The Asian citrus psyllid (ACP), Diaphorina citri, is a major pest of citrus since it is the only known vector of ‘Candidatus Liberibactor’ species, the bacterium associated with citrus greening disease.  Since control of the psyllid is the only effective defense so far against citrus greening, and heavy reliance upon pesticides is not sustainable, an RNA interference (RNAi) strategy for ACP control was investigated. RNA interference is an innate immune response triggered by the cellular uptake of double stranded RNA (dsRNA) and studies were conducted to determine if ACP could be killed by oral uptake of dsRNA that target essential ACP transcripts. An artificial diet system was designed that facilitated the ingestion of dsRNA complementary to genes involved in digestion. Using this system, ACP mortality was observed as a result of oral uptake of dsRNAs targeting an apparent essential ACP gene and this mortality was shown to be dose responsive, reaching a maximum of 37% at high dsRNA concentrations.  There was also what appeared to be sequence independent ACP toxicity of large doses of dsRNA, concentrations above 48 ng/uL in the diet.  However, this sequence independent mortality was not as high as that observed for the targeted ACP gene, never rising above 17%.   These results provide support for the concept that RNAi could be adapted for use as a control strategy for the ACP.

Diaphorina citri Kuwayama, the Asian citrus psyllid (ACP), is a devastating citrus pest due to its transmission of a phloem-limited bacterial pathogen, Candidatus Liberibacter asiaticus, that causes citrus greening. Psyllid control is a major part of effective greening disease management, and our research targets perturbation of insect juvenile hormone metabolism as a new psyllid control strategy.  Previous studies have shown that application of a juvenile hormone (JH) analogue, pyriproxyfen, is known to produce ovicidal/nymphicidal effects, morphological abnormalities, and reduced fecundity in ACP adults. These observations prompted us to identify JH biosynthetic and degradative pathways as targets for biologically-based control strategies, including RNA interference, as alternatives to heavy reliance on broad-spectrum pesticides. First, candidate genes/cDNAs encoding the JH metabolic enzymes, juvenile hormone acid methyl transferase (JHAMT) and juvenile hormone esterase, were identified through computational analysis of the D. citri genome. Second, JHAMT cDNA was cloned, expressed in E. coli and a functional protein was purified. This JHAMT had a high affinity for substrates leading to JHI and JHIII synthesis, making it plausible that both juvenoids are present in the ACP. Results are discussed with respect to mechanism(s) of juvenile hormone biosynthesis/catabolism in the ACP and targeting this process as an interdiction point for a bio-rational ACP control strategy.

Citrus greening disease is a serious bacterial disease of citrus worldwide and is vectored by the Asian citrus pysllid (Diaphorina Citri).  The only effective control strategy includes vigorous control of the psyllid, primarily through heavy reliance on pesticides. As a more sustainable and environmentally friendly method of psyllid control, we evaluated a RNA interference (RNAi) approach based on psyllid oral uptake of dsRNA molecules that target specific psyllid genes.  This approach is based on the finding that cellular uptake of dsRNAs, that match the sequence of essential genes, results in down regulation of those genes and can lead to cell/organism death.  These dsRNA molecules were introduced into the psyllids through feeding on citrus engineered to express the dsRNA using a Citrus tristeza virus as a paratransgenesis vector.  Increased toxicity was observed when adult psyllids were fed on citrus producing dsRNA targeting either gut protease genes.  No increased psyllid toxicity was observed in psyllids fed on citrus producing green fluorescent protein (GFP) dsRNA.  Toxicity related to specific psyllid gene knockout will be discussed.  These results suggest that RNAi-based control may be a viable alternative to current pesticide use for control of psyllids and all phloem feeding pests.