Acetylcholinesterase (AChE) is a key enzyme that terminates impulse transmission by rapidly hydrolyzing the neurotransmitter acetylcholine at cholinergic synapses. Previous studies have discovered a transiently opening channel referred to as the “back door” in Torpedo californica AChE. Previously, we observed that substituting the Tyr391 residue with a Phe residue significantly decreased the catalytic efficiency of recombinant Apis mellifera AChE1 (AmAChE1), while the reverse substitution restored it. Interestingly, substitution of the Tyr391 residue with a Phe residue in AmAChE1 disrupted the formation of the backdoor, while the reverse substitution restored it. This finding suggests that the Tyr-to-Phe substitution impairs backdoor formation, thereby leading to a significant reduction in the catalytic activity of AmAChE1. This serves as one of the driving forces for the functional transition from AmAChE1 to AmAChE2. In this experiment, we also confirmed the gradual restoration and increase in AChE activity by substituting Phe391 in AmAChE1 with Ser, Trp, Thr, Ile, Asn, and Tyr residues through kinetic assay and molecular dynamics simulation.

Steaming is a method that has traditionally been used for medicinal plant extraction. This study investigated nitrite oxide production, ferrous ion chelating activity, α-glucosidase, xanthine oxidase, and acetylcholinesterase inhibitory activities of ethanol, acetone and hot-water extracts of Codonopsis lanceolata prepared by steaming seven times. MTT assay showed that each extract was non-toxic up to a concentration of 700 μg/mL confirming that there was no cytotoxicity in all extracts. The α-glucosidase, xanthine oxidase, and acetylcholinesterase inhibitory activities exhibited by the hot-water extract obtained from steaming seven times were higher (83.1%) than the other extracts. Higher production of nitrite oxide and better ferrous chelating activity was recorded with hot-water extract compared to ethanol and acetone extracts. These results indicated that more steaming of Codonopsis lanceolata extracts would be required to validate the possibility of developing antioxidants. Also, further study is needed to determine if the components present in the tested extracts might be useful in the prevention of Alzheimer's disease. These results showed that hot-water extracts may be useful for their antioxidant and the production inhibitory activity of nitrite oxide. It will be helpful in the investigation of the constituent analysis of the steam-processed product of Codonopsis lanceolata.

Molecular diagnostic markers are necessary for establishing highthroughput screening systems to support insecticide-resistant population management. Here, we identified single amino acid substitution mutations related to carbamate resistance in Laodelphax striatellus Fallén type-1 acetylcholinesterase (Lsace1) using carbofuran-selected strains. The phenotypic resistance profiles of the final selection strain (SEL9) compared to the susceptible strain revealed a 14-fold higher resistance ratio based on topical application, 1.2-fold higher general esterase activity, and 4.3- fold higher acetylcholinesterase insensitivity based on the 50% inhibitory concentration (I50), suggesting that insensitivity of the target site could occur as a resistance factor. Comparison of the nucleotide sequences of Lsace1 of five strains (SUS, SEL0, SEL3, SEL6, and SEL9) revealed two amino acid substitutions (F330Y and F331H). To understand the roles of these mutations, we determined the allele frequency of both point mutations in the selected strains using quantitative sequencing methods. In addition, several quantitative genotypic traits (e.g., gene copy numbers and transcript levels of Lsace1, Lsace2, and LS.CarE1) were assessed. A correlation analysis of genotypic and phenotypic traits revealed strong correlations between resistance level and I50 with F331H allele frequency. Interestingly, the F331H mutation was negatively correlated with transcript levels of Lsace1, suggesting that selection pressure might result in a reduction of the target gene. Overall, the F331H mutation and reduced mRNA are important factors in the development of carbamate resistance. Furthermore, the point mutation can be used to monitor rapid carbofuran resistance in conjunction with molecular diagnostic methods such as quantitative sequencing.

The honey bee soluble acetylcholinesterase 1 (AmAChE1) is overexpressed under the overwintering and brood rearing-suppressed conditions. To investigate the role of AmAChE1 in regulating acetylcholine (ACh) titer, ACh concentrations both in the head (neuronal) and abdomen (non-neuronal) were analyzed. ACh titer was significantly lower in both tissues of worker bees under the overwintering and brood rearing-suppressed conditions compared to control bees. The expression levels of another two factors that regulate ACh titer, choline acetyltransferase (AmAChT) and acetylcholinesterase 2 (AmAChE2), were not altered as judged by qPCR and native PAGE, suggesting that the lower ACh titer was mainly regulated by AmAChE1. For precise verification of AmAChE1 as an ACh titer regulator, honey bees were put under brood rearing-suppressed condition to induce AmAChE1 and injected AmAChE1 dsRNA to knock down the gene. The ACh titer of AmAChE1-knocked down honey bees was 1.9 and 2.6 folds higher than that of control bees in head and abdomen, respectively. Taken together, in spite of its extremely low catalytic activity, the overexpression of AmAChE1 is likely to be related with the low level of ACh homeostasis, perhaps via ACh sequestration, under brood rearingsuppressed condition, and likely induce metabolic changes through ACh receptors-related pathways.

Small hive beetle (Aethina tumida) (SHB) is an invasive species to most northern hemisphere countries, including Korea. In an attempt to obtain basic information for efficient management of SHB, genes encoding conventional insecticide targets [voltage-sensitive sodium channel α-subunit (VSSC) and acetylcholinesterase (AChE)] were annotated and characterized following the analysis of whole transcriptomes of adults and larvae. A single VSSC gene was identified but no apparent mutations associated with pyrethroid resistance were detected. Genes encoding two AChEs (AtAChE1 and AtAChE2) were identified from the SHB transcriptome. AtAChE1 was determined to be the main catalytic enzyme, thereby being a toxicologically more relevant target. No apparent mutations associated with resistance to organophosphorus and carbamate insecticides was identified in the AtAChE1 gene, whereas the S238G mutation, originally identified from the Colorado potato beetle, was detected in the AtAChE2 gene.

The honey bee soluble acetylcholinesterase 1 (AmAChE1) is overexpressed under the overwintering and brood rearing-suppressed conditions. To investigate the role of AmAChE1 in regulating acetylcholine (ACh) titer, ACh concentrations in both the head (central nervous system) and abdomen (peripheral nervous system) were analyzed. ACh titer was significantly lower in both tissues of worker bees under the overwintering and brood rearing-suppressed conditions compared to control bees. Interestingly, the expression levels of choline acetyltransferase (AmChAT) and molecular marker genes of immune systems were significantly reduced in honey bee head under the same conditions. Taken together, ACh titer appears to be reduced via a cooperative interaction of the AmAChE1 overexpression and AmChAT underexpression and to be linked to reduced inmmune responses under the overwintering and brood rearing-suppressed conditions. The roles of AmAChE1 (with little catalytic activity) and AmChAT in the ACh homeostasis and signaling was discussed in the contexts of immune response and longevity regulation in honey bees.

There are two different types of acetylcholinesterase (AChE1 and AChE2) in the western honeybee as in most of insects. It is suggested that soluble AmAChE1 might be related with a stress response as judged from its elevated expression level in honey bee workers when brood rearing was suppressed. In this study, to ensure the nature of AmAChE1 responding to stress factors, the expression patterns of AmAChE1 following heat shock, brood rearing suppression and chemical treatments (Imidacloprid and fluvalinate) were investigated. Also, several heat shock protein (hsp) genes (hsp10, hsp60, hsp70 and hsp90) known as general stress markers were tested as positive references. Heat shock induced expression of every tested hsp along with AmAChE1. In brood rearing-suppressed worker bees, 7 days old bees showed much higher expression level of AmAChE1 and hsp90 compared to control honey bees. However, treatment of imidacloprid and fluvalinate did not induce any apparent overexpression of these genes. These results confirm that both HSP and AmAChE1 genes generally respond to temperature and brood rearing suppression and further suggest that AmAChE1 can serve as a potential biomarker along with hsps for the detection of stress in honey bee colonies.

Recently, the existence of non-neuronal, soluble AChEs with non-classical functions, such as stress response and chemical defense, has been reported in both vertebrates and invertebrates. With this in mind, it is intriguing to hypothesize that fat body is a main tissue to express non-neuronal AChE at least in some insects. As an initial step for the systematic approach to investigate the distribution of non-neuronal AChEs in insect fat body and to elucidate their physiological functions, we have selected 12 different insect species across different orders and isolated fat body tissues from them. Then, the presence or absence of AChE and its solubility nature were determined by native polyacrylamide gel electrophoresis in conjunction with western blot analysis insect-specific AChE1 and 2 antibodies. Among 12 insects examined, soluble AChE1 was determined to be expressed in fat bodies of insects involving honey bees, brown plant hoppers, dynastid beetles, lice, etc, AChE2 in fruit flies, bed bugs, mealworm beetles. However, no AChE was detected in fat bodies of the remaining two species American cockroaches and dragonflies Our findings clearly show that AChE is widely distributed in the fat body tissue of diverse insect species. More extensive investigation on in a wider variety of insect species would be necessary to deduce the evolutionary origin of fat body-specific AChE, which would be the ancestor of AChE with non-neuronal function.

Acetylcholinesterase (AChE) is a target enzyme of organophosphate (OP) and carbamate (CB) insecticides. Point mutation in AChE is one of the major mechanisms of OP and CB resistance. Recently, we investigated soluble AChEs abundantly expressed in the non-neuronal tissues of the honey bee Apis mellifera and fruit fly Drosophila melanogaster. Pre-incubation of OP and CB insecticides with honey bee soluble AChE showed a significant reduction in the inhibition of catalytic AChE activity. In the fruit fly, expression of soluble AChE was induced by insecticide exposure, and the wild-type fly expressing both soluble and membrane-anchored AChEs was more tolerant to insecticide than the transgenic fly expressing only membrane-anchored AChE. These findings suggested that soluble form of AChE is possibly involved in chemical defense against xenobiotics, including insecticides.

A series of conserved point mutations in acetylcholinesterase (AChE) confer resistance to organophosphorus and carbamate insecticides in most arthropod pests. However, the mutations associated with reduced sensitivity to insecticides usually results in the reduction of catalytic efficiency and leads to a fitness disadvantage. To compensate for the reduced catalytic activity, overexpression of neuronal AChE appears to be necessary, which is achieved by a relatively recent duplication of the AChE gene (ace) as observed in the two-spotted spider mite and other insects. Unlike the cases with overexpression of neuronal AChE, the extensive generation of soluble AChE is observed in some insects either from a distinct non-neuronal ace locus or from a single ace locus via alternative splicing. The production of soluble AChE in the fruit fly is induced by chemical stress. Soluble AChE acts as a potential bioscavenger and provides tolerance to xenobiotics, suggesting its role in chemical adaptation during evolution.

Acetylcholinesterase (AChE) is an enzyme for hydrolyzing neurotransmitter acetylcholine. Soluble form of AChE is generated via alternative splicing and functions as a bioscavenger in Dropsophila melanogaster. In this study, effects of ethanol and acetic acid on the soluble AChE expression were investigated. Treatment of ethanol and acetic acid results in over-expression of soluble AChE in the abdomen in a dose-dependent manner. However, no apparent change in AChE expression was observed in the head. Our finding suggests that the soluble AChE is involved in chemical defense against high concentration of ethanol, which is a common by-product from fermented food，and acetic acid, the main metabolite of ethanol. Thus, high level of ethanol and acetic acid resistance in D. melanogaster appears to be evolved via the induction mechanism of soluble AChE expression.

Acetylcholinesterase 1 (AmAChE1) has low catalytic activity and is abundantly expressed in both neuronal and non-neuronal tissues. In previous experiments, we observed that AmAChE1 is rarely expressed in summer while highly expressed in winter. Through additional experiments, the expression of AmAChE1 was suggested to be associated with brood rearing status. Under the assumption that abnormal suppression of brood rearing activity may result in stressful condition in honey bee social community, it was further suggested that AmAChE1 is likely involved in stress management particularly during winter. We hypothesized that the increased docility usually observed in overwintering bees is likely an outcome of stress management in colony, which is mediated by AmAChE1 expression. To verify this, worker bees expressing abundant AmAChE1 were collected in early winter and injected with Amace1 dsRNA to knockdown Amace1. Then, the behavioral activity of the bees was investigated using the EthoVison video tracking system. Honey bees injected with Amace1 dsRNA showed significantly increased motility, which was strongly correlated with the suppressed expression level of AmAChE1 in the abdomen. No apparent reduced expression of AmAChE1 in the head was observed perhaps due to the limited efficacy of RNA interference in the blood-brain-barrier. Our finding suggests that behavioral activity can be regulated, at least, by AmAChE1 expression level in non-neuronal tissue (i.e., fatbody) perhaps via metabolic alteration.