BackgroundDNA 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 FindingsFrom 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 beetle's genome, and all samples showed successful amplifications.Conclusion/SignificanceThese 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.

RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.

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.

Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are grown widely for pest control, 1 but insect adaptation can reduce their efficacy. [2][3][4][5][6] The genetically modified Bt toxins Cry1AbMod and Cry1AcMod were designed to counter insect resistance to native Bt toxins Cry1Ab and Cry1Ac. 7 Previous results suggested that the modified toxins would be effective only if resistance was linked T A B A S H N I K E T A L ., N A T U R E B I O T E C H N O L O G Y 2 9 : 1 2 ( D E C E M B E R 2 0 1 1 )2 with mutations in genes encoding toxin-binding cadherin proteins. 7 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.The toxins produced by Bt kill some major insect pests but cause little or no harm to people and most other organisms. 8 Bt toxins have been used in sprays for decades and in transgenic plants since 1996 (ref. 6). Transgenic corn and cotton producing Bt toxins were planted on >58 million hectares worldwide in 2010 (ref. 1). The primary threat to the longterm efficacy of Bt toxins is the evolution of resistance by pests. [2][3][4][5][6] Many insects have been selected for resistance to Bt toxins in the laboratory, and some populations of at least eight crop pests have evolved resistance to Bt toxins outside of the laboratory, including two species resistant to Bt sprays and at least six species resistant to Bt crops. [2][3][4][5][6][9][10][11][12][13] The most widely used Bt toxins are crystalline proteins in the Cry1A family, particularly Cry1Ab in transgenic Bt corn and Cry1Ac in transgenic Bt cotton, which kill some lepidopteran larvae. 3 Cry1A toxins bind to the extracellular domains of cadherin, aminopeptidase, and alkaline phosphatase in larval midgut membranes. 14,15 Disruption of Bt toxin binding to midgut receptors is the most common general mechanism of insect resistance. 9 Mutations in the genes encoding midgut cadherins that bind Cry1Ac are linked with resistance in at least three lepidopteran pests of cotton, [16][17][18] but such cadherin mutations are not the primary cause of many other cases of field-and laboratory-selected resistance. 9,19,20 Although some aspects of the mode of action of Bt toxins remain unresolved, extensive evidence shows that after Cry1A protoxins are ingested by larvae, they are solubilized in the gut and cleaved by mid-gut proteases such as trypsin to yield activated 60-kD monomeric toxins that bind with membrane-associated receptors. 14,15 The signaling model suggests that after protease-activated monomeric toxins bind to ca...

"Parental RNA interference of genes involved in embryonic development of the western corn rootworm, Diabrotica virgifera virgifera LeConte" (2015). Faculty Publications: Department of Entomology. 420.