Large-Scale Monitoring of the Frequency of Ryanodine Receptor Target-Site Mutations Conferring Diamide Resistance in Brazilian Field Populations of Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

Fall armyworm (FAW), Spodoptera frugiperda, is an important lepidopteran pest in the Americas, and recently invaded the Eastern Hemisphere. In Brazil, FAW is considered the most destructive pest of corn and cotton. FAW has evolved resistance to many insecticides and Bacillus thuringiensis (Bt) proteins. Here, a large-scale monitoring was performed between 2019 and 2021 to assess diamide insecticide susceptibility in more than 65 FAW populations sampled in corn and cotton. We did not detect a significant shift in FAW susceptibility to flubendiamide, but a few populations were less affected by a discriminating rate. F2 screen results of 31 selected FAW populations across regions confirmed that the frequency of diamide resistance alleles remained rather stable. Two laboratory-selected strains exhibited high resistance ratios against flubendiamide, and cross-resistance to anthranilic diamides. Reciprocal crosses indicated that resistance is autosomal and (incompletely) recessive in both strains. F1 backcrosses suggested monogenic resistance, supported by the identification of an I4734M/K target-site mutation in the ryanodine receptor (RyR). Subsequent genotyping of field-collected samples employing a TaqMan-based allelic discrimination assay, revealed a low frequency of RyR I4790M/K mutations significantly correlated with phenotypic diamide resistance. Our findings will help to sustainably employ diamides in FAW resistance management strategies across crops.

Detection of a ryanodine receptor target-site mutation in diamide insecticide resistant fall armyworm, Spodoptera frugiperda.

BACKGROUND: Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), a major lepidopteran pest in Latin and North America, has very recently invaded the continents of Africa and Asia. FAW has evolved resistance to different insecticides and transgenic corn expressing Bacillus thuringiensis (Bt) toxins. Here, we investigated the extent and mechanisms of resistance to diamide insecticides in a Brazilian field-collected FAW strain selected using chlorantraniliprole. RESULTS: Continuous laboratory selection of a field-collected FAW strain with chlorantraniliprole resulted in resistance ratios of 225-fold and > 5400-fold against chlorantraniliprole and flubendiamide, respectively, when compared with a susceptible strain. Pre-exposure to different synergists known to inhibit detoxification enzymes did not result in significantly increased larval toxicity, suggesting a minor role for metabolic resistance. Sequencing of the FAW ryanodine receptor (RyR) C-terminal domains II to VI revealed a single nucleotide polymorphism, resulting in a I4734M mutation recently said to confer target-site resistance to diamides in lepidopteran pests. Genotyping by pyrosequencing of field-collected FAW larvae sampled in the 2018 crop season suggests a low resistance allele frequency. Furthermore, we developed a fluorescent polymerase chain reaction (PCR)-based allelic discrimination assay for rapid genotyping of field-collected FAW samples, because diamides are increasingly used in Bt-/non-Bt corn. CONCLUSIONS: Recently, the identified RyR mutation has been shown to confer field resistance in other lepidopteran pests such as diamondback moth, tomato leafminer and striped rice stem borer. The developed PCR-based allelic discrimination assay will help to monitor the frequency and future spread of diamide resistance allele in FAW field populations and help to implement appropriate resistance management measures. © 2019 Society of Chemical Industry.

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.

Inheritance and fitness costs of Spodoptera frugiperda (Lepidoptera: Noctuidae) resistance to spinosad in Brazil.

BACKGROUND: Spodoptera frugiperda is a pest of economically important crops in South America. In Brazil, this species is considered the most destructive pest of maize. Use of spinosyn insecticides in insect resistance management (IRM) has been one strategy to control this pest. In this study, we selected a strain of S. frugiperda resistant to spinosad and evaluated the inheritance and fitness costs of the resistance. RESULTS: Estimated LC50 (concentration required to kill 50% of larvae) values were 0.011 and 9.80 µg cm-2 for the spinosad-susceptible (Sus) and -resistant (Spin-res) strains, respectively. This represents an 890-fold resistance ratio. LC50 values for reciprocal crosses were 0.18 and 0.14 µg cm-2 , indicating that resistance to spinosad is an autosomal incompletely recessive trait. Backcrosses of the F1 progeny from reciprocal crosses with the parental Spin-res strain showed a polygenic effect. The estimated minimum number of independent segregations was ∼ 2.45, indicating that resistance to spinosad is associated with multiple genes. In greenhouse assays, third-instar larvae from the Spin-res strain showed >92% survival on spinosad-treated maize. By contrast Sus and reciprocal crosses exhibited 0% and <5% survival, respectively, indicating that resistance is recessive. Life history studies to investigate the fitness cost of resistance revealed a 41% reduction in the rate of survival to adulthood and a 49% lower reproductive rate in the Spin-res strain compared with the Sus strain. CONCLUSIONS: The autosomal, incompletely recessive and polygenic resistance to spinosad in S. frugiperda and the fitness costs associated with this resistance can be exploited in IRM strategies to preserve the lifetime of spinosad for control of S. frugiperda in Brazil. © 2017 Society of Chemical Industry.

Effective dominance of resistance of Spodoptera frugiperda to Bt maize and cotton varieties: implications for resistance management.

The resistance of fall armyworm (FAW), Spodoptera frugiperda, has been characterized to some Cry and Vip3A proteins of Bacillus thuringiensis (Bt) expressed in transgenic maize in Brazil. Here we evaluated the effective dominance of resistance based on the survival of neonates from selected Bt-resistant, heterozygous, and susceptible (Sus) strains of FAW on different Bt maize and cotton varieties. High survival of strains resistant to the Cry1F (HX-R), Cry1A.105/Cry2Ab (VT-R) and Cry1A.105/Cry2Ab/Cry1F (PW-R) proteins was detected on Herculex, YieldGard VT PRO and PowerCore maize. Our Vip3A-resistant strain (Vip-R) exhibited high survival on Herculex, Agrisure Viptera and Agrisure Viptera 3 maize. However, the heterozygous from HX-R × Sus, VT-R × Sus, PW-R × Sus and Vip-R × Sus had complete mortality on YieldGard VT PRO, PowerCore, Agrisure Viptera, and Agrisure Viptera 3, whereas the HX-R × Sus and Vip-R × Sus strains survived on Herculex maize. On Bt cotton, the HX-R, VT-R and PW-R strains exhibited high survival on Bollgard II. All resistant strains survived on WideStrike, but only PW-R and Vip-R × Sus survived on TwinLink. Our study provides useful data to aid in the understanding of the effectiveness of the refuge strategy for Insect Resistance Management of Bt plants.

Biological activity of Bt proteins expressed in different structures of transgenic corn against Spodoptera frugiperda / Atividade biológica de proteínas Bt expressas em diferentes estruturas de milho transgênico em Spodoptera frugiperda

ABSTRACT: Spodoptera frugiperda (J. E. Smith) is the main target pest of Bt corn technologies, such as YieldGard VT PRO(tm) (Cry1A.105/Cry2Ab2) and PowerCore(tm) (Cry1A.105/Cry2Ab2/Cry1F). In this study, it was evaluated the biological activity of Bt proteins expressed in different plant structures of YieldGard VT PRO(tm) and PowerCore(tm) corn against S. frugiperda . Complete mortality of S. frugiperda neonates was observed on leaf-disc of both Bt corn technologies. However, the mortality in silks and grains was lower than 50 and 6%, respectively. In addition, more than 49% of the surviving larvae in silks and grains completed the biological cycle. However, all life table parameters were negatively affected in insects that developed in silks and grains of both Bt corn events. In summary, the low biological activity of Bt proteins expressed on silks and grains of YieldGard VT PRO(tm) and PowerCore(tm) corn can contribute to the resistance evolution in S. frugiperda populations.

RESUMO: Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae ) é a principal praga-alvo dos eventos de milho YieldGard VT PRO(tm) (Cry1A.105/Cry2Ab2) e PowerCore(tm) (Cry1A.105/Cry2Ab2/Cry1F). Para subsidiar o manejo de resistência, avaliou-se a atividade biológica das proteínas de Bt expressas em diferentes estruturas dessas tecnologias de milho Bt contra S. frugiperda . Em discos de folha de milho YieldGard VT PRO(tm) e PowerCore(tm), houve mortalidade completa de neonatas de S. frugiperda . No entanto, em estilo-estigmas e grãos, a mortalidade foi inferior a 50% e 6%, respectivamente. Em adição, mais de 49% das larvas sobreviventes em estilos-estigmas e grãos completaram o ciclo biológico. No entanto, todos os parâmetros de tabela de vida de fertilidade foram negativamente afetados nos insetos que se desenvolveram em estilos-estigmas e grãos de milho Bt. A baixa atividade biológica das proteínas Bt expressas em estilos-estigmas e grãos de milho YieldGard VT PRO(tm) e PowerCore(tm) pode contribuir para a evolução de resistência em populações de S. frugiperda .

Selection and characterization of resistance to the Vip3Aa20 protein from Bacillus thuringiensis in Spodoptera frugiperda.

BACKGROUND: Spodoptera frugiperda is one the main target pests of maize events expressing Vip3Aa20 protein from Bacillus thuringiensis (Bt) in Brazil. In this study, we selected a resistant strain of S. frugiperda on Bt maize expressing Vip3Aa20 protein and characterized the inheritance and fitness costs of the resistance. RESULTS: The resistance ratio of the Vip3Aa20-resistant strain of S. frugiperda was >3200-fold. Neonates of the Vip3Aa20-resistant strain were able to survive and emerge as fertile adults on Vip3Aa20 maize, while larvae from susceptible and heterozygous strains did not survive. The inheritance of Vip3Aa20 resistance was autosomal recessive and monogenic. Life history studies to investigate fitness cost revealed an 11% reduction in the survival rate until adult stage and a ∼50% lower reproductive rate of the Vip3Aa20-resistant strain compared with susceptible and heterozygous strains. CONCLUSION: This is the first characterization of S. frugiperda resistance to Vip3Aa protein. Our results provide useful information for resistance management programs designed to prevent or delay resistance evolution to Vip3Aa proteins in S. frugiperda. © 2016 Society of Chemical Industry.

Near-Isogenic Cry1F-Resistant Strain of Spodoptera frugiperda (Lepidoptera: Noctuidae) to Investigate Fitness Cost Associated With Resistance in Brazil.

Field-evolved resistance to Cry1F maize in Spodoptera frugiperda (J.E. Smith) populations in Brazil was reported in 2014. In this study, to investigate fitness costs, we constructed a near-isogenic S. frugiperda-resistant strain (R-Cry1F) using Cry1F-resistant and Cry1F-susceptible strains sharing a close genetic background. A near-isogenic R-Cry1F strain was obtained by eight repeated backcrossings, each followed by sib-mating and selection among resistant and susceptible strains. Fitness cost parameters were evaluated by comparing the biological performance of resistant, susceptible, and heterozygous strains on artificial diet. Fitness parameters monitored included development time and survival rates of egg, larval, pupal, and egg-to-adult periods; sex ratio; adult longevity; timing of preoviposition, oviposition, and postoviposition; fecundity; and fertility. A fertility life table was also calculated. The near-isogenic R-Cry1F strain showed lower survival rate of eggs (32%), when compared with Sus and reciprocal crosses (41 and 55%, respectively). The number of R-Cry1F insects that completed the life cycle was reduced to ∼25%, compared with the Sus strain with ∼32% reaching the adult stage. The mean generation time (T) of R-Cry1F strain was ∼2 d shorter than R-Cry1F♂×Sus♀ and Sus strains. The reproductive parameters of R-Cry1F strain were similar to the Sus strain. However, fewer females were produced by R-Cry1F strain than R-Cry1F♀×Sus♂ and more females than R-Cry1F♂×Sus♀. In summary, no relevant fitness costs are observed in a near-isogenic Cry1F-resistant strain of S. frugiperda, indicating stability of resistance to Cry1F protein in Brazilian populations of this species in the absence of selection pressure.