We investigated the molecular and kinetic properties of two acetylcholinesterases (AmAChE1 and AmAChE2) from the Western honey bee, Apis mellifera. Western blot analysis revealed that AmAChE2 has most of catalytic activity rather than AmAChE1, further suggesting that AmAChE2 is responsible for synaptic transmission in A. mellifera, in contrast to most other insects. AmAChE2 was predominately expressed in the ganglia and head containing the central nervous system (CNS), while AmAChE1 was abundantly observed not only in the CNS but also in the peripheral nervous system/non-neuronal tissues. Both AmAChEs exist as homodimers; the monomers are covalently connected via a disulfide bond under native conditions. However, AmAChE2 was associated with the cell membrane via the glycophosphatidylinositol anchor, while AmAChE1 was present as a soluble form. The two AmAChEs were functionally expressed with a baculovirus system. Kinetic analysis revealed that AmAChE2 has approximately 2,500-fold greater catalytic efficiency toward acetylthiocholine and butyrylthiocholine than AmAChE1, supporting the synaptic function of AmAChE2. In addition, AmAChE2 likely serves as the main target of the organophosphate (OP) and carbamate (CB) insecticides as judged by the lower IC50 values against AmAChE2 than against AmAChE1. When OP and CB insecticides were pre-incubated with a mixture of AmAChE1 and AmAChE2, a significant reduction in the inhibition of AmAChE2 was observed, suggesting a protective role of AmAChE1 against xenobiotics. Taken together, based on their tissue distribution pattern, molecular and kinetic properties, AmAChE2 plays a major role in synaptic transmission, while AmAChE1 has non-neuronal functions, including chemical defense.

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Acetylcholinesterase (AChE) plays a pivotal role in synaptic transmission in the cholinergic nervous system of most animals, including insects. Insects possess duplicated AChE gene loci (ace1 vs. ace2) encoding two distinct AChEs (AChE1 and AChE2). A phylogenetic analysis suggested that the last common ancestor of two aces shared its origin with Platyhelminthes. In addition, the ace duplication event likely occurred after the divergence of Protostomian but before the split of Ecdysozoa. The ace1 lineage exhibited a significantly lower evolutionary rate (d and dN/dS ratio) than the ace2 lineage, suggesting that the ace1 lineage has retained the essential function of synaptic transmission following its duplication. Therefore, the putative functional transition from ace1 to ace2 observed in some Hymenopteran insects appears to be a local and relatively recent event. The amino acid sequence comparison and three-dimensional modeling of insect AChEs identified a few consistent differences in the amino acid residues in functionally crucial domains between two AChEs, which are likely responsible for the functional differentiation between two AChEs. A unique amino acid substitution causing a dramatic reduction in the catalytic activity of AChE1 in some Hymenopteran insects was suggested to be responsible for the aforementioned functional transition of ace.

Potato virus Y (PVY) (Potyviridae: potyvirus) is a serious emerging virus affecting seed potato worldwide. It affects the seed potato by transmitting non‐persistently via aphids. Sometimes this virus induces symptomless infection and is hard to detect in potato. So, it requires a specific and quick diagnosis for efficient examination. Recently, a reverse‐transcription polymerase chain reaction (RT‐PCR)‐based PVY detection method has been developed from plant as well as insect vectors. However, it is a complex and time consuming method. Here, we developed a simple PVY detection method that uses boiling for releasing the viral RNA from aphid stylets, and amplification by PVY‐specific primers located in the viral coat protein gene. The method is suitable for various strains. This simplified method could save time compared to earlier detection methods because of the simplified RNA extraction step. Following this procedure, we tested this one‐step RT‐PCR‐based PVY detection method by using three PVY vectoring aphid species (Myzus persicae, Aphis gossypii and Macrosiphum euphorbiae) as well as other sucking insects such as thrips, Frankliniella occidentalis. The reliability of a newly developed primer set was suitable for RT‐PCR and procedures were successfully demonstrated for virus detection. This PVY detection method is rapid, easy to use and suitable for large‐scale testing in laboratories of seed potato, and could potentially be applied to virus‐free seed potato production.