Evolution of resistance threatens sustainability of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). The fall armyworm (Spodoptera frugiperda) is a devastating pest of corn in the Western Hemisphere initially controlled by transgenic Bt corn producing the Cry1Fa insecticidal protein (event TC1507). However field-evolved resistance to TC1507 was observed in Puerto Rico in 2007 and has subsequently been reported in a number of locations in North and South America. Early studies on Puerto Rico fall armyworm populations found that the resistance phenotype was associated with reduced expression of alkaline phosphatase. However, in this work we show that field-evolved resistance to Cry1Fa Bt corn in Puerto Rico is closely linked to a mutation in an ATP Binding Cassette subfamily C2 (ABCC2) gene that functions as a Cry1Fa receptor in susceptible insects. Furthermore, we report a DNA-based genotyping test used to demonstrate the presence of the resistant (SfABCC2mut) allele in Puerto Rico populations in 2007, coincident with the first reports of damage to TC1507 corn. These DNA-based field screening data provide strong evidence that resistance to TC1507 in fall armyworm maps to the SfABCC2 gene and provides a useful molecular marker for detecting the SfABCC2mut allele in resistant fall armyworm.

Transgenic maize plants expressing dsRNA targeting western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) v‐ATPase subunit C mRNA for RNAi provided significant root protection from WCR larval feeding damage in greenhouse assays compared to negative controls. Transcribed hairpin dsRNA in WCR‐resistant maize plants was present as both intact hairpin‐derived dsRNA and plant‐processed siRNA. Therefore, the ability of dsRNA and siRNA targeting Dv v‐ATPase CmRNA to cause an RNAi response was studied in both WCR larvae and adults. In 9‐day diet‐based feeding assays, dsRNA of at least 60 bp in length resulted in high levels of larval mortality. In contrast, 15‐, 25‐ or 27‐bp dsRNAs or pooled 21‐bp siRNAs did not cause mortality of exposed larvae. When larvae were fed with diet overlaid with siRNAs, Dv v‐ATPase C transcript levels did not change. Conversely, when WCR larvae were fed with diet overlaid with 184‐bp dsRNA, the mRNA level was reduced by >20‐fold relative to yfp dsRNA negative control. Similarly, 184‐bp dsRNA caused 100% mortality of WCR adults, whereas the mortality of adults fed on diet treated with siRNAs was similar to the negative control. Feeding adults with siRNAs on diet did not affect the level of Dv v‐ATPase CmRNA transcripts, whereas adults fed with the 184‐bp dsRNA showed approximately 35‐fold reduction in the target mRNA level. Similar results were obtained with the WCR adults injected with 184‐bp dsRNA or 21‐bp siRNA. These results suggest that only long dsRNA or hairpin‐derived dsRNA is effective in causing lethal knock‐down of Dv v‐ATPase CmRNA. These results have implications for efficacious plant‐delivered dsRNA for the protection of transgenic maize from WCR feeding damage and for the risk assessment of transgenic maize expressing insecticidal dsRNA.

dInsecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae.

Western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is a serious insect pest in the major corn growing areas of North America and in parts of Europe. WCR populations with resistance to Bacillus thuringiensis (Bt) toxins utilized in commercial transgenic traits have been reported, raising concerns over their continued efficacy in WCR management. Understanding the modes of action of Bt toxins is important for WCR control and resistance management. Although different classes of proteins have been identified as Bt receptors for lepidopteran insects, identification of receptors in WCR has been limited with no reports of functional validation. Our results demonstrate that heterologous expression of DvABCB1 in Sf9 and HEK293 cells conferred sensitivity to the cytotoxic effects of Cry3A toxins. The result was further validated using knockdown of DvABCB1 by RNAi which rendered WCR larvae insensitive to a Cry3A toxin. However, silencing of DvABCB2 which is highly homologous to DvABCB1 at the amino acid level, did not reduce the sensitivity of WCR larvae to a Cry3A toxin. Furthermore, our functional studies corroborate different mode-of-actions for other insecticidal proteins including Cry34Ab1/35Ab1, Cry6Aa1, and IPD072Aa against WCR. Finally, reduced expression and alternatively spliced transcripts of DvABCB1 were identified in a mCry3A-resistant strain of WCR. Our results provide the first clear demonstration of a functional receptor in the molecular mechanism of Cry3A toxicity in WCR and confirmed its role in the mechanism of resistance in a mCry3A resistant strain of WCR.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important maize pest throughout most of the U.S. Corn Belt. Bacillus thuringiensis (Bt) insecticidal proteins including modified Cry3Aa and Cry34/35Ab1 have been expressed in transgenic maize to protect against WCR feeding damage. To date, there is limited information regarding the WCR midgut target sites for these proteins. In this study, we examined whether a cadherin-like gene from Diabrotica virgifera virgifera (DvvCad; GenBank accession # EF531715) associated with WCR larval midgut tissue is necessary for Cry3Aa or Cry34/35Ab1 toxicity. Experiments were designed to examine the sensitivity of WCR to trypsin activated Cry3Aa and Cry34/35Ab1 after oral feeding of the DvvCad dsRNA to knockdown gene expression. Quantitative real-time PCR confirmed that DvvCad mRNA transcript levels were reduced in larvae treated with cadherin dsRNA. Relative cadherin expression by immunoblot analysis and nano-liquid chromatography - mass spectrometry (nanoLC-MS) of WCR neonate brush border membrane vesicle (BBMV) preparations exposed to DvvCad dsRNA confirmed reduced cadherin expression when compared to BBMV from untreated larvae. However, the larval mortality and growth inhibition of WCR neonates exposed to cadherin dsRNA for two days followed by feeding exposure to either Cry3Aa or Cry34/35Ab1 for four days was not significantly different to that observed in insects exposed to either Cry3Aa or Cry34/35Ab1 alone. In combination, these results suggest that cadherin is unlikely to be involved in the toxicity of Cry3Aa or Cry34/35Ab1 to WCR.