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.

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.

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.

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.

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.

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.

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

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.