Diamondback Moth Resistance Research Articles

Regional changes of maximum dose insecticide responses in diamondback moth (Lepidoptera: Plutellidae) populations from Georgia and Florida, USA.

Maximum dose bioassays were conducted to assess the efficacy of multiple registered active ingredients for diamondback moth (DBM), Plutella xylostella (L.), control in Georgia and Florida from 2021 to 2023 as a follow-up to an earlier study. Low efficacy (<40% mortality) was recorded for the highest labeled rate of Bacillus thuringiensis var. kurstaki strain ATBS-351 in Georgia, as well as chlorantraniliprole, cyantraniliprole, and cyclaniliprole in Georgia and Florida. The active ingredients with the highest efficacy (>80% mortality) in both states were naled, emamectin benzoate, and spinetoram. Independent analysis of data by state indicated that the efficacy of bifenthrin, chlorantraniliprole, cyantraniliprole, tolfenpyrad, and methomyl was higher in Florida populations than in Georgia populations. In addition, a comparison of these data to a recent DBM maximum dose survey in the same region suggested that these DBM populations have rapidly developed high levels of resistance to cyantraniliprole and cyclaniliprole. This work provides growers in the region with a recent ranking of insecticide efficacy that documents the loss of control for certain active ingredients, which assists pest managers in the planning of ongoing insecticide rotations for DBM resistance management.

A comprehensive assessment of insecticide resistance mutations in source and immigrant populations of the diamondback moth Plutella xylostella (L.).

The diamondback moth (DBM) Plutella xylostella has developed resistance to almost all insecticides used to control it. Populations of DBM in temperate regions mainly migrate from annual breeding areas. However, the distribution pattern of insecticide resistance of DBM within the context of long-distance migration remains unclear. In this study, we examined the frequency of 14 resistance mutations for 52 populations of DBM collected in 2010, 2011, 2017 and 2018 across China using a high-throughput KASP genotyping method. Mutations L1041F and T929I conferring pyrethroid resistance, and mutations G4946E and E1338D conferring chlorantraniliprole resistance were near fixation in most populations, whereas resistant alleles of F1020S, M918I, A309V and F1845Y were uncommon or absent in most populations. Resistance allele frequencies were relatively stable among different years, although the frequency of two mutations decreased. Principal component analysis based on resistant allele frequencies separated a southern population as an outlier, whereas the immigrants clustered with other populations, congruent with the migration pattern of northern immigrants coming from the Sichuan area of southwestern China. Most resistant mutations deviated from Hardy-Weinberg equilibrium due to a lower than expected frequency of heterozygotes. The deviation index of heterozygosity for resistant alleles was significantly higher than the index obtained from single nucleotide polymorphisms across the genome. These findings suggest heterogeneous selection pressures on resistant mutations. Our results provide a picture of resistant mutation patterns in DBM shaped by insecticide usage and migration of this pest, and highlight the widespread distribution of resistance alleles in DBM. © 2022 Society of Chemical Industry.

MAPK-mediated transcription factor GATAd contributes to Cry1Ac resistance in diamondback moth by reducing PxmALP expression.

The benefits of biopesticides and transgenic crops based on the insecticidal Cry-toxins from Bacillus thuringiensis (Bt) are considerably threatened by insect resistance evolution, thus, deciphering the molecular mechanisms underlying insect resistance to Bt products is of great significance to their sustainable utilization. Previously, we have demonstrated that the down-regulation of PxmALP in a strain of Plutella xylostella (L.) highly resistant to the Bt Cry1Ac toxin was due to a hormone-activated MAPK signaling pathway and contributed to the resistance phenotype. However, the underlying transcriptional regulatory mechanism remains enigmatic. Here, we report that the PxGATAd transcription factor (TF) is responsible for the differential expression of PxmALP observed between the Cry1Ac susceptible and resistant strains. We identified that PxGATAd directly activates PxmALP expression via interacting with a non-canonical but specific GATA-like cis-response element (CRE) located in the PxmALP promoter region. A six-nucleotide insertion mutation in this cis-acting element of the PxmALP promoter from the resistant strain resulted in repression of transcriptional activity, affecting the regulatory performance of PxGATAd. Furthermore, silencing of PxGATAd in susceptible larvae reduced the expression of PxmALP and susceptibility to Cry1Ac toxin. Suppressing PxMAP4K4 expression in the resistant larvae transiently recovered both the expression of PxGATAd and PxmALP, indicating that the PxGATAd is a positive responsive factor involved in the activation of PxmALP promoter and negatively regulated by the MAPK signaling pathway. Overall, this study deciphers an intricate regulatory mechanism of PxmALP gene expression and highlights the concurrent involvement of both trans-regulatory factors and cis-acting elements in Cry1Ac resistance development in lepidopteran insects.

A feasibility trial of genomics-based diagnosis detecting insecticide resistance of the diamondback moth.

Insecticide resistance management has been key for crop protection for over 70 years and is increasingly important because the development of new active ingredients has decreased in recent years. By monitoring the development of resistance in a timely manner, we can effectively prolong insecticide efficacy. Genomic-based diagnosis can reliably predict resistance development if information on resistant mutations against major pesticides is available. Here, we developed a feasibility trial of genomics-based diagnosis of insecticide resistance in diamondback moth (Plutella xylostella) populations in Nagano Prefecture, Japan. Amplicon sequencing analyses using a next-generation sequencer (Illumina MiSeq) for major insecticides, including diamides, pyrethroids, Bacillus thuringiensis (Bt) toxin (Cry1Ac), organophosphates, and spinosyns, were conducted. Mutations related to the resistance of pyrethroids, organophosphates, and diamides (flubendiamide and chlorantraniliprole) prevailed, while those of a diamide (cyantraniliprole), Bt (Cry1Ac), and spinosyns were scanty, suggesting that they are still effective. The results of the genomics-based diagnosis were generally concordant with the results of bioassays. Resistance development tendencies were generally uniform across Nagano. An insecticide-resistance management campaign can be conducted in Nagano Prefecture with a quick genomic-based diagnosis in early spring while bioassay is the only option for monitoring resistances whose mutations are unavailable. Our study is the first step in the future management of insecticide resistance in all significant pests. © 2022 Society of Chemical Industry.

Monitoring the development of resistance in diamondback moth (DBM) Plutella xylostella (L.) to diamide group of insecticides

The field resistance level in Plutella xylostella to diamide insecticides, viz., flubendiamide 20 WG at 50 g a.i. ha-1, chlorantraniliprole 18.5 SC at 10 g a.i. ha-1 and cyantraniprole 10.26 OD at 600 g a.i. ha-1 was assayed at four locations in Tamil Nadu, India viz., Coimbatore, Krishnagiri, Oddanchatram and Kotagiri. Susceptibility of these populations varied and exhibited significant resistance towards these insecticides. The highest level of resistance to flubendiamide, chlorantraniliprole and cyantraniprole in terms of resistance ratio (fold increase in LC50 over the susceptible laboratory population) was registered in the P. xylostella population collected from Krishnagiri (9.73), Kotagiri (9.15) and Krishnagiri (5.47), respectively.

Bacillus thuringiensis and Chlorantraniliprole Trigger the Expression of Detoxification-Related Genes in the Larval Midgut of Plutella xylostella.

Background: Diamondback moth (DBM), Plutella xylostella (L.), has developed resistance to many insecticides. The molecular mechanism of DBM resistance to Bt-G033A combined with chlorantraniliprole (CL) remains undefined.Methods: In this study, field-resistant strains of Plutella xylostella to three pesticides, namely, Bacillus thuringiensis (Bt) toxin (Bt-G033A), CL, and a mixture of Bt + CL, were selected to evaluate the resistance level. Additionally, transcriptomic profiles of a susceptible (SS-DBM), field-resistant (FOH-DBM), Bt-resistant (Bt-DBM), CL-resistant (CL-DBM), and Bt + CL-resistant (BtC-DBM) strains were performed by comparative analysis to identify genes responsible for detoxification.Results: The Bt-G033A was the most toxic chemical to all the DBM strains among the three insecticides. The comparative analysis identified 25,518 differentially expressed genes (DEGs) between pairs/combinations of strains. DEGs were enriched in pathways related to metabolic and catalytic activity and ABC transporter in resistant strains. In total, 17 metabolic resistance-related candidate genes were identified in resistance to Bt-G033A, CL, and Bt + CL by co-expression network analysis. Within candidate genes, the majority was upregulated in key genes including cytochrome P450, glutathione S-transferase (GST), carboxylesterase, and acetylcholinesterase in CL- and BtC-resistant strains. Furthermore, aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, trypsin, and ABC transporter genes were eminent as Bt-resistance-related genes. Expression patterns of key genes by the quantitative real-time PCR (qRT-PCR) proved the credibility of transcriptome data and suggest their association in the detoxification process.Conclusion: To date, this study is the most comprehensive research presenting functional transcriptome analysis of DBM using Bt-G033A and CL combined insecticidal activity.

Profiling of MicroRNAs in Midguts of Plutella xylostella Provides Novel Insights Into the Bacillus thuringiensis Resistance.

The diamondback moth (DBM), Plutella xylostella, one of the most destructive lepidopteran pests worldwide, has developed field resistance to Bacillus thuringiensis (Bt) Cry toxins. Although miRNAs have been reported to be involved in insect resistance to multiple insecticides, our understanding of their roles in mediating Bt resistance is limited. In this study, we constructed small RNA libraries from midguts of the Cry1Ac-resistant (Cry1S1000) strain and the Cry1Ac-susceptible strain (G88) using a high-throughput sequencing analysis. A total of 437 (76 known and 361 novel miRNAs) were identified, among which 178 miRNAs were classified into 91 miRNA families. Transcripts per million analysis revealed 12 differentially expressed miRNAs between the Cry1S1000 and G88 strains. Specifically, nine miRNAs were down-regulated and three up-regulated in the Cry1S1000 strain compared to the G88 strain. Next, we predicted the potential target genes of these differentially expressed miRNAs and carried out GO and KEGG pathway analyses. We found that the cellular process, metabolism process, membrane and the catalytic activity were the most enriched GO terms and the Hippo, MAPK signaling pathway might be involved in Bt resistance of DBM. In addition, the expression patterns of these miRNAs and their target genes were determined by RT-qPCR, showing that partial miRNAs negatively while others positively correlate with their corresponding target genes. Subsequently, novel-miR-240, one of the differentially expressed miRNAs with inverse correlation with its target genes, was confirmed to interact with Px017590 and Px007885 using dual luciferase reporter assays. Our study highlights the characteristics of differentially expressed miRNAs in midguts of the Cry1S1000 and G88 strains, paving the way for further investigation of miRNA roles in mediating Bt resistance.

Utilizing local plant extracts to control Diamondback moth in round cabbage

Diamondback moth (Plutella xylostella L.) with abbreviation DBM is a detrimental pest that mostly attacks cabbage and other vegetables in Brassicaceae family. The build-up of resistance in DBM due to continuous and indiscriminate use of synthetic insecticides have refocused farmer’s perspectives to plant based extracts. Plant extracts are generally compatible with natural enemies of pests therefore are considered eco-friendly. This study was done to evaluate the push effect of Garlic (Allium sativum L.), Chili (Capsicum frutescens L.) and Seaweed extract on the activity of DBM. All three treatments; chili extract, garlic extract, and seaweed extract were generally effective in controlling DBM than the reference group (control treatment). Separation of means by Student-newman-keuls (SNK) test showed that Diamondback moth (DBM) abundance under seaweed extract was significantly low (2.18±0.32, p< 0.05) followed by chili extract (3.22±0.43, p< 0.05). There was no significant difference in DBM abundance under garlic extract and control treatment. For defoliation, only control treatment recorded a significant increase in DBM population (5.40±0.64, p< 0.05). The other three plant extracts had similar low non-significant abundances (p> 0.05). Data on LAI did not show any significant differences in all the treatments meaning treatments did not affect LAI (p> 0.05). This study found that both seaweed and chili extracts were effective in controlling P. xylostella in round cabbage. Both extracts are recommended for use since they differ in their mechanism pathways to protect crops.

Antimicrobial peptides are not involved in Plutella xylostella resistance to Cry1Ac

AbstractPrevious studies on Bacillus thuringiensis (Bt) resistance in the diamondback moth (DBM) Plutella xylostella have often focused on receptor proteins in the gut. In recent years, many studies have indicated that insect resistance to Bt is related to the immune system. Moreover, our group demonstrated that the expression of peptidoglycan recognition protein (PGRP) genes, which are located upstream of the insect humoral immune system signalling pathway, is significantly different between Cry1Ac‐resistant and Cry1Ac‐susceptible strains of DBM. Thus, whether antimicrobial peptides (AMPs), which are the major downstream effectors of the insect humoral immune system signalling pathway, are also related to the resistance of DBMs to Bt is a question worthy of in‐depth study. In the present study, we sought to (a) clone the CDS of AMP genes expressed in DBMs and (b) explore the effect of the cloned AMPs on the biological characteristics and Bt resistance of DBMs. Here, we revealed three kinds of AMPs (PxDef, PxMor and PxCec), and each of their full‐length CDSs was cloned. The recombinant PxDef, PxMor or PxCec proteins, which were expressed in a prokaryotic expression system, have a significant protective effect on DBMs in response to the complex microbial environment of the outside world but have no significant effect on the resistance of DBMs to Cry1Ac toxin. We believe that AMPs are not suitable as the primary entry point for studying the relationship between the immune system of DBMs and their resistance to Bt. This result will enable investigation of breakthrough ideas for further elucidating the mechanism of Bt impact on the insect immune system.

Mode of inheritance of chlorpyrifos resistance in diamondback moth

Diamondback moth is one of the destructive insect pests of brassicaceous vegetable crops worldwide with increased insecticide resistance. Chlorpyrifos is one of the most widely-used organophosphate insecticides to control various pests including P. xylostella. The insecticide resistance in Plutella xylostella can be best managed by perceiving CPF resistance inheritance. We designed this experiment to evaluate CPF resistance's genetics of two genetically pure lines of P. xylostella, namely insensitive AChE1-carrying Kar-R resistant strain and Gu-S wild-type strain. A considerable difference was observed among LC50 values of F1 (Kar-R♀ × Gu-S♂) and F1′ (Kar-R♂ × Gu-S♀) strains due to reciprocal crosses between each resistant strain and susceptible strains. The degree of dominance values were 0.58 and 0.75, respectively suggesting an autosomal and partially dominant resistance. The χ2 goodness-of-fit test results and measuring the number of genes which segregate in CPF-resistant strain suggested that several genetic factors control the resistance.

Ryanodine receptor mutations (G4946E and I4790K) differentially responsible for diamide insecticide resistance in diamondback moth, Plutella xylostella L.

This study examined diamondback moth (Plutella xylostella) strains showing high-level resistance to cyantraniliprole (KA17 strain) and to flubendiamide and chlorantraniliprole (KU13 strain). The LC50 value of the KA17 strain against cyantraniliprole was ca. 100-fold higher than that of the KU13 strain. The KA17 strain also exhibited high-level resistance to chlorantraniliprole and flubendiamide equivalent to those in the KU13 strain. The KU13 strain showed a higher LC50 value against cyantraniliprole than the susceptible strains. However, the LC50 value of the KU13 strain against cyantraniliprole was below the agriculturally recommended concentration. Subsequent QTL analysis using ddRAD-seq identified the resistance responsible regions of the KA17 and KU13 strains with different diamide resistance profiles. Ryanodine receptor (RyR) gene was included in the identified regions. Single nucleotide polymorphism calling in the RyR gene using RNA-seq found previously reported G4946E (amino acid mutation from glycine to glutamic acid at amino acid position 4946) and novel I4790K (amino acid mutation from isoleucine to lysine at amino acid position 4790) mutations, respectively, in the RyR of the KU13 and KA17 strains. Functional significance of I4790K in the resistance was confirmed in calcium imaging of the human embryonic kidney 293T cell line expressing Bombyx mori RyR with the mutation. This reporting is the first describing I4790K as a fundamental mechanism responsible for the resistance to the diamides including cyantraniliprole. From this study, we also report up-regulated expression of some degradation enzymes and that of the RyR gene in the KA17 and KU13 strains based on results of RNA-seq data analysis.

Behavioral response and adaptive cost in resistant and susceptible Plutella xylostella to Chlorantraniliprole

Diamides have been used worldwide to manage the diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), however some strains showed resistance to these molecules. Also, pheromone traps could be used to manage this pest, hence reducing the use of insecticides in the field. Resistant DBM strains may have biological disadvantages in comparison to susceptible strains in areas without sprays, including reduction in fitness or behavioral changes. Therefore, the aim of this study was to investigate whether DBM strains resistant to chlorantraniliprole showed adaptive costs that could alter male attraction to the sex pheromone, in comparison to susceptible strains in the laboratory and semi-field conditions. First, the LC1, LC10, LC25, and LC50 of DBM to chlorantraniliprole were established, which were 0.003, 0.005, 0.007, and 0.011 mg a.i. liter-1, and 5.88, 24.80, 57.22, and 144.87 mg a.i. liter-1 for the susceptible and resistant strains, respectively. Development and reproduction of DBM strains subjected to those concentrations were compared. Later, male response to the sex pheromone was investigated in a Y-tube in the laboratory and in a greenhouse to pheromone traps. Resistant DBM strain showed an adaptive cost in comparison to the susceptible strain that can result in a delay in population growth in the field when selection pressure is absent. Conversely, resistant males have no olfactory response alteration in comparison to susceptible males, consistently at 3 (P = 0.6848) and 7 days (P = 0.9140) after release, suggesting that pheromone traps continue to be a viable alternative to manage DBM in an IPM system.

Combining the high-dose/refuge strategy and self-limiting transgenic insects in resistance management-A test in experimental mesocosms.

The high‐dose/refuge strategy has been the primary approach for resistance management in transgenic crops engineered with Bacillus thuringiensis toxins. However, there are continuing pressures from growers to reduce the size of Bt toxin‐free refugia, which typically suffer higher damage from pests. One complementary approach is to release male transgenic insects with a female‐specific self‐limiting gene. This technology can reduce population sizes and slow the evolution of resistance by introgressing susceptible genes through males. Theory predicts that it could be used to facilitate smaller refugia or reverse the evolution of resistance. In this study, we used experimental evolution with caged insect populations to investigate the compatibility of the self‐limiting system and the high‐dose/refuge strategy in mitigating the evolution of resistance in diamondback moth, Plutella xylostella. The benefits of the self‐limiting system were clearer at smaller refuge size, particularly when refugia were inadequate to prevent the evolution of resistance. We found that transgenic males in caged mesocosms could suppress population size and delay resistance development with 10% refugia and 4%–15% initial resistance allele frequency. Fitness costs in hemizygous transgenic insects are particularly important for introgressing susceptible alleles into target populations. Fitness costs of the self‐limiting gene in this study (P. xylostella OX4139 line L) were incompletely dominant, and reduced fecundity and male mating competitiveness. The experimental evolution approach used here illustrates some of the benefits and pitfalls of combining mass release of self‐limiting insects and the high‐dose/refuge strategy, but does indicate that they can be complementary.

Mode of inheritance of Indoxacarb resistance in diamondback moth, Plutella xylostella (L.) and cross resistance to different groups of pesticides

The development of insecticidal resistance in diamondback moth (DBM) Plutella xylostella has immediate implications for its management. In this study, we examined the mode of inheritance of Indoxacarb resistance in P. xylostella. The indoxacarb-resistant strain (Px-R) was obtained through continuous laboratory selection with increasing doses of indoxacarb in each generation. At the 14th generation of selection, the resistance ratio of Px-R strains was 238 over the susceptible strain (Px-S). The mode of inheritance to Indoxacarb in P. xylostella was examined performing standard reciprocal crosses between Px-R and Px-S and response of Px-R, Px-S and F1 hybrid progenies to Indoxacarb through leaf dip bioassay. The degree of dominance (D) and heritability (h) of F1 hybrid progeny ranged from −0.001 to 0.0012 and 0.499 to 0.506, respectively. The Indoxacarb resistance was appeared to be autosomal and inherited as a semi-dominant trait. The Px-R strain of P. xylostella showed little cross resistance to cypermethrin and there was no cross resistance to other pesticides viz., chlorpyrifos, spinosad, karanjin, xentari (Bta-Cry1C) and MVP-II (Cry1Ac). Since the Indoxacarb resistance inherited as a semi-dominant trait in P. xylostella, the sub lethal doses and frequent use of indoxacarb should be avoided for the management of P. xylostella. Moreover, Px-R of P. xylostella showed positive cross resistance to synthetic pyrethroids (cypemethrin), therefore indoxacarb and synthetic pyrethroids should not be recommended together for management of P. xylostella.

Molecular evaluation of diamide resistance in diamondback moth (Lepidoptera: Yponomeutidae) populations using quantitative sequencing

A method to estimate proportions of the G4946E mutation in the ryanodine receptor gene, a major mechanism of diamide insecticide resistance, was developed for Plutella xylostella Linnaeus populations using quantitative sequencing (QS). Using the QS-based method, the relationship between proportions of the G4946E mutation and mortalities for diamide (flubendiamide) was examined using field-collected populations. Results showed that the populations might be classifiable as diamide resistant.

Global identification of microRNAs associated with chlorantraniliprole resistance in diamondback moth Plutella xylostella (L.)

The diamondback moth (DBM), Plutella xylostella (L.), is one of the most serious cruciferous pests and has developed high resistance to most insecticides, including chlorantraniliprole. Previous studies have reported several protein-coding genes that involved in chlorantraniliprole resistance, but research on resistance mechanisms at the post-transcription level is still limited. In this study, a global screen of microRNAs (miRNAs) associated with chlorantraniliprole resistance in P. xylostella was performed. The small RNA libraries for a susceptible (CHS) and two chlorantraniliprole resistant strains (CHR, ZZ) were constructed and sequenced, and a total of 199 known and 30 novel miRNAs were identified. Among them, 23 miRNAs were differentially expressed between CHR and CHS, and 90 miRNAs were differentially expressed between ZZ and CHS, of which 11 differentially expressed miRNAs were identified in both CHR and ZZ. Using miRanda and RNAhybrid, a total of 1,411 target mRNAs from 102 differentially expressed miRNAs were predicted, including mRNAs in several groups of detoxification enzymes. The expression of several differentially expressed miRNAs and their potential targets was validated by qRT-PCR. The results may provide important clues for further study of the mechanisms of miRNA-mediated chlorantraniliprole resistance in DBM and other target insects.

Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae)

Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.

Diamondback moth resistance to insecticides and its management in the Sigatoka Valley, Fiji

Diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae) is a serious cabbage pest in Fiji. Excessive insecticide use has led to resistance to all commonly used insecticides, including the recently introduced anthranilic diamide, rynaxypyr. Grower surveys and laboratory bioassays show that the limited range of insecticides available in Fiji results in their overuse and contributes to field resistance. In 2009, 95% of cabbage farmers used pyrethroids against DBM (resistance ratio (RR) to deltamethrin = 41-191), 68% used indoxacarb (RR=40-89), 28% used lufenuron (RR<10) and 23% used organophosphates (RR not determined). Use of Bacillus thuringiensis (Bt) (8% of growers) and rynaxypyr (5%) was limited and field populations were highly susceptible to both insecticides. By 2012, insecticide use had changed markedly, rynaxypyr was used by 41% of growers, pyrethroids by 76%, indoxacarb by 46% but Bt was not used at all. This resulted in RRs for pyrethroids and indoxacarb falling to 12-30 and 1-26, respectively, but the RR for rynaxypyr increased to 13. In the absence of selection in the laboratory, resistance to rynaxypyr remained static over 10 generations but increased markedly (RR=52) within just two generations when exposed to the insecticide. This rapid development of stable field resistance to rynaxypyr is a significant threat to its sustainable use. Field trials show that resistant field populations can be effectively managed by strategic applications of Bt. The approach to making Bt readily available to Fijian farmers is discussed and progress on the development and implementation of a national insecticide resistance management strategy presented.

CDNA cloning and characterization of the 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 carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a full-length cDNA of carboxylesterase pxCCE016b was cloned and exogenously expressed in Escherichia coli at the first time, which contained a 1693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa 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 (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of the pxCCE016b was significantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore, pxCCE016b 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, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1 293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfully with prokaryotic cell. Western blot analysis showed that it was coded by pxCCE016b. All the above findings provide important information for further functional study, although we are uncertainty whether the pxCCE016b gene is actually involved in chlorantraniliprole resistance.

Discovery of Organophosphate Resistance-Related Genes Associated With Well-known Resistance Mechanisms of Plutella xylostella (L.) (Lepidoptera: Plutellidae) by RNA-Seq.

Pesticide resistance poses many challenges for pest control, particularly for destructive pests such as diamondback moths ( Plutella xylostella ). Organophosphates have been used in the field since the 1950s, leading to selection for resistance-related gene variants and the development of resistance to new insecticides in the diamondback moth. Identifying actual and potential genes involved in resistance could offer solutions for control. This study established resistant diamondback moth strains from two different collections using mevinphos. Two sets of transcriptome sequencing (RNA-Seq) data were generated for pairs of mevinphos-resistant versus susceptible (wild-type) strains. One susceptible strain containing 14 giga base pairs was assembled into a reference-based assembly using published scaffold sequences as reference. Differential expression data between resistant and susceptible strains revealed 944 transcripts (803 with annotations) showing upregulation and 427 transcripts (150 with annotations) showing downregulation. Around 6.8% of the differential expression transcripts (65) could be categorized as associated with well-known resistance mechanisms such as penetration, detoxification, and behavior response; of these 65 transcripts, 38 showed upregulation, and 12 relating to penetration were upregulated when the transcripts of 19 cytochrome P450s, 2 zeta-class glutathione S-transferases, and 4 ATP-binding cassette transporters showed upregulation. In addition, 11 groups of transcripts related to olfactory perception appeared to be downregulated in trade-off situations. Quantitative polymerase chain reaction expression results were consistent with RNA-Seq data. Possible roles of these differentially expressed genes in resistance mechanisms are discussed in this study.