The involvement of clathrin‐mediated endocytosis and two Sid‐1‐like transmembrane proteins in double‐stranded RNA uptake in the Colorado potato beetle midgut

Abstract

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double-stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient-1 (Sid-1) transmembrane channel-mediated uptake and clathrin-mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi-of-RNAi experiments, we demonstrated the contribution of two different sid-1-like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H(+) ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid-1-like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well-devised reporter system in any future experiments on cellular dsRNA uptake.

Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata , but not in the desert locust, Schistocerca gregaria

“…Apart from degradation in the digestive system, other research has shown the importance of functional uptake mechanisms for dsRNA. In insects, the primary mode of cellular uptake of dsRNA appears to be scavenger receptor-mediated endocytosis (Cappelle et al, 2016;Ivashuta et al, 2015;Li et al, 2015;Saleh et al, 2006;Ulvila et al, 2006;Wynant et al, 2014;Xiao et al, 2015;Yoon et al, 2016). Interestingly, also in adults of S.…”

“…It is characterized by the ability to quickly develop resistance to a wide range of chemical pesticides, urging the need to develop alternative management strategies (Zhu et al, 2011). Several studies have shown that CPB is able to take up dsRNA from the gut lumen and subsequently induce a potent systemic RNAi response, suggesting that RNAi could be a feasible novel control method for this insect pest (Cappelle et al, 2016;Zhu et al, 2011). Until now no high nuclease activity has been demonstrated in the gut juice of CPB.…”

RNAi Efficiency, Systemic Properties, and Novel Delivery Methods for Pest Insect Control: What We Know So Far

“…Therefore, environmental RNAi needs cooperation between Sid-1 and Sid-2 proteins. The Sid-1 genes seem to be present in most insect species, but so far no Sid-2 genes have been found in insect species whose genomes have been sequenced (e.g., Tomoyasu et al, 2008; Xu and Han, 2008; Zha et al, 2011; Cappelle et al, 2016). A phylogenetic analysis suggested that Sid-1 like genes in Tribolium may not be orthologous to Sid-1 , but rather to the C. elegans Tag-130 gene which is not associated in systemic RNAi in nematodes (Tomoyasu et al, 2008).…”

Uptake and impact of natural diet-derived small RNA in invertebrates: Implications for ecology and agriculture

“…However, the importance of the endocytic pathway has been shown in the oriental fruit fly 88 and both SID-like proteins and endocytosis are important in the dietary uptake of sRNA in in the Colorado potato beetle. 89 These data suggest that endocytosis may be a common mechanism for sRNA uptake by the cells of the digestive tract.…”

“…However, as pointed out in the recent study by Capelle et al, both pathways have rarely been examined in a given insect species making comprehensive comparison difficult. 89 In the tick and the desert locust, the scavenger receptor has been implicated in endocytosis-mediated spread, 138,139 which may indicate that this receptor is commonly used for this mechanism; however, other receptors yet to be discovered may also be employed for sRNA recognition. In addition, cells of different tissues within an organism can have different potential for uptake.…”