Characterization of field evolved resistance to transgenic Cry1Fa maize in Spodoptera frugiperda (J. E. Smith)

Transgenic Bt crops expressing Cry and Vip toxins from Bacillus thuringiensis (Bt) have been increasingly planted to manage insect pest damage on agricultural crops. The high adoption of Bt-based insecticidal technologies suggests an increase selection pressure for the evolution of resistance in insect populations. So far, nine insect species have developed field evolved resistance to Bt crops, yet the mechanisms involved in field evolved resistance are unknown. In the present study, the resistance mechanism in field evolved resistance to maize producing Cry1Fa in Spodoptera frugiperda collected in fields from Puerto Rico was characterized. High levels of resistance to Cry1Fa have been observed in S. frugiperda with recessive and autosomal mode inheritance. Binding experiments showed the reduced binding of Cry1Fa toxin to brush border membranes of resistant (456) larvae compared to susceptible (Benzon) larvae. The same binding reduction was observed for Cry1A toxins, but not for Cry1Ca toxin. This reduced binding signifies the modification of a common Cry1Fa-Cry1A toxin binding site. Comparison of receptor protein levels revealed reduced alkaline phosphatase (ALP) levels in resistant compared to susceptible larvae. This reduced expression of ALP phenotype was linked to Cry1Fa resistance in S. frugiperda. In cross-resistance studies using bioassays, reduced susceptibility to Cry1Ab and Cry1Ac toxins was detected and no differences in susceptibility to purified Cry1Bb, Cry1Ca, and Cry1Da toxins or Xentari WG and Dipel ES pesticidal formulations compared to susceptible larvae was detected. The cross-resistance patterns observed in these bioassays are in agreement with data from competition experiments indicating an altered binding site for Cry1A and Cry1Fa toxins in 456 larvae. The only difference detected in fitness cost studies was a significant increase in the larval developmental time in resistant insects, which could result in emergence asynchrony between susceptible and resistant moths. The lack of fitness costs was also supported by stable resistance after 12 generations of rearing in the absence of a selective (transgenic maize) agent. This work is the first study on field level resistance to a Bt crop. Results from this study will help to understand resistance mechanisms responsible for field-level resistance and formulate improved resistance management practices

Reduced Levels of Membrane-Bound Alkaline Phosphatase Are Common to Lepidopteran Strains Resistant to Cry Toxins from Bacillus thuringiensis

Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests

BBMV from Cry1Ac-resistant <i>H. virescens</i> larvae display reduced ALP levels.

<p>(A) BBMV proteins from <i>H. virescens</i> strains as indicated were used in specific ALP or APN activity assays. Different letters indicate statistically significant differences (p<0.05; Holm-Sidak method) among the samples. (B) Western blot analysis of HvmALP, APN, or HevCaLP in BBMV proteins from <i>H. virescens</i> strains: Lane 1, YDK; lane 2, YHD2-B; lane 3, CXC; lane 4, KCBhyb.</p

Reduced ALP activity correlates with reduced levels of HaALP transcripts in Cry1Ac-resistant <i>H. armigera</i> larvae.

<p>BBMV proteins from susceptible (96S) and Cry1Ac-resistant (BtR) <i>H. armigera</i> strains were used in specific ALP (A) or APN (B) activity assays. Different letters indicate statistically significant differences (p<0.05; Holm-Sidak method) among the samples. C) Mean relative transcript quantity of HaALP1 and HaALP2 isoforms. Data shown are the mean transcript quantity relative to the 96S sample with the highest transcript amounts from three independent biological replicates for each HaALP isoform and strain. All reactions were performed with triplicate technical replicates. Bars denote standard error of the mean. Different letters indicate significant differences (P<0.05; Holm-Sidak method).</p

Reduced ALP activity correlates with reduced levels of mALP in BBMV from Cry1Fa-resistant <i>S. frugiperda</i> larvae.

<p>BBMV proteins from susceptible (Mon and Ben) and Cry1Fa-resistant (456 and 512) strains of <i>S. frugiperda</i> were used in specific ALP (A) or APN (B) activity assays. Different letters indicate statistically significant differences (p<0.05; Holm-Sidak method) among the samples. C) Detection of mALP in BBMV from susceptible (Mon and Ben) and resistant (456 and 512) strains of <i>S. frugiperda</i> using Western blotting. Lane 1, Mon, lane 2, 456, lane 3, 512, lane 4, Ben. Arrow points to ALP protein band.</p

Reduced levels of HvmALP1 and HvmALP2 transcripts in Cry1Ac-resistant <i>H. virescens</i> larvae relative to YDK as detected by qRT-PCR.

<p>Data shown are the mean transcript quantity relative to the YDK sample with the highest transcript amounts from three independent biological replicates for each HvmALP isoform and strain. All reactions were performed with triplicate technical replicates. Bars denote standard error of the mean; different letters on each bar indicate significant differences (P<0.05; Holm-Sidak method).</p

Detection of reduced HvmALP expression in Cry-resistant strains of <i>H. virescens</i> using quantitative differential proteomic analysis (2D-DIGE).

<p>BBMV proteins from susceptible (YDK) and resistant (YHD2-B, CXC, and KCBhyb) larvae were fluorescently labeled and the corresponding sub-proteome analyzed using 2D-DIGE. A representative gel image is presented with arrows pointing to the four HvmALP spots detected with lower expression levels. The identity of these spots as HvmALP was obtained by peptide mass fingerprinting, de novo sequencing, and Western blotting with specific antisera (data not shown). The standardized log abundance for each spot in all three BBMV samples (labeled with Cy3 or Cy5) from each strain used is shown. Differences in protein levels between HvmALP spots in BBMV from susceptible and resistant larvae were statistically significant (p<0.001; Student T-test). HvmALP expression levels among BBMV samples from resistant larvae were not significantly different.</p