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Molecular characterization of Cry1F resistance in fall armyworm, Spodoptera frugiperda from Brazil

Fall armyworm, Spodoptera frugiperda (J.E. Smith) is a major lepidopteran pest of maize in Brazil and its control particularly relies on the use of genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins such as Cry1F. However, control failures compromising the efficacy of this technology have been reported in many regions in Brazil, but the mechanism of Cry1F resistance in Brazilian fall armyworm populations remained elusive. Here we investigated the molecular mechanism of Cry1F resistance in two field-collected strains of S. frugiperda from Brazil exhibiting high levels of Cry1F resistance. We first rigorously evaluated several candidate reference genes for normalization of gene expression data across strains, larval instars and gut tissues, and identified ribosomal proteins L10, L17 and RPS3A to be most suitable. We then investigated the expression pattern of ten potential Bt toxin receptors/enzymes in both neonates and 2nd instar gut tissue of Cry1F resistant fall armyworm strains compared to a susceptible strain. Next we sequenced the ATP-dependent Binding Cassette subfamily C2 gene (ABCC2) and identified three mutated sites present in ABCC2 of both Cry1F resistant strains: two of them, a GY deletion (positions 788–789) and a P799 K/R amino acid substitution, located in a conserved region of ABCC2 extracellular loop 4 (EC4) and another amino acid substitution, G1088D, but in a less conserved region. We further characterized the role of the novel mutations present in EC4 by functionally expressing both wild type and mutated ABCC2 transporters in insect cell lines, and confirmed a critical role of both sites for Cry1F binding by cell viability assays. Finally, we assessed the frequency of the mutant alleles by pooled population sequencing and pyrosequencing in 40 fall armyworm populations collected from maize fields in different regions in Brazil. We found that the GY deletion being present at high frequency. However we also observed many rare alleles which disrupt residues between sites 783–799, and their diversity and abundance in field collected populations lends further support to the importance of the EC4 domain for Cry1F toxicity.

Diverse patterns of constitutive and inducible overexpression of detoxifying enzyme genes among resistant Aphis glycines populations

Understanding the mechanisms of pyrethroid resistance is essential to the effective management of pesticide resistance in Aphis glycines Matsumura (Hemiptera: Aphididae). We mined putative detoxifying enzyme genes in the draft genome sequence of A. glycines for cytochrome oxidase P450 (CYP), glutathione-S-transferase (GST) and esterases (E4 and carboxylesterases-CES). Aphids from clonal populations resistant to pyrethroids from three sites in Minnesota, USA, were screened against a diagnostic LC99 concentration of either λ-cyhalothrin or bifenthrin and detoxifying enzyme genes expression in survivors was analyzed by qPCR. Their expression profiles were compared relative to a susceptible clonal population. We found 61 CYP (40 full-length), seven GST (all full-length), seven E4 (five full-length) and three CES (two full-length) genes, including 24 possible pseudogenes. The detoxifying enzymes had different expression profiles across resistant aphid populations, possibly reflecting differences in the genetic background and pyrethroid selection pressures as the number of constitutively overexpressed detoxifying enzyme genes was correlated with the level of resistance. Our findings will strengthen the understanding of the pyrethroid resistance mechanisms in A. glycines.

Reduced cadherin expression associated with resistance to Bt toxin Cry1Ac in pink bollworm

Better understanding of the molecular basis of resistance is needed to improve management of pest resistance to transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Here we analyzed resistance of the pink bollworm (Pectinophora gossypiella) to Bt toxin Cry1Ac, which is used widely in transgenic Bt cotton. Field‐evolved practical resistance of pink bollworm to Cry1Ac is widespread in India, but not in China or the United States. Previous work with laboratory‐ and field‐selected pink bollworm indicated that resistance to Cry1Ac is caused by changes in the amino acid sequence of a midgut cadherin protein (PgCad1) that binds Cry1Ac in susceptible larvae.

Divergent molecular evolution in glutathione S-transferase conferring malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel)

Insect glutathione S-transferases (GSTs) are important in insecticide detoxification and Insect-specific GSTs, Epsilon and Delta, have largely expanded in insects. In this study, we functionally expressed and characterized an epsilon class GST gene (BdGSTe8), predominant in the adult Malpighian tubules of Bactrocera dorsalis. This gene may be associated with malathion resistance based on transcriptional studies of resistant and susceptible strains. RNA interference-mediated knockdown of this gene significantly recovered malathion susceptibility in the adults of a malathion-resistant strain, and overexpression of BdGSTe8 enhanced resistance in transgenic Drosophila. Analysis of BdGSTe8 polymorphism showed that several point mutations may be associated with metabolic resistance to malathion. A cytotoxicity assay in Escherichia coli indicated that both of the recombinant BdGSTe8 proteins may play a functional role in protecting cells from toxicity. The allele of BdGSTe8-B conferred higher levels of malathion detoxification capability. Liquid chromatography and ultra-performance liquid chromatography–tandem mass spectrometry analysis showed that the BdGSTe8-A allele did not metabolize malathion directly. However, the BdGSTe8-B allele was involved in the direct metabolism of malathion, which was caused by a mutation in V128A. Further analysis of the sequence suggests that BdGSTe8 evolved rapidly. It maybe play the role of a backup gene and could become a new gene in the future in order to retain the ability of detoxification of malathion, which was driven by positive selection. These results suggest that divergent molecular evolution in BdGSTe8 has played a role in metabolic resistance to malathion in B. dorsalis.

Multiple detoxification genes confer imidacloprid resistance to Sitobion avenae Fabricius

The English grain aphid, Sitobion avenae Fabricius, is one of the major wheat pests distributed worldwide. Insecticide application is typically effective for controlling S. avenae, However, S. avenae has developed resistance to multiple insecticides. Thus, it is crucial to elucidate resistance mechanisms in S. avenae. In this study, the mechanisms of resistance to imidacloprid in S. avenae and the risk of cross resistance to other insecticides were investigated. An imidacloprid resistant strain (SA-R) of S. avenae was established via continuous imidacloprid selection with 42.7-fold resistance compared with the susceptible population (SA-S). There was cross resistance in SA-R between imidacloprid and other insecticides tested (acetamiprid, abamectin, chlorpyrifos, and omethoate), with ratios ranging from 10.7- to 24.7-fold higher than SA-S. S, S, S-tributyl phosphorotrithioate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM) had synergism with imidacloprid in the SA-R strain with 2.85-, 3.59-, and 2.14-fold reductions, respectively, in LC50 values. Additionally, the activities of cytochrome P450 (P450), glutathione-S-transferases (GST), and carboxylesterase (COE) were significantly higher by 2.69-, 1.42-, and 1.39-fold in the SA-R strain than in the SA-S strain. Based on the S. avenae transcriptome database, 11 P450, two COE, four GST, and two esterase (EST) genes that were significantly induced by imidacloprid were selected to compare gene expression levels between SA-R and SA-S. Fourteen detoxification genes of the 19 tested from the SA-R strain were overexpressed compared to those of the SA-S strain. Silencing of CYP6A14-1, CYP307A1, GST1-1-1, and COE2 by dsRNA feeding resulted in increased susceptibility of SA-R aphids to imidacloprid. These results provide evidence that detoxification genes including CYP6A14-1, CYP307A1, GST1-1-1, and COE2 contribute to resistance to imidacloprid in S. avenae, which was confirmed by RNA interference (RNAi) feeding.

Laboratory selection, cross‐resistance, and estimations of realized heritability of indoxacarb resistance in Phenacoccus solenopsis (Homoptera: Pseudococcidae)

Cotton mealybug Phenacoccus solenopsis is a pest of cotton, vegetables, ornamentals, and medicinal plants. In many parts of the world P. solenopsis has been managed by integration of cultural, mechanical, biological and chemical methods, but in Pakistan the use of insecticide sprays has resulted in the development of resistance to some insecticides. In this study indoxacarb resistance was investigated by selecting a P. solenopsis population under laboratory conditions for many generations. The cross‐resistance potential of indoxacarb resistance with other chemistries and the realized heritability of indoxacarb resistance were also evaluated.

Biochemical mechanisms and molecular analysis of fenvalerate resistant population of Spodoptera litura (Fabricius)

Spodoptera litura (Fabricius) is an important polyphagous insect pest of several host plants worldwide and has developed resistance to many insecticides. The present study aimed at analysing the effect of selection with fenvalerate on a resistant field population from Sangrur, Punjab, India (SGR) through synergist and biochemical assays. Following selection with fenvalerate for 14 generations, the selected population (SGR-Sel) was analysed through bioassays to assess changes in levels of resistance, synergism and detoxification enzyme activities. Resistance ratio increased from 12.61 to 52.81 fold when compared to the laboratory susceptible strain (H-Sus). Synergism of piperonyl butoxide (6.19 fold) and triphenyl phosphate (5.88) was observed with SGR-Sel, which suggested the involvement of mainly monooxygenases (mixed function oxidases or MFOs) and esterases in resistance to fenvalerate. A strong positive correlation (r = 0.98) was observed between MFO activity and fenvalerate resistance in SGR-Sel, the activity was 4.26- and 1.84- fold that of H-Sus and SGR. Similarly, esterase activity was 3.70- and 1.73- fold that of H-Sus and SGR populations, with a strong positive correlation between esterases and fenvalerate resistance (r = 0.97). Alignment between COI sequences of SGR-Sel and SGR identified the existence of a single base/nucleotide polymorphism at position 421 represented by replacement of C in SGR with T in SGR-Sel. In the translated protein sequence, serine in SGR was replaced by phenylalanine in SGR-Sel population at position 139.

ABCC2 participates in the resistance of Plutella xylostella to chemical insecticides

The ABCC2 protein of Plutella xylostella is an important target of Cry1A toxins from Bacillus thuringiensis (Bt), but whether this protein is involved in the resistance of P. xylostella to other insecticides remains unclear. In this study, the abcc2 gene of P. xylostella was cloned and the expression levels of Pxabcc2 in susceptible and resistant strains were investigated. ABCC2 was found to be expressed 3.2–6.7-fold higher in the resistant strain than in the susceptible strain; in the surviving P. xylostella, ABCC2 levels were significantly higher when treated with indoxacarb, avermectin, and beta-cypermethrin. We constructed a stable ABCC2-expressing HEK-293 cell line to reveal the contribution of ABCC2 to insecticide resistance. The avermectin and chlorfenapyr sensitivities of the stably-transfected cell line were significantly lower than those of the control cells. The intracellular avermectin concentration was significantly lower in the stably-transfected cell line than in the control cells after four hours of exposure. This study shows that up-regulated ABCC2 expression is related to insecticide resistance in P. xylostella. Moreover, we used RNA interference technology to reduce ABCC2 levels in P. xylostella. Down-regulating ABCC2 expression did not significantly affect avermectin or chlorfenapyr resistance in P. xylostella. We speculate that increased ABCC2 expression can enhance metabolic resistance in P. xylostella. This study also provides new insights into cross-resistance between B. thuringiensis toxins and chemical insecticides.

Field‐evolved resistance by western corn rootworm to Cry34/35Ab1 and other Bacillus thuringiensis traits in transgenic maize

Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) are widely planted to manage agricultural insect pests. However, widespread adoption of Bt crops has led to the evolution of Bt resistance. The western corn rootworm, Diabrotica virgifera virgifera, is among the most serious pests of maize in the midwestern United States and is currently managed with Bt maize. To date, there is evidence of field‐evolved resistance to all Bt toxins used to manage this pest. While western corn rootworm resistance to Cry3Bb1, and the closely related mCry3A and eCry3.1Ab traits, is widely distributed within the Midwest, fewer cases of Cry34/35Ab1 resistance have been observed, and planting of Cry34/35Ab1 maize is one of the methods used to manage Cry3‐resistant rootworm.

Assessment of insecticide resistance in Diamondback Moth (Plutella xylostella L.) on Cabbage

Investigations on assessment of insecticide resistance in diamondback moth, Plutella xylostella, Linnaeus on cabbage were undertaken at Department of Agricultural Entomology, Post Graduate Institute, MPKV, Rahuri during 2011-2013. Insecticide usage pattern in Ahmednagar, Dhule, Pune and Nashik locations against diamondback moth on cabbage was also studied. Survey results indicated that farmers relied mostly on chemical insecticides to control the diamondback moth on cabbage. The insecticides viz., chlorpyriphos, quinalphos, profenophos, cypermethrin, lambda-cyhalothrin, flubendiamide and chlorantraniliprole were the most widely used for its control. It was also observed that the most cabbage growers from all four locations followed routine or calendar spraying pattern. Majority of the farmers did sprayings at an interval of 6 to 10 days giving maximum 6 to 8 sprays. Toxicity of certain conventional insecticides against P. xylostella in western Maharashtra was studied. The populations collected from Ahmednagar, Dhule, Nashik and Pune locations were tested using leaf dip technique of bioassay. Based on the LC50 values, cartap hydrochloride was found to be most toxic against DBM population from different locations with LC50 values ranging from 0.3233 to 0.4384 mg a.i. ml.-1 followed by lambda-cyhalothrin and chlorpyriphos with LC50 values of 0.5812 to 0.7179 and 1.1619 to 1.6513 mg a.i. ml-1, respectively. Fenvalerate was found to be least effective against all the field populations with the highest LC50 values ranging from 1.9966 to 2.3478 mg a.i. ml-1 followed by quinalphos with LC50 values ranging from 1.2330 to 1.7351 mg a.i. ml-1. In the present study, P. xylostella population collected from all four locations recorded varied levels of resistance ranged from 3.73 to 104.81-fold. Maximum level of resistance was observed in fenvalerate (89.13-104.81-fold) followed by lambda cyhalothrin (69.19-85.46-fold), quinalphos (38.41-54.05-fold) and chlorpyriphos (21.92-31.16-fold). Very low level of resistance was recorded by cartap hydrochloride (3.73-5.06-fold). Baseline susceptibility studies with newer insecticides were conducted by Potters spray tower method. The results revealed that chlorantraniliprole was highly toxic to P. xylostella with lowest (0.0039 mg a.i. ml-1) LC50 value followed by spinosad, indoxacarb and chlorfenapyr with LC50 values of 0.0119, 0.0348 and 0.0873 mg a.i. ml-1, respectively. Novaluron was the least toxic with highest LC50 value of 0.0921 mg a.i. ml-1. Based on LC50 values, the descending order of toxicity of insecticides was chlorantraniliprole > spinosad > indoxacarb > chlorfenapyr > novaluron. The level of resistance recorded by chlorantraniliprole, spinosad, indoxacarb, chlorfenapyr and novaluron were 3.90, 5.41, 7.73, 12.65 and 16.45-fold, respectively when compared with susceptible strain.

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Artigos anteriores

 

Subsídios para a Implementação de um Programa de Manejo da Resistência de Spodoptera frugiperda (Lepidoptera: Noctuidae) a Inseticidas na Cultura do Algodão.
Celso Omoto (ESALQ/University of São Paulo, Brazil). 

 

Insecticide Resistance Management of Spodoptera frugiperda (Lepidoptera: Noctudiae) in Brazilian Cornfields.
Celso Omoto (ESALQ/University of São Paulo, Brazil).

 

Modo de Ação de Inseticidas e Acaricidas
Paula G. Marçon (V ENFRUTE)

 

Princípios e Práticas de Manejo da Resistência de Pragas a Pesticidas
Celso Omoto (ENFRUTE)

 

Resistência de Pragas a Inseticidas no MIP na Cultura do Milho.
Celso Omoto (ESALQ/University of São Paulo, Brazil) - Congresso de Milho e Sorgo.

 

Avanços na Implementação de Programas de Manejo da Resistência de Pragas a Pesticidas no Brasil
Advances in the implementation of Pesticide Resistance Management Programs in Brazil
Celso Omoto (Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, ESALQ-USP)

 

Manejo da Resistência de Pragas a Inseticidas
Celso Omoto - Professor Doutor do Departamento de Entomologia, Fitopatologia e Zoologia Agrícola da ESALQ/USP.

 

Mesa Redonda: "Manejo da Resistência de Pragas a Agrotóxicos"
Manejo de Resistência de Insetos em Plantas Geneticamente Modificadas
Insect Resistance Management Applied for Geneticaly Modified Crop.
O. D. Fernandes - Departamento de Tecnologia da Monsanto do Brasil Ltda.

 

Manejo de Resistência de Insetos a Inseticidas na Dow Agrosciences:
Desafios e Realizações
L. A. Pavan, Dow Agrosciences - Estação Experimental

 

Bases for an Insecticide Resistance Management Programs in Brazil.
C Omoto (superscript: 1) - Depto. de Entomologia, Fitopatologia e Zoologia Agrícola, ESALQ-USP
R. N. C. Guedes (superscript: 2) - Depto. de Biologia Animal, Universidade Federal de Viçosa
L. A. Pavan (superscript: 3) Dow AgroSciences

 

Programa IRAC_BR para o Manejo de Resistência de Spodoptera frugiperda
(Lepidoptera: Noctudiae) a Inseticidas na Cultura do Milho no Brasil.
L.A. Pavan, Dow Agrosciences - Estação Experimental

 

Programa Glogal de Manejo Preventivo de Resistência de Pragas ao Insenticida INDOXACARB
P. C. R. G. Marçon - Dupont do Brasil S. A.

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Comitê Brasileiro de Ação à Resistência a Inseticidas - IRAC-BR - 2020

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