Sublethal effects of an indoxacarb enantiomer insecticide on Plutella xylostella caterpillar and Chrysoperla sinica predator.

Plutella xylostella (L.) is a migratory species and an important insect pest of cruciferous crops worldwide, and Chrysoperla sinica (Tjeder) is a predaceous insect of agricultural and forest pests in the field. Indoxacarb has two enantiomers: (+)-S-indoxacarb and (-)-R-indoxacarb. This study was conducted to clarify the selective toxicity and sublethal effects of both enantiomers on P. xylostella and C. sinica. The (+)-S-indoxacarb isomer had greater acute toxicity to P. xylostella and C. sinica, while (-)-R-indoxacarb had less toxicity to P. xylostella and low toxicity to C. sinica. Lethal concentration 25 % (LC25) of (+)-S-indoxacarb had significant effects on the development, population, and fecundity of P. xylostella and C. sinica. The LC25 concentration of (-)-R-indoxacarb had a significant effect on the oviposition of P. xylostella. The field recommended concentration of (-)-R-indoxacarb significantly affected the pupal stage, adult survival rate, oviposition, and larval survival rate of C. sinica. Both enantiomers could significantly affect the search efficiency, successful attack rate, prey handling time, and maximum predation of C. sinica larvae, and the effects of (+)-S-indoxacarb alone were greater than those of (-)-R-indoxacarb. This study provided evidence of the different selective toxicity, sublethal effects of indoxacarb enantiomers on P. xylostella and C. sinica, which of the results could provide a basis for more rational use of indoxacarb in ecosystems.

Sublethal effects of tolfenpyrad on the development, reproduction, and predatory ability of Chrysoperla sinica.

The lacewing, Chrysoperla sinica, is a predaceous insect that is important in crop pest management. Chemical pesticides have adversely impacted predaceous insect species. Here we studied the effect of tolfenpyrad on C. sinica. The acute toxicity of tolfenpyrad to the second-instar larvae was determined and indicated that tolfenpyrad is a medium-risk insecticide. Sublethal concentrations (LC10, LC20, and LC30) of tolfenpyrad had effects on the development, reproduction, and predatory ability of C. sinica. When the second-instar larvae of C. sinica were exposed to sublethal concentrations of tolfenpyrad, the activities of protective enzymes, such as superoxide dismutase, peroxidase, and catalase, and detoxification enzymes, including carboxylesterase, glutathione-S-transferase, and P450 monooxygenases, were increased with exposure time. The second-instar larvae of C. sinica exposed to sublethal concentrations of tolfenpyrad exhibited an oxidative stress response that increased the levels of malondialdehyde and reactive oxygen species (ROS). Within 48-120 h after treatment, the contents of mitochondrial respiratory chain complex I and adenosine triphosphate in the second-instar larvae were decreased. This resulted in an imbalance between the production and clearance of ROS and caused cellular damage.

Insecticide resistance and resistance mechanisms in the melon aphid, Aphis gossypii, in Shandong, China.

The melon aphid, Aphis gossypii, is an important pest of vegetables. Insecticide resistance in A. gossypii has increased due to the frequent use of insecticides. We studied the levels and mechanisms of A. gossypii resistance to imidacloprid, acetamiprid and lambda-cyhalothrin here. The resistance levels of the three insecticides in 20 populations of A. gossypii varied. When compared to the susceptible strain (Lab-SS), there were two moderate resistance (MR) populations and nine low resistance (LR) populations to imidacloprid, respectively, two MR populations and two LR populations to acetamiprid, respectively, and, five MR populations and two LR populations to λ-cyhalothrin, respectively. Gene mutation detection in the MR level populations showed arginine to threonine substitution (R81T) in three populations and lysine to glutamine substitution (K264E) in the nicotinic acetylcholine receptor (nAChR) ß1 subunit in one population, respectively. No valine to isoleucine substitution (V62I) was found in the nAChR ß1 subunit in any of the tested populations. The leucine to phenylalanine substitution (L1014F) in sodium channel α subunit was found in five MR populations. The relative expression of the CYP6CY13 gene was significantly upregulated in the Daiyue and Shenxian populations. The CYP6CY14 gene was significantly upregulated in Daiyue, Dongchangfu, Shenxian, Mengyin and Anqiu populations. The CYP6CY19 gene was significantly upregulated in the Dongchangfu and Mengyin populations. The relative expressions of the esterase E4 or FE4 genes were significantly upregulated in most of the MR populations. These results provide insight into the current insecticide resistance of A. gossypii and may contribute to more effective resistance management strategies.

Reduced fecundity and regulation of reproductive factors in flubendiamide-resistant strains of Plutella xylostella.

Diamondback moth (DBM), Plutella xylostella, is an important pest of crucifers worldwide. The extensive use of flubendiamide has led to the development of resistance in field populations and reports of control failures. In this study, the lab-selected (Rf) and field-collected (Rb) flubendiamide-resistant strains of P. xylostella with LC50 resistance ratios of 1890-fold and 1251-fold, respectively, were used, as well as a lab-reared flubendiamide-susceptible strain (S). The results showed that the fecundity of the Rf and Rb-resistant strains was significantly lower than that of S strain. The contents of vitellin and transcripts of P. xylostella vitellogenin (PxVg) and P. xylostella vitellogenin receptor (PxVgR) genes in the Rf and Rb strains were significantly higher than those of S strains at 0-48 h after adult eclosion. At 96 h after eclosion, the content of vitellin in the Rf and Rb strains did not differ significantly from those of S strains, whereas transcripts of the PxVg and PxVgR genes in the Rf and Rb strains were significantly lower than that of the S strain. The content of the juvenile hormone III (JH III), ß-ecdysone (20E), and the gene expression level of P. xylostella methoprene tolerant (PxMet) in the Rf and Rb strains were significantly higher than that of the S strain. The activity of trehalase was significantly higher in the Rf and Rb strains than that of the S strain in the first to the third instar larvae, whereas in the fourth instar larvae, there was no significantly difference in the three strains. At different times after adult eclosion, the differences in trehalase activity were erratic between the strains. The transcripts of P. xylostella trehalase (PxTre) gene in the Rf and Rb strains were significantly higher than that of the S strain in most developmental stages. Here, we report differences in fecundity between flubendiamide-resistant and susceptible strains of P. xylostella and discuss gene expression of several reproductive factors, which provides a possible explanation for the mechanism of fecundity reduction concurrent with flubendiamide-resistance in P. xylostella.

Increased Responses of Phenoloxidase in Chlorantraniliprole Resistance of Plutella xylostella (Lepidoptera: Plutellidae).

The diamondback moth (Plutella xylostella, DBM) is an important pest of cruciferous vegetables. The use of chlorantraniliprole has been essential in the management of the DBM. However, in many countries and areas, DBM has become highly resistant to chlorantraniliprole. Three different DBM strains, susceptible (S), chlorantraniliprole-selected (Rc), and field-collected (Rb) resistant strains/populations were studied for the role of phenoloxidase in resistance development to the insecticide. By assaying the activity of phenoloxidase (PO) in the three different DBM strains, the results showed that the PO activity in the Rc strain was increased significantly compared with the S strain. The synergistic effects of quercetin showed that the resistant ratio (RR) of the QRc larvae to chlorantraniliprole was decreased from 423.95 to 316.42-fold compared with the Rc larvae. Further studies demonstrated that the transcriptional and translational expression levels of PxPPO1 (P. xylostella prophenoloxidase-1 gene) and PxPPO2 (P. xylostella prophenoloxidase-2 gene) were increased to varying degrees compared with the S strain, such as the transcriptional expression levels of PxPPO2 were 24.02-fold that of the S strain. The responses of phenoloxidase were significantly different in chlorantraniliprole-resistant DBM.

Mixtures of fluopyram and abamectin for management of Meloidogyne incognita in tomato.

The southern root-knot nematode (RKN), Meloidogyne incognita, causes significant damage to vegetable production and is a major problem in greenhouse tomatoes. The effect of a combination of fluopyram and abamectin, at a mass ratio of 1:5, was studied for RKN control. Pot trials showed that fluopyram, abamectin, and their combination at three dosages increased the height, stem diameter, root fresh weight, shoot fresh weight, and the root length of tomato plants. The RKN control efficacy of the 1:5 combination at 450 g a.i./ha was 74.06% at 30 days after transplanting (DAT), and the control efficacy of the combination at 337.5 and 450 g a.i./ha differed significantly from those of other treatments at 60 DAT. The root-galling index (RGI) control efficacy of the combination at 450 g a.i./ha and of fluopyram (41.7% SC) only at 450 g a.i./ha were better than the control efficacies of other treatments, and these two treatments significantly increased root activity. Field trial results showed that the soil nematode control efficacy was similar to that of the pot trials at 30 and 60 DAT. The RGI control efficacy of the combination at 337.5 and 450 g a.i./ha and of fluopyram (41.7% SC) only at 450 g a.i./ha differed significantly from those of the two other treatments. The tomato yields of the 1:5 combination at 450 g a.i./ha were increased by 24.07 and 23.22% compared to the control in field trials during two successive years. The combination of fluopyram and abamectin provides good nematode measure, and it can increase tomato yields. It provides an effective solution for the integrated management of southern RKN.

Flubendiamide resistance and Bi-PASA detection of ryanodine receptor G4946E mutation in the diamondback moth (Plutella xylostella L.).

The extensive application of flubendiamide has led to increasingly prominent development of resistance in diamondback moth, Plutella xylostella. Here we report that the moderate and high level resistance to flubendiamide was identified in a laboratory-selected and two field-collected strains of P. xylostella. The resistance ratios were tested in the lab-selected resistant strains (R), and two field strains (BY and ZC). Compared with the S strain, the R strain showed extended larval development time, decreased pupation rate, emergencing rate, and male adult longevity. The realized heritability (h(2)=0.135) implies the high risk of flubendiamide resistance development in P. xylostella. A Bi-PASA (bi-directional PCR amplification of specific allele)-based method was successfully developed to detect the point mutation (G4946E) potentially causing flubendiamide resistance in diamondback moth, in which different fragments 866 bp + 340 bp, 866 bp+568 bp, and 866 bp+568 bp+340 bp were presented in SS, RR and RS stains, respectively. The predominant genotype was 83.33% SS homozygote in the S strain, 80.77% RR homozygote in ZC population, and 73.08% RS heterozygote in BY population, respectively. Current results showed the significant correlation between the frequencies of the allele carrying G4946E mutation (51.92%, 55.77% and 90.38% for R, BY and ZC, respectively) and the resistance ratios (40.72, 24.24 and 1779.24-folds for R, BY and ZC, respectively) in the three strains/populations. In addition, the relative PxRyR mRNA transcript level in the R strain was 2.938 ± 0.53 folds as compared with the S strain (1.0-fold).

Chlorantraniliprole resistance in the diamondback moth (Lepidoptera: Plutellidae).

The wide application of chlorantraniliprole, which selectively targets insect ryanodine receptors (RyR), for control of the diamondback moth, Plutella xylostella (L.), has led to increasingly prominent development of resistance to this insecticide. Although much work has been carried out on the structure and function of RyR, the molecular mechanisms of resistance to chlorantraniliprole in diamondback moth still needs further investigation. P. xylostella strains with medium and high resistance to chlorantraniliprole were obtained by laboratory selection and field collection. The biological activity of chlorantraniliprole against the third-instar larvae of susceptible and resistant strains was tested, and resistance development and biological fitness were investigated. The realized heritability (h2) of resistance showed the diamondback moth has a high risk of resistance to chlorantraniliprole. RyR transcript levels were lower in resistant strains than in susceptible strains, indicating that decreased expression of PxRyR may be associated with chlorantraniliprole resistance in P. xylostella. A 4,400 bp fragment of the RyR cDNA, which encodes most of the functional domains of RyR, was cloned and characterized from four strains (S, F18, BY, and ZC). A 14 amino acid (Q4546-S4559) deletion was found in three resistant strains (F18, BY, and ZC). A point mutation resulting in a glycine to glutamate substitution, as reported in a previously published article, was also found in the carboxyl-terminal region of two resistant strains (BY and ZC). These results indicated that decreased transcriptional level of RyR mRNA and combined with the site mutation might be related to chlorantraniliprole resistance in P. xylostella.

Sublethal effects and reproductive hormesis of emamectin benzoate on Plutella xylostella.

The diamondback moth (DBM), Plutella xylostella L., is an important pest of cruciferous vegetables, and population control mainly depends on chemical pesticides. Emamectin benzoate is a highly effective insecticide used for controlling DBM. However, it is unknown how the sublethal effects of low concentration residues of emamectin benzoate on DBM. So the population development sublethal effects of emamectin benzoate, at LC5, LC10, and LC20 with concentrations of 0.014 mg/L, 0.024 mg/L and 0.047 mg/L, respectively, on adult DBM and their progeny were investigated in this study. The pupal weight, pupal period, female fecundity, and vitellin content of the F0 DBM generation increased significantly compared to the control. And the single female oviposition number of DBM was increased by 20.21% with LC20 treatment. The pupation rate, adult longevity and ovariole length of the treatment groups decreased significantly. The fecundity of DBM in the treatment groups increased, and this increased the population by a presumptive 13.84%. Treatment also led to the shortening of ovarioles and the reduction of egg hatching, and increased pupal weight in the F1 generation. We concluded that the effects of sublethal/low concentration emamectin benzoate on the different life stages of DBM were variable, and the reproductive hormesis on DBM adults were attractive findings.

Acute lethal and sublethal effects of four insecticides on the lacewing (Chrysoperla sinica Tjeder).

The lacewing, Chrysoperla sinica, is an important predatory insect, which plays an important role in the integrated pest management of agroforestry pests. However, the extensive use of insecticides negatively affects C. sinica. The acute toxicity, risk level, and, sublethal effects on growth and production, predation ability, protective enzyme activity and genotoxicity of four insecticides: indoxacarb, emamectin benzoate, imidacloprid and lambda-cyhalothrin to C. sinica were studied. The results showed that all four insecticides had lethal toxicity to larvae of C. sinica. Among them, emamectin benzoate had the highest toxicity with LC50 value of 7.41 mg/L. The insecticides also had different effects on the growth and reproduction of C. sinica, of which lambda-cyhalothrin had the greatest impacts. Even at a very low LC1 concentration (3.37 mg/L), it had strong impacts on the growth, reproduction and predatory ability of C. sinica. The four insecticides also caused a decrease in the predatory ability of the lacewing, of which lambda-cyhalothrin had the greatest effect. During the larval stage, the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were significantly decreased by the four insecticides. At the pupal and adult stages, the effects of the four insecticides on the activities of protective enzymes were different, and the activities of SOD, CAT and POD decreased or increased. Indoxacarb and lambda-cyhalothrin exposure induced DNA damage in the haemocytes of C. sinica and produced obvious genotoxicity. These results provide important scientific basis for the rational use of these insecticides and the protection and utilization of lacewing.

Chromosomal deletions mediated by CRISPR/Cas9 in Helicoverpa armigera.

Helicoverpa armigera, cotton bollworm, is one of the most disastrous pests worldwide, threatening various food and economic crops. Functional genomic tools may provide efficient approaches for its management. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, dependent on a single guide RNA (sgRNA), has been used to induce indels for targeted mutagenesis in cotton bollworm. However, genomic deletions may be more desirable to disrupt the function of noncoding genes or regulatory sequences. By injecting two sgRNAs with Cas9 protein targeting different exons, we obtained predictable genomic deletions of several hundred bases. We achieved this type of modification with different combinations of sgRNA pairs, including HaCad and HaABCC2. Our finding indicated that CRISPR/Cas9 can be used as an efficient tool to engineer genomes with chromosomal deletion in H. armigera.

Resistance to Diamide Insecticides in Plutella xylostella (Lepidoptera: Plutellidae): Comparison Between Lab-Selected Strains and Field-Collected Populations.

Diamondback moth, Plutella xylostella (L.; Lepidoptera: Plutellidae), is an important pest of crucifers worldwide. The extensive use of diamide insecticides has led to P. xylostella resistance and this presents a serious threat to vegetable production. We selected chlorantraniliprole (Rf) and flubendiamide (Rh) resistance strains of P. xylostella with resistance ratios of 684.54-fold and 677.25-fold, respectively. The Rf and Rh strains underwent 46 and 36 generations of lab-selection for resistance, respectively. Low cross resistance of Rh to cyantraniliprole was found. Cross resistance to chlorfenapyr, tebufenozid, and indoxacarb was not found in Rf and Rh strains. The P. xylostella ryanodine receptor gene (PxRyR) transcripts level in the Rf and Rh strains was up-regulated. Except for Rf34 and Rh36, PxRyR expression in all generations of Rf and Rh selection gradually increased with increasing resistance. Two resistant populations were field-collected from Guangzhou Baiyun (Rb) and Zengcheng (Rz) and propagated for several generations without exposure to any pesticide had higher PxRyR expression than the susceptible strain (S). In the S strain, PxRyR expression was not related to the resistance ratio. Gene sequencing found that the RyR 4946 gene site was glycine (G) in the S, Rf, and Rh strains, and was glutamate (E) with 70% and 80% frequency in the Rb and Rz populations, respectively. The 4946 gene site was substituted by valine (V) with the frequency of 30% and 20% in Rb and Rz populations, respectively. These results increase the understanding of the mechanisms of diamide insecticide resistance in P. xylostella.

Inhibition kinetics of cabbage butterfly (Pieris rapae L.) larvae phenoloxidase activity by 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone.

Phenoloxidase (PO) is a key enzyme in insect development, responsible for catalyzing the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay in air-saturated solutions and the kinetic behavior of PO from Pieris rapae (Lepidoptera) larvae in the oxidation of L-tyrosine (a monophenol) and L-DOPA (l-3, 4-dihydroxyphenylalanine) (a diphenol) was studied. The inhibitory effects of 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone (3-H-4-MBT) on the monophenolase and diphenolase activities of PO were also studied. The results show that 3-H-4-MBT can inhibit both the monophenolase and diphenolase activities of PO. The lag period of L-tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activities of the enzyme sharply decreased. The inhibitor was found to be noncompetitively reversible with a K I (K I = K IS) of 0.30 micromol/L and an estimated IC50 of 0.14 +/- 0.02 micromol/L for monophenolase and 0.26 +/- 0.04 micromol/L for diphenolase. In the time course of the oxidation of L-DOPA catalyzed by the enzyme in the presence of different concentrations of 3-H-4-MBT, the rate decreased with increasing time until a straight line was approached. The microscopic rate constants for the reaction of 3-H-4-MBT with the enzyme were determined.

Molecular Characterization and Bioinformatics Analysis of a Prophenoloxidase-1 (PPO1) in Plutella xylostella.

Phenoloxidase (PO) is an important enzyme in insect life, which is involved in important physical functions, such as defensive encapsulation and melanization of foreign organisms and wound healing. In this study, we obtained a cDNA sequence of 2838 bp with 2049 open reading frames encoding 682 amino acids. The protein sequence deduced from the cDNA has high homology with the known PPO1 sequences of other lepidopterous insects. There were three conserved regions, including the two copper-binding sites characteristic of arthropod PPOs. The whole PxPPO1 DNA was also obtained with 7202 bp when the five fragments were stitched together and the overlapping sequences were deleted. The PxPPO1 DNA consists of 11 introns and 12 exons, and the homology is 99.9% when the exons are compared with the above cDNA. Moreover, the gene expression levels were also determined by semiquantitative polymerase chain reaction (PCR), Western blotting, and real-time quantitative PCR; the results indicated that PxPPO1 transcripts in the eggs and the fourth instar larvae were more abundant, followed by the second and the third instar larvae, prepupae, and pupa.

Quantitative structure-activity relationship studies of mushroom tyrosinase inhibitors.

Here, we report our results from quantitative structure-activity relationship studies on tyrosinase inhibitors. Interactions between benzoic acid derivatives and tyrosinase active sites were also studied using a molecular docking method. These studies indicated that one possible mechanism for the interaction between benzoic acid derivatives and the tyrosinase active site is the formation of a hydrogen-bond between the hydroxyl (aOH) and carbonyl oxygen atoms of Tyr98, which stabilized the position of Tyr98 and prevented Tyr98 from participating in the interaction between tyrosinase and ORF378. Tyrosinase, also known as phenoloxidase, is a key enzyme in animals, plants and insects that is responsible for catalyzing the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the bioactivities of 48 derivatives of benzaldehyde, benzoic acid, and cinnamic acid compounds were used to construct three-dimensional quantitative structure-activity relationship (3D-QSAR) models using comparative molecular field (CoMFA) and comparative molecular similarity indices (CoMSIA) analyses. After superimposition using common substructure-based alignments, robust and predictive 3D-QSAR models were obtained from CoMFA (q2 = 0.855, r2 = 0.978) and CoMSIA (q2 = 0.841, r2 = 0.946), with 6 optimum components. Chemical descriptors, including electronic (Hammett sigma), hydrophobic (pi), and steric (MR) parameters, hydrogen bond acceptor (H-acc), and indicator variable (I), were used to construct a 2D-QSAR model. The results of this QSAR indicated that pi, MR, and H-acc account for 34.9, 31.6, and 26.7% of the calculated biological variance, respectively. The molecular interactions between ligand and target were studied using a flexible docking method (FlexX). The best scored candidates were docked flexibly, and the interaction between the benzoic acid derivatives and the tyrosinase active site was elucidated in detail. We believe that the QSAR models built here provide important information necessary for the design of novel tyrosinase inhibitors.

3D-QSAR and molecular docking studies of benzaldehyde thiosemicarbazone, benzaldehyde, benzoic acid, and their derivatives as phenoloxidase inhibitors.

Phenoloxidase (PO), also known as tyrosinase, is a key enzyme in insect development, responsible for catalyzing the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. Inhibition of PO may provide a basis for novel environmentally friendly insecticides. In the present study, we determined the inhibitory activities and IC50 values of 57 compounds belonging to the benzaldehyde thiosemicarbazone, benzaldehyde, and benzoic acid families against phenoloxidase from Pieris rapae (Lepidoptera) larvae. In addition, the inhibitory kinetics of 4-butylbenzaldehyde thiosemicarbazone against PO was measured in air-saturated solutions for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA). The results indicated that the compound is a reversible noncompetitive inhibitor. The bioactivity results were used to construct three-dimensional quantitative structure-activity relationship (3D-QSAR) models using two molecular field analysis techniques: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). After carrying out superimposition using common substructure-based alignment, robust and predictive 3D-QSAR models were obtained from CoMFA (q2=0.926, r2=0.986) and CoMSIA (q2=0.933, r2=0.984) with six optimum components. The 3D-QSAR model built here will provide hints for the design of novel PO inhibitors. The molecular interactions between the ligands and the target were studied using a flexible docking method (FlexX). The best scored candidates were docked flexibly, and the interaction between the representative compound 4-butylbenzaldehyde thiosemicarbazone and the active site was elucidated in detail.