Twelve Lamiaceae plant essential oils (EOs) and their components were evaluated for their fumigant and contact toxicities. Inhibition of AChE by the major components of active EOs was also assessed. Strong fumigant toxicity was observed from EOs of Mentha piperita and Perilla frutescens. Menthone and menthol, and perilla aldehyde were identified as major components of the above two EOs, respectively. The LC50 (mg/L) values of M. piperita, P. frutescens, menthone, menthol and perilla aldehyde were 3.87, 2.44, 5.76, 1.88, and 0.99 against male, and 4.10, 3.31, 5.13, 1.94, and 1.15 against female, respectively. Strong contact toxicity was observed from the EOs of Satureja montana and Thymus zygis. Thymol and carvacrol were major components of these two EOs. The LD50 (μg/fly) values for contact toxicity of S. montana, T. zygis, thymol and carvacrol were 2.95, 2.93, 1.63, and 1.30 for male, and 4.59, 5.09, 2.68, and 2.60 for female, respectively. Among the five major components, perilla aldehyde showed most active inhibition activity against AChE of both sexes of SWD.

이산화염소는 병원미생물에 대한 소독제로 사용되고 있다. 최근 저곡해충에 대한 이산화염소의 산화적 스트레스에 의한 살충력이 확인되 었다. 그러나 이산화염소의 산화적 스트레스에 의한 대상 곤충의 체내 분자 종말점에 대해서는 알려지지 않았다. 본 연구는 화랑곡나방(Plodia interpunctella)의 아세틸콜린에스터레이즈가 이산화염소의 분자표적으로 가정하고 노출에 따른 이 효소의 활성을 분석하였다. 아세틸콜린에스 터레이즈 활성은 화랑곡나방 발육 시기에 따라 상이했다. 화랑곡나방 5령 유충에서 치사를 일으킬 수 있는 이산화염소 농도 처리는 아세틸콜린에 스터레이즈 활성을 뚜렷하게 증가시켰다. 또한 훈증제 형태의 이산화염소 처리에서도 아세틸콜린에스터레이즈 활성 증가가 유발되었다. 화랑곡 나방 5령 유충은 음성주광성을 보이는 데, 이산화염소 처리는 이 선천성 행동을 교란하였다. 이러한 결과는 아세틸콜린에스터레이즈가 이산화염 소 처리에 따른 산화적 스트레스의 분자표적 가운데 하나라는 것을 제시하고 있다.

The acetylcholinesterase 1 (AmAChE1) of the honey bee is known to be abundantly expressed both in the central and peripheral nervous systems. AmAChE1 exists mostly in the soluble form with little catalytic activity and has non-neuronal functions. Our preliminary observation showed that AmAChE1 expression fluctuated between the forages and nurses. A more systematic expression profiling of AmAChE1 over a year cycle on a monthly basis revealed that AmAChE1 was predominantly expressed during the winter months with being moderately expressed during the rainy summer time. However, no significant difference in AmAChE1 expression was noticed between the nurse and forager workers. Interestingly, AmAChE1 expression was inhibited when bees were allowed for brooding by placing overwintering bee hives in strawberry green houses with the supplement of pollen diets whereas it was resumed when the bee hives were removed from the green houses, thereby suppressed brooding. To confirm whether brooding status is a main determining factor for the suppression of AmAChE1 expression, active bee hives were placed in a screen tent, thereby hindering foraging, until brooding was completely suppressed, and then allowed to restore brooding by removing the screen. The AmAChE1 expression in the head was up-regulated when brooding was suppressed whereas its expression was down-regulated when brooding was resumed. These finding demonstrates that AmAChE1 expression in the central nervous system (i.e., head) is related with brooding status of honey bee. To understand the connection between the AmAChE1 expression and other pathways related with brooding, currently in progress are the analyses of head transcriptomes of honey bee workers with or without their brooding suppressed.

The habitat of the cockroach varies relative to species. The German cockroach (Blattella germanica) lives in human dwelling while B. nipponica lives in mountainous region. Even though, these two species have similar morphology, their habitats and walking speed were different. Underlying this fact, we hypothesized that habitats might influence on the walking speed via altering particularly nervous system including acetylcholinesterase (EC3.1.1.7, AChE). Full length ORFs were cloned from each species and sequenced. Sequence analyses showed that both genes in each species possess typical features of ace, and that Bgace1 and Bnace1. Those features are orthologous to the insect ace1 whereas Bgace2 and Bnace2 are to the insect ace2. Some SNPs were observed but Bgace1 and 2 showed high similarity (99%) with Bgace1 and 2, respectively. Multiple AChE bands were identified by native PAGE and IEF from three tissues (head, leg and other body) and their expression pattern was almost identical between two species. However, Esterase expression patterns were significantly different. Furthermore, length as well as detailed structure of antenna, leg and tail cerci was also significantly different. Taken together, various factors such as sensory organ detailed morphology and esterases are responsible for habitat and walking speed difference.

Recently, the expression of acetylcholinesterase1 (AChE1) in honeybee worker has been found to be seasonally fluctuated. Seasonal investigation on the AChE1 expression profiles revealed that it is abundantly expressed in January but its expression was completely abolished in February in both head and abdomen. In an attempt to predict the physiological function of seasonally expressed AChE1, proteomic analysis of honeybee worker was conducted using the samples collected in January and February. Total protein samples separately extracted from the head and abdomen of honeybee forager were compared by 2-D electrophoresis (2-DE). More than 2-fold differences in expression patterns between the two different samples were observed in 50 and 85 protein spots in the head and abdomen, respectively. Among them, 20 protein spots showing >17-fold differences in expression between the two different samples of the head were identified by mass spectrometry. Most of the proteins were identified to be the major royal jelly protein (MRJP) families (e.g., MRJP, MRJP2 and MRJP3), which are known to be expressed in nurse bees during brooding season, and their expression was significantly higher in January than in February. This result was unexpected because brooding usually began in the study site apiary during February and the worker bees used for analysis were assumed to be foragers (old workers). Thus, current findings suggest, though speculative, that the workers collected in January may function as nurses despite their old ages in January or that MRJPs may have other not-yet-characterized functions, which is apart from the conventionally known roles. Finally, possible association of MRJPs with AChE1 was discussed.

Coprinellus miaceus, belongs to Coprinaceae of Agaricales, Basidiomycota, has been used for an edible purposes in asian countries. This experiment was initiated to evaluate the free radical scavenging, free radical scavenging, anti-acetylcholinesterase, anti-inflammatory and α-glucosidase inhibitory activities of C. micaceus fruiting bodies extracted with methanol and hot water. In 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, the scavenging activities of methanol and hot water extracts were lower than that of positive control, BHT. The chelating effects of methanol and hot water extracts were significantly higher than positive control, BHT at the concentrations of 0.125-2.0 mg/mL. In the reducing power assay, methanol and hot water extracts exhibited the lower activities than the positive control, BHT at the 0.125-0.2 mg/mL concentration. In the HPLC analysis of phenolic acids profile of the mushroom fruiting bodies, 4 phenolic compounds including procatechuic acid, chlorogenic acid, (-)-epicatechin, and naringin were detected. The tyrosinase inhibitory activities of methanol and hot water extracts were 91.33% and 91.99% at 2.0 mg/mL concentration, while the inhibitory activity of kojic acid, the positive control, was 99.61%. Nitric oxide (NO) production in lipopolysaccahride (LPS) activated RAW 264.7 cells were inhibited by the methanol extract in a concentration dependent manner. In the acetylcholinesterase (AChE) inhibitory activity assay, methanol and hot water extracts of the mushroom inhibited the AChE by 94.64% and 74.19%, respectively at the 1.0 mg/mL concentration, whereas galanthamine, the standard drug inhibited the AChE activity by 97.80% at the same concentration. The methanol and hot water extracts of the mushroom inhibited the α-glucosidase activity by 62.26% and 67.59%, respectively at the 2.0 mg/mL concentration, while acarbose, the positive control inhibited the α-glucosidase activity by 81.81% at the same concentration. Therefore, it is concluded that fruiting bodies of C. micaceus contained natural antioxidant, anti-inflammatory, anti-acetylcholinesterase and α-glucosidase inhibitory substances which might be used for promoting human health.

Lentinus giganteus is a edible mushroom cultivated in Asian countries. The present study was initiated to evaluate the anti-inflammatory, anti-acetylcholinesterase, anti-α-glucosidase, and free radical scavenging activities from fruiting bodies of L. giganteus extracted with methanol and hot water. The free radical scavenging activities of methanol and hot water extracts on 1,1-diphenyl-2-picrylhydrazyl (DPPH) were 92.26% and 90.17% at 2.0 mg/mL concentration, respectively and comparable with positive control, BHT. The chelating activities of methanol and hot water extracts were significantly higher than the positive control tested. The reducing power of methanol and hot water extracts showed lower activities compared to positive control, BHT. The phenolic and flavonoid contents of hot water extract were 1.56 μg/mg and 24.35 μg/mg, respectively. Nitric oxide (NO) produced by lipopolysaccahride (LPS) activated RAW 264.7 cells were significantly inhibited by treatment of methanol and hot water extracts. The methanol extract inhibited the acetylcholinesterase (AChE) activity by 91.19% at the 2.0 mg/mL concentration, whereas galanthamine, the standard drug, inhibited the AChE activity by 97.80%. The hot water extracts inhibited the α-amylase and α-glucosidase activities by 78.86% and 80.78%, respectively at the 2.0 mg/mL concentration, while acarbose, the positive control, inhibited the activities by 89.91% and 81.81%, respectively at the same concentration. Therefore, it is concluded that fruiting bodies of L. giganteus contain antioxidant, anti-inflammatory, anti-cholinesterase, and anti-diabetic substances, which can be used for natural health food for promoting human health.

Acetylcholinesterase (AChE, EC 3.1.1.7) plays a vital role in neurotransmission in both vertebrates and invertebrates. AChE is a key enzyme in the insect nervous system, in which the cholinergic system is essential. In addition to its classical synaptic functions, AChE is recently known to play other non-neuronal roles. In vertebrates, only one AChE has been shown to be involved in both neuronal and non-neuronal functions, whereas two different AChEs have been identified from various insect species, and seem to play respective neuronal and non-neuronal functions. In honey bee, for instance, membrane-anchored AChE2 is reported to be responsible for classical synaptic functions, while soluble AChE1 plays non-neuronal functions. In contrast to most insect species expressing two AChEs, Cyclorrhaphan flies are known to possess only a single ace locus. In Cyclorrhapha possessing only one AChE, multiple forms (molecular variation) of Drosophila melanogaster AChE (DmAChE) were recently suggested to be generated via alternative splicing. Among various molecular forms, membrane-anchored dimer has high enzyme activity, whereas soluble monomer is abundantly expressed without catalytic activity. Interestingly, expression of the soluble DmAChE was induced by chemical stress. Based on the results, it can be hypothesized that the generation of multiple isoforms of AChE2, particularly the soluble forms, may have provided alternative protein copies for AChE1, replaced the functions of AChE1 and eventually allowed the loss of AChE1 in Cyclorrhaphan flies.

This study was initiated to investigate antioxidant, anti-acetylcholinesterase, and xanthine oxidase inhibitory activities and properties of fruiting bodies, mycelia, and fermentation culture filtrates from Phellinus igniarius. The contents of total phenols and flavonoid of fruit bodies, mycelia, and culture filtrate were 15.35-1.36 mg/g, 10.35-7.85 mg/g, and 8.25-5.36 mg/g. The 1,1- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging abilities of the extracts from the fruiting bodies, mycelia, and culture filtrates were 90.25-95.60%, 78.82-85.24%, and 76.32-82.50% at 50-400 μg/mL, respectively. The chelating ability of fruiting body extract on ferrous ions was higher than those of mycelia and culture filtrates tested. The anti-acetylcholinesterase inhibitory activity of the fruiting body extract at 400 μg/mg exhibited 91.10% on AChE, which is lower than that of positive control, galanthamine (94.82%). The xanthine oxidase inhibitory activities of the fruiting bodies, mycelia, and culture extract were 85.47%, 78.13%, and 72.49% at 400 μg/mL, respectively. Overall, the fruiting body extract has better anti-acetylcholinesterase, antioxidant and xanthine oxidase inhibitory activities than those from mycelia and culture filtrate.

본 연구에서는 기와층버섯의 자실체에서 메탄올과열수를 이용해 추출한 물질의 항산화와 항염증, 항아세틸콜린에스테라제(anti-acetylcholinesterase)의 효과를 탐색하였다. DPPH 라디칼 소거능, 환원력 및철 이온 제거능 등을 이용해 항산화 효과를 측정한결과 양성대조군으로 사용한 BHT나 토코페롤에 비해 낮았지만 다른 종류의 버섯에 비해 효과가 우수한것을 확인하였다. 철 이온을 제거하는 항산화 실험에서 기와층버섯의 메탄올 추출물의 효과는 양성대조군인 BHT나 토코페롤에 비해 월등하게 높아서 기와층버섯 자실체의 추출물은 높은 항산화 효과를 지닌것으로 나타났다. 기와층버섯의 염증저해 효과 실험에서는 배양 중인 RAW 264.7 대식세포에 자실체의메탄올과 열수추출물을 각각 전 처리 한 후 염증매개물질인 LPS를 투여하여 추출물의 NO 생성 저해효과를 조사하였다. 실험 결과, 처리한 추출물의 농도가 증가함에 따라 생성된 NO의 양이 현저하게 감소하는 경향을 나타내었다. 또한 기와층버섯의 추출물을 이용해 carrageenan에 의해 흰쥐 뒷발에 유도된부종을 저해하는 실험에서는 투여한 추출물의 농도가 증가함에 따라 흰쥐의 뒷발에 유도된 부종의 용적도 농도 의존적으로 감소되었다. 따라서 기와층버섯자실체에 함유된 항산화, 항염증 및 항아세틸콜린에스테라제 성분은 천연 항산화제, 소염제 및 알츠하이머병 치료에 이용이 가능할 것으로 사료되었다.

Acetylcholinesterase (AChE) plays a pivotal role in the synaptic transmission in the cholinergic nervous system of most animals, including insects. Insects have two different ace (ace1 and ace2) loci that encode two distinct AChEs (AChE1 and AChE2), which were originated by duplication events long before the radiation of insects. However, little is known about when the ace duplication occurred and how each duplicated ace locus has evolved to retain the original functions. In this study, we conducted phylogenetic analysis for cholinesterase genes from all the lower animals with their genome sequenced together with all known arthropod ace1 and ace2, including those from a number of insects that were newly cloned. Among several independent duplications in lower animal lineages, one duplication event found in platyhelminthes appeared to be the direct origin of arthropod ace1 and ace2. Comparison of the evolutionary distance (d) of two aces from different insect groups relative to those from common ancestors revealed that ace1 has evolved with a significantly slower rate compared to ace2, suggesting that the ace1 lineage has maintained relatively more essential functions following duplication. When the dN/dS ratio was compared between ace1 and ace2 within different insect orders, ace2 was determined to have received relatively more positive selection pressure in Diptera and Hymenoptera whereas the same was true for ace1 in Coleoptera, Hemiptera and Lepidoptera. Along with the relatively more decreased d value for ace2, such an increased dN/dS ratio for ace2 in Diptera and Hymenoptera implied the incidence of functional transition of ace1 to ace2. Our findings should provide with new insights into the evolution of two insect AChEs: when they were generated and how they retain and gain the neuronal functions.

Fumigant and contact toxicities of 11 Myrtaceae plant essential oils and their constituents against adult male and female Blattella germanica were evaluated. Of 11 Myrtaceae plant essential oils, Eucalyptus polybractea, E. smithii, E. radiata, E. dives, E. globulus, and Melaleuca uncinata, showed 100% fumigant toxicity against adult male German cockroaches at a concentration of 7.5 mg/liter air concentration. In contact toxicity tests, E. polybractea, E. smithii, E. radiata, E. dives, E. globulus, M. dissitiflora, and M. uncinata produced strong insecticidal activity against adult male and female German cockroaches. Of the essential oil constituents, terpinolene, α-terpinene, and terpinen-4-ol demonstrated strong fumigant toxicity against adult male and female B. germanica. Eugenol, isoeugenol, methyl eugenol, and terpinen-4-ol showed strong contact toxicity against adult male B. germanica. The toxicity of the constituent blends identified from M. dissitiflora essential oils indicated that terpinen-4-ol were major contributor to the fumigant activity or contact toxicity of the artificial blend. Only isoeugenol exhibited inhibition activity against male acetylcholinesterase. IC50values of isoeugenol were 0.22 mg/mL against male acetylcholinesterase.

Inhibition of acetylcholinesterase (AChE) is one of the important modes of action available for the control of insects. An assessment was made of the electric eel AChE inhibitory activity of 55 endophytic fungi isolated from the leaves of Huperzia serrata collected in Eunshi, Hubei Province, China. Of 55 mycelial extracts and 55 ethyl acetate-soluble fractions from liquid culture of the fungi, the ethyl acetate-soluble fraction of strain ES056 exhibited strong AChE inhibitory activity (88%) at a concentration of 100 μg/mL. Based on morphological characteristics and nuclear ribosomal DNA ITS sequence analysis, this fungus showed greatest similarity to members of the order Eurotiales and was shown to be most closely related to members of the Aspergillus aculeatus group. It was designated Aspergillus sp. MY056. This strain merits further study as a potential anti-AChE product.

Most insect possess two AChEs (i.e., AChE1 and AChE2 encoded by ace1 and ace2 gene, respectively). Due to its higher transcription level and responsibility for insecticide resistance, it is believed that AChE1 is likely the main catalytic AChE, between two AChEs of the insects having both AChE1 and AChE2 studied to date. However, Cyclorrhaphan flies have only AChE2, suggesting the evolutionary scenarios for the takeover of ace2 and the loss of ace1 during Diptera evolution (Huchard et al., 2006). Therefore, it is essential to investigate the evolutionary distribution of AChE1 and AChE2 in Insecta. In this study, among 100 insect species examined, we found 33 species expressing AChE2 as the catalytic major enzyme by Western blotting using AChE1- and AChE2 specific antibodies. These findings are contrary to the common expectation that AChE1 is dominant synaptic enzyme in all insect species with the exception of Cyclorrhapha. In this study, we compared the characteristics of both AChE1 and AChE2 from the German cockroach and the honey bee, in which AChE1 and AChE2 were expressed as major catalytic enzyme, respectively. In addition, we presented a unique case of a damselfly, which expresses both AChEs exhibiting almost identical enzymatic activities, along with the fruit fly, where AChE1 is not present but multiple forms of AChE2 appear to replace the function of AChE1. The current study will provide valuable insights into the evolution of AChE.

Substrate affinity and insecticide sensitivity of acetylcholinesterase (AChE) from Daphnia magna S., Bombyx mori L., Musca domestica L., Myzus persicae S., Anguilla anguilla L., Cyprinus carpio L., Oryzias latipes T.&S., Homo sapiens L., Bos taurus L. were tested. The Km values of M. domestica AChE to acetylthiocholine (ATCh), propionylthiocholine (PTCh), butyrylthiocholine (BTCh) were 57.3 μM, 13.4 μM and 85.9 μM respectively, which were lower than those of A. anguilla, C. carpio, O. latipes, H. sapiens and B. taurus. In nontarget organisms, the I50 values of AChE to fenitroxon and DDVP were 1.5×10-6 M~7.8×10-5 M and 2.4×10-6 M~1.1×10-4 M respectively, thus they have lower sensitivity compared with M. domestica. The I50 value of M. persicae AChE to pirimicarb was 1.3×10-8 M, which was higher sensitivity compared with other test animals except D. magna. The I50 values of D. magna AChE to fenitroxon, DDVP, carbaryl, eserine, pirimicarb were 5.2×10-10 M~2.1×10-8 M, which were higher sensitivity compared with the other test animals used for this study. cDNA of Daphnia magna AChE precursor was sequenced and compared with those of Musca domestica, Drosophila melanogaster and Torpedo californica.

Most insects possess two different acetylcholinesterases (AChE1 and AChE2) but it remains unknown which AChE plays the major role in synaptic transmission. To investigate the evolutionary distribution of AChE1 and AChE2, the AChE with the main catalytic function in several insect species belonging to 18 representative orders was determined by native-PAGE in conjunction with Western blotting using AChE1- and AChE2-specific antibodies. Among the 98 insect species examined, AChE1 was expressed as the main enzyme in 65 species across diverse taxa. In the remaining species, however, AChE2 was expressed as the major enzyme. These findings are contrary to the common expectation that AChE1 is major enzyme in most insects, with the exception of Cyclorrhapha, and further demonstrate that the specialization of AChE2 as the main enzyme or the replacement of AChE1 function with AChE2 function were rather common events, having multiple independent origins during insect evolution. It was hypothesized that the generation of multiple AChE2 isoforms via alternative splicing has allowed the loss of ace1during the functional replacement of AChE1 with AChE2 in Cyclorrhapha. However, the presence of AChE2 as the main AChE in higher social Hymenoptera provides an example of the functional replacement of AChE1 with AChE2 without the loss of ace1. The current findings should provide valuable insights into which AChE has evolved to perform synaptic functions and to become the main insecticide target in different species

I conducted experiments in Drosophila to investigate the consequences of altered acetylcholinesterase (AChE) activity in the nervous system. In ace hypomorphic mutant larvae, the amount of ace mRNA and the activity of AChE both in vivo and in vitro were significantly reduced compared with those of controls. Reduced Ace in Drosophila larvae resulted in significant down-regulation of branch length and the number of boutons in Type 1 glutamatergic neuromuscular junctions (NMJs). These defects in ace hypomorphic mutant larvae were suppressed when Musca domestica AChE was transgenically expressed. Because AChE inhibitors are utilized for medications for Alzheimer’s disease, we investigated whether pharmacological inhibition of AChE activity induced any synaptic defects. I found that controls exposed to a sublethal dose of DDVP phenocopied the synaptic structural defects of the ace hypomorphic mutant. These results suggest that down-regulation of AChE activity, regardless of whether it is due to genetic or pharmacological manipulations, results in altered synaptic architecture. This study suggests that exposure to AChE inhibitors for 6-12 months may induce altered synaptic architectures in human brains with Alzheimer’s diseases, similar to those reported here. These changes may underlie or contribute to the loss of efficacy of AChE inhibitors after prolonged treatment.

Twenty four fractions by solvent extraction and/or acid precipitation from fruit body and culture broth of Inonotus obliquus were prepared, and their inhibitory effect against acetylcholinesterase (AChE) was investigated. Among these fractions, acid (1 M HCl) precipitates from cell-free culture broth and fruit body exhibited the highest inhibitory effect on AChE in vitro. Acid precipitates inhibited AChE activity in a concentration-dependant manner and IC50 values of both acid precipitates were 0.53 mg/mL. The inhibition pattern was general non-competitive inhibition. The energetic parameters were also determined by dual substrate/temperature design. Both acid precipitates increased the values of Ea, ΔH, ΔG and ΔH* decreasing the value of ΔS for AChE. The results implied that the acid precipitates from I. obliquus increased the thermodynamic barrier, leading to the breakdown of ES complex and the formation of products as inhibitory mechanism.

A carbofuran-resistant strain (CAS) showed ca. 41.1- and 15.1-fold resistance compared to a susceptible strain (SUS) and a non-selected field strain (FM), respectively. Enhanced activities of carboxylesterase and P450 were found as ca. 3- and 1.6-fold higher in CAS strain, suggesting these enzymes play a minor role in carbofuran resistance. Interestingly, the insensitivity of acetylcholinesterase (AChE) to carbofuran was revealed to be ca. 5.5- and 3.7-fold higher in CAS strain compared to, indicating that AChE insensitivity mechanism is associated with carbofuran resistance. In the western blot analysis, two kinds of AChEs were found and type-1 AChE (Nlace1) was identified as the major AChE in N. lugens. The open reading frame of Nlace1 is composed of 2,106 bp (ca. 78 Kd) and revealed 52.5% and 24.3% identity compared with Nephotettix cincticeps and Drosophila melanogaster, respectively. In the screening of point mutations, four amino acid substitutions (G119A, F/S330Y, F331I and H332L) were identified in the CAS strain that likely contribute to the AChE insensitivity. The allele frequencies of these mutations increased in the survived populations following the selection by LC50 of carbofuran, confirming that they are in fact associated with reduced sensitivity to carbofuran in N. lugens. These point mutation can be useful for the monitoring of resistance levels in conjunction with QS methods.

Almost all insect species possess two different acetylcholinesterases (AChE1 and AChE2) but it still remains unknown which AChE plays a major role in synaptic transmission. To predict the evolutionary distributions of functional AChE, relative amount and activity of two AChEs were investigated by native-PAGE in conjunction with Western blotting using AChE1- and AChE2-specific antibodies. Among 39 insect species examined, AChE1 was expressed as the main enzyme in 26 insect species across diverse taxa, suggesting that AChE1 likely plays a more crucial role in these insects. In contrast, AChE2 was predominantly expressed in remaining insect species, including paleopteran insects, suggesting that the replacement of AChE1 function with AChE2 is an old event. As expected, only AChE2 was detected in Cyclorrhaphan flies, supporting the notion that AChE2 in Cyclorrhapha has completely replaced the physiological functions of AChE1 during the evolution of Diptera. Taken together, contrary to the common belief that AChE1 is the major enzyme in most insects, many insect species across various taxa employ AChE2 as the main AChE, suggesting that functional transition from AChE1 to AChE2 has occurred rather universally and randomly with multiple independent origins within the class Insecta. This finding should provide valuable insights into which AChE has evolved to undertake the synaptic function and how functional diversification of AChE has occurred.