Optimizing dsRNA concentration for gene silencing in Asian citrus psyllid, Diaphorina citri

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an important invasive pest of citrus and vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease. RNA interference (RNAi) is a powerful tool for managing insect pests. There is great interest in the potential of dsRNA biopesticides to control D. citri infestations in citrus to reduce the spread of CLas. The efficiency of RNAi, or optimal gene silencing, in a target insect depends on several parameters, including the concentration of dsRNA that is ingested. The objective of this study was to determine the optimal dsRNA concentration required to knock down gene expression in adult D. citri, by 3-days post-dsRNA ingestion. Four concentrations of dsRNA, 10, 50, 100 and 200 ng. μL-1, were tested for knockdown of Clathrin heavy chain (CHC) gene expression. The dsRNA was delivered to adult D. citri in a 20% sucrose solution, given a 48-hr ingestion-access. Post-ingestion, surviving psyllids were transferred to the new shoots of orange jasmine (Murraya paniculata) plants for a 3-day incubation period. The efficiency of CHC silencing was positively correlated with dsRNA concentration, with 100 and 200 ng/μL-1 giving the highest knockdown (P > 0.05) in CHC gene expression averaging 40% and 44%, respectively,  compared with non-target dsRNA_Luciferase control. No significant difference observed in ACP mortality with non-target dsRNA_Luciferase compared to water as negative control. Among the four concentrations tested, 100 ng / μL-1, the dsRNA treatment resulted in optimal RNAi for CHC in D. citri adults.