Azole selenourea disrupted the midgut and caused malformed development of Plutella xylostella

Chemical insecticides targeting the digestive system of diamondback moth(DBM),Plutella xylostella,have not been developed.The discovery of an insecticide with novel mode of action is a challenge for the control of DBM.In this study,a class of selenium-and difluoromethyl-modified azoles(fluoroazole selenoureas,FASU)were designed and synthesized for the control of DBM.Of these azoles,compound B4 showed the highest insecticidal activity against DBM.The LC50of third-and second-instar larvae reached 32.3 and 4.6μg mL^(–1),respectively.The midgut tissue of larvae was severely disrupted,and the larval intestinal tissue was dotted with unique red spots after treatment with compound B4.Compound B4 led to disintegration of the peritrophic matrix,swelling of the midgut epithelium,fracture of the microvilli,and extensive leakage of cellular debris in the midgut lumen.Surviving larvae grew very slowly,and the larval duration was significantly prolonged after exposure to compound B4 at sublethal doses(LC10,LC25and LC50).Furthermore,the pupation rate,emergence rate and pupae weight were significantly decreased.Compound B4 also induced abnormal pupae,causing adults to be trapped in the cocoon or failure to fly due to twisted wings.These results demonstrated that FASU could reduce the population of DBM in sublethal doses.FASU is the first synthetic insecticidal lead compound that has been shown to disrupt the midgut tissue of the larvae of DBM,and its mode of action totally differs from that of commercial chemical insecticides.

CRISPR/Cas9-based functional characterization of Px ABCB1 reveals its roles in the resistance of Plutella xylostella(L.)to Cry1Ac,abamectin and emamectin benzoate

The identification of functional midgut receptors for pesticidal proteins produced by Bacillus thuringiensis(Bt)is critical for deciphering the molecular mechanism of Bt resistance in insects.Reduced expression of the PxABCB1 gene was previously found to be associated with Cry1Ac resistance in the diamondback moth,Plutella xylostella(L.).To directly validate the potential receptor role of PxABCB1 and its contribution to Bt Cry1Ac toxicity in P.xylostella,we used CRISPR/Cas9 to generate a homozygous knockout ABCB1KO strain with a 5-bp deletion in exon 3 of its gene.The ABCB1KO strain exhibited a 63-fold resistance to Cry1Ac toxin compared to the parental DBM1Ac-S strain.Intriguingly,the ABCB1KO strain also exhibited significant increases in susceptibility to abamectin and emamectin benzoate.No changes in susceptibility to various other Bt Cry proteins or synthetic insecticides were observed.The knockout strain exhibited no significant fitness costs.Overall,our study indicates that PxABCB1 can protect the insect against avermectin insecticides on one hand,while on the other hand it facilitates the toxic effect of the Bt Cry1Ac toxin.The results of this study will help to inform integrated pest management approaches against this destructive pest.

Integrated Assessment of Trapping Efficiency of Colored Sticky Cards and Sex Pheromone on Liriomyza spp.and Plutella xylostella

[Objective] To evaluate the control effect of yellow sticky cards and sex pheromone on Plutella xylostella and Liriomyza spp.,which can provide reference for large area extension of the technique.[Method] The integrated assessment of trapping efficiency of colored sticky cards and sex pheromone on Liriomyza spp.and Plutella xylostella was conducted by field surveys and structured interview in Tonghai County,Yunnan Province,China.[Result] The results showed that yellow sticky cards and sex pheromone have strong power of trapping Liriomyza spp.and Plutella xylostella（3 414±720 and（219±157） head/piece,respectively）;the control cost by the usage of pesticide is the highest,（10 099.5±2 752.5） yuan/hm^2,yellow sticky cards and sex pheromone takes the second place,（1 125.0±465.0） yuan/hm^2,the control cost by the usage of yellow sticky cards is the lowest,（450.0 ±186.0）yuan/hm^2.Without the usage of yellow sticky cards and sex pheromone,pesticide application times and costs are（15.0±2.7） times and（12 070.5±2 136.0） yuan/hm^2;combined with usage of yellow sticky cards and sex pheromone,pesticide application times and costs reduce by 5.7 times and 4 618.5 yuan/hm^2.The ratio of trapped beneficial insects and target pests was 1 ∶1 131,which showed that the negative effect of yellow sticky cards and sex pheromone on the non-target insects was very limited.[Conclusion] The trapping approach has become popular among all the local farmers.Looking at the above factors,the trapping technology has strong application prospect and promotion value in pest control field.

Determination of Resistance to Seven Insecticides in Plutella xylostella L. in Fields of Northern Hunan

The resistance of field populations of Plutella xylostella, from the three vegetable producing areas （Nianyuxu Town of Yueyang City, Canggang Town of Changde City and Shatou Town of Yiyang City） in northern Hunan, to seven insecticides was determined using leaf dipping method in door. The results showed that Plutella xylostella showed an extremely high-level resistance to beta-cypermethrin （resistance ratio, RR=257.13）, a high-level resistance to abamectin （RR=135.15） and indoxacarb （RR=103.08） and a moderate-level resistance to chlorfenapyr and emamectin benzoate. But Plutella xylostella is relatively sensitive to chlorantraniliprole and Bacillus thuringiensis （Bt）. Therefore, the prevention of Plutella xylostella in northern Hunan should focus on the alternative use of chlorfenapyr, emamectin benzoate, chlorantraniliprole and Bacillus thuringiensis and avoid the use of beta-cypermethrin so as to delay the generation and development of resistance to insecticides in Plutella xylostella.

A Mutant of PlxyCSP1 Protein in Plutella xylostella

[Objective] This study aimed to explore the effects of cysteine （Cys58） on the characteristics of PlxyCSP1 in P. Iostella combined with pesticide compounds. [Method] Cys58 of PlxyCSP1 was mutated into Trp58 by using overlap extension PCR method, and PlxyCSP1-M2 mutant was obtained. Expression vector was con- structed and the protein was detected by western blot. [Result] Expression vector pET32a-PlxyCSP1-M2 was constructed to express the 35kDa weight protein. [Conclusion] PlxyCSP1 mutant protein has been expressed successfully in prokaryotic ex- pression system.

Optimization of Artificial Rearing Methods for Plutella xylostella L.(Plutellidae)and Its Susceptibility to Five Types of Insecticides

[Objective] The research aimed to optimize laboratory artificial rearing methods for Plutella xylostella L. and to select out effective insecticides against this pest species. [Method] Brassica chinensis seedlings were cultivated in the laboratory at （25±1）℃, 60-75% RH, and a photoperiod of 14：10 （L：D） h. The green blow molding papers were used to collect eggs, and Brassica oleracea seedlings were cultivated to breed Plutella xylostella L. until the insects were ready for use. Mean-while, the susceptibility of 3rd instar larvae of Plutella xylostella L. to five types of insecticides was tested by leaf-dipping method. [Result] Plutella xylostella L. larvae were found well-grown and uniform in developmental stages with average pupa weight of 5.4 mg, pupa emergence percentage of 94.0 % and egg number of 135.1 per female. The LCso values of 3rd instar Plutella xylostella L. larvae against 200 g/L chlorantraniliprole SC, 20% flubendiamide WG, 150 g/L indoxacarb EC, 200 g/L tebufenozide SC and 1.8% abamectin EC, were 0.152, 0.223, 1.151, 93.340 and 0.128 mg/L, respectively. [Conclusion] This rearing method is easy to be implemented and of great practicality, suitable for mass propagation of P. xylostella L. Abamectin, chlorantraniliprole and flubendiamide showed high insecticidal activities, so these three insecticides can be used alternately for the field prevention and control of Plutella xylostella L.

饲养温度对小菜蛾(Plutella xylostella)AchE和CarE活力的影响

在湿度定义为干、湿球温度差为3℃、光照强度10000-12000 lx、光照周期14L:10 D条件下,探索饲养温度(X:24-36℃)之变化对小菜蛾(Plutella xylostella)4龄幼虫乙酰胆碱酯酶(AchE)和羧酸酯酶(CarE)活力(Y)的影响效应。实验结果表明,活力指数随着饲养温度的升高而降低,两者之间存在显著的线性相关关系:温度与乙酰胆碱酯酶(AchE)活力两者间的相关关系函数式为Y=0．3327-0．0068X;与羧酸酯酶(CarE)活力的为Y=3．1278-0．0780X。

Biochemical Mechanism of Chlorantraniliprole Resistance in the Diamondback Moth, Plutella xylostella Linnaeus

The insecticide chlorantraniliprole exhibits good efifcacy and plays an important role in controlling the diamondback moth, Plutella xylostella Linnaeus. However, resistance to chlorantraniliprole has been observed recently in some ifeld populations. At present study, diamondback moths with resistance to chlorantraniliprole （resistant ratio （RR） was 82.18） for biochemical assays were selected. The assays were performed to determine potential resistance mechanisms. The results showed that the selected resistant moths （GDLZ-R） and susceptible moth could be synergized by known metabolic inhibitors such as piperonyl butoxide （PBO）, triphenyl phosphate （TPP） and diethyl-maleate （DEM） at different levels （1.68-5.50-fold and 2.20-2.89-fold, respectively）, and DEM showed the maximum synergism in both strains. In enzymes assays, a high level of glutathione-S-transferase （GST） was observed in the resistant moth, in contrast, moths that are susceptible to the insecticide had only 1/3 the GST activity of the resistant moths. The analysis of short-term exposure of chlorantraniliprole on biochemical response in the resistant strain also showed that GST activity was signiifcantly elevated after exposure to a sub-lethal concentration of chlorantraniliprole （about 1/3 LC50, 12 mg L-1） 12 and 24 h, respectively. The results show that there is a strong correlation between the enzyme activity and resistance, and GST is likely the main detoxiifcation mechanism responsible for resistance to chlorantraniliprole in P. xylostella L., cytochrome P450 monooxygenase （P450） and carboxy-lesterase （CarE） are involved in to some extent.

Sub-Lethal Effects of Fenvalerate on the Development, Fecundity, and Juvenile Hormone Esterase Activity of Diamondback Moth, Plutella xylostella (L.)

The toxicities of fenvalerate （20% EC） to the 3rd instar larvae of diamondback moth （DBM）, Plutella xylostella （L.）, reared on three host plants viz., radish, oilseed rape, and cabbage were tested. The LC50 values of fenvalerate to the 3rd instar larvae of DBM varied with host plants, however, there wasn＇t any significant difference among them （P〉 0.05）. Similarly, DBM fed on three host plants had same pupal weight, pupal period, pupation rate, adult emergence rate, female ratio, and fecundity. The activity of juvenile hormone esterase （JHE, EC 3.1.1.1） in the 3rd instar larvae of DMB did not significantly vary with host plants, either. These suggested that DBM had similar fitness on the three host plant species. When fed on the host plants pretreated with fenvalerate at the concentrations equivalent to LC20, LC50 and LC50, the pupation rate, pupal weight, adult emergence rate, female ratio, fecundity, and JHE activity of the tested insects were declined as compared with insects in control treatments fed on the same host plant species. Furthermore, the pupal period of the tested insects was extended after fenvalerate treatment. The decrease in JHE activity after fenvalerate treatment in the tested insects could partly explain the changes in the mentioned growth parameters. Whether the role of fenvalerate in the inhibition of JHE activity could serve as a new way to control DBM needs further investigation.

Ultrastructure of the sensilla on antennae and mouthparts of larval and adult Plutella xylostella (Lepidoptera:Plutellidae)

Plutella xylostella （L.） （Plutellidae） is an important agricultural pest throughout the world. In this study, the morphology of antennal and mouthpart sensilla in the larvae and adults of P. xylostella （L.） was observed by using a scanning electron microscope. The larval antennae possess six sensilla basiconica, two sensilla chaetica and one sensillum styloconicum. Larval mouthparts possess six types of sensilla： sensilla chaetica, sensilla digitiformia, sensilla epipharyngeal, sensilla basiconica, sensilla styloconica and sensilla placodea. In the adult, seven types of sensilla are found on the antennae in males and six types of sensilla （sensilla basiconica absent） occur in females. Sexual dimorphism is also found in the number and size of these sensilla on the antennae of adults. We describe for the first time the five types of sensilla on the mouthparts of the adult of P. xylostella. This study provides useful information for further research into the function of these sensilla, and better understanding the behavioral mechanisms involved in pest control.

Inhibitory Effects of Kojic Acid on Phenoloxidase of Diamondback Moth Plutella xylostella

The effects of kojic acid on phenoloxidase (PO) of Plutella xylostella were investigated, when it had been partially purified by 40% saturated [(NH4)2SO4] and Sephadex G-100 gel filtration. Kojic acid showed inhibitory effects on both monophenolase and o-diphenolase activity of the PO. The inhibitor concentrations leading to 50% (IC50) activity lost were estimated to be 0.07 mmol L-1 for monophenolase and 1 mmol L-1 for diphenolase, respectively. Kojic acid can also prolong the lag time of PO for oxidation of L-tyrosine. The inhibition kinetics were analyzed by Lineweaver-Burk plots and kojic acid was found to be a reversible competitive inhibitor with the Ki of 0.47 mmol L-1. The ability of kojic acid to inhibit the enzyme activity may be associated with its ability to chelate copper at the active site. In addition, the iron ion was found to shorten the lag time obviously, but have no important effect on the monophenolase activity.

Effects of Various Degrees of Antennal Ablation on Mating and Oviposition Preferences of the Diamondback Moth,Plutella xylostella L.

Using scanning electron microscopy, we investigated the distribution of the trichoid, basiconic, and coeloconic sensilla on the antennae of the diamondback moth （DBM;Plutella xylostella）. The trichoid sensilla were the most abundant sensory organ, and the male moth antennae host signiifcantly more trichoid sensilla than female moth antennae. Conversely, basiconic and coeloconic sensilla were found more frequently on female than on male antennae. We performed experiments with various degrees of antennal ablation and demonstrated that DBM antennae played a key role in the control of mating and oviposition. We found that neither oviposition preference nor mating behaviors changed signiifcantly when less than 1/4 of both antennae were removed. However, there was a signiifcant behavioral change when the antennae were ablated by more than half. As the length of the antenna was shortened, the successful mating rate decreased and mating peak was delayed. An otherwise consistent host preference for oviposition was eliminated when both antennae were completely removed. Furthermore, we found that the number of trichoid sensilla was positively correlated with mating rate and oviposition preference. However, the numbers of basiconic and coeloconic sensilla were not correlated with mating rate and mating peak, but highly correlated with oviposition preference. Taken together, our results indicate that antennal sensory information plays a critical role in the mating and oviposition behaviors of this economically important pest.

Sublethal Effects of Metaflumizone on Plutella xylostella (Lepidoptera: Plutellidae)

Metaflumizone is a novel sodium channel blocker insecticide, which has been registered for controling the diamondback moth （DBM）, Plutella xylostella （Lepidoptera： Plutellidae） in China. It is unavoidable for DBMs to be exposed to sublethal dose of metaflumizone in brassicaceous vegetable field. Sublethal effects of metaflumizone at LC15 （1.06 mg L-1） and LCz5 （2.03 mg L-1） on the biological characteristics were investigated. Pupation rate, pupal period and pupal weight were significantly declined comparing with the control （P=0.05） when third instar larvae were exposed to LC15 and LC25 of metaflumizone. Comparing the control group to LC15 and LC25 treatment groups, there were significant differences in the development periods of eggs, first instar larvae, pupae and total preoviposition period. The peak of age-specific fecundity in LC15 and LC25 treatment groups was lagged behind control group. Mean values of intrinsic rate of increase （rm）, net reproductive rate （R0） and finite rate of increase （λ） observed from the LCl5 and LC25 treatment groups were lower than those from the control group. But mean values of gross reproduction rate （GRR） was not different. The mean generation time （T） of the treatment groups was prolonged. Whether sublethal doses of metaflumizone could stimulate reproduction in the long term needs further investigation on continuous generations.

Antifeedant Activity and Active Ingredients Against Plutella xylostella from Momordica charantia Leaves

With the bioguided fractionation of the ethanol extracts from the leaves of Momordica charantia, we obtained two most active compounds against the feeding of the diamondback moth, Plutella xylostella larvae. The antifeedant activity of momordicine Ⅰ and momordicine Ⅱ against the second and the third instar larvae of Plutella xylostella were tested using leaf discs of cabbage in the laboratory. The results showed that momordicin Ⅰ and momordicin Ⅱ had significant antifeedant activity on the larvae of P. xylostella, and momordicin Ⅱ was more active than momordicin Ⅰ. The concentrations for 50% antifeedant effects （AFC50） of momordicin Ⅱ against the second and the third instar larvae of P. xylostella were 76.69 and 116.24μg mL^-1, whereas that of momordicin I was 144.08 and 168.42μg mL^-1, respectively. In addition, momordicin Ⅰ and momordicin Ⅱ had significant inhibitive effect on the rate of weight gain and survival of P. xylostella larvae.

High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rAChE,23.2 U mL-1in supernatant）was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate.The maximum production of r ACh E reached about 11.8 mg of the enzyme protein per liter of culture.The r ACh E was first precipitated with ammonium sulfate（50%saturation）and then purified with procainamide affinity column chromatography.The enzyme was purified 12.1-fold with a yield of 22.8%and a high specific activity of 448.3 U mg-1.It was sensitive to inhibition by methamidophos and pirimicarb,the calculated 50% inhibitory concentration（IC50）values of the two pesticides were 0.357 and 0.888 mg L-1,respectively,and the calculated 70% inhibitory concentration（IC70）values were 0.521 and 0.839 mg L-1,respectively.The results suggested that it has a potential application in the detection of pesticide residues.

Microbial control of diamondback moth, Plutella xylostella L. （Lepidoptera： Yponomeutidae） using bacteria （Xenorhabdus nematophila） and its metabolites from the entomopathogenic nematode Steinernema carpocapsae

Cells and cell-free solutions of the culture filtrate of the bacterial symbiont, Xenorhabdus nematophila taken from the entomopathogenic nematode Steinernema carpocapsae in aqueous broth suspensions were lethal to larvae of the diamondback moth Plutella xylostella. Their application on leaves of Chinese cabbage indicated that the cells can penetrate into the insects in the absence of the nematode vector. Cell-free solutions containing metabolites were also proved as effective as bacterial cells suspension. The application of aqueous suspensions of cells of X.nematophila or solutions containing its toxic metabolites to the leaves represents a possible new strategy for controlling insect pests on foliage.

Characterization of carboxylesterase PxαE8 and its role in multi-insecticide resistance in Plutella xylostella(L.)

Carboxylesterase(CarE)was considered as important phase-I detoxifying enzymes which participated in detoxification of different types of insecticides.Up-regulation of CarE genes has been proved playing a major role in insecticide resistance in many pest insects,but its involvement in resistance to insecticides in Plutella xylostella has been rarely reported.In this study,a CarE cDNA named PxαE8 was identified in P.xylostella,which has an open reading frame of 1599 nucleotides and putatively encodes 532 amino acids.The investigation of spatial expression profiles of PxαE8 revealed that it was expressed in all developmental stages,especially in larvae and adults.The body part/tissue-specific expression profiles showed that the PxαE8 mainly expressed in fat body,malpighian tubule and hemolymph of larvae.Further,the relative expression of PxαE8 in two multi-resistant field populations,Hainan(HN)and Guangdong(GD)populations,was found 24.4-and 15.5-fold higher than that in susceptible population,respectively.Knockdown of PxαE8 by RNA interference dramatically increased the mortalities of larvae of HN population treated with LC_(50) of beta-cypermethrin and phoxim by 25.3 and 18.3%,respectively.These results suggested that up-regulation of PxαE8 was involved in resistance to both beta-cypermethrin and phoxim in P.xylostella,which shed light on further understanding of molecular mechanisms of multi-insecticide-resistance in P.xylostella and other pest insects.

cDNA cloning and characterization of t he carboxylesterase pxCCE016b from the diamondback moth, Plutella xylostella L.

Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced c arboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth（DBM）. However, t he related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a full-length c DNA of carboxylesterase pxCCE016 b was cloned and exogenously expressed in Escherichia coli at the first time, which contained a 1 693 bp open reading frame（ORF） and encoded a protein of 542 amino acids. Sequence analysis showed that this c DNA has a predicted mass of 61.56 k Da and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features： a type-B carboxylesterase signature 2（EDCLYLNVYTK）, a type-B carboxylesterase serine active site（FGGDPENITIFGESAG） and the catalytic triad（S er186, Glu316, and His444）. The real-time quantitative PCR（q PCR） analysis showed that t he expression level of the p x CCE016 b was significantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore, pxCCE016 b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, c hlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016 b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector p ET-pxCCE016 b was constructed with the most coding region（1 293 bp） and large number of soluble recombinant proteins（less than 48 k Da） were expressed successfully with prokaryotic cell. Western blot analysis showed that it was coded by pxCCE016 b. All the above findings provide important information for further f unctional study, although we are uncertainty whether the pxCCE016 b gene is actually i nvolved in chlorantraniliprole resistance.

Electrophysiological responses of Plutella xylostella to 9 different plant volatiles

Plutella xylostella is the main pest of cruciferae plants in the worldwide fields.The volatiles released from host plants serve vital roles in host-finding and oviposition behavior.Electroantennograms were recorded from male and female Plutella xylostella to 9 different plant volatiles(cabbage Brassica oleracea L.var.capitata,baby bokchoi Brassica chinensis L.,broccoli Brassica oleracea L.var.botrytis L.,Chinese cabbage Brassica pekinensis Rupr.,Radish Raphanus sativus L.,towel gourd Luffa Cylindrica Roem.,eggplant Solanum melongena L.,tomato Solanum lycopersicum,pepper Capsicum annuum L.) in healthy/injured status.The statistic analysis show there is a significant difference of EAG relative values between cruciferae and non-cruciferae volatiles.The EAG relative values of injured plant volatiles to both male and female changed a lot during the tests.Multiple-ANOVA analysis represents cross factors(sex,host-plant,plant status) showed a great interactive impacts to the EAG-RV.

Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera:Plutellidae)

Laccase（EC 1.10.3.2）is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction,which generally includes two types of laccase,Lac1 and Lac2.Lac1 oxidizes toxic compounds in the diet,and Lac2 is known to play an important role in melanizing the insect exoskeleton.In this study,we cloned and sequenced the cDNA of the diamondback moth,Plutella xylostella Lac2（PxLac2）,from the third instar larvae using polymerase chain reaction（PCR）and rapid amplification of cDNA ends techniques.The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp.PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa.Moreover,we determined the expression levels of PxLac2 in different stages by quantitative PCR（qPCR）.The results indicated that PxLac2 was expressed differently in different stages.We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae.We also investigated the enzymatic properties of laccase,which had optimal activity at pH 3.0 and at 35°C.Under these optimal conditions,laccase had a Michaelis constant（Km）of 0.97 mmol L-1,maximal reaction speed（Vm）of 56.82 U mL-1,and activation energy（Ea）of 17.36 kJ mol-1 to oxidize2,2＇-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid ammonium salt）.Type II copper enhanced laccase activity below 0.8mmol L-1 and reduced enzyme activity above 0.8 mmol L-1 with an IC50 concentration of 1.26 mmol L-1.This study provides insights into the biological function of laccase.