Temporal And Spatial Expression Of Acetylcholinesterase In The Bumblebee Bombus Hypocrita (Hymenoptera: Apidae)

Bumblebee is one of the most crucial pollinators for the endangered, alpine plants and greenhousecrops. Bombus hypocrita （Hymenoptera: Apidae） is one of the dominant bumblebees in China, and iswidely used as one of the most crucial pollinators in greenhouse due to easy mass-rearing, strongpopulation and effective pollinating performance. However, the application of insecticides wasconsidered as one of the most reseaons for declining of the bumblebees. Organophosphate andcarbamate insecticides are the most widely used pesticides, which taget in acetylcholinesterase （AChE）.In order to properly protect and utilize bumblebee, we studied AChE in Bombus hypocrita. We usedenzyme activity determination and real time PCR to indentify the spatial and temporal expressioncharacteristics of AChE in B. hypocrita. In addition, the inhibition and toxicities of severalorganophosphate and carbamate insecticides on B. hypocrita were conducted by in vitro inhibiton assaysand bioassays.The results are as follows:（1） Optimization of conditions for assaying activity of AChE in B. hypocrita. The results showed thatenzyme concentration0.25g protein/L, substrate concentration0.8mmol/L, pH7.5, reactiontemperature40℃, and reaction time5min were the optimal conditions. The five factors allextremely significantly impact the assaying of AChE in the head of B. hypocrita. The effect sizeswith the order are enzyme concentration, pH, temperature, substrate concentration and reactiontime.（2） The distribution of AChE activity and ace2gene mRNA relative expression were conducted for thespatial expression characteristics. The tissue distribution results showed that the head AChEactivity （63.55%） was significantly higher than the thorax （18.44%） and abdomen （18.01%）.Further study presented that the AChE in the head subcellular distribution of AChE activity wasmainly located in the mitochondrial layer, followed by the microsomal layer, and the solution ofcytoplasm and nucleus and cell debris layer. In addition, the results of the ace2（GenBankaccession: JQ359006） relative expression for the different tissues showed that the head ace2relative expression level was the highest. The thorax ace2expression was52%of the head,abdomen14%of the head. These results suggest that AChE is mainly distributed in the nervoussystem developed in the head of B. hypocrita, expecially in the subcellular membrane organelles.The specific activity of AChE and ace2gene relative expression of different developmentalstages showed the temporal characteristics. The results showed during the developmental stages,AChE activity for larvae was the lowest and began to increase after emergence. During thedevelopmental stages, AChE activity for5thday,20thday and30thday were higher than others. Onthe other hand, AChE mRNA was detected lowly in the larvae and pupae stage of the workers byreal-time PCR method. The highest expression level was in the20thday old adult workers,following by the10th,5th, and25th. We make conclusion that the adult stage had the highest AChE specific activity and the highest ace2mRNA expression during the development of B. hypocrita,but no significant difference was found among the different ages of adults from0thday ofemergence to the30thday, which means that no significance were found between the foragers andnurses of B. hypocrita.（3） In vitro inhibition assays showed that the inhibition concentration (IC₅₀) of phoxim, triazophos,propoxur and profenofos in turn weakened and the inhibiton were enhanced. In addition, bioassaysshowed that the lethal concentration (LC₅₀) of these four insecticides was enhanced and thetoxicities were weakened. After exposure to the sublethal dose of the four insecticides, the AChEactivities of the heads in B. hypocrita decreased compared to the control groups. We makeconclusion that organophosphate and carbamate insecticides on B. hypocrita have a significantdose-response relationship, and the trends of LC₅₀and IC₅₀are similar.