Non-steroidal Ecdysone Agonist Research Articles

Gonadotrophic effects of RH-5849, a non-steroidal ecdysone agonist on the rice moth Corcyra cephalonica St. (Lepidoptera: Galleriinae)

In several areas throughout the world, the eggs of the fictitious host insect, Corcyra cephalonica, are used to produce multiple natural enemies, such as Trichogramma species, which are widely utilized for biological control of lepidopterous pests on diverse crops in more than 30 nations. The purpose of this study was to determine the efficacy of RH-5849, a non-steroidal ecdysone mimic, on the gonadotrophic parameters of the rice moth, Corcyra cephalonica. The separated II instar larva received the compound topically on the abdomen dorsum by means of Hamilton micro syringe in acetone. Five replicates (50 insect/replicate) were used per each dosage. Similar replicates were used for the control insects without any treatment. The mean number of eggs laid by a female moth was worked out on the basis of the total number of eggs laid by ten females. In a dose-dependent way, the sterility percentage was enhanced while the hatching percentage was decreased. The sterility indices were shown to be dose-dependent, according to current data. It should not be forgotten that the hatching percentages reversed this effectuation tendency (egg viability).

Receptor-binding affinity and larvicidal activity of tetrahydroquinoline-type ecdysone agonists against Aedes albopictus.

Tetrahydroquinolines (THQs), a class of nonsteroidal ecdysone agonists, are good candidates for novel mosquito control agents because they specifically bind to mosquito ecdysone receptors (EcRs). We have recently performed quantitative structure–activity relationship (QSAR) analyses of THQs to elucidate the physicochemical properties important for the ligand–receptor interaction. Based on previous QSAR results, here, we newly synthesized 15 THQ analogs with a heteroaryl group at the acyl moiety and evaluated their binding affinity against Aedes albopictus EcRs. We also measured the larvicidal activity of the combined set of previously and newly synthesized compounds against A. albopictus to examine the contribution of receptor-binding to larvicidal activity. Multiple regression analyses showed that the binding affinity and the molecular hydrophobicity of THQs are the key determinants of their larvicidal activity.

Field resistance of Plutella xylostella (Lepidoptera: Plutellidae) to lufenuron: Inheritance and lack of cross-resistance to methoxyfenozide

Plutella xylostella (L.), commonly known as diamondback moth, is one of the most important pests of Brassicaceae in Brazil. This study aimed to assess the susceptibility of P. xylostella and pattern of resistance inheritance to lufenuron, an insect growth regulator (IGR) that impairs the insect molting, as well as the cross-resistance to methoxyfenozide (Nonsteroidal ecdysone agonist). Concentration-mortality bioassays were performed using the leaf dipping method towards field collected populations and genotypes used in the genetic crossings. Populations of this pest, from the mesoregion of Pernambuco (Brazil), were highly resistant to lufenuron. The recommended concentration of lufenuron caused more than 80% mortality of larvae of two populations [Piedade (PDD) and Recife (REC-S)]. Methoxyfenozide caused more than 80% mortality of larvae of all assessed populations, suggesting no cross-resistance between insect growth regulators. The population of Bezerros (BZR-R) was the most resistant (11,283-fold) to lufenuron compared with REC-S population (standard reference). Resistance to lufenuron was inherited as autosomal, incompletely recessive, and monofactorial trait, suggesting that a single gene or a set of genes tightly linked is involved in resistance. Methoxyfenozide is an alternative molecule suggested for the control of P. xylostella in areas where resistance to lufenuron has evolved. Furthermore, information provided in this work should fine-tune insecticide resistance management (IRM) programs to delay the evolution of resistance to chitin synthesis inhibitors like lufenuron, where resistance frequency is incipient.

친환경적 살충제 개발을 위한 비스테로이드성 Ecdysone 작용제들의 화학구조 특성 비교 연구

The relationship between physicochemical and molecular structural properties of non-steroidal ecdysone agonists and their bioactivity was investigated in this study. 2 Dibenzoylhydrazines including RH 5849 and tebufenozide, 1 non-dibenzoylhydrazine, DTBHIB and 1 naturally found agonist, 8-O-acetylharpagide were selected and their molecular physicochemical parameter values such as Log P, hydrophilic-lipophilic balance(H.L.B.), hydrogen bond(H.B.), dipole moment(D.M.), and connectivity/kappa index and distribution of electronic charge were calculated/drawn using computer program to be compared. DTBHIB showed physicochemical and steric properties more similar to dibenzoylhydrazines than 8-O-acetylharpagide. Overally, non-steroidal ecdysone agonists showed an increasing tendency of bioactivity with the increase of their hydrophobicity within a certain range. It was also shown that dibenzoylhydrazines and DTBHIB share the common distribution pattern of molecular electronic charge which is offered by -C=O linked with the left benzene ring. These results suggest that bioactivity of non-steroidal ecdysone agonists is affected combinedly by physico-chemical properties such as hydrophilicity of the ligand, position and steric orientation of functional groups and distribution of electronic charge in the molecule, not only by the number of -OH in the molecule and resulting possibility of hydrogen bond with receptors.

MiR-189942 regulates fufenozide susceptibility by modulating ecdysone receptor isoform B in Plutella xylostella (L.)

Although dibenzoylhydrazine-type non-steroidal ecdysone agonists, such as fufenozide, have an excellent performance record, the emergence of resistance could severely compromise the efficacy of these compounds in integrated pest management programs. To investigate possible mechanisms of resistance, we investigated the regulation of the expression of the PxEcR-B gene encoding the ecdysone receptor isoform B (PxEcR-B), which is the specific target of fufenozide in P. xylostella. Bioinformatics analysis revealed a putative miR-189942 binding site in the 3’-UTR of PxEcR-B mRNA. In a PxEcR-B 3’-UTR luciferase reporter system, miR-189942 downregulated the luciferase activity, and these effects were abolished by a deletion mutation in the putative miR-189942 binding site. Moreover, at 96 h after treatment with an agomir (mimic) or antagomir (inhibitor) of miR-189942, PxEcR-B expression was decreased by 71 ± 4% and increased by 4.19- fold respectively. Furthermore, overexpression or knockdown of miR-189942 changed the sensitivity of P. xylostella to fufenozide in vivo but had no influence on the sensitivity to chlorantraniliprole, which does not target PxEcR-B. These data indicate that miR-189942 suppressed PxEcR-B expression via binding at the 3’-UTR of PxEcR-B, thus increasing the tolerance of P. xylostella to fufenozide. These findings provide empirical evidence of the involvement of miRNAs in the regulation of insecticide resistance.

Asymmetric synthesis of tetrahydroquinoline-type ecdysone agonists and QSAR for their binding affinity against Aedes albopictus ecdysone receptors.

Tetrahydroquinolines (THQs) are a class of non-steroidal ecdysone agonists that specifically bind to mosquito ecdysone receptors (EcR). The THQ scaffold contains two chiral centers at the C-2 and C-4 positions, resulting in four stereoisomers. We have previously shown that the (2R,4S)-isomers are the most biologically active; however, the lack of a practical synthetic method for these isomers has hampered further structure-activity studies. In this study, a chiral phosphoric acid-catalyzed Povarov reaction was employed to develop a facile asymmetric synthesis of THQs with a (2R,4S)-configuration, which allowed the preparation of a 40-compound library of enantiopure THQs. Evaluation of their binding affinity against Aedes albopictus EcR, followed by quantitative structure-activity relationship (QSAR) analyses, uncovered the physicochemical properties of THQs that are important for the ligand-receptor interaction. The most potent THQ derivative was twofold more active than the molting hormone, 20-hydroxyecdysone. The QSAR results provide valuable information for the rational design of novel mosquito-specific ecdysone agonists. © 2018 Society of Chemical Industry.

Activity of a selective insecticide (methoxyfenozide) against two mosquito species (Culex pipiensandCuliseta longiareolata): toxicological, biometrical and biochemical study

AbstractMethoxyfenozide is an insect growth disruptor belonging to the class of nonsteroidal ecdysone agonists. In the present study, methoxyfenozide (23% emulsifiable concentrate) is evaluated against newly molted fourth‐instar larvae ofCulex pipiensL. andCuliseta longiareolataMacquart (Diptera: Culicidae), aiming to investigate its possible effects on growth and development. Larvae are exposed for 24 h under standard laboratory conditions in accordance with World Health Organization recommendations. The product is found to exhibit insecticidal activity against the two tested mosquito species with a concentration–response relationship. Moreover, based on the lethal concentrations determined, methoxyfenozide is slightly more toxic againstC. pipiensthanC. longiareolata. Furthermore, fourth‐instar larva of the two mosquito species are treated using two lethal concentrations (LC50and LC90) of methoxyfenozide, and then individual survivals (larvae, pupae and adults) from larval treatment are subjected to a biometrical and biochemical study. The compound is found to interfere with growth by reducing the larval and pupal development duration. Moreover, the body volume and the main biochemical contents (proteins, carbohydrates and lipids) from different stages are affected. The overall results suggest that the ecdysone agonist under investigation interferes with the development process and has potential for mosquito control.

Toxicological and morphological effects of tebufenozide on Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae

The velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), is an important soybean pest in the Americas. Tebufenozide, a novel nonsteroidal ecdysone agonist is used to control this pest. Bioassays were conducted to assess tebufenozide toxicity and their ultrastructural effects on midgut of A. gemmatalis. The toxicity, survivorship, behavior response, and respiration rate for A. gemmatalis larvae after exposure to tebufenozide were evaluated. Also, A. gemmatalis larvae were treated with LC50 obtained from tebufenozide and changes were observed on their midgut cells after 24, 48 and 96 h. Tebufenozide was toxic to A. gemmatalis (LC50 = 3.86 mg mL−1 and LC90 = 12.16 mg mL−1) and survivorship was 95% for adults that had not been exposed to tebufenozide, decreasing to 52% with LC50 and 27% with LC90 estimated value. Damage to midgut cells was increased with exposure time. These cells show damaged striated border with release of protrusions to the midgut lumen, damaged nuclear membrane and nucleus with condensed chromatin and increase in amount of autophagic vacuoles. Mitochondria were modified into nanotunnels which might be an evidence that tebufenozide induces damage to cells, resulting in cell death, proved by immunofluorescence analyses. This insecticide also caused paralysis movement with change in homeostasis and compromised larval respiration. Thus, sublethal exposure to tebufenozide is sufficient to disturb the ultrastructure of A. gemmatalis midgut, which might compromise insect fitness, confirming tebufenozide a possible controlling insecticide.

Residue analysis and dietary exposure risk assessment of tebufenozide in stem lettuce (Lactuca sativa L. var. angustana Irish)

Tebufenozide, a newly-developed nonsteroidal ecdysone agonist, is in pre-regulation phase (before approval for use) on stem lettuce in China. Aiming at the safe application of tebufenozide, the dissipation and terminal residue trials on stem lettuce were performed under good agricultural practice (GAP). The dissipation trials shown that tebufenozide was rapidly degraded in stem lettuce, with half-lives of 5.0–8.2 days. Pre-regulation dietary exposure risk assessments were evaluated to recommend maximum residue limits (MRLs) based on risk quotients (RQ) method. Relevant toxicological parameters including ADI (acceptable daily intake) and ARfD (acute reference dose) were applied to assess the potential dietary exposure risk. The results indicated the chronic dietary exposure risk probability (RQc) of tebufenozide ranged from 36.4% to 70.0%. The acute dietary exposure risk probability (RQa) of tebufenozide was 2.88%–8.49% in lettuce stems and 14.0%–20.0% in lettuce leaves, respectively. On the basis of supervised field trial data and dietary exposure risk assessment results, the MRLs of tebufenozide were recommended as 3 mg/kg for lettuce stems and 10 mg/kg for lettuce leaves, respectively. The results demonstrated that the dietary exposure risk of tebufenozide used in stem lettuce under GAP was negligible and would not pose unacceptable health risk to Chinese consumers.

Quantitative structure–activity relationship of substituted imidazothiadiazoles for their binding against the ecdysone receptor of Sf-9 cells

Imidazothiadiazoles (ITDs) are a class of potent nonsteroidal ecdysone agonists with larvicidal activity. Previously, we performed the Hansch–Fujita type of quantitative structure–activity relationship (QSAR) analysis for ITD analogs (Yokoi et al., Pestic. Biochem. Physiol.2015, 120, 40–50). The activity was reasonably explained by hydrophobicity and electronegativity of substituents on the imidazothiadiazole ring system. However, the limited data points (n = 8) hampered the examination of other physicochemical parameters. In the present study, we expanded the library of ITD congeners and evaluated their receptor-binding affinity using intact Sf-9 cells. The QSAR analysis for the expanded set revealed the significance of the third physicochemical parameter, the negative steric effect for long substituents. We also evaluated the larvicidal activity of the synthesized compounds against Spodoptera litura; however, it was not correlated to the binding affinity. The results obtained here suggests that the pharmacokinetic properties must be improved to enhance the larvicidal activity of ITDs.

New reporter gene assays for detecting natural and synthetic molting hormone agonists using yeasts expressing ecdysone receptors of various insects.

Synthetic nonsteroidal ecdysone agonists, a class of insect growth regulators (IGRs), target the ecdysone receptor (EcR), which forms a heterodimer with ultraspiracle (USP) to transactivate ecdysone response genes. These compounds have high binding affinities to the EcR–USP complexes of certain insects and their toxicity is selective for certain taxonomic orders. In the present study, we developed reporter gene assay (RGA) systems to detect molting hormone (ecdysone) activity by introducing EcR–USP cDNA and a bacterial lacZ reporter gene into yeast. EcR and USP were derived from the insect species of three different taxonomic orders: Drosophila melanogaster (Diptera), Chilo suppressalis (Lepidoptera), and Leptinotarsa decemlineata (Coleoptera). Transcriptional coactivator taiman (Tai) cDNA cloned from D. melanogaster was also used in this RGA system. This yeast RGA system responded to various EcR ligands in a dose‐dependent and ecdysteroid‐specific manner. Furthermore, the insect order‐selective ligand activities of synthetic nonsteroidal ecdysone agonists were linearly related to their binding activities, which were measured against in vitro translated EcR–USP complexes. Our newly established yeast RGA is useful for screening new molting hormone agonists that work selectively on target insects.

Dissipation and metabolism of tebufenozide in cabbage and soil under open field conditions in South China

The novel nonsteroidal ecdysone agonist tebufenozide is capable of controlling a wide range of lepidopteran pests. However its intensive use in vegetables has raised concerns towards the safety of food and environment. Here, we developed an easy and reliable method to analyze tebufenozide in cabbage and soil by reversed-phase high performance liquid chromatography (HPLC). The average recoveries of tebufenozide ranged from 72.01% to 101.10% with the relative standard deviations (RSD)<6%, and the LOD and LOQ were 0.02µgg−1 and 0.05µgg−1, respectively. According to the dissipation study, the half-lives of tebufenozide were 2.96 and 4.08 d in cabbage and 4.95–7.70d in soil, respectively. The final residues were determined below the maximum residue limit (MRL) (0.5mgkg−1) after a pre-harvest interval (PHI) of 7d. Moreover, its major metabolites were identified by liquid chromatography tandem mass spectrometry (LC–MS/MS) on LTQ-Orbitrap XL, which leading to the first report of the degradation pathway of tebufenozide in cabbage. The present study is expected to provide basic data for the use guidance and safety evaluation of tebufenozide in agricultural crops and environment.

Structure–activity relationship of imidazothiadiazole analogs for the binding to the ecdysone receptor of insect cells

Diacylhydrazines are the first non-steroidal ecdysone agonists, and five compounds are used as insecticides in agriculture. After the discovery of diacylhydrazine-type compounds, numerous non-steroidal structures were reported as ecdysone agonists. Among various ecdysone agonists, imidazothiadiazoles are reported to be very potent in vitro; however, the experimental detail for the structure identification and bioassays are not stated in the paper (Holmwood and Schindler, Bioorg. Med. Chem. 17, 4064–4070, 2009). In our present study, we synthesized 18 imidazothiadiazole-type compounds and confirmed the chemical structures by spectrometric analyses. The binding activity of the synthesized compounds to the ecdysone receptor was evaluated in terms of the concentration required for 50% inhibition of [3H]ponasterone A incorporation [IC50 (M)] into lepidopteran (Sf-9), coleopteran (BCRL-Lepd-SL1), and dipteran (NIAS-AeAl2) cells. 6-(2-Chlorophenyl)-2-(trifluoromethyl)imidazo[2,1-b] [1,3,4]-thiadiazol-5-yl)acrylamide analogs with CONHR (secondary amide) were very potent against Sf-9 cells, but further alkylation (tertiary amide: CONR2) decreased the activity dramatically. Additionally, a primary amide analog (CONH2) was inactive. The activity also decreased 150-fold by the saturation of olefin region of the acrylamide moiety. In addition, various substituents were introduced at the 2-position of the imidazothiadiazole ring to disclose the physicochemical properties of the substituents which are important for receptor binding. The activity increased by 7500-fold with the introduction of the CF2CF2CF3 group compared to the unsubstituted compound against Sf-9 cells. Quantitative structure–activity relationship analysis for these substituents indicated that hydrophobic and electron-withdrawing groups were favorable for binding. Some of the compounds with strong receptor binding activity showed good larvicidal activity against Spodoptera litura. In contrast, the binding affinity of imidazothiadiazole analogs was low or not observed against dipteran and coleopteran cells.

EFFECTS OF THE NONSTEROIDAL ECDYSONE AGONIST, TEBUFENOZIDE, ON LIVER CYP ACTIVITIES

The non-steroidal ecdysone agonist tebufenozide was orally administered to Wistar rats for 5 days to examine their effects on the liver cytochrome P450 (CYP) activities. Also Michaelis-Menten kinetics of the metabolic reactions catalyzed by liver CYPs were studied after adding tebufenozide to the assay system to investigate its possible inhibitory effects on liver CYPs activities. These reactions included ethoxyresorufin O-deethylation, tolbutamide hydroxylation, bufuralol 1-hydroxylation, and midazolam 1,4-hydroxylation, for CYP1A, 2C, 2D, and 3A activities, respectively. Results revealed no any significant alteration in the activities of the tested microsomal enzymes after oral administration of tebufenozide. none of the examined CYPs was inhibited by tebufenozide as the Ki values were very high (more than 9000 M). We can conclude that tebufenozide is considered safe and can be a good alternate for the traditional insecticides.

Tebufenozide resistance is associated with sex-linked inheritance in Plutella xylostella.

The diamondback moth (DBM), Plutella xylostella (L.), is a major pest of cruciferous crops. Tebufenozide, a novel nonsteroidal ecdysone agonist, exhibits good efficacy and has played an increasingly important role in the control of Lepidopteran pests in China. For its resistance management, the genetic basis of tebufenozide resistance was studied using a laboratory selected resistant strain of DBM (resistant ratio, RR = 268). A series of crosses with laboratory susceptible and resistant strains revealed that tebufenozide resistance in this pest was partially biased toward female heredity, with a large difference in RR for F1 (RR = 29) and rF1 progeny (RR = 147). The dominance calculated for these 2 cross progeny was -0.788 and 0.09, respectively. Further analysis showed that the susceptible male and female larvae were similar in their sensitivity to tebufenozide, but the resistant female larvae showed significantly higher resistance than the resistant male larvae. The heredity of tebufenozide resistance in DBM might be linked with the W sex chromosome, which suggested that DBM has the ability to develop high levels of resistance to tebufenozide. This is the first report of sex-linked inheritance of tebufenozide resistance in P. xylostella (L.).

Cloning and functional analysis of the ecdysteroid receptor complex in the opossum shrimp Neomysis integer (Leach, 1814)

In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan.Major results were: (i) cDNAs encoding the ecdysteroid receptor (EcR) and the retinoid-X-receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likelihood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacostracan clade (bootstrap percentage=75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the common shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein–protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to tebufenozide at a concentration of 100μg/L, and no effects against the molting process and nymphal development were scored.In conclusion, the in vitro cell reporter assay, based on NiEcR-LBD/CrcRXR-LBD heterodimerization in Hi5 cells and validated with the natural ecdysteroid hormone PonA, represents a useful tool for the screening of putative EDCs. As a test example for non-steroidal ecdysone agonist insecticides, tebufenozide had no negative effects on NiEcR/RXR receptor dimerization in vitro, nor on the molting process and nymphal development of N. integer at the tested concentration (100μg/L) in vivo.

Low field-relevant tebufenozide concentrations affect reproduction in Chironomus riparius (Diptera: Chironomidae) in a long-term toxicity test

A few studies reporting the effects of tebufenozide, a non-steroidal ecdysone agonist that mimics natural moulting hormones in chironomids exist in the literature. However, nothing is known about its chronic or multigenerational effects on the reproduction of aquatic insects, although tebufenozide is present in aquatic ecosystems. Here, we investigated the chronic toxicity of tebufenozide in two successive generations of Chironomus riparius using nominal concentrations that ranged from 4 to 26.2 μg/L. We started the test from the first instar larvae in the parental (P) generation, quantifying life cycle parameters (emergence, sex ratio, development rate, fecundity and fertility) until the emergence in the subsequent F1 generation. Results showed a reduction in reproduction and a significant decrease in male developmental rate of midges for all treatments, in the F1 generation compared with the P generation (paired t test; p<0.001). Two-way analysis of variance revealed a significant exposure × generation effect on male fraction with male fraction increasing (P generation) or decreasing (F1 generation) with increasing exposure. These effects on C. riparius underline the importance of conducting long-term studies with environmentally relevant concentrations to investigate population-level endpoints for endocrine disrupting chemicals.

Sequencing and structural homology modeling of the ecdysone receptor in two chrysopids used in biological control of pest insects

In insects, the process of molting and metamorphosis are mainly regulated by a steroidal hormone 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) that specifically bind to the ecdysone receptor ligand-binding domain (EcR-LBD). Currently, several synthetic non-steroidal ecdysone agonists, including tebufenozide, are commercially available as insecticides. Tebufenozide exerts its activity by binding to the 20E-binding site and thus activating EcR permanently. It appears that subtle differences in the architecture among LBDs may underpin the differential binding affinity of tebufenozide across taxonomic orders. In brief, first we demonstrated the harmlessness of tebufenozide towards Chrysoperla externa (Ce). Then, a molecular analysis of EcR-LBD of two neuropteran insects Chrysoperla carnea and Ce was presented. Finally, we constructed a chrysopid in silico homology model docked ponasterone A (PonA) and tebufenozide into the binding pocket and analyzed the amino acids indentified as critical for binding to PonA and tebufenozide. Due to a restrict extent in the cavity at the bottom of the ecdysone-binding pocket a steric clash occurred upon docking of tebufenozide. The absence of harm biological effect and the docking results suggest that tebufenozide is prevented of any deleterious effects on chrysopids.

The stability and biochemical basis of fufenozide resistance in a laboratory-selected strain of Plutella xylostella

Fufenozide, a novel non-steroidal ecdysone agonist, exhibits good efficacy and plays an important role in controlling lepidopteran pests in China. A high level of resistance to fufenozide (320.3-fold) in Plutella xylostella (L.) has been selected under laboratory conditions. In this study, the stability and biochemical basis of resistance to fufenozide were investigated. The resistance ratio to fufezonide declined substantially from 320.3- to 20.5-fold in the fufenozide resistant strain (JSR) when the selection pressure was removed for six consecutive generations. However, the susceptibility to fufezonide could not be fully restored after 28 generations without exposure to the compound. Increased relative fitness (0.87) was associated with the rapid reversal of resistance in the resistance decaying strain (JSRD). The enzymatic analyses showed reduced specific activities of monooxygenase (MO), glutathione S-transferases (GSTs), carboxylesterase and aryl-acylamidase in the JSRD strain, whereas the activity of chitinase increased. The current findings suggest the selected resistance to fufenozide in P. xylostella is not stable, and the four detoxifying enzymes including MO, GSTs, carboxylesterase, and aryl-acylamidase may be involved. For resistance management proposes, insecticide rotation should be a viable option to delay the occurrence of fufenozide resistance in the field.

Cross‐resistance patterns and fitness in fufenozide‐resistant diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

Fufenozide is a novel non-steroidal ecdysone agonist with good efficacy against diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae). At present, it is widely applied for the control of a range of lepidopterous pests in China. This study compared the activities of fufenozide and 12 other insecticides against unselected and fufenozide-selected strains of DBM to examine potential patterns of cross-resistance. The relative fitness of the fufenozide-selected strain was assessed to provide information pertinent to insecticide resistance management. Compared with the susceptible strain (JSS), the fufenozide-resistant strain (JSR) showed high cross-resistance to dibenzoylhydrazines and benzoylphenylureas, low cross-resistance to abamectin and no cross-resistance to organophosphates, carbamates and pyrethroids. JSR had a lower reproductive ability and a relative fitness of 0.5 compared with JSS. P. xylostella has the potential to develop resistance to fufenozide, albeit at the expense of fitness. Cross-resistance between the same and other classes of insecticides is of concern, and should be a key consideration when implementing fufenozide-based control strategies for this species.