Double-stranded DNA reduces dsRNA degradation in the saliva and significantly enhanced RNAi-mediated gene silencing in Halyomorpha halys

Abstract

The invasive pest Halyomorpha halys (Hemiptera: Pentatomidae) poses a significant threat to agriculture, necessitating effective control methods beyond chemical pesticides. Our research explores RNA interference (RNAi) as a targeted gene silencing approach for H. halys population management. However, the variable efficacy of RNAi across insect orders, particularly in hemipteran insects like H. halys, poses challenges. Ex vivo degradation assays revealed rapid degradation of double-stranded RNA (dsRNA) in H. halys (Hh) saliva and extracts of salivary glands across several growth stages, attributed to the high expression of the DNA/RNA non-specific nuclease (HhNSE). A key discovery from our research was that double-stranded DNA (dsDNA) can act as a protective agent, increasing the stability of dsRNA in saliva probably by competitive inhibition of HhNSE. Based on the well-established lethality of silencing the gene encoding the heavy chain of clathrin (HhCHC) in insects, this gene was chosen as a target to test the functionality of our dsDNA-based formulation for enhancing dsRNA-mediated gene silencing. Our in-vivo experiments showed increased HhCHC silencing after 72 hours of feeding initiation with a mixture of dsRNA-CHC and dsDNA, as opposed to dsRNA alone. This discovery indicates potential for enhancing the efficiency of orally delivered dsRNA through formulations based on dsDNA. Although the injection of dsRNA-CHC resulted in near-total mortality, the dsDNA formulation did not significantly enhance mortality rates when fed together with dsRNA-CHC. This emphasises the necessity for further investigation into additional factors beyond nuclease activity, such as the understanding of dsRNA uptake and release mechanisms within the gut epithelial cells of H. halys. Nevertheless, our study opens avenues for developing cost-effective formulations to enhance RNAi efficacy in H. halys and perhaps other insects where nucleases hinder dsRNA delivery, representing a promising solution for sustainable pest control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/617619v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1517e13org.highwire.dtl.DTLVardef@12c4977org.highwire.dtl.DTLVardef@8b243borg.highwire.dtl.DTLVardef@f79eca_HPS_FORMAT_FIGEXP M_FIG C_FIG