## 3 Time Course and Current Status of Field-Evolved Resistance  
In 2009, farmers in Iowa observed high levels of feeding injury by western corn rootworm to maize producing Cry3 Bb1 [3]. Subsequent plant-based bioassays found that this feeding injury was associated with Cry3 Bb1 resistance by western corn rootworm [3]. Additional cases of Cry3 Bb1 resistance were identified in Iowa in 2010 [38]. In 2011, field populations were sampled from several fields that had high levels of feeding injury from western corn rootworm to either Cry3 Bb1 maize or mCry3A maize, and results of plant-based bioassays revealed resistance to both mCry3A and Cry3 Bb1, and cross-resistance between these Bt toxins [23]. In 2012, field populations were sampled across the northern half of Iowa from fields, where high levels of feeding injury to Cry3 Bb1 maize were observed, and plant-based bioassays demonstrated that cross-resistance between Cry3 Bb1 and mCry3A also extended to eCry3.1 Ab [39]. Fields that were sampled in these studies were typified by a node or more of feeding injury to roots of Bt maize, and in some cases, more than two nodes, with each node of roots lost translating to a 15% to 17% reduction in yield [10; 11]. This means that Bt resistance by western corn rootworm has practical significance for farmers by substantially reducing yield. It is noteworthy that field populations evaluated from 2009 to 2012 were not resistant to Cry34/35 Ab1 [32; 38; 39]. However, resistance to the Cry3 traits meant that the IRM advantage of pyramiding had been greatly reduced when Cry34/35 Ab1 was placed in a pyramid with a Cry3 trait. Resistance to Cry3 maize by western corn rootworm is not limited to Iowa and has been documented in Illinois, Minnesota, Nebraska and North Dakota [40; 41; 42; 43]. All four states have areas of intensive maize production, with fields commonly planted to maize for several consecutive years [44]. Additionally, some of these populations were tested for cross-resistance to other Bt traits, and similar to research from Iowa, cross-resistance was found among Cry3 Bb1, mCry3A and eCry3.1 Ab [40; 41]. Field-evolved resistance in some of these states appears to have occurred at a similar time as the observation of Bt resistance in Iowa, suggesting that Bt resistance evolved independently in several locations throughout the Corn Belt. The presence of cross-resistance among Cry3 Bb1, mCry3A and eCry3.1 Ab may be due to the structural similarities among these three-domain Bt toxins, such that genetic changes conferring resistance to one of the Cry proteins likely confer resistance to the others [23; 31; 39]. By contrast, Cry34/35 Ab1 is a binary toxin and differs structurally from three-domain toxins, and thus also likely has a mechanism of toxicity that is independent of the Cry3 toxins [31; 39]. To date, little is known about the mechanistic basis of Bt resistance in western corn rootworm [14]. However, the potential for western corn rootworm to develop Cry34/35 Ab1 resistance appears similar to that of Cry3 Bb1. For example, laboratory selection experiments have shown that western corn rootworm developed Cry34/35 Ab1 resistance after three to seven generations of selection [45; 46], which is similar to past selection studies with Cry3 Bb1 maize [47; 48]. As suggested by these laboratory selection experiments, following field-evolved resistance to Cry3 maize, studies provided evidence that resistance to Cry34/35 Ab1 had emerged in field populations of western corn rootworm. In 2013, fields in Iowa were sampled where high levels of feeding injury from western corn rootworm were observed for Cry34/35 Ab1 maize (>2 nodes of injury on average) and for maize pyramided with Cry34/35 Ab1 and a Cry3 toxin (>1 node of injury on average).