Murugesan Rangasamy

Efficacy of genetically modified Bt toxins against insects with different genetic mechanisms of resistance

Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are grown widely for pest control, but insect adaptation can reduce their efficacy. The genetically modified Bt toxins Cry1AbMod and Cry1AcMod were designed to counter insect resistance to native Bt toxins Cry1Ab and Cry1Ac. Previous results suggested that the modified toxins would be effective only if resistance was linked with mutations in genes encoding toxin-binding cadherin proteins. Here we report evidence from five major crop pests refuting this hypothesis. Relative to native toxins, the potency of modified toxins was >350-fold higher against resistant strains of Plutella xylostella and Ostrinia nubilalis in which resistance was not linked with cadherin mutations. Conversely, the modified toxins provided little or no advantage against some resistant strains of three other pests with altered cadherin. Independent of the presence of cadherin mutations, the relative potency of the modified toxins was generally higher against the most resistant strains.

Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles

Background DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction. Methodology/Principal Findings From individual western corn rootworm beetles, Diabrotica virgifera virgifera, DNA extractions by the SDS method, CTAB method, DNAzol® reagent, Puregene® solutions and DNeasy® column were compared in terms of DNA quantity and quality, cost of materials, and time consumed. Although all five methods resulted in acceptable DNA concentrations and absorbance ratios, the SDS and CTAB methods resulted in higher DNA yield (ng DNA vs. mg tissue) at much lower cost and less degradation as revealed on agarose gels. The DNeasy® kit was most time-efficient but was the costliest among the methods tested. The effects of ethanol volume, temperature and incubation time on precipitation of DNA were also investigated. The DNA samples obtained by the five methods were tested in PCR for six microsatellites located in various positions of the beetles genome, and all samples showed successful amplifications. Conclusion/Significance These evaluations provide a guide for choosing methods of DNA extraction from western corn rootworm beetles based on expected DNA yield and quality, extraction time, cost, and waste control. The extraction conditions for this mid-size insect were optimized. The DNA extracted by the five methods was suitable for further molecular applications such as PCR and sequencing by synthesis.

Parental RNA interference of genes involved in embryonic development of the western corn rootworm, Diabrotica virgifera virgifera LeConte

RNA interference (RNAi) is being developed as a potential tool for insect pest management and one of the most likely target pest species for transgenic plants that express double stranded RNA (dsRNA) is the western corn rootworm. Thus far, most genes proposed as targets for RNAi in rootworm cause lethality in the larval stage. In this study, we describe RNAi-mediated knockdown of two developmental genes, hunchback (hb) and brahma (brm), in the western corn rootworm delivered via dsRNA fed to adult females. dsRNA feeding caused a significant decrease in hb and brm transcripts in the adult females. Although total oviposition was not significantly affected, there was almost complete absence of hatching in the eggs collected from females exposed to dsRNA for either gene. These results confirm that RNAi is systemic in nature for western corn rootworms. These results also indicate that hunchback and brahma play important roles in rootworm embryonic development and could provide useful RNAi targets in adult rootworms to prevent crop injury by impacting the population of larval progeny of exposed adults. The ability to deliver dsRNA in a trans-generational manner by feeding to adult rootworms may offer an additional approach to utilizing RNAi for rootworm pest management. The potential to develop parental RNAi technology targeting progeny of adult rootworms in combination with Bt proteins or dsRNA lethal to larvae may increase opportunities to develop sustainable approaches to rootworm management involving RNAi technologies for rootworm control.

Estimating the frequency of Cry1F resistance in field populations of the European corn borer (Lepidoptera: Crambidae)

BACKGROUND Transgenic corn hybrids that express toxins from Bacillus thuringiensis (Bt) have suppressed European corn borer populations and reduced the pest status of this insect throughout much of the US corn belt. A major assumption of the high-dose/refuge strategy proposed for insect resistance management and Bt corn is that the frequency of resistance alleles is low so that resistant pests surviving exposure to Bt corn will be rare. RESULTS The frequency of resistance to the Cry1F Bt toxin was estimated using two different screening tools and compared with annual susceptibility monitoring based on diagnostic bioassays and LC50 and EC50 determinations. An F1 screening approach where field-collected individuals were mated to a resistant laboratory strain and progeny were assayed to determine genotype revealed that resistance alleles could be recovered even during the first year of commercially available Cry1F corn (2003). Estimates of frequency from 2003-2005 and 2006-2008 indicated that, although allele frequency was higher than theoretical assumptions (0.0286 and 0.0253 respectively), there was no indication that the frequency was increasing. Similar estimates in 2008 and 2009 using an F2 screening approach confirmed the presence of non-rare resistance alleles (frequency ≈ 0.0093 and 0.0142 for 2008 and 2009, respectively). The results of both screening methods were in general agreement with the observed mortality in diagnostic bioassays and LC50 and EC50 determinations. CONCLUSIONS These results are consistent with previous modeling results, suggesting that the high-dose/refuge strategy that is in place for Bt corn may be effective in delaying resistance evolution even when a relatively high frequency of resistance alleles exists.

Long dsRNA but not siRNA initiates RNAi in western corn rootworm larvae and adults

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.

RNAi as a management tool for the western corn rootworm, Diabrotica virgifera virgifera

The western corn rootworm (WCR), Diabrotica virgifera virgifera, is the most important pest of corn in the US Corn Belt. Economic estimates indicate that costs of control and yield loss associated with WCR damage exceed

A combinatorial bidirectional and bicistronic approach for coordinated multi-gene expression in corn

US 1 billion annually. Historically, corn rootworm management has been extremely difficult because of its ability to evolve resistance to both chemical insecticides and cultural control practices. Since 2003, the only novel commercialized developments in rootworm management have been transgenic plants expressing Bt insecticidal proteins. Four transgenic insecticidal proteins are currently registered for rootworm management, and field resistance to proteins from the Cry3 family highlights the importance of developing traits with new modes of action. One of the newest approaches for controlling rootworm pests involves RNA interference (RNAi). This review describes the current understanding of the RNAi mechanisms in WCR and the use of this technology for WCR management. Further, the review addresses ecological risk assessment of RNAi and insect resistance management of RNAi for corn rootworm.

Systemic RNAi in western corn rootworm, Diabrotica virgifera virgifera, does not involve transitive pathways

Transgene stacking in trait development process through genetic engineering is becoming complex with increased number of desired traits and multiple modes of action for each trait. We demonstrate here a novel gene stacking strategy by combining bidirectional promoter (BDP) and bicistronic approaches to drive coordinated expression of multi-genes in corn. A unidirectional promoter, Ubiquitin-1 (ZMUbi1), from Zea mays was first converted into a synthetic BDP, such that a single promoter can direct the expression of two genes from each end of the promoter. The BDP system was then combined with a bicistronic organization of genes at both ends of the promoter by using a Thosea asigna virus 2A auto-cleaving domain. With this gene stacking configuration, we have successfully obtained expression in transgenic corn of four transgenes; three transgenes conferring insect (cry34Ab1 and cry35Ab1) and herbicide (aad1) resistance, and a phiyfp reporter gene using a single ZMUbi1 bidirectional promoter. Gene expression analyses of transgenic corn plants confirmed better coordinated expression of the four genes compared to constructs driving each gene by independent unidirectional ZmUbi1 promoter. To our knowledge, this is the first report that demonstrates application of a single promoter for co-regulation of multiple genes in a crop plant. This stacking technology would be useful for engineering metabolic pathways both for basic and applied research.

RNAi induced knockdown of a cadherin-like protein (EF531715) does not affect toxicity of Cry34/35Ab1 or Cry3Aa to Diabrotica virgifera virgifera larvae (Coleoptera: Chrysomelidae).

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double‐stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA‐dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi‐mediated knockdown of Dv v‐ATPase C mRNA throughout the WCR gut and other tissues using high‐sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v‐ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.

Gene silencing in Tribolium castaneum as a tool for the targeted identification of candidate RNAi targets in crop pests

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.