Table 2. Comparison of RNAi efficiency among ECB developmental stages when various delivery methods and RNAi-enhancing reagents were employed.

Unfed larvae were starved for 24 h prior to dsRNA delivery. Fed larvae were allowed to feed normally (i.e., not starved) prior to dsRNA delivery. Non-feeding stages do not feed as part of their natural developmental cycle.

RNAi Bioassays
Delivery method	Stage	Feeding status	Reagent	Significant suppression of OnLgl	Phenotype(s)	Results
Oral	First-instar larvae	Unfed	50 mM Zn2+	No	None	Fig. S-10
			6 mM EDTA	No	EDTA reduced weight by 1.7-fold at 6 d (6 mM vs 0 mM)	Figs. S-7, S-8
			1% FB28	No	FB28 reduced weight by 2.7-fold at 6 d (1% vs 0%)	Figs. S-12, S-13
			Lipo	No	None	Fig. S-11
	Second-instar larvae	Fed	10 & 20 mM Zn2+	68%, or 3.2-fold decrease at 6 d (dsOnLgl vs dsGFP)	None	Figs. 2, S-9
Injection	Second-instar larvae	Fed	10 & 20 mM Zn2+	No	None	Fig. S-21
	Third-instar larvae	Fed	25 mM Zn2+	32%, or 1.5-fold decrease at 3 d (Zn2+ dsOnLgl vs Zn2+ dsGFP)	None	Fig. 4, S-19
			50 mM Zn2+	No	None	Fig. S-20
			Meta	57%, or 2.3-fold decrease at 6 d (naked dsOnLgl vs naked dsGFP)	Meta decrease survivorship by 1.25-fold & weight by 1.24-fold at 6 d (Meta dsRNA vs naked dsRNA)	Figs. 3, S-14
	Wandering (fifth-instar) larvae	Nonfeeding	Meta	No*	None	Fig. S-15
	Pupae	Nonfeeding	Lipo	No	None	Fig. S-18
			Meta	No	None	Fig. S-16
	Adults	Unfed	Meta	No, 1.4-fold increase at 3 d (dsOnLgl vs dsGFP)	None	Fig. S-17

^*Significant differences were detected between some treatment groups but could not be meaningfully interpreted. Abbreviations: mM, millimolar; Lipo, Lipofectamine RNAiMax; Meta, Metafectene Pro; EDTA; ethylenediaminetetraacetic acid; Zn²⁺, nuclease inhibitor; FB28, fluorescent brightener 28; h, hour; d, day.