Functional characterization of two acetylcholinesterase genes in the brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy), using heterologous expression and RNA interference

- We cloned the cDNA sequence of two AChE genes, Tcace1 and Tcace2 (encoding TcAChE1 and TcAChE2) from brown citrus aphid.
- The potential differences in enzymatic function were characterized by heterologous expression of the two genes in Sf9 cells.
- The recombinant TcAChE1 showed greater sensitivities to all ten tested insecticides.
- Silencing of Tcace1 and Tcace2 significantly increased susceptibility of T. citricida to malathion and carbaryl.

Acetylcholinesterase (AChE) is the primary target of organophosphate- and carbamate-based insecticides. We sequenced the full-length cDNAs of two AChE genes from the brown citrus aphid Aphis (Toxoptera) citricidus (Kirkaldy). These two genes, Tcace1 and Tcace2, which encode TcAChE1 and TcAChE2, respectively, had a shared amino acid identity of 29% and were highly similar to other insect ace1 and ace2 genes, respectively, having specific functional motifs. Potential differences in enzymatic function were characterized by the heterologous expression of the two genes using a baculovirus system in Sf9 insect cells. Both of the recombinant AChEs had high specific activities for three typical substrates, acetylthiocholine iodide, butyrylthiocholine iodide, and propinylthiocholine iodide. TcAChE1 had a lower Michaelis-Menten constant value and a higher maximal reaction velocity than recombinant TcAChE2, indicating a higher affinity for substrates and greater catalytic efficiency, respectively. Bioassays showed a greater sensitivity of recombinant TcAChE1 to the 10 tested insecticides. Silencing of Tcace1 and Tcace2 by RNA interference significantly increased the susceptibility of A. citricidus to malathion and carbaryl; however, silencing Tcace1 resulted in a higher mortality rate than silencing Tcace2. Additionally, the specific enzyme activity decreased more after silencing Tcace1 than after silencing Tcace2. Thus, TcAChE1 plays a major role in postsynaptic neurotransmission in A. citricidus.

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