provided an additional opportunity for A. apis infection. Furthermore, the fact that A. apis is so widespread in the globe and its viable spores can be found in stored honey, pollen, pollen capsules/ tablets, used hive components, used beekeeping tools and equipments, and possibly in soil around infected apiaries for more than 15 years, makes the possibility of its eradication most unlikely. Even though management strategies, chemicals and the use of selected chalkbrood resistant bees have all been shown to have some benefits so far, safe control of the disease have been noticed to be difficult. Thus, genetic engineering works for better manipulation of the fungus, molecular investigation of pathogenesis pathways of A. apis, and possible identification of strategies to break the pathway through the identification of responsible genes for its pathogenesis shall take current issue of research for better understanding of chalkbrood.

Xue F.,Chinese Academy of Agricultural Sciences | Li W.,Chinese Academy of Agricultural Sciences | Wubie A.J.,Chinese Academy of Agricultural Sciences | Wubie A.J.,Institute of Amhara Agricultural Research | And 4 more authors.
Biological Control | Year: 2015

Ascosphaera apis (Onygenales: Ascosphaeraceae) is a chalkbrood causing pathogen in honeybees. Recently, chemicals have been used against this disease. Consequently, the abuse of chemicals has caused serious impacts on the environment and honey quality and on honeybee colonies themselves. Therefore, the search for biological control options should be the priority for controlling chalkbrood disease. In this experiment, we employed restriction enzyme mediated integration (REMI) to construct mutants (transformants) with growth inhibition on A. apis in honeybees using the Trichoderma atroviride T23 wild strain. The integration of the plasmid pBluescript KS-EH1 into the REMI mutants was confirmed by PCR and Southern hybridization. As a result, ten single-copied T. atroviride T23 transformants were successfully identified. Furthermore, culture filtrates of the transformants, their wild strain and Shabaijing (a commercial fungicide, ethyl ethylsulfonothiolate) were tested to compare their in vitro growth inhibition capability on A. apis. The results demonstrated that transformants Tat5 and Tat6 had the highest growth inhibition activity with the minimal fungicidal concentration (v/v) of 3.5-4.5%. Thus, the Trichoderma transformants constructed by REMI could be used as potential biological agents against the growth of fungal diseases in bee hives. Further refinement and strategy development for the integrative use of this result are vital future plans. © 2015 Elsevier Inc.

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