## 2 RNAi in the Western Corn Rootworm
### dsRNA Uptake  
To elicit an RNAi response, dsRNA must first enter the cell. In WCR, current evidence points to two putative uptake mechanisms: systemic RNA interference defective-like proteins (SID-like or SIL) and clathrin-mediated endocytosis (CME). In _C. elegans_, various SID proteins facilitate dsRNA movement across biological membranes [57; 58; 59; 60; 61]. Coleopteran homologs of _C. elegans sid_ genes are labeled _sid-like (sil)_ due to more substantial homology with _tag-130/chup-1_, a protein involved in cellular cholesterol import, than with SID proteins themselves [62]. At least two _sils_ are found within the WCR genome (GenBank: PXJM0000000.2), _silA_ (LOC114327414) and _silC_ (LOC114340333). In WCR larvae, knockdown of each gene individually reduced the phenotype of a second dsRNA application (targeting _DvEbony_), implicating _silA_ and _silC_ as putative routes for dsRNA uptake in WCR [63]. However, in adult WCR, _silA_ silencing did not affect _V-ATPase_ knockdown after secondary exposure to _DvV-ATPase_ dsRNA. Despite this, mortality levels typically associated with a _DvV-ATPase_ dsRNA exposure were reduced in _silA_ knockdown treatments, while silencing of _silC_ alone and in combination with _silA_ had no effect on _V-ATPase_ silencing or mortality [64]. Due to these ambiguous results, more evidence is required to determine if _silA_ and _silC_ are indeed involved in dsRNA uptake in WCR. In _C. elegans, tag-130/chup-1_, the gene most similar to cooleopteran _sils_, plays no role in systemic RNAi [62]; therefore, it is conceivable that _silA_ and _silC_ may have a function unrelated to dsRNA uptake. In addition, some insects with systemic RNAi responses lack SID homologs within their genome, suggesting the existence of alternative routes of cellular uptake [65]. The clearest evidence for dsRNA uptake in WCR is clathrin-mediated endocytosis (CME) [64]. CME is a form of endocytosis by which macromolecules such as proteins, lipids, and pathogens enter a cell in a clathrin-dependent manner [66; 67]. CME requires cell surface receptors to recognize ligands and initiate internalization [68]. In WCR, silencing of key CME factors _clathrin-heavy chain (chc)_, _ vacuolar H+ATPase 16 kDa submit (vha16)_, and _clathrin adaptor protein AP50 (AP50)_ led to a reduction in marker dsRNA effect, while knockdown of _ADP ribosylation factor-like 1 (Arf72A)_ and _small GTPase Rab7_ did not produce the same effect [64]. Both AP50 and CHC are required for clathrin-coated pit formation, in which these proteins congregate around the intracellular portion of a receptor and allow for endosomal development [69]. Vha16 is also necessary for endosomal maturation by regulating vesicle acidification [70]. Interestingly, knockdown of _chc_ only slightly reduced silencing caused by a marker dsRNA in adult WCR [64], while a similar assay using _T. castaneum_ larvae found much a stronger effect on marker gene silencing [71]. The same experiment also identified Rab7 as an essential component in dsRNA uptake in _T. castaneum_[71], while in WCR, _rab7_ knockdown did not show result in a reduction in the silencing of the marker gene [64]. Similarly, results from experiments with Colorado potato beetle, _Leptinarsa decmelineata_, larvae mirrored those of _T. castaneum_[65]. Divergent results between cooleopterans, _D. v. virgifera_ (Chrysomelidae), _L. decmelineata_ (Chrysomelidae), and _T. castaneum_ (Tenebrionidae) could be due to physiological differences between species or from variable responses between life stages. Results suggesting involvement of _silA_ and silC_ in the RNAi mechanism in _T. castaneum_, _L. decmelineata_, and WCR were found using the larval life stage. However, similar experiments carried out with WCR adults were not definitive. Expression of core RNAi genes was found to change based on the larval stage in _L. decmelineata_, affecting dsRNA efficacy [72] This finding provides an argument for life stage-dependent dsRNA uptake.