## 4 Field Efficacy of RNAi for Insect Control, Insect Resistance Management, and RNAi Resistance
### Rootworm Resistance to RNAi  
As in previous _Bt_ PIPs, selection pressure from continuous exposure to dsRNA will eventually promote resistance evolution. Mechanisms of resistance to dsRNA were initially postulated to include degradation of dsRNA in the gut, reduced dsRNA uptake, alteration in proteins involved in dsRNA transport or formation of the RISC complex, loss of siRNA recognition by the RISC complex, mutation of the target gene, or systemic spreadfailure [76; 134; 160]. Identifying the resistance mechanism to _DvSnf7_ dsRNA in WCR will inform effective IRM strategies to extend product durability if resistance is related to dsRNA processing, which may confer cross-resistance to other RNAi traits. Efforts to evaluate the multigenerational effect of _DvSnf7_ in field-collected insects resulted in the development of a resistant colony. [PERSON] et al. [75] collected WCR adults emerging from areas planted with transgenic maize expressing _DvSnf7_ dsRNA. Field-collected beetles were crossed with a non-diapausing WCR colony and exposed to _DvSnf7_ dsRNA for eight generations, creating a population with \(\geq\)130-fold resistance to _DvSnf7_ dsRNA [75]. Reciprocal crosses determined that _DvSnf7_ dsRNA resistance was recessive, monogenic, and autosomal [75]. Resistance resulted from reduced uptake of dsRNA in gut cells and, interestingly, was found to be non-sequence specific. Cross-resistance to dsRNAs targeting _v-ATPase A_, _COPI B_ (Coatomer Subunit beta), and _mov34_ (26s proteasome) was identified, indicating adaptation occurred within the RNAi mechanism itself. Due to the development of cross-resistance to a variety of dsRNA targets in this study, it is possible dsRNA represents a single MOA in WCR [75]. However, it is yet unknown how insects will respond and adapt to dsRNA under field conditions. Further studies with other RNAi traits will provide a better understanding of potential resistance mechanisms that might exist in the field. Because _DvSnf7_ will not be released as a single trait product, it was important to identify the chromosomal location of resistance alleles and compare with current knowledge on the location of resistance alleles to the _Bt_ traits in SmartStax(r) PRO. The Cry3 Bb1 resistance gene(s) is located on linkage group 8 (LG8) [161] and the resistance locus for _DvSnf7_ dsRNA is located on linkage group 4 (LG4) [75]. The fact that resistance causative elements are located on different chromosomes supports experimental evidence showing a lack of cross-resistance between _DvSnf7_ dsRNA and Cry3 Bb1 [75; 148]. Therefore, resistance to both traits would have to develop independently and would have a lower probability of occurring than if a single trait or MOA were used. Similar work in _L. decemlineata_ furthers our understanding of how dsRNA resistance could develop in WCR. A laboratory-derived dsRNA-resistant _L. decemlineata_ cell line and colony were recently established [105; 162]. In both cases, cross-resistance to multiple dsRNAs was found, supporting results observed in WCR [75]. In dsRNA-resistant WCR, non-specific dsRNA uptake was disrupted, conferring resistance to all sequences tested. This fits with the transcriptional analysis of _L. decemlineata_ cells, wherein genes related to uptake, _clathrin light chain_, _vha55_, _silA_, and the novel dsRNA binding protein _staufenC_, were downregulated in resistant cells [105]. However, while resistance in the WCR colony was narrowed to one locus in the genome, the _L. decemlineata_ dsRNA-resistant colony displayed polygenic inheritance. This indicates that various mechanisms relating to dsRNA uptake could adapt to intense selection pressure. Given that resistance alleles have been detected in natural WCR field populations and those detected to date may confer cross-resistance to other dsRNA sequences, monitoring changes of susceptibility of field populations will be necessary to preserve RNAi technology.