Identification and characterization of ace1-type acetylcholinesterase likely associated with organophosphate resistance in Plutella xylostella

Recent studies have revealed that non-Cyclorrhapha insects possess two acetylcholinesterases (AChEs): Drosophila Ace-orthologous (o-Ace) and -paralogous (p-Ace) forms. In these insects, p-Ace is considered as a neural target of insecticides because mutations conferring insecticide insensitivity have been consistently identified in equivalent forms from various insects. To clarify the functional differentiation between the two AChE isoforms, we characterized the expression of two AChE genes in nerve cell-rich and -poor tissues of German cockroaches. The two AChE genes were expressed at comparable levels in both the nerve cord and head. Two AChE isoforms with mutually discriminatory fenitroxon sensitivities were exhibited in the nerve cord, indicating that o-Ace and p-Ace function in the neurons. pAce was mainly expressed in the nerve cord and head, while o-Ace was ubiquitously expressed at a comparable level. Moreover, o-Ace transcripts as well as AChE activity were detected in the fat body where no nervous tissue is observed. No AChE activity was detected in the hemolymph. These results show that p-Ace is better as an insecticidal target because of its abundant transcripts in nervous tissue. In addition, the roles of o-Ace, which is present ubiquitously without secretion into the hemolymph in insects, are discussed.

Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Differential tissue distribution of two acetylcholinesterase transcripts in the German cockroach, Blattella germanica

Mizuno

Komagata

et al. 2007

“…Recent studies on AChE gene expression in some insect species indicate that AO-AChE gene expression is much lower than the AP-AChE gene in the insect body (Baek et al, 2005;Mizuno et al, 2006). Insensitivity appears to depend on a low level of expression of AO-AChE in the insect body as compared to AP-AChE.…”

Section: Discussionmentioning

confidence: 99%

Biochemical properties of recombinant acetylcholinesterases with amino acid substitutions in the active site

Kozaki²,

Tomita³

et al. 2007

Several amino acid substitutions causing insensitivity have been found in the active site of Ace-paralogous acetylcholinesterase (AP-AChE); Gly119Ser (Culex pipiens, Anopheles gambiae), Ala201Ser (Aphis gossypii), Phe290Val (Nephotettix cincticeps), Ser331Phe (Myzus persicae, A. gossypii), Phe331Cys (Tetranychus urticae) and Phe331Trp (Cx. tritaeniorhynchus). To confirm the responsibility of these substitutions to the insensitivity, the six substitutions were introduced into the AP-AChE cDNA of Cx. tritaeniorhynchus resistant strain (Toyama), and their biochemical properties were examined by using a baculovirus-insect cell system. The substitution Gly119Ser gave the enzyme a high level of insensitivity to carbamate insecticides but a slight insensitivity to organophosphates. On the other hand, an amino acid substitution Phe331Trp located in the acyl pocket induced a very high level of insensitivity to organophosphates and ten times lower insensitivity to carbamates. The amino acid replacement appear to render the acyl pocket less hydrophobic and smaller, and then alter the accessibility of the substrates and inhibitors to this site.

“…5Ј RACE or 3Ј RACE were also tried with gene-specific primers of the mosquito AChE, but a specific band was not obtained. It has been reported that there are long UTR promoting erroneous fusions occur during PCR in marginal regions of the AChE gene in P. xylostella (Ni et al, 2003;Baek et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…gambiae (Weill et al, 2003), An. albimanus (Weill et al, 2004), C. tritaeniorhynchus (Nabeshima et al, 2004), M. persicae (Nabeshima et al, 2003), Aphis gossypii (Andrews et al, 2004;Toda et al, 2004) and Plutella xylostella (Baek et al, 2005). Recent studies of several insect species have shown that the expression level of the AP-AChE gene was much higher than that of the AO-AChE gene, and suggest that AP-AChE plays a main role in terminating synaptic transmission.…”

Section: Introductionmentioning

confidence: 99%

cDNA identification and gene expression of two types of acetylcholinesterases in a cultured cell line of Aedes albopictus, compared to mosquito whole body extracts

Mizuno

Tomita

Kasai

et al. 2006

Two cDNA sequences containing open reading frames encoding two types of acetylcholinesterase (AChE) precursors, Ace-orthologous AChE (AO-AChE) and Ace-paralogous AChE (AP-AChE), with 635 residues and 702 residues, respectively, were determined in Aedes albopictus by PCR-based techniques. The partial and whole cDNAs for AOAChE and AP-AChE, respectively, were also sequenced from a cultured cell line of mosquito, NIAS-AeAl-2, derived from Ae. albopictus neonatal larvae. Comparing the sequences of the respective AChEs between cultured cells and mosquito whole body extracts, many nucleotide polymorphisms were found in both AChE cDNAs but only one amino acid substitution. The expression level of AChE genes in the cultured cells was low, 1/2 and 1/7 that of the mosquito body for AO-AChE and AP-AChE, respectively. The expression ratio of AO-AChE/AP-AChE was 1/2 in the cultured cells. AP-AChE genes was more highly expressed than AO-AChE genes in both cultured cells and the mosquito body. In an enzyme assay, the activity of AChE per protein in the cultured cells was 1/8 that of the mosquito body. The function of AChE in cultured cells is discussed based on the present and previous papers.