Molecular characterization of Cry1F resistance in fall armyworm, Spodoptera frugiperda from Brazil
Graphical abstract
Introduction
Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a polyphagous lepidopteran pest species, causing significant damage in several economically important crops, particularly maize in Brazil (Ivan Cruz, 1995; Barros et al., 2010). The control of this pest has relied mainly on synthetic insecticides, which led to the evolution of resistance to different chemical classes (Diez-RodríGuez and Omoto, 2001; Carvalho et al., 2013; Nascimento et al., 2016; Okuma et al., 2017; Bolzan et al., 2019). Currently, the main measure to control fall armyworm in Brazil involves the use of genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins (O. Bernardi et al., 2015).
Since the introduction of transgenic maize expressing Cry1F in 2009, the Bt technology has been adopted in large scale year-round production in Brazil, with limited refuge areas of non-Bt plants (Horikoshi et al., 2016). Subsequently, the presence of the Cry1F protein in both maize and cotton products contributed to the evolution of Cry1F-resistance in fall armyworm, which was first reported in 2014 (Farias et al., 2014).
The inheritance of Cry1F resistance in Brazil has been described for S. frugiperda as (incompletely) recessive, autosomal and monogenic (Farias et al., 2014; Leite et al., 2016; Santos-Amaya et al., 2016a), and many recent studies have shown cross-resistance among Cry1F, Cry1A.105, Cry1Ac and Cry1Ab (Vélez et al., 2013; D. Bernardi et al., 2015; Santos-Amaya et al., 2016b; Burtet et al., 2017). Field-evolved resistance of fall armyworm to Cry1F in Argentina was characterized as autosomal and incompletely recessive (Chandrasena et al., 2018). In order to implement reliable resistance management strategies, it is important to understand the molecular mechanism of Bt resistance. A broadly accepted model of Bt toxicity is that once the crystalline inclusions containing the Cry proteins are ingested by the insect, they act in a sequential manner on different targets in the insect midgut. The Cry proteins have to be solubilized and processed to an active toxin. By crossing the peritrophic matrix, the activated toxins then interact with different enzymes and receptors, resulting in pore formation, osmotic cell lysis and insect death (Bravo et al., 2007; Adang et al., 2014). A number of proteins have been reported as receptors for the Cry toxins, including aminopeptidase N (APN), cadherin (CAD), alkaline phosphatases (ALP) and ATP-binding cassette (ABC) transporters (Bravo et al., 2007). A well-known Bt toxin resistance mechanism is the reduction of Cry toxin binding to their specific midgut receptors, by changes in the expression level and/or mutations (Bravo et al., 2007; Heckel et al., 2007). Many studies have indicated a major role of ABC transporter subfamily C2 (ABCC2) in mediating the insertion of Cry toxins into the midgut membrane of lepidopteran species (Gahan et al., 2010). Mutations in the ABCC2 transporters have been linked to Cry1-type resistance in many lepidopteran pests (Gahan et al., 2010; Atsumi et al., 2012; Park et al., 2014; Xiao et al., 2015), including S. frugiperda (Banerjee et al., 2017; Flagel et al., 2018). The resistance to Cry1F in fall armyworm populations from Puerto Rico has been linked to an insertion of two nucleotides in the ABCC2 gene, which lead to a premature stop codon and consequently a non-functional receptor for the Bt toxin (Banerjee et al., 2017; Flagel et al., 2018).
Nevertheless, until now the mechanism of Cry1F resistance in fall armyworm populations from Brazil is unknown. Therefore, understanding the molecular basis is critical to develop effective resistance management programs and sustain the Bt technology (Tabashnik et al., 2013). In the present study we elucidated the molecular mechanism of Cry1F resistance in S. frugiperda from Brazil. For this purpose we first selected stable reference genes with low expression variance among strains, larval stages and gut tissue. We then investigated in one susceptible and two Cry1F-resistant fall armyworm strains the expression pattern of known receptors/enzymes involved in Bt toxin mode of action. Next we screened for mutations in full length sequences of ABCC2 and characterized the functional role of non-synonomous mutations using cell toxicity assays. Finally, we assessed the frequency of the mutant alleles in fall armyworm populations recently collected from maize fields in different regions in Brazil by different technologies.
Section snippets
Fall armyworm strains
Three S. frugiperda strains, Sf_Bra (susceptible to Cry1F), Sf_Cor and Sf_Des (field-resistant to Cry1F) were collected in Brazil according to Table S1. The insects were reared under controlled conditions (25 ± 1 °C, 55 ± 5% relative humidity) in the laboratory on standard noctuid artificial diet without exposure to any Bt toxin or synthetic insecticides. For pyrosequencing genotyping larvae were collected at ten sites in Brazilian non-Bt maize fields in 2017–2018, and preserved in alcohol
Bioassays and genetics of resistance
Both Sf_Cor and Sf_Des strains showed high levels of resistance to Cry1F (Fig. 1A), when compared to the susceptible strain, Sf_Bra (Table S5). On the other hand, the bioassay results against Xentari™ showed very low resistance ratios (RR ≤ 5-fold) for the Cry1F-resistant strains most likely not relevant under applied aspects, i.e. almost exhibiting a similar susceptibility to this Bt-based product as the susceptible strain Sf_Bra (Fig. 1B). Reciprocal crosses of strains Sf_Des and Sf_Bra
Discussion
One of the best-known resistance mechanisms to Bt toxins is the reduced binding to specific midgut receptors, due to reduced expression or target-site mutations (Bravo et al., 2007; Heckel et al., 2007). To characterize the Cry1F resistance mechanism in S. frugiperda populations from Brazil, we first analyzed the expression pattern of genes involved in the Bt mode of action among resistant (Sf_Cor and Sf_Des) and susceptible (Sf_Bra) strains. This analysis required a comprehensive validation of
Declaration of competing interest
Some of the authors are employed by Bayer AG. Bayer AG manufactures maize containing the Cry1Fa and Cry1A.105 proteins. This work was partially financed by Bayer AG. The authors declare no additional conflicts of interest.
Acknowledgements
We greatly appreciate the excellent support by Dr. Christian Baden and the Pest Control Entomology Lab. We thank Katharina Kurkuris for excellent technical support on the cytotoxicity assays.
References (69)
A method of computing the effectiveness of an insecticide
J. Econ. Entomol.
(1925)- et al.
Diversity of Bacillus thuringiensis crytal toxins and mechanism of action
- et al.
Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets
Cancer Res.
(2004) - et al.
Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori
Proc. Natl. Acad. Sci.
(2012) - et al.
Mechanism and DNA-based detection of field-evolved resistance to transgenic Bt corn in fall armyworm (Spodoptera frugiperda)
Sci. Rep.
(2017) - et al.
Development of Spodoptera frugiperda on different hosts and damage to reproductive structures in cotton
Entomol. Exp. Appl.
(2010) - et al.
Parallel evolution of Bacillus thuringiensis toxin resistance in Lepidoptera
Genetics
(2011) - et al.
Trimmomatic: a flexible trimmer for Illumina sequence data
Bioinformatics
(2014) - et al.
Selection and characterization of the inheritance of resistance of Spodoptera frugiperda (Lepidoptera: Noctuidae) to chlorantraniliprole and cross-resistance to other diamide insecticides: resistance of Spodoptera frugiperda to diamide insecticides
Pest Manag. Sci.
(2019) - et al.
Dominance of insecticide resistance presents a plastic response
Genetics
(1996)
Insecticide resistance and dominance levels
J. Econ. Entomol.
Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control
Toxicon
Managing fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), with Bt maize and insecticides in southern Brazil: managing S. frugiperda with Bt maize and insecticides
Pest Manag. Sci.
Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda
PLoS One
Characterization of field-evolved resistance to Bacillus thuringiensis-derived Cry1F delta-endotoxin in Spodoptera frugiperda populations from Argentina
Pest Manag. Sci.
A lagarta-do-cartucho na cultura do milho
Embrapa – Embrapa Milho E Sorgo Sete Lagoas
Cross-resistance between Cry1 proteins in fall armyworm (Spodoptera frugiperda) may affect the durability of current pyramided Bt maize hybrids in Brazil
PLoS One
Fitness costs of Cry1F resistance in two populations of fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from Puerto Rico and Florida
J. Invertebr. Pathol.
Herança da resistência de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) a lambda-cialotrina
Neotrop. Entomol.
Extracellular loop structures in silkworm ABCC transporters determine their specificities for Bacillus thuringiensis Cry toxins
J. Biol. Chem.
Field-evolved resistance to Cry1F maize by Spodoptera frugiperda (Lepidoptera: Noctuidae) in Brazil
Crop Protect.
Mutational disruption of the ABCC2 gene in fall armyworm, Spodoptera frugiperda, confers resistance to the Cry1Fa and Cry1A.105 insecticidal proteins
Sci. Rep.
An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin
PLoS Genet.
Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges
Sci. Rep.
The diversity of Bt resistance genes in species of Lepidoptera
J. Invertebr. Pathol.
Soft sweeps: molecular population genetics of adaptation from standing genetic variation
Genetics
Soft sweeps and beyond: understanding the patterns and probabilities of selection footprints under rapid adaptation
Methods Ecol. Evol.
Constitutive activation of the midgut response to Bacillus thuringiensis in Bt-resistant Spodoptera exigua
PLoS One
Effective dominance of resistance of Spodoptera frugiperda to Bt maize and cotton varieties: implications for resistance management
Sci. Rep.
Lack of relevant cross-resistance to Bt insecticide XenTari in strains of Spodoptera frugiperda (J. E. Smith) resistant to Bt maize
J. Invertebr. Pathol.
Fitness costs associated with field-evolved resistance to Bt maize in Spodoptera frugiperda (Lepidoptera: Noctuidae)
J. Econ. Entomol.
Field-evolved mode 1 resistance of the fall armyworm to transgenic Cry1Fa-expressing corn associated with reduced Cry1Fa toxin binding and midgut alkaline phosphatase expression
Appl. Environ. Microbiol.
Structural basis of substrate recognition by the Multidrug resistance protein MRP1
Cell
Reduced levels of membrane-bound alkaline phosphatase are common to Lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis
PLoS One
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