Table 2.

Practical considerations when designing eRNAi‐based SIT strategies

eRNAi is dose‐dependent.	The potency of gene silencing correlates with dsRNA concentration and period of exposure (Zhou et al., 2008; Tian et al., 2009, 2015; Singh et al., 2013; Asokan et al., 2014; Yu et al., 2014; Li et al., 2015b; Ulrich et al., 2015; Whyard et al., 2015; Rebijith et al., 2016). A period of latency between dsRNA administration and gene silencing is common in larvae and adults, which is consistent with a threshold effect. Reported latency periods include: 12 h in Helicoverpa armigera (Tian et al., 2015) and Aedes aegypti (Coy et al., 2012), 24 h in Mamestra configurata (Toprak et al., 2013) and Bactrocera dorsalis (Zheng et al., 2015), 7 days in Spodoptera exigua (Tian et al., 2009), and 12 days in Solenopsis invicta (Choi et al., 2012). It is of note that Choi et al. (2012) did not feed dsRNA to insects directly but via a secondary worker individual, and this may have diluted dsRNA somewhat leading to an extended latency period.
Taxonomy cannot be used to reliably predict sensitivity to eRNAi or the latency period between dsRNA uptake and gene silencing.	Equivalent eRNAi methods can produce disparate results even when different biotypes (Li et al., 2015a), or subpopulations (Sugahara et al., 2017) of the same species are targeted. Within the Lepidoptera, H. armigera is capable of eliciting a robust eRNAi response (Xiong et al., 2013), but Spodoptera frugiperda is recalcitrant to eRNAi (Ivashuta et al., 2015). Working on hemipteran and dipteran models, Coleman et al. (2015) and Yi et al. (2014) observed a 4‐day period of latency between dsRNA administration and gene silencing. Another dipteran (A. aegypti) exhibited gene silencing after 12 h of feeding with dsRNA in solution (Coy et al., 2012). These discrepancies may be eliminated if equivalent feeding protocols are used.
The sensitivity of a gene to RNAi has not been fully assessed unless the entire mRNA molecule has been targeted for knockdown.	When all variables remain constant, variation in RNAi potency is likely to be due to regional susceptibility of mRNAs to cleavage. The strength of gene knockdown can vary greatly when multiple genes are targeted in the same insect using identical methods (Pridgeon et al., 2008; Li et al., 2011; Singh et al., 2013; Toprak et al., 2013; Killiny et al., 2014; Taracena et al., 2015).
Insects may become more tolerant to dsRNA with aging.	A diminution in the efficiency of RNAi with age has been suggested by Tian et al. (2015) and is supported by evidence that silencing appears more efficient in neonates than in late stage larvae (Zhu et al., 2011; Toprak et al., 2013). Furthermore, Coleman et al.'s (2015) observation that silencing is longer lived in nymphs than in adults suggests that fully developed insects are less sensitive to dsRNA.
Optimum eRNAi delivery methods must be determined by trial and error in most insect species.	Yang & Han (2014) found that feeding H. armigera with transgenic bacteria induced more efficient eRNAi than feeding with naked dsRNA. However, naked dsRNA elicited more robust gene silencing than did bacterial feeding in B. dorsalis (Li et al., 2011). Zhu et al. (2011) report that three of five genes were knocked down more efficiently using a bacterial system in Leptinotarsa decemlineata, but that the remaining two were more efficiently silenced by naked dsRNA.
When inducing eRNAi the capacity for systemic RNAi is critical if target genes lie beyond gut tissue.	The systemic RNAi capacity of various insects has been assessed by targeting chitin synthase genes specific to the exoskeleton (Tian et al., 2009; Zhang et al., 2010; Singh et al., 2013). Other examples of studies that targeted genes distal to gut tissue include the silencing of ebony in Diabrotica virgifera vigifera (Miyata et al., 2014) and Rhodnius heme binding protein (RHBP) in Rhodnius prolixus (Taracena et al., 2015). Ivashuta et al. (2015) suggest that systemic RNAi in D. v. virgifera is facilitated by transport of dsRNAs of >60 bp long.
Parental RNAi requires further analysis to determine whether it can be effective for SIT.	A robust systemic RNAi response enables the silencing of genes in germ cells (parental RNAi; pRNAi). When germ cells are affected by pRNAi, gene expression can be limited in zygotes and developing insects (Zwier et al., 2012; Paim et al., 2013; Coleman et al., 2015; Khajuria et al., 2015). A simple application of pRNAi in pest management would be to reduce future insect populations via embryonic lethal gene silencing (Khajuria et al., 2015). For use with SIT, sexual differentiation genes could be targeted in the mothers of target insects (Shukla & Palli, 2012). dsRNA delivery methods may drastically affect the potency of pRNAi. Zheng et al. (2015) report that eRNAi silencing of sex peptide receptor in B. dorsalis limited eclosion rates of their progeny, whereas Peng et al. (2015) describe that silencing the transformer gene by microinjection in this species has no effect on progeny. The disparity in pRNAi efficiency between these delivery methods might be due to the fact that eRNAi would have consistently supplied flies with dsRNA during the development of germ cells, whereas expression of transformer would have been only transiently reduced by microinjection.
Insects may become less sensitive to dsRNA over time.	Working with B. dorsalis, Li et al. (2015b) demonstrated that eRNAi potency was reduced following a series of exposures to dsRNA. The effect was dose‐dependent, was not gene specific, and lasted for up to 20 days following primary exposure. Refractoriness only occurred following targeting of endogenous genes, which suggests a role in immune priming. Li et al. (2015b) suggest that flies may become refractory to dsRNA when genes that mediate endocytosis are downregulated. Bactrocera dorsalis has also been reported to upregulate the expression of target genes following exposure to dsRNA (Li et al., 2011). Therefore, reduced eRNAi potency may be due to synergistic overexpression of target genes along with downregulation of endocytic mediators.
Endocytic pathways and SID transport proteins may work synergistically in eRNAi.	Both endocytosis and SID mediated dsRNA transport facilitate eRNAi in L. decemlineata (Cappelle et al., 2016).
Insects may become more sensitive to eRNAi if dsRNA is vectored in nanoparticles. The performance of various nanoparticle technologies for use with RNAi is reviewed in Liao et al. (2016).	Nanoparticle‐based delivery of dsRNA may serve dual purposes: (1) enhancing passage of dsRNA across the gut, and (2) prolonging the effect of RNAi via slow release of dsRNA. Whyard et al. (2009) utilized Lipofectamine 2000 and Cellfectin liposomal nanoparticles to successfully vector dsRNA to Drosophila melanogaster, even though this species is reported as eRNAi incompetent. Liposomal vectoring achieved ca. 50% gene knockdown. Recently, Drosophila suzukii (Matsumura) has also been reported as recalcitrant to feeding with naked dsRNA, but sensitive if molecules are vectored in liposomes (Taning et al., 2016). Chitosan nanoparticles can vector molecules to Anopheles gambiae and A. aegypti (Zhang et al., 2010, 2015b; Mysore et al., 2015), although mosquito larvae (Figueira‐Mansur et al., 2013; Singh et al., 2013; Whyard et al., 2015) and adults (Pridgeon et al., 2008; Coy et al., 2012) also demonstrate eRNAi when exposed to naked dsRNA. Potent eRNAi has been demonstrated in A. aegypti larvae using carbon quantum dot (CQD) nanoparticles (Das et al., 2015).
Data regarding the simultaneous knockdown of genes via administration of multiple dsRNAs are conflicting.	Simultaneous silencing of genes has been reported to enhance the potency of RNAi in A. aegypti using a bacterial feeding approach (Whyard et al., 2015). A plant feeding study of Myzus persicae and Bactericera cockerelli also suggested that targeting genes simultaneously may induce a synergistic effect (Tzin et al., 2015). Zhang et al. (2015a) and Ulrich et al. (2015) report simultaneous silencing in coleopteran models actually dilutes the potency of RNAi. When feeding combinations of dsRNAs to D. suzukii, Taning et al. (2016) found the potency of RNAi was enhanced for some target gene combinations but not others.