## 2 RNAi in the Western Corn Rootworm
### Silencing Mechanism  
The siRNA pathway is initiated when dsRNA within the cytoplasm is cleaved into multiple siRNAs by the RNase III family ribonuclease Dicer-2 (Dcr-2) [77; 78; 79]. Dcr-2 and the dsRNA-binding protein R2D2 form a complex to bind and cleave dsRNA [80]. siRNAs are 21-23 bp long duplexes with a 3' two-nucleotide overhang [81; 82; 83]. A single copy of _dcr-2_ has been annotated in the WCR genome (LOC114339627). Knockdown of _dcr-_2 in WCR adults prevented the silencing of a marker dsRNA, indicating the requirement of Dcr-2 in the WCR RNAi response to exogenous dsRNA [84; 85]. Knockdown of _dcr-_2 in female WCR  
Figure 1: (**a**) Overview of the known RNAi uptake and processing mechanism in the western corn rootworm; (**b**) list of dsRNA gene targets active against the western corn rootworm subsequently transformed into maize. led to a reduction in oviposition and silencing during the third instar negatively affected adult emergence [86]. However, knockdown of _dcr_-2 in neonates did not affect larval growth or development [87]. R2D2 not only plays a role in dsRNA binding but is also vital for loading siRNAs into the RNA-induced silencing complex (RISC) [88; 89; 90]. One copy of _r2d2_, expressing three isoforms, has been identified in WCR (LOC114342393), and silencing in WCR females led to a reduction in egg-laying capacity [86]. Based on a homology search within the WCR genome, one more _r2d2_ gene may exist; however, functional evaluation is lacking. After cleavage by Dcr-2, siRNAs are loaded onto the RISC by the RISC loading complex (RLC), comprised of Dcr-2, R2D2 [90; 91], and the TATA-Box Binding Protein Associated Factor 11 (TAF11) [92]. Each siRNA is individually bound to an RNase H-like endonuclease, Argonaute-2 (Ago-2) [93; 94], within the RISC. Argonaute proteins then separate siRNA duplexes into two single-stranded RNAs, a guide and a passenger strand, after which the passenger strand is degraded and the guide is retained [93; 95]. Once the RISC/guide-siRNA complex locates a single-stranded RNA target through base-pair matching, silencing commences via mRNA hydrolysis [96; 81; 97]. Two silencing requirements include a catalytically active Argonaute protein, which does the actual degradation, and a sequence match between the guide siRNA and the target mRNA [98]. Data from _T. castaneum_ suggest a greater than 80% sequence identify with target mRNA is required for an efficient RNAi response [97] and in _D. melanogaster_, 19 bp homology with mRNA led to effective target silencing [99]. After initial hydrolyzation of the mRNA target by Argonaute, the remaining nucleic acid is either degraded by endoribonucleases, exoribonucleases, or translated into incomplete proteins [100]. Multiple _ago2s_ may be present in the WCR genome; however, current research has focused on one gene (LOC114327218). Knockdown of _ago2_ prevented the downregulation of genes targeted by dsRNA, indicating the requirement of AGO2 for the RNAi response in WCR. Knockdown of _ago2_ did not affect WCR development or survival [63; 85]. The core RNAi silencing mechanism is relatively well conserved, and data from WCR have generally matched that of other insects. However, while key aspects of the pathway have been evaluated in WCR, many putative steps related to dsRNA and siRNA movement within the cytoplasm remain unstudied. For example, in _D. melanogaster_, the molecular chaperone complex Hsc70-Hsp90 facilitates the loading of siRNAs into Ago2 [101], and the small RNA methyltransferase HUA Enhancer 1 (HEN-1) activates the RISC through siRNA methylation [102]. Whether homologs exist with similar functions in WCR is unknown. Furthermore, crosstalk between the siRNA, miRNA, and piRNA RNAi pathways does exist. For example, silencing of _r2d2_ and _ago2_ negatively impacted miRNA processing in second instar WCR [86]. In _L. decemlineata_, Ago1 (miRNA) and Aubergine (piRNA) are involved in the dsRNA response [103], and the dsRNA binding protein Loquacious participates in both the siRNA and miRNA pathways in _D.