Duplication of acetylcholinesterase gene in diamondback moth strains with different sensitivities to acephate.

This study examined the acetylcholinesterase 1 gene (AChE1) in Plutella xylostella strains with different sensitivities to acephate. Multiple haplotypes of the gene were found in the field-collected strains including distinct haplotypes carrying one or both previously reported mutations (A298S and G324A). Moreover, sequencing results indicated the presence of duplicated copies of the gene in the field-collected strains. No correlation was found between copy numbers of AChE1 and levels of resistance to acephate suggesting that extensive AChE1 duplication is not a major resistance factor at least in some P. xylostella strains. Proportions of the A298S and G324A mutations showed no correlation with levels of resistance to acephate. This suggests that acephate resistance of P. xylostella is complex and cannot be evaluated based on the AChE1 copy number or proportions of the resistance mutations alone.

Identification and expression of caspase-1 gene under heat stress in insecticide-susceptible and -resistant Plutella xylostella (Lepidoptera: Plutellidae).

A caspase gene in Plutella xylostella (DBM) was identified firstly and named Px-caspase-1. It had a full-length of 1172 bp and contained 900 bp open reading frame that encoded 300 amino acids with 33.6 kDa. The deduced amino acid of Px-caspase-1 had two domain profile including caspase_p20 (position 61-184) and caspase_p10 (position 203-298) (i.e. the big and small catalytic domains), and the highly conserved pentapeptide QACQG in caspase_p20 domain (the recognized catalytic site of caspases). Being highly homologous to effector caspase genes in other insect and mammalian species, Px-caspase-1 was thought to be an effector caspase gene. Heat stress could result in significant mortality increase on adult DBM. Px-caspase-1 mRNA expression and caspase-3 enzyme activity (a effector caspase) were elevated with age and heat treatment. And, heat stress facilitated the procession of Px-caspase-1 expression. Significantly higher mRNA transcription levels were found in a chlorpyrifos-resistant DBM strain, as compared to those in insecticide-susceptible DBM. The results indicated that high temperature could significantly promote apoptosis process resulting in an the increased DBM mortality rate, and that insecticide-susceptible DBM had a significantly higher physiological fitness at high temperatures than insecticide-resistant DBM.

RNA interference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin.

We have previously reported that a cytochrome P450, CYP6BG1, from Plutella xylostella was found to be overexpressed in 4th instars of a permethrin resistant strain and inducible in the susceptible counterpart. The findings suggested potential involvement of CYP6BG1 in permethrin resistance, hence warranted a functional analysis. To assess the functional link of the gene to permethrin resistance, we adopted RNA interference-mediated gene silencing (RNAi) by dsRNA droplet feeding. Here, real time PCR analyses show that oral delivery of dsRNA can efficiently reduce the expression of CYP6BG1. Knockdown of CYP6BG1 transcript was evident in midgut and larval tissues enclosed in carcass. As a consequence of knockdown, a significant reduction in resistance of larvae fed CYP6BG1 dsRNA was observed after 24 and 48h of exposure to permethrin. In addition, CYP6BG1 dsRNA feeding to larvae led to reduced total P450 activities of microsomal preparations toward model substrates p-nitroanisole and benzyloxyresorufin. These results indicate that the overexpressed CYP6BG1 participate in enhanced metabolism of permethrin, thereby, resistance. The knockdown of a non-overexpressed P450, CYP6BF1v4, from the same resistant P. xylostella strain did not lead to changes in the level of resistance to permethrin, supporting further the specific involvement of CYP6BG1 in the resistance.

Hydrolysis of acetylthiocholine iodide and reactivation of phoxim-inhibited acetylcholinesterase by pralidoxime chloride, obidoxime chloride and trimedoxime.

The hydrolysis of acetylthiocholine iodide (ATCh) by pralidoxime chloride (2-PAM Cl), trimedoxime (TMB(4)) and obidoxime chlpride (LUH(6)) was studied at pH 5.8-8.0 and incubation temperature from 5 to 40 degrees C in vitro. Significant ATCh hydrolysis by 2-PAM Cl, TMB(4) and LUH(6) was found, with the exceptions of those at pH 7.0, 6.2 and 5.8 at 5 degrees C and those at pH 6.2 and 5.8 at 15 degrees C. The hydrolysis by TMB(4) and LUH(6) was significantly stronger than that by 2-PAM Cl. The hydrolysis increased with increasing pH, incubation temperature and three oxime or ATCh concentration. Significant hydrolysis of ATCh by the three oximes could be found when the terminal concentration of oxime was higher than 0.01 mM at pH 7.0 and 7.4 at 30 and 37 degrees C. However, no hydrolysis of natural substrate (acetylcholine iodide) by the three oximes was found when very high terminal concentrations of oximes were used. In addition, the three oximes displayed an extraordinary efficiency in the reactivation of phoxim-inhibited acetylcholinesterase (AChE) from fish (Carassius auratus) or rabbit (Oryctolagus cuniculus domestic) brain in vitro. Parallel to the level of ATCh hydrolysis by the oximes, TMB(4) and LUH(6) displayed significantly higher reactivation efficiency than 2-PAM Cl to phoxim-inhibited AChE. And, the extent of reactivation by 2-PAM Cl was also lower than the other two. Plausible antidotal actions of the oximes against organophosphate poisoning AChE and erroneously high estimation of AChE activity by the Ellman method were discussed.

Insecticide susceptibility of surviving Cotesia plutellae (Hym: Braconidae) and Diaeretiella rapae (M'Intosh) (Hym: Aphidiidae) as affected by sublethal insecticide dosages on host insects.

The effects of sublethal dosages of insecticides applied to Plutella xylostella L. (Lepidoptera: Yponomeutidae) and Lipaphis erysimi Kaltenbach (Homoptera: Aphidiidae) on the insecticide susceptibility of the surviving endoparasitoids, Cotesia plutellae Kurdjumov (Hymenoptera: Braconidae) and Diaeretiella rapae (M'Intosh) (Hymenoptera: Aphidiidae), were studied in Shangjie, Minhou, China. The susceptibility to methamidophos and the sensitivity of acetylcholinesterase (AChE) to methamidophos and dichlorvos in the adults of host insects were substantially lower than those in the two parasitoids. The host insects were treated with sublethal dosages of methamidophos in P. xylostella and of methamidophos and avermectin in L. erysimi. The cocoon formation in the two parasitoids decreased significantly, from 35.0% (control) to 13.0% (with methamidophos treatment) for C. plutellae; from 20.6% (control) to 9.0% (with methamidophos treatment) and from 24.3% (control) to 16.7% (with avermectin treatment) for D. rapae. The susceptibility to methamidophos of the resultant emerging adults of the two parasitoids was found to be significantly lower than that of the control when the parasitoids were left in contact with the same dosages of methamidophos. The average AChE activity inhibition by methamidophos and dichlorvos in 34-60 adults of the two parasitoids that emerged from the treatments (15.1% and 31.8% respectively for C. plutellae, and 21.1% and 26.9% for D. rapae) was also significantly lower than those of the controls (55.4% and 48.3% respectively for C. plutellae, and 42.9% and 51.7% for D. rapae). The bimolecular rate constant (k(i)) values of AChE to methamidophos and dichlorvos in the adults of parasitoids without the insecticide treatment were 1.78 and 1.56 times as high as those that emerged from the host insects treated with methamidophos for C. plutellae, and 1.91 and 1.66 times as high as those in the case of D. rapae. It is suggested that there is a difference in AChE sensitivity to insecticides between the resultant emerging parasitoids with and without insecticide pretreatment. Furthermore, the introduction of the insecticides to the host insects could be an important factor in the insecticide resistance development of the endoparasitoids. The natural selectivity would favour the parasitoids that had developed an insensitivity to the insecticide(s).

Insecticide toxicity and synergism by enzyme inhibitors in 18 species of pest insect and natural enemies in crucifer vegetable crops.

The toxicities of three enzyme inhibitors and their synergistic effects on four insecticides were studied by using the dry film method on field populations of 18 species of insects collected in Jianxin and Shanjie, China, from 2003 to 2005. Meanwhile, the inhibitory effects of these enzyme inhibitors on the activities of acetylcholinesterases (AChE), carboxyesterases (CarE) and glutathione-S-transferases (GST), in vivo, were also studied. In general, triphenyl phosphate (TPP) and diethyl maleate (DEM) showed low toxicities to six herbivorous pest insects, four ladybirds and eight parasitoids. Piperonyl butoxide (PB) exhibited low toxicities to the herbivorous pest insects and ladybirds, but high toxicities to the eight parasitoids. The tolerance to the insecticides in 11 pest insects and natural enemies was mainly associated with the tolerance to PB. PB showed the highest synergism on methamidophos, fenvalerate, fipronil and avermectin in nine species of pest insects and natural enemies. In general, TPP and DEM showed significant synergisms to these four insecticides in four parasitoid species. However, in contrast to their effects on the parasitoids, the synergistic effects of TPP and DEM on the four insecticides by TPP and DEM against four pest insects and one ladybird varied depending on the insect species and enzyme inhibitor. Activity of AChE, CarE or GST could be strongly inhibited, in vivo, by PB, TPP or DEM, depending on the insect species and enzyme inhibitors. From the results obtained in this study, mixed-function oxidase (MFO) was thought to play the most critical role in insect tolerances to the tested insecticides in the field. Low competition existed in the evolution of insecticide resistance in the field populations of parasitoids, as compared with herbivorous pest insects and ladybirds. Possible causes of the high synergistic effects of PB on the four classes of insecticides, based on multiattack on the activity of CarE, GST or AChE in the insect species, are also discussed.

Monooxygenase activity in methidathion resistant and susceptible populations of Amblyseius womersleyi (Acari: Phytoseiidae).

The purpose of this study was to evaluate the cytochrome P450-dependent monooxygenase activities in methidathion resistant and susceptible strains of Amblyseius womersleyi Schicha. Artificial laboratory selections for resistance and susceptibility to methidathion were performed in an organophosphate resistant strain of A. womersleyi (Kanaya strain). Selections for susceptibility were also performed in a susceptible strain of this predaceous mite (Ishigaki Strain). After the selection process, the LC(50) of methidathion for the selected strains of A. womersleyi were 816 mg/l (Kanaya R), 4.61 mg/l (Kanaya S) and 1.59 mg/l (Ishigaki S). The monooxygenase activities were determined biochemically by the O-deethylation of 7-ethoxycoumarin (7-EC). The monooxygenase activity in adult females of Kanaya R strain (51.1 pmol/30 min/mg protein) was 3.60- and 5.42-fold higher than the activity observed for Kanaya S and Ishigaki S strains, respectively. Significant correlation between monooxygenase activity and LC(50) (mg/l) of methidathion was observed analyzing 16 populations of A. womersleyi with different susceptibilities to methidathion. Monooxygenase activity was also evaluated in different life stages (egg, larva, protonymph, deutonymph and adult) of A. womersleyi. The lowest activity was observed for the larval stage, which presented the highest susceptibility to methidathion. Protonymph, deutonymph and adult presented the highest monooxygenase activities. These stages were the most tolerant to methidathion. Monooxygenase activities of the Kanaya R strain were higher than of the Kanaya S strain in all developmental stages. The present study can be helpful for the implementation of a program involving release of insecticide-resistant populations of A. womersleyi in the field. The monooxygenase activity determination is easier and quicker than the estimation of LC(50), requiring fewer mites.

Seasonal changes of methamidophos susceptibility and biochemical properties in Plutella xylostella (Lepidoptera: Yponomeutidae) and its parasitoid Cotesia plutellae (Hymenoptera: Braconidae).

Methamidophos resistance and acetylcholinesterase (AChE) insensitivity to methamidophos, dichlorvos, and carbofuran were determined in the field populations of Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) and its parasitoid Cotesia plutellae Kurdjumov (Hymenoptera: Braconidae) collected from the corresponding hosts between October 1998 and December 2003 in Fuzhou and Minhou, Fijian, China. Resistance levels to methamidophos and AChE insensitivity to the three insecticides in the two species of insects were high during autumn and spring and low during summer. Resistance to methamidophos was 15.3- and 12.6-fold higher in resistant F0 parents of P. xylostella and C. plutellae than in their susceptible F11 progeny, respectively. The bimolecular rate constant (k(i)) values of AChE to methamidophos, dichlorvos, and carbofuran were 4.6-, 6.3-, and 7.7-fold higher in F11 progeny of P. xylostella, and 3.7-, 4.5-, and 3.7-fold higher in F11 progeny of C. plutellae than those in their F0 parents, respectively. Compared with susceptible F11 progeny, the resistance ratios for methamidophos were 4.2-29.8 and 3.8-13.1 in 21 field populations of P. xylostella and C. plutellae, respectively. The k(i) values of AChE to methamidophos, dichlorvos, and carbofuran were 2.0-21.6-, 3.6-9.5-, and 2.6-9.2-fold higher in F11 progeny of P. xylostella, and 1.8-7.6-, 1.9-4.6-, and 2.2-7.6-fold higher in F11 progeny of C. plutellae than those in 21 field populations, respectively. Significant correlative variations of methamidophos resistance as well as significant correlative variations of k(i) values of AChE to insecticides between the two species of insects also were found in space and time. The k(i) values of AChE to insecticides in C. plutellae were far higher than those in P. xylostella. There were no obvious differences in the Km and Vmax of AChE between F0 parents and F11 progeny of P. xylostella and C. plutellae, respectively. But carboxylesterase activity was 1.6-fold higher in F0 parents of C. plutellae than in F11 progeny, and glutathione S-transferase activity was 1.5-fold higher in F0 parents of P. xylostella than in F11 progeny. The results suggested that the AChE insensitivity to insecticides might play the most important role in methamidophos resistance in the two species of insects. From these results, a spatial and temporal correlative evolution of methamidophos resistance and insensitive AChE was found to exist between P. xylostella and C. plutellae.

Effects of synergists on toxicity of six insecticides in parasitoid Diaeretiella rapae (Hymenoptera: Aphidiidae).

The resistance to and the effects of synergists on the toxicity of six insecticides in Diaeretiella rapae (M'Intosh) (Hymenoptera: Aphidiidae), a parasitoid of vegetable aphid collected in Jianxin at Fuzhou-City, Fujian, China, were studied. In comparison with susceptible F21 progeny, the resistance ratios in resistant F0 parents were 27.6 for methamidophos, 20.8 for fipronil, 47.5 for avermectin, 3.3 for fenvalerate, 4.5 for cypermethrin, and 74.7 for imidacloprid. Piperonyl butoxide (PB), triphenyl phosphate (TPP), and diethyl maleate (DEM) were chosen to be applied in susceptible F21 progeny, as well as in resistant F11 progeny and F0 parents. Significant synergistic effects on the toxicity of the six insecticides were found by using PB, TPP, and DEM in F0 parents; on methamidophos, avermectin, and imidacloprid by PB, TPP, and DEM in F11 progeny; on fipronil by PB and DEM in F11 progeny; and on fenvalerate and cypermethrin by PB in F11 progeny. PB also showed significant synergism on the six insecticides in susceptible F21 progeny, although the synergism was far less in F21 progeny than those in resistant F0 parents. TPP and DEM showed little or no synergistic effects on the toxicity of the six insecticides in F21 progeny. Compared with TPP and DEM, the highest synergistic ratios of PB for methamidophos, fipronil, avermectin, fenvalerate, cypermethrin, and imidacloprid were observed in F0 parents, and F11 and F21 progeny. The resistance levels to methamidophos, fipronil, avermectin, fenvalerate, and cypermethrin could be inhibited strongly by applying PB in F0 parents. From the results, oxidative degradation is believed to play a critical role in resistance to methamidophos, fipronil, avermectin, fenvalerate, and cypermethrin in D. rapae. To a lesser extent, hydrolytic reactions also were partially involved in the resistance to these five insecticides by using the synergists PB, TPP, and DEM. However, although high synergism of PB, TPP, and DEM on imidacloprid was found, the resistance levels to imidacloprid remained high in the presence of PB, TPP, and DEM. The mediated detoxification of oxidative degradation and hydrolytic reactions was thought to be involved in the resistance to imidacloprid in F0 parents.

Molecular phylogeny of the Eichhorni group of Delias Hübner, 1819 (Lepidoptera: Pieridae).

The eichhorni group lies within the genus Delias (Lepidoptera: Pieridae) which has markedly diversified aposematic wing markings. The phylogenetic relationships among all species of the eichhorni group, representatives of each of the other 21 species groups of Delias butterflies, and some related genera were analyzed based on nucleotide sequences of the mitochondrial NADH dehydrogenase subunit 5 gene. A supplemental study using the nuclear elongation factor-1alpha (EF-1alpha) gene was also carried out. The results are compared with those of morphological studies. Our results confirm the monophyly of the eichhorni group and suggest the monophyly of the genus Delias. They also indicate phylogenetic intragroup relationships, particularly the division of the eichhorni complex into groups I and II. Moreover, they also indicate that the initial diversification of the eichhorni group involved separation of the D. catisa + D. toxopei clade, followed by the divergence of other species including the eichhorni complex. Based on these findings, it is supposed that this group first appeared close to or within the western mountain range of New Guinea Island (135 degrees 30(')-140 degrees E) where D. catisa, D. toxopei, and representatives of other species cohabit.