The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this paper, we investigated the molecular structure, tissue distribution patterns, and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was detected by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of sequestration and hydrolysis of acetylcholine (ACh)/choline (Ch) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. Kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions as a sequestering enzyme specific to ACh (not to Ch) by having a very strong affinity to ACh but an extremely long turnover time.

We analyzed molecular and enzymatic properties of three cholinesterases (ChEs; ClAChE1, ClAChE2 and ClSChE) from Cimex lectularius. The ClAChE1 and ClAChE2 were generally present as a membrane-anchored dimeric insoluble form in the brain and ganglia. In the case of ClSChE, monomeric and dimeric soluble forms were observed. To investigate enzymatic properties, three ChEs were functionally expressed using baculovirus expression system. ClAChE1 revealed a significantly higher activity than ClAChE2 to acetylthiocholine iodide (ATChI) substrate. Kinetic analysis using two choline substrates (ATChI and butyrylthiocholine iodide) demonstrated that ClAChE2 had higher catalytic efficiency but lower substrate specificity than ClAChE1. Inhibition assay was conducted by using three inhibitors (BW284C51, eserine, Iso-OMPA) and two insecticides (chlorpyrifos-methyl and carbaryl). Two ClAChEs revealed high sensitivities to BW284C51, eserine, chlorpyrifos-methyl and carbaryl, but were not sensitive to Iso-OMPA. This inhibition profile confirmed that both ClAChEs are categorized as ChEs. Interestingly, the salivary specific cholinesterase did not show any measurable activities to choline substrates, confirming its non-synaptic function in C. lectularius

The impact of transgenic Bt maize plant contained Cry1F was evaluated on the oat aphid Rhopalosiphum padi as a non-target insect species. Slightly reduced rates of survival and alata vivipar production were observed on Bt maize than on the non-Bt maize. In addition, slightly low preference to Bt maize plant was observed. Aphid fecundity, measured as the number of offspring produced for 7 days, was higher on Bt maize than on non-Bt maize but not different significantly. ELISA test using Cry1F-antibody revealed that 26% of Cry1F protein compared to the positive control was detected from the whole body of R. padi when the insects were fed Bt maize for 50 days, showing that R. padi can carry Cry1F protein to the higher trophic level when exposed to Bt maize. Taken together, the Bt maize plant is not likely to cause any negative side impacts on non-target insect R. padi but Bt toxin can be transferred to higher predators via R. padi as it carries the toxin.

Western blot analysis using acetylcholinesterase (AChE)-specific antibody was conducted to determine whether AChE gene (Tuace) duplication actually results in overproduction of AChE in Tetranychus urticae (TuAChE). The protein quantities of TuAChE in seven field-collected mite populations were precisely correlated with the copy numbers. To investigate the effects of each mutation on AChE insensitivity and possible fitness cost, eight variants of TuAChE were in vitro expressed using the baculovirus expression system. Kinetic analysis revealed that the Ala391Thr mutation did not change kinetic properties of AChE, whereas the Gly228Ser and Phe439Trp mutations significantly increased the insensitivity to monocrotophos. Moreover, when the Gly228Ser and Phe439Trp mutations are present together, insensitivity increased over a thousand-fold, showing that both mutations confer resistance in a synergistic manner. Presence of the mutations, however, reduced catalytic efficiency of AChE considerably, suggesting an apparent fitness cost in monocrotophosresistant mites. Reconstitution of the multiple copies of AChE having different compositions of mutations revealed that the catalytic efficiencies of the six-copy and two-copy AChEs (resembling the AD and PyriF strains of mite, respectively) were still lower but comparable to that of wildtype AChE. These finding clearly suggested that multiple rounds of Tuace duplication was needed to compensate the reduced catalytic activity of AChE caused by mutations.

Large amounts of genetically modified (GM) grains, including maize, cotton and soybean, have been imported to Korea for food, feed and processing (FFP). To evaluatethe environmental impacts, particularly on non-target insects, of FFP GM grains of unknown source, it is a prerequisite to identify Cry protein types in the test GM grains and to establish proper risk assessment protocols. Imported GM maize grains were randomly obtained and their Cry toxins were analyzed by ELISA using Cry1A, Cry1F, and Cry3A antibodies. Since all tested GM maize grains contained Cry1A, Tenebrio molitor, a non-lepidopteran species, was selected as a non-target insect species. A domestic maize strain was used as a non-GM control, which did not show any differences in major nutritional composition from the GM maize grain. Slightly increased survival rate and head capsule width of T. molitor larvae were observed when reared on GM maize powder, demonstrating no sub-chronic adverse effects of GM maize on T. molitor larvae. Head capsule width of T. molitor neonate increased steadily from hatch to 70-day-old, regardless of being fed Bt or non-Bt maize. ELISA test using Cry1A-antibody revealed that concentration of Cry1A protein slowly increased in the whole body of T. molitor from 0 to 50 post-feeding days when the insects were fed GM maize but rapidly decreased within 5 days when Bt maize-fed larvae were transferred to non-Bt maize, showing that the Cry toxin is not accumulated inside the body of T. molitor once the exposure source is removed. In addition, no Cry protein was detected in the hemolymph of the larvae reared on Bt maize, suggesting little possibility of Cry toxin exposure to higher tropic level. Taken together, the imported GM-maize grains is not likely to cause any side impacts on non-target insect T. molitor.