## 3 RNAi Traits for Rootworm Control
### Target Site Discovery
#### 3.2.2 Parental and Reproductive RNAi  
Naturally, most RNAi trait targets have focused on those that result in mortality due to the need to protect maize roots from damage by WCR larval feeding. However, given the susceptibility of WCR to environmental RNAi and the ability of RNAi-mediated gene suppression to spread systemically within WCR after ingestion, additional complementary approaches to WCR management can be explored. Parental RNAi is a new concept where adult oral exposure to dsRNA targeting embryonic development genes leads to effects on fecundity or effects within the offspring [115, 130, 131]. Two genes, _brahma_ and _hunchback_, significantly impacted egg viability after female beetles were exposed to corresponding target gene dsRNA in diet feeding assays [115] (Figure 1b). _Brahma_ (_DvBrm_) encodes an SWI/SNF ATP-dependent chromatin remodeler and _hunchback_ (_DvHb_) encodes a zinc finger transcription factor [115]. Larvae from unhatched eggs laid by _DvHb_ dsRNA-treated adult females primarily displayed deformation of abdominal and thoracic segmentation. This phenotype is consistent with the role of _DvHb_ as an essential regulatory gene in the anterior-posterior patterning of insect embryos. No embryo development was observed from unhatched eggs laid by female beetles treated with _DvBrm_ dsRNA. Additional chromatin remodeling ATPases including _chd-1_, _iswi-1_, _mi-2_, and _iswi2_ were tested and generated a similar genotype to that observed with _DvBrm_[131]. These results demonstrated that adult exposure to dsRNA can manifest in their offspring [115, 130, 131]. However, the results obtained from diet-based assays did not translate effectively to _in planta_ experiments with adults feeding on maize vegetative tissue expressing _DvBrm_ or _DvHb_ (data not published).  
[PERSON] et al. [132] targeted two reproductive genes, _DvVgr_ and _DvBol_, to determine the impact of suppression on insect fecundity (Figure 1b). While oral feeding of _DvVgr_ and _DvBol_ dsRNA in diet-based assays using WCR larvae resulted in a reduction in fecundity, this trend did not translate to _in planta_ results, a discrepancy possibly explained by the reduced expression of dsRNA found in transgenic plants compared to diet-based assays. A significant reduction in fecundity was only observed when WCR larvae fed on roots of transgenic maize plants expressing _DvBol_ hairpin RNA. _bol_ encodes an RNA-bindingprotein whose homolog in _Drosophila_ impacts male spermatogenesis. Unexposed adult female WCR showed a significant reduction in egg-lay number and percentage of egg hatch after mating with adult male WCR that fed on transgenic maize roots expressing _DvBol_ hairpin RNA. This resulted in an overall net reduction in fecundity between 84-95%. However, only a modest reduction in the number of eggs laid per female was observed from mated WCR that emerged after feeding on _DvVgr_ hairpin RNA-expressing maize roots during the larval stage. These results reveal that RNAi targeting reproductive genes in WCR adults can persist throughout the development cycle after initial exposure as larvae and significantly impact adult fecundity. This phenomenon could be explained by Internally transmitted dsRNAs [140] or RNA-directed DNA or histone methylation [141]. However, these studies were performed in _T. castaneum_ and _C. elegans_ and _D. melanogaster_, respectively, and further exploration is necessary to unveil the specific mechanism allowing parental and reproductive RNAi in WCR.  
The development of parental and reproductive RNAi as potential RNAi trait MOA represents a truly novel RNAi-based approach to mitigating WCR damage in the field by reducing or suppressing rootworm populations (Figure 1b). Combining different RNAi trait MOAs may be explored to manage rootworm populations further and slow the development of resistance to RNAi and the pyramided insecticidal proteins to protect yields from this devastating maize pest.