vATPase-A gene silencing through orally delivered dsRNA in Asian citrus psyllid, Diaphorina citri

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a damaging, invasive pest of citrus and the vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the putative causal agent of citrus greening disease. RNA-interference (RNAi) is a lucrative biopesticidal solution for controlling pest insects and vectors of plant pathogens. In particular, 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 or ‘penetrance’ of RNAi in a target insect depends on several parameters, including the concentration (dose) of dsRNA ingested. Experiments were conducted to determine the optimal dsRNA concentration required to knockdown gene expression in D. citri nymphs and adult, 3-days post-dsRNA ingestion-access period (IAP). Three dsRNA concentrations, 100, 200 and 500 ng/ul, were tested for knockdown of vacuolar-ATPase-A (vATPase) gene expression. Adult and nymphs of D. citri were given a 48-hr ingestion-access period (IAP) on dsRNA presented in a 20% sucrose solution. Post-IAP, psyllids were transferred to the young shoots of Murraya paniculate L. (Jack) plants for a 5-day feeding-access period. Significant knockdown (P < 0.05) of vATPase-A gene expression ranged from 22-25% for adults and 22-40% for nymphs, over the range of 100-500 ng/ul dsRNA, compared to the non-target control dsRNA, luciferase. Based on the qPCR results, dsRNA reduced gene expression in both adult and nymphs, with nymphs exhibiting greater knockdown. dsRNA knockdown of vATPase-A gene expression was more pronounced at the two high doses in both life stage.