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.

RNA interference (RNAi) technology based on feeding double-stranded RNA (dsRNA) has been employed for the control of insect pests. In general, strong lethal effects have been observed when feeding RNAi is applied to chewing insects. However, the efficacy of feeding RNAi for sap-sucking insects has not been reported to be limited most likely due to the reduced rate of dsRNA translocation into the plant sap. In this experiment, therefore, we tested whether the long-hairpin RNA (lhRNA) structure, which mimics the viroid, can improve its translocation within plant tissues, thereby increasing lethality of target gene, when compared with dsRNA structure. Either lhRNA of dsRNA structure (75 ng/ul) of vacuolar ATP synthase subunit A (V-ATPase) gene was delivered via rice seedling to Nilaparvata lugens, which is one of the major sucking insects on rice, and mortality was measured until 60 h post-treatment. Treatment of the lhRNA and dsRNA of V-ATPase gene caused increased mortality over time compared with eGFP-treated control, reaching the maximum level at 48 h post-treatment, and the mortality was significantly higher in lhRNA treatment than in dsRNA treatment. Gene silencing of target gene was confirmed at 24 h and 48 h post-treatment. In summary, treatment of lhRNA resulted in significantly higher mortalities than that of dsRNA, suggesting that delivery of lhRNA has an apparent advantage over dsRNA in exerting RNAi-induced lethality.

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, asignificant 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.