Yoshiaki Kono

An amino acid substitution on the second acetylcholinesterase in the pirimicarb-resistant strains of the peach potato aphid, Myzus persicae.

cDNAs encoding two acetylcholinesterases (AChEs) were isolated from the peach potato aphid, Myzus persicae. MpAChE1 was orthologous and MpAChE2 was paralogous with the ace of Drosophila melanogaster. The deduced amino acid sequence of MpAChE1 cDNA was identical between the pirimicarb susceptible and resistant strains. However, a single amino acid substitution of Ser431Phe on MpAchE2 was found in the pirimicarb resistant strains. This substitution was located in the acyl pocket of the enzyme and was thought to alter the ligand specificity.

Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite, Tetranychus urticae

Acetylcholinesterase (AChE) from two-spotted spider mites, Tetranychus urticae was compared between an organophosphate susceptible (TKD) and a resistant (NCN) strain. The AChE of TKD had lower affinity to acetylthiocholine and propionylthiocholine than that of NCN, and the inhibition of AChE by DDVP, ambenonium, eserine and n-methyl-eserine showed that NCN was more insensitive than TKD. AChE cDNA sequence was determined, and the 687 amino acids of primary structure were deduced. There were six replacements of amino acid residues in TKD and two in NCN. #F331(439)C was the only substitution unique to NCN, however, this mutation existed homozygously in only two out of nine mites. This residue is one of the gorge lining components, and #F331(439)C might act an important role in the sensitivity of AChE to the inhibitors.

Fipronil modulation of glutamate-induced chloride currents in cockroach thoracic ganglion neurons.

Fipronil is the first phenylpyrazole insecticide introduced for pest control. It is effective against some insects that have become resistant to other insecticides, and exhibits low mammalian toxicity. Although fipronil is known to block GABA receptors, the mechanisms of its selective toxicity and efficacy against insects with dieldrin-resistant GABA receptors are not fully understood. We studied the effects of fipronil on the inhibitory glutamate receptor-chloride channel complex, which is found only in invertebrates. Glutamate-activated chloride currents were recorded from neurons isolated from cockroach thoracic ganglia using the whole-cell patch clamp technique. When glutamate was applied to a neuron, it evoked inward currents with an EC50 of 36.8 +/- 3.0 microM and a Hill coefficient of 1.56 +/- 0.17. The similarity between the reversal potential and the calculated chloride equilibrium potential indicated that glutamate-induced currents were carried by chloride ions. Fipronil suppressed the glutamate-induced peak currents in a dose-dependent manner with an IC50 of 0.73 +/- 0.27 microM and a Hill coefficient of 0.68 +/- 0.15. The current decay phases were greatly prolonged after fipronil application in a concentration-dependent manner. Picrotoxinin (PTX) at 100 microM slightly suppressed glutamate-induced currents to 87.8 +/- 3.7% of the control, and dieldrin at 100 microM had no effect (96.7 +/- 3.1%). AP5 and CNQX, mammalian glutamate receptor antagonists, were without effect on glutamate-induced Cl- currents. It is concluded that the potent blocking action of fipronil against glutamate-gated chloride channels may contribute to the higher toxicity against insects than mammals, as well as the efficacy against insects resistant to other insecticides.

Genetics and mechanisms of permethrin resistance in the YPER strain of house fly

Abstract A strain of house fly was collected from the Third Yumenoshima Island in Japan and selected with the pyrethroid insecticide permethrin. Resistance to permethrin evolved to >18,400-fold in the selected (YPER) strain, and the mechanisms and genetics of resistance in this strain were examined. Permethrin resistance was decreased by pretreatment with piperonyl butoxide or 2-propynyl 2,3,6-trichlorophenyl ether, but not S , S , S -tributylphosphorotrithioate or diethylmaleate. The level of total cytochromes P450 was 2.7-fold increased, and the level of cytochrome b 5 was 1.5-fold increased, in YPER compared to the susceptible CS strain. These results suggest P450 monooxygenases, but not hydrolases or glutathione S -transferase, are a mechanism of resistance in the YPER strain. Analysis of the para -homologous sodium channel α-subunit gene in YPER indicates this strain has the super-kdr allele. Permethrin resistance in the YPER strain was inherited as a multigenic and incompletely recessive trait. A factorial analysis of resistance in the YPER strain indicated the relative contribution to resistance by the different autosomes was 2>3>5⩾1. Comparison of YPER with three other strains of house fly having high levels of permethrin resistance (LPR, NG98, and ALHF) indicates the genetic basis of this trait is variable between populations. This indicates there may be a greater difficulty for the development of diagnostic tools that could be used reliably over large areas to monitor pyrethroid resistance in house flies than was previously thought.

Comparative linkage map development and identification of an autosomal locus for insensitive acetylcholinesterase-mediated insecticide resistance in Culex tritaeniorhynchus

A comparative linkage map for Culex tritaeniorhynchus was constructed based on restriction fragment length polymorphism markers using cDNAs from Aedes aegypti. Linear orders of marker loci in Cx. tritaeniorhynchus were identical to Culex pipiens wherein chromosomes 2 and 3 reflect whole‐arm rearrangements compared to A. aegypti. However, the sex determination locus in Cx. tritaeniorhynchus maps to chromosome 3, in contrast to Cx. pipiens and Ae. aegypti where it is located on chromosome 1. Our results indicate that insensitive acetylcholinesterase (AChE)‐mediated organophosphate resistance is controlled by a single major gene (AChER) on chromosome 2, while the AChE structural gene (Ace) is located on chromosome 1. No evidence for a second Ace gene was observed, even under very low stringency hybridization conditions.

Triterpenoid and caffeic acid derivatives in the leaves of ragweed, Ambrosia artemisiifolia L. (Asterales : Asteraceae), as feeding stimulants of Ophraella communa LeSage (Coleoptera : Chrysomelidae)

Summary.The leaf beetle Ophraella communa infestsnalmost exclusively Ambrosia artemisiifolia in the fields ofnJapan, even though it normally feeds on several Asteraceousnplants. A filter paper bioassay showed that the feeding ofnO. communa is strongly stimulated by methanolic extracts ofnA. artemisiifolia. The feeding stimulants for O. communa havenbeen isolated from methanolic extracts of A. artemisiifolia.nα-Amyrin acetate, β-amyrin acetate, 5-caffeoylquinic acidn(chlorogenic acid) and 3,5-dicaffeoylquinic acid fromnA. artemisiifolia have been identified as feeding stimulantsnfor O. communa. Triterpenoid derivativesn(α-amyrin acetate or β-amyrin acetate) and caffeic acid derivatives (3,n5-dicaffoylquinic acid or 5-caffeoylquinic acid) showednfeeding stimulant activity when mixed together.n

Identification of a hemocyte membrane protein of the silkworm, Bombyx mori, which specifically binds to bacterial lipopolysaccharide

Abstract An in vitro system with isolated hemocytes of the silkworm, Bombyx mori (B. mori) was developed to examine the induction mechanism of insect antibacterial proteins by bacterial lipopolysaccharide (LPS). The gene expression of B. mori cecropin B, a representative antibacterial protein, was triggered by LPS in this in vitro system. To identify LPS-binding site(s) of the hemocytes, the [ 125 I]LPS binding assay to the hemocytes was performed in vitro . The amount of [ 125 I]LPS bound to hemocytes increased proportionately with the increase of incubation time and LPS dose. The binding was strongly inhibited by excess unlabeled LPS or lipid A, indicating that the binding of [ 125 I]LPS to hemocytes contains a highly specific reaction. Moreover, the specific binding could not be detected with Bm-N4 cells in which cecropin B gene expression was not induced by LPS, suggesting that the LPS binding is specific for LPS responsive cells. The LPS binding was fully sensitive to the proteinase K treatment of intact hemocytes, suggesting that a protein(s) located on the surface of hemocytes is involved in the LPS binding. Fluorescein isothiocyanate (FITC) conjugated-LPS binding assay demonstrated that this compound mainly binds to granular cells rather than other hemocytes under our assay conditions. Affinity-labeling with photoreactive-LPS allowed the identification of a 11 kDa LPS-binding protein in hemocytes, which might relate to the specific membrane receptor for LPS.

Molecular Characterization of Two Acetylcholinesterase cDNAs in Pediculus Human Lice

Abstract Two cDNA sequences encoding Drosophila Ace-orthologous and -paralogous acetylcholinesterase precursors (AO- and AP-AChE precursors, respectively), were identified from the body louse, Pediculus humanus humanus L. In vitro inhibition studies with an insecticide-susceptible body louse strain exhibited a simplex inhibitory response of AChE. The I50 values of fenitroxon and carbaryl were estimated to be 2.2 and 1.9 &mgr;M for the susceptible lice, respectively. The mRNA level of AP-AChE gene was 3.1- and 9.3-fold higher than that of AO-AChE gene in the abdomen and the combined parts of the head and thorax, respectively, suggesting, due to its abundance, the potential significance of the AP-AChE isoform in Pediculus human lice in association with the efficacy of AChE-targeting pediculicides.

Chromosomal localization of amplified esterase genes in insecticide resistant Culex mosquitoes

Abstract The esterase B gene is amplified 32-fold in an organophosphorus insecticide resistant strain, Shinjuku, of Culex pipiens molestus. The amplified gene was revealed to cluster in single extended chromosomal region (ECR) of chromosome 2 by fluorescence in situ hybridization on salivary gland chromosomes. The ECR appeared to be homogeneously non-staining with DAPI, orcein or Giemsa stains. In the F1 heterozygotes between Shinjuku and an insecticide susceptible strain, chromosome 2 showed an apparent asynapsis in the ECR derived from Shinjuku, but otherwise paired well. The results suggest that the process of DNA amplification does not involve large chromosomal rearrangements. Restriction map of the co-amplified sequence with esterase B gene in Shinjuku strain is unique compared to those reported in C. pipiens complex, supporting an independent origin of the DNA amplification.

Oxydemeton-Methyl Resistance, Mechanisms, and Associated Fitness Cost in Green Peach Aphids (Hemiptera: Aphididae)

Abstract Susceptibility to oxydemeton-methyl and imidacloprid, and the inhibitory effects of oxydemeton-methyl and some organophosphate compounds on acetylcholinesterase (AChE) and carboxylesterase activity were studied in two populations (Karaj and Rasht) of green peach aphids, Myzus persicae (Sulzer). Results show that the Karaj population was resistant to oxydemeton-methyl but susceptible to imidacloprid. The esterase activity of the resistant and susceptible populations suggests that one of the resistance mechanisms to oxydemeton-methyl was esterase-based. The inhibition assay shows that the AChE of the Karaj population is less sensitive to oxydemeton-methyl and paraoxon derivatives. Regarding the paraoxon derivatives, the smaller paraoxon side chain is more potent against the modified AChE than against the AChE from the susceptible strain. Fertility life table parameters of green peach aphid populations resistant and susceptible to oxydemeton-methyl also were studied under laboratory conditions. The standard errors of the population growth parameters were calculated using the Jackknife method. Results showed that susceptible strain exhibits a significantly higher rm than the resistant strain, probably because the resistant strain had a higher generation time than the susceptible strain. These results suggested that the resistant Karaj strain may be less fit than the susceptible strain.